Scientific Bibliography

Nanobiology and Cancer Nanotechnology
Nanotechnology Devices and Nanomaterials
Nanotechnology-Enabled Therapeutics Development and Delivery
Cancer Diagnostics and Biosensors
Nanotechnologies in Advanced Imaging
Environment, Health and Safety

Nanotechnology - Enabled Therapeutics Development and Delivery

2007   2006   2005   2004   2003   2002

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2007

Carbon nanotube-enhanced thermal destruction of cancer cells in a noninvasive radiofrequency field.
Gannon CJ, Cherukuri P, Yakobson BI, Cognet L, Kanzius JS, Kittrell C, Weisman RB, Pasquali M, Schmidt HK, Smalley RE, Curley SA.
Eur J Pharm Sci. 2007 Nov;32(3):159-68.
[ expand abstract ]

BACKGROUND.: Single-walled carbon nanotubes (SWNTs) have remarkable physicochemical properties that may have several medical applications. The authors have discovered a novel property of SWNTs-heat release in a radiofrequency (RF) field-that they hypothesized may be used to produce thermal cytotoxicity in malignant cells. METHODS.: Functionalized, water-soluble SWNTs were exposed to a noninvasive, 13.56-megahertz RF field, and heating characteristics were measured with infrared thermography. Three human cancer cell lines were incubated with various concentrations of SWNTs and then treated in the RF field. Cytotoxicity was measured by fluorescence-activated cell sorting. Hepatic VX2 tumors in rabbits were injected with SWNTs or with control solutions and were treated in the RF field. Tumors were harvested 48 hours later to assess viability. RESULTS.: The RF field induced efficient heating of aqueous suspensions of SWNTs. This phenomenon was used to produce a noninvasive, selective, and SWNT concentration-dependent thermal destruction in vitro of human cancer cells that contained internalized SWNTs. Direct intratumoral injection of SWNTs in vivo followed by immediate RF field treatment was tolerated well by rabbits bearing hepatic VX2 tumors. At 48 hours, all SWNT-treated tumors demonstrated complete necrosis, whereas control tumors that were treated with RF without SWNTs remained completely viable. Tumors that were injected with SWNTs but were not treated with RF also were viable. CONCLUSIONS.: The current results suggested that SWNTs targeted to cancer cells may allow noninvasive RF field treatments to produce lethal thermal injury to the malignant cells. Now, the authors are developing SWNTs coupled with cancer cell-targeting agents to enhance SWNT uptake by cancer cells while limiting uptake by normal cells.

Enhanced cytotoxicity of monoclonal anticancer antibody 2C5-modified doxorubicin-loaded PEGylated liposomes against various tumor cell lines.
Elbayoumi TA, Torchilin VP.
Eur J Pharm Sci. 2007 Nov;32(3):159-68.
[ expand abstract ]

Doxorubicin-loaded long-circulating liposomes (Doxil, ALZA Corp.) were additionally modified with the nucleosome-specific monoclonal antibody 2C5 (mAb 2C5) recognizing a broad variety of tumor cells via the tumor cell surface-bound nucleosomes. These mAb 2C5-modified PEGylated liposomes demonstrated 3-8-fold increase in the in vitro binding and internalization by multiple cancer cell lines of diverse origins (murine LLC, 4T1, C26 and human BT-20, MCF-7, and PC3), as shown by flow cytometry (FACS) and epi and confocal microscopy. As a result, mAb 2C5-modified Doxil demonstrated significantly higher cytotoxicity towards various cancer cells, including those resistant to doxorubicin, than all control preparations. The specific internalization of the mAb 2C5-Doxil into cytosol, along with the nuclear localization of their drug load, inside the target cancer cells were mainly responsible the superior anticancer activity. The IC50 values of mAb 2C5-Doxil with various murine and human cancer cells were 5-8-fold lower than those of control doxorubicin-loaded liposomes, Doxil or Doxil modified with a nonspecific IgG.

Polymer-caged lipsomes: a pH-responsive delivery system with high stability.
Lee SM, Chen H, Dettmer CM, O'Halloran TV, Nguyen ST.
J Am Chem Soc. 2007 Dec 12;129(49):15096-7.
[ expand abstract ]

Polymer-incorporated liposomes were prepared from preformed liposomes and a cholesterol-functionalized poly(acrylic acid) additive via a simple drop-in procedure. These modified liposomes possess surface-active carboxylate groups that can be cross-linked with telechelic 2,2'-(ethylenedioxy)bis(ethylamine) linkers, resulting in polymer-caged liposomes (PCLs) that are highly stable and have tunable pH-sensitive responses.

Microfluidic self-assembly of tumor spheroids for anticancer drug discovery.
Wu LY, Di Carlo D, Lee LP.
Biomed Microdevices. 2007 Oct 27; [Epub ahead of print].
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Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since it may provide a better model than monolayer culture of in vivo tumors. Moreover, continuous dynamic perfusion allows the establishment of physiologically relevant drug profiles to exposed spheroids. Here we present a physiologically inspired design allowing microfluidic self-assembly of spheroids, formation of uniform spheroid arrays, and characterizations of spheroid dynamics all in one platform. Our microfluidic device is based on hydrodynamic trapping of cancer cells in controlled geometries and the formation of spheroids is enhanced by maintaining compact groups of the trapped cells due to continuous perfusion. It was found that spheroid formation speed (average of 7 h) and size uniformity increased with increased flow rate (up to 10 mul min(-1)). A large amount of tumor spheroids (7,500 spheroids per square centimeter) with a narrow size distribution (10 +/- 1 cells per spheroid) can be formed in the device to provide a good platform for anticancer drug assays.

A Cellular Trojan Horse for Delivery of Therapeutic Nanoparticles into Tumors.
Choi MR, Stanton-Maxey KJ, Stanley JK, Levin CS, Bardhan R, Akin D, Badve S, Sturgis J, Robinson JP, Bashir R, Halas NJ, Clare SE.
Nano Lett. 2007 Dec 12;7(12):3759-3765.
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Destruction of hypoxic regions within tumors, virtually inaccessible to cancer therapies, may well prevent malignant progression. The tumor's recruitment of monocytes into these regions may be exploited for nanoparticle-based delivery. Monocytes containing therapeutic nanoparticles could serve as "Trojan Horses" for nanoparticle transport into these tumor regions. Here we report the demonstration of several key steps toward this therapeutic strategy: phagocytosis of Au nanoshells, and photoinduced cell death of monocytes/macrophages as isolates and within tumor spheroids.

Temperature-sensitive poly(N-(2-hydroxypropyl)methacrylamide mono/dilactate)-coated liposomes for triggered contents release.
Paasonen L, Romberg B, Storm G, Yliperttula M, Urtti A, Hennink WE.
Bioconjug Chem. 2007 Nov-Dec;18(6):2131-6.
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We prepared thermosensitive poly( N-(2-hydroxypropyl)methacrylamide mono/dilactate) (pHPMA mono/dilactate) polymer and studied temperature-triggered contents release from polymer-coated liposomes. HPMA mono/dilactate polymer was synthesized with a cholesterol anchor suitable for incorporation in the liposomal bilayers and with a cloud point (CP) temperature of the polymer slightly above normal body temperature (42 degrees C). Dynamic light scattering (DLS) measurements showed that whereas the size of noncoated liposomes remained stable upon raising the temperature from 25 to 46 degrees C, polymer-coated liposomes aggregated around 43 degrees C. Also, noncoated liposomes loaded with calcein showed hardly any leakage of the fluorescent marker when heated to 46 degrees C. However, polymer-coated liposomes showed a high degree of temperature-triggered calcein release above the CP of the polymer. Likely, liposome aggregation and bilayer destabilization are triggered because of the precipitation of the thermosensitive polymer above its CP onto the liposomal bilayers, followed by permeabilization of the liposomal membrane. This study demonstrates that liposomes surface-modified with HPMA mono/dilactate copolymer are attractive systems for achieving temperature-triggered contents release.

Multifunctional poly(d,l-lactide-co-glycolide)/montmorillonite (PLGA/MMT) nanoparticles decorated by Trastuzumab for targeted chemotherapy of breast cancer.
Sun B, Ranganathan B, Feng SS.
Biomaterials. 2008 Feb;29(4):475-86.
[ expand abstract ]

This paper continued our earlier work on the poly(d,l-lactide-co-glycolide)/montmorillonite nanoparticles (PLGA/MMT NPs), which were further decorated by human epidermal growth factor receptor-2 (HER2) antibody Trastuzumab for targeted breast cancer chemotherapy with paclitaxel as a model anticancer drug. Such a NP system is multifunctional, which formulates anticancer drugs with no harmful adjuvant, reduces the side effects of the formulated anticancer drug, promotes synergistic therapeutic effects, and achieves targeted delivery of the therapy. The paclitaxel-loaded PLGA/MMT NPs were prepared by a modified solvent extraction/evaporation technique, which were then decorated with Trastuzumab. The effects of the surface decoration on particle size and size distribution, surface morphology, drug encapsulation efficiency, as well as the drug release kinetics, were investigated. The NP formulation exhibited a biphasic drug release with a moderate initial burst followed by a sustained release profile. The surface decoration speeded the drug release. Surface chemistry analysis was conducted by X-ray photoelectron spectroscopy (XPS), which confirmed the presence of Trastuzumab on the NP surface. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the stability of the antibody in the NP preparation process. Internalization of the coumarin-6-loaded PLGA/MMT NPs with or without the antibody decoration by both of Caco-2 colon adeno carcinoma cells and SK-BR-3 breast cancer cells was visualized by confocal laser scanning microscopy and quantitatively analyzed, which shows that the antibody decoration achieved significantly higher cellular uptake of the NPs. The results of in vitro cytotoxicity experiment on SK-BR-3 cells further proved the targeting effects of the antibody decoration. Judged by IC50 after 24h culture, the therapeutic effects of the drug formulated in the NPs with surface decoration could be 12.74 times higher than that of the bare NPs and 13.11 times higher than Taxol((R)).

Tumor-selective vessel occlusions by platelets after vascular targeting chemotherapy using paclitaxel encapsulated in cationic liposomes.
Strieth S, Nussbaum CF, Eichhorn ME, Fuhrmann M, Teifel M, Michaelis U, Berghaus A, Dellian M.
Int J Cancer. 2008 Jan 15;122(2):452-60.
[ expand abstract ]

Paclitaxel encapsulated in cationic liposomes (EndoTAG-1) significantly impairs tumor growth by a significant reduction of functional tumor microcirculation and induction of endothelial cell apoptosis. The aim of the study was to analyze whether platelet activation within the tumor microcirculation contributes to the antivascular effects of vascular targeting chemotherapy using EndoTAG-1. In vitro, FACS analysis revealed a significant activation of platelets upon treatment with EndoTAG-1. In vivo, using A-Mel-3 tumors in Syrian Golden hamsters equipped with dorsal skinfold chamber preparations, the contribution of platelets to the antivascular effects of EndoTAG-1 was evaluated by fluorescence and laser-scanning microscopy. Immediately after a single treatment with EndoTAG-1 or cationic liposomes devoid of paclitaxel, an increase of platelet adherence in tumor microvessels was observed. This was accompanied by an acute impairment of the microcirculation within the treated tumors leading to reduced tumor perfusion. After repetitive therapy, an increase of platelet adherence and subsequent tumor microvessel occlusions occurred only after treatment with EndoTAG-1. Comparing to "tumor free" normal tissue controls these microthromboses were tumor selective. Significantly disbalancing the coagulation system within tumors by targeted induction of microthromboses within the tumor microcirculation appears to be an important mechanism of EndoTAG-1 therapy.

Oligonucleotide Loading Determines Cellular Uptake of DNA-Modified Gold Nanoparticles.
Giljohann DA, Seferos DS, Patel PC, Millstone JE, Rosi NL, Mirkin CA.
Nano Lett. 2007 Dec;7(12):3818-21.
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The cellular internalization of oligonucleotide-modified nanoparticles is investigated. Uptake is dependent on the density of the oligonucleotide loading on the surface of the particles, where higher densities lead to greater uptake. Densely functionalized nanoparticles adsorb a large number of proteins on the nanoparticle surface. Nanoparticle uptake is greatest where a large number of proteins are associated with the particle.

Impact of tumor-specific targeting on the biodistribution and efficacy of siRNA nanoparticles measured by multimodality in vivo imaging.
Derek W. Bartlett, Helen Su, Isabel J. Hildebrandt, Wolfgang A. Weber, Mark E. Davis.
Proc Natl Acad Sci U S A. 2007 September 25; 104(39): 15549–15554.
[ expand abstract ]

Targeted delivery represents a promising approach for the development of safer and more effective therapeutics for oncology applications. Although macromolecules accumulate nonspecifically in tumors through the enhanced permeability and retention (EPR) effect, previous studies using nanoparticles to deliver chemotherapeutics or siRNA demonstrated that attachment of cell-specific targeting ligands to the surface of nanoparticles leads to enhanced potency relative to nontargeted formulations. Here, we use positron emission tomography (PET) and bioluminescent imaging to quantify the in vivo biodistribution and function of nanoparticles formed with cyclodextrin-containing polycations and siRNA. Conjugation of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid to the 5' end of the siRNA molecules allows labeling with 64Cu for PET imaging. Bioluminescent imaging of mice bearing luciferase-expressing Neuro2A s.c. tumors before and after PET imaging enables correlation of functional efficacy with biodistribution data. Although both nontargeted and transferrin-targeted siRNA nanoparticles exhibit similar biodistribution and tumor localization by PET, transferrin-targeted siRNA nanoparticles reduce tumor luciferase activity by ˜50% relative to nontargeted siRNA nanoparticles 1 d after injection. Compartmental modeling is used to show that the primary advantage of targeted nanoparticles is associated with processes involved in cellular uptake in tumor cells rather than overall tumor localization. Optimization of internalization may therefore be key for the development of effective nanoparticle-based targeted therapeutics.

Active nanodiamond hydrogels for chemotherapeutic delivery.
Huang H, Pierstorff E, Osawa E, Ho D.
Nano Lett. 2007 Nov;7(11):3305-14.
[ expand abstract ]

Nanodiamond materials can serve as highly versatile platforms for the controlled functionalization and delivery of a wide spectrum of therapeutic elements. In this work, doxorubicin hydrochloride (DOX), an apoptosis-inducing drug widely used in chemotherapy, was successfully applied toward the functionalization of nanodiamond materials (NDs, 2-8 nm) and introduced toward murine macrophages as well as human colorectal carcinoma cells with preserved efficacy. The adsorption of DOX onto the NDs and its reversible release were achieved by regulating Cl- ion concentration, and the NDs were found to be able to efficiently ferry the drug inside living cells. Comprehensive bioassays were performed to assess and confirm the innate biocompatibility of the NDs, via real-time quantitative polymerase chain reaction (RT-PCR), and electrophoretic DNA fragmentation as well as MTT analysis confirmed the functional apoptosis-inducing mechanisms driven by the DOX-functionalized NDs. We extended the applicability of the DOX-ND composites toward a translational context, where MTT assays were performed on the HT-29 colon cancer cell line to assess DOX-ND induced cell death and ND-mediated chemotherapeutic sequestering for potential slow/sustained released capabilities. These and other medically relevant capabilities enabled by the NDs forge its strong potential as a therapeutically significant nanomaterial.

Quantum dot-aptamer conjugates for synchronous cancer imaging, therapy, and sensing of drug delivery based on bi-fluorescence resonance energy transfer.
Bagalkot V, Zhang L, Levy-Nissenbaum E, Jon S, Kantoff PW, Langer R, Farokhzad OC.
Nano Lett. 2007 Oct;7(10):3065-70.
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We report a novel quantum dot (QD)-aptamer(Apt)-doxorubicin (Dox) conjugate [QD-Apt(Dox)] as a targeted cancer imaging, therapy, and sensing system. By functionalizing the surface of fluorescent QD with the A10 RNA aptamer, which recognizes the extracellular domain of the prostate specific membrane antigen (PSMA), we developed a targeted QD imaging system (QD-Apt) that is capable of differential uptake and imaging of prostate cancer cells that express the PSMA protein. The intercalation of Dox, a widely used antineoplastic anthracycline drug with fluorescent properties, in the double-stranded stem of the A10 aptamer results in a targeted QD-Apt(Dox) conjugate with reversible self-quenching properties based on a Bi-FRET mechanism. A donor-acceptor model fluorescence resonance energy transfer (FRET) between QD and Dox and a donor-quencher model FRET between Dox and aptamer result when Dox intercalated within the A10 aptamer. This simple multifunctional nanoparticle system can deliver Dox to the targeted prostate cancer cells and sense the delivery of Dox by activating the fluorescence of QD, which concurrently images the cancer cells. We demonstrate the specificity and sensitivity of this nanoparticle conjugate as a cancer imaging, therapy and sensing system in vitro.

Apoptotic epidermal growth factor (EGF)-conjugated block copolymer micelles as a nanotechnology platform for targeted combination therapy.
Lee H, Hu M, Reilly RM, Allen C.
Mol Pharm. 2007 Sep-Oct;4(5):769-81.
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The overexpression of epidermal growth factor receptor (EGFR) in human epithelial cancers has been associated with aggressive disease, poor patient prognosis, and a high incidence of metastases. In the present study, block copolymer micelles are conjugated with epidermal growth factor (EGF), which acts as both a targeting ligand for the drug carrier and an apoptotic factor against EGFR-overexpressing cancers. Drug-free EGF-conjugated micelles are shown to result in cell-cycle arrest at the G 1 phase and subsequent induction of cell-type-specific apoptosis in EGFR-overexpressing breast cancer cells as demonstrated by flow cytometric analysis. EGF delivered as EGF-conjugated micelles was found to be 13-fold more potent than free EGF; the IC 50 was decreased from 0.98 +/- 0.1 nM for free EGF to 0.076 +/- 0.01 nM for EGF micelles. The apoptotic micelles, however, are non-antiproliferative to cells expressing a low level of EGFR, suggesting that the apoptotic micelles have minimal or no toxicity against normal healthy tissues. Ellipticine, a chemotherapeutic agent, was loaded into the EGF-micelles after it had been shown, using the combination index-isobologram equation, to act synergistically with EGF. A 10-fold increase in EGF content in the ellipticine-loaded micelles lowered the IC 50 of ellipticine in EGFR-overexpressing breast cancer cells by more than 18-fold. The EGF-micelles have the potential to be further pursued as a versatile nanotechnology platform for targeted delivery of a wide range of chemotherapeutic agents as a combination therapy for the treatment of EGFR-overexpressing cancers.

Synthetic nano-LDL with paclitaxel oleate as a targeted drug delivery vehicle for glioblastoma multiforme.
Nikanjam M, Gibbs AR, Hunt CA, Budinger TF, Forte TM.
J Control Release. 2007 Dec 20;124(3):163-71.
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The low density lipoprotein (LDL) receptor has been shown to be upregulated in GBM tumor cells in vitro and is therefore a potential molecular target for the delivery of therapeutic agents. A synthetic nano-LDL (nLDL) particle was developed as a drug delivery vehicle targeted to GBM cells by incorporating a lipophilic prodrug, paclitaxel oleate, into the particle. Nano-LDL containing paclitaxel oleate (nLDL-PO) was constructed by combining a synthetic peptide containing a lipid binding motif and the LDL receptor (LDLR) binding domain of apolipoprotein B-100 with a lipid emulsion consisting of phosphatidyl choline, triolein, and paclitaxel oleate. Paclitaxel oleate incorporated into the core of the lipid particle. nLDL-PO cell survival in GBM cell lines was found to be time, concentration, and cell line dependent. Cell killing was observed with short drug incubations and exhibited saturation at 6 h. nLDL-PO cell survival improved in the presence of the LDL receptor inhibitor, suramin, demonstrating that the drug was delivered via the LDL receptor. Collectively, these data strongly suggest that the synthetic nano-LDLs can incorporate lipophilic drugs and are capable of killing GBM cells. nLDL-PO has the potential to serve as a selective drug delivery vehicle for targeting GBM tumors via the LDL receptor.

Radiosensitization of paclitaxel, etanidazole and paclitaxel+etanidazole nanoparticles on hypoxic human tumor cells in vitro.
Jin C, Bai L, Wu H, Tian F, Guo G.
Biomaterials. 2007 Sep;28(25):3724-30.
[ expand abstract ]

Paclitaxel and etanidazole are hypoxic radiosensitizers that exhibit cytotoxic action at different mechanisms. The poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles containing paclitaxel, etanidazole and paclitaxel+etanidazole were prepared by o/w and w/o/w emulsification-solvent evaporation method. The morphology of the nanoparticles was investigated by scanning electron microscope (SEM). The drug encapsulation efficiency (EE) and release profile in vitro were measured by high-performance liquid chromatography (HPLC). The cellular uptake of nanoparticles for the human breast carcinoma cells (MCF-7) and the human carcinoma cervicis cells (HeLa) was evaluated by transmission electronic microscopy and fluorescence microscopy. Cell viability was determined by the ability of single cell to form colonies in vitro. The prepared nanoparticles were spherical shape with size between 80 and 150 nm. The EE was higher for paclitaxel and lower for etanidazole. The drug release was controlled over time. The cellular uptake of nanoparticles was observed. Co-culture of the two tumor cell lines with drug-loaded nanoparticles demonstrated that released drug effectively sensitized hypoxic tumor cells to radiation. The radiosensitization of paclitaxel+etanidazole nanoparticles was more significant than that of single drug-loaded nanoparticles.

Enhanced retention of the alpha-particle-emitting daughters of Actinium-225 by liposome carriers.
Sofou S, Kappel BJ, Jaggi JS, McDevitt MR, Scheinberg DA, Sgouros G.
Bioconjug Chem. 2007 Nov-Dec;18(6):2061-7.
[ expand abstract ]

Targeted alpha-particle emitters hold great promise as therapeutics for micrometastatic disease. Because of their high energy deposition and short range, tumor targeted alpha-particles can result in high cancer-cell killing with minimal normal-tissue irradiation. Actinium-225 is a potential generator for alpha-particle therapy: it decays with a 10-day half-life and generates three alpha-particle-emitting daughters. Retention of (225)Ac daughters at the target increases efficacy; escape and distribution throughout the body increases toxicity. During circulation, molecular carriers conjugated to (225)Ac cannot retain any of the daughters. We previously proposed liposomal encapsulation of (225)Ac to retain the daughters, whose retention was shown to be liposome-size dependent. However, daughter retention was lower than expected: 22% of theoretical maximum decreasing to 14%, partially due to the binding of (225)Ac to the phospholipid membrane. In this study, Multivesicular liposomes (MUVELs) composed of different phospholipids were developed to increase daughter retention. MUVELs are large liposomes with entrapped smaller lipid-vesicles containing (225)Ac. PEGylated MUVELs stably retained over time 98% of encapsulated (225)Ac. Retention of (213)Bi, the last daughter, was 31% of the theoretical maximum retention of (213)Bi for the liposome sizes studied. MUVELs were conjugated to an anti-HER2/neu antibody (immunolabeled MUVELs) and were evaluated in vitro with SKOV3-NMP2 ovarian cancer cells, exhibiting significant cellular internalization (83%). This work demonstrates that immunolabeled MUVELs might be able to deliver higher fractions of generated alpha-particles per targeted (225)Ac compared to the relative fractions of alpha-particles delivered by (225)Ac-labeled molecular carriers.

Anti-CD166 single chain antibody-mediated intracellular delivery of liposomal drugs to prostate cancer cells.
Roth A, Drummond DC, Conrad F, Hayes ME, Kirpotin DB, Benz CC, Marks JD, Liu B.
Mol Cancer Ther. 2007 Oct;6(10):2737-46.
[ expand abstract ]

Targeted delivery of small-molecule drugs has the potential to enhance selective killing of tumor cells. We have identified previously an internalizing single chain [single chain variable fragment (scFv)] antibody that targets prostate cancer cells and identified the target antigen as CD166. We report here the development of immunoliposomes using this anti-CD166 scFv (H3). We studied the effects of a panel of intracellularly delivered, anti-CD166 immunoliposomal small-molecule drugs on prostate cancer cells. Immunoliposomal formulations of topotecan, vinorelbine, and doxorubicin each showed efficient and targeted uptake by three prostate cancer cell lines (Du-145, PC3, and LNCaP). H3-immunoliposomal topotecan was the most effective in cytotoxicity assays on all three tumor cell lines, showing improved cytotoxic activity compared with nontargeted liposomal topotecan. Other drugs such as liposomal doxorubicin were highly effective against LNCaP but not PC3 or Du-145 cells, despite efficient intracellular delivery. Post-internalization events thus modulate the overall efficacy of intracellularly delivered liposomal drugs, contributing in some cases to the lower than expected activity in a cell line-dependent manner. Further studies on intracellular tracking of endocytosed liposomal drugs will help identify and overcome the barriers limiting the potency of liposomal drugs.

Gold Nanorods mediate tumor cell death by compromising membrane integrity.
L. Tong, Y. Zhao, T. B. Huff, M. N. Hansen, A. Wei, J.X. Cheng.
Adv Materials. 2007;19(20):3136 – 3141.
[ expand abstract ]

No abstract available (Citation link)

Bio-nano-informatics: an integrated information management system for personalized oncology.
Stokes TH, Phan J, Quo CF, Nie S, Wang MD.
Conf Proc IEEE Eng Med Biol Soc. 2006;1:3325-8.
[ expand abstract ]

The Emory-Georgia Tech Nanotechnology Center for Personalized and Predictive Oncology is one of the eight national centers funded by the National Cancer Institute (NCI). Its overall goal is to combine nanotechnology and biocomputing with clinical oncology for personalized detection, diagnosis and treatment of human cancer. Within this large-scale and multifaceted center, a key challenge is how to integrate and manage data and resources. Here we have developed an "intelligent" information system for data management, interpretation, and for translation of new results to clinical applications.

Multifunctional poly(d,l-lactide-co-glycolide)/montmorillonite (PLGA/MMT) nanoparticles decorated by Trastuzumab for targeted chemotherapy of breast cancer.
Sun B, Ranganathan B, Feng SS.
Biomaterials. 2008 Feb;29(4):475-86.
[ expand abstract ]

This paper continued our earlier work on the poly(d,l-lactide-co-glycolide)/montmorillonite nanoparticles (PLGA/MMT NPs), which were further decorated by human epidermal growth factor receptor-2 (HER2) antibody Trastuzumab for targeted breast cancer chemotherapy with paclitaxel as a model anticancer drug. Such a NP system is multifunctional, which formulates anticancer drugs with no harmful adjuvant, reduces the side effects of the formulated anticancer drug, promotes synergistic therapeutic effects, and achieves targeted delivery of the therapy. The paclitaxel-loaded PLGA/MMT NPs were prepared by a modified solvent extraction/evaporation technique, which were then decorated with Trastuzumab. The effects of the surface decoration on particle size and size distribution, surface morphology, drug encapsulation efficiency, as well as the drug release kinetics, were investigated. The NP formulation exhibited a biphasic drug release with a moderate initial burst followed by a sustained release profile. The surface decoration speeded the drug release. Surface chemistry analysis was conducted by X-ray photoelectron spectroscopy (XPS), which confirmed the presence of Trastuzumab on the NP surface. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the stability of the antibody in the NP preparation process. Internalization of the coumarin-6-loaded PLGA/MMT NPs with or without the antibody decoration by both of Caco-2 colon adeno carcinoma cells and SK-BR-3 breast cancer cells was visualized by confocal laser scanning microscopy and quantitatively analyzed, which shows that the antibody decoration achieved significantly higher cellular uptake of the NPs. The results of in vitro cytotoxicity experiment on SK-BR-3 cells further proved the targeting effects of the antibody decoration. Judged by IC50 after 24h culture, the therapeutic effects of the drug formulated in the NPs with surface decoration could be 12.74 times higher than that of the bare NPs and 13.11 times higher than Taxol((R)).

Organically modified silica nanoparticles with covalently incorporated photosensitizer for photodynamic therapy of cancer.
Ohulchanskyy TY, Roy I, Goswami LN, Chen Y, Bergey EJ, Pandey RK, Oseroff AR, Prasad PN.
Nano Lett. 2007 Sep;7(9):2835-42.
[ expand abstract ]

We report a novel nanoformulation of a photosensitizer (PS), for photodynamic therapy (PDT) of cancer, where the PS molecules are covalently incorporated into organically modified silica (ORMOSIL) nanoparticles. We found that the covalently incorporated PS molecules retained their spectroscopic and functional properties and could robustly generate cytotoxic singlet oxygen molecules upon photoirradiation. The synthesized nanoparticles are of ultralow size ( approximately 20 nm) and are highly monodispersed and stable in aqueous suspension. The advantage offered by this covalently linked nanofabrication is that the drug is not released during systemic circulation, which is often a problem with physical encapsulation. These nanoparticles are also avidly uptaken by tumor cells in vitro and demonstrate phototoxic action, thereby highlighting their potential in diagnosis and PDT of cancer.

Tumour cell toxicity of intracellular hyperthermia mediated by magnetic nanoparticles.
Wilhelm C, Fortin JP, Gazeau F.
J Nanosci Nanotechnol. 2007 Aug;7(8):2933-7.
[ expand abstract ]

Intracellular hyperthermia is a process by which malignant cells can be selectively killed by heat generated by nanomediators located inside the cell. Here we show that maghemite anionic nanoparticles are efficiently captured by human prostatic tumor cells (PC3) and concentrate within intracellular vesicles. When submitted to an alternative magnetic field, maghemite nanocrystals generate heat from the cell inside, inducing a temperature elevation of eight degree in a loose pellet of 20 million magnetically labeled cells. We demonstrate that this heating modality was as lethal as external waterbath heating. A one hour AC magnetic field (700 kHz-31 mT) exposure of the magnetically labeled cells killed 44% of the cells. Interestingly, more than 80% of the cells were killed after being submitted twice to the magnetic field. Finally, when magnetic cells coexist with non magnetic ones, the same proportions of cells were damaged for both populations, after magnetic field exposure. These findings pave the way for an efficient cell killing mediated by intracellular magnetic hyperthermia.

Dendrimer-modified magnetic nanoparticles enhance efficiency of gene delivery system.
Pan B, Cui D, Sheng Y, Ozkan C, Gao F, He R, Li Q, Xu P, Huang T.
Cancer Res. 2007 Sep 1;67(17):8156-63.
[ expand abstract ]

Magnetic nanoparticles (MNP) with a diameter of 8 nm were modified with different generations of polyamidoamine (PAMAM) dendrimers and mixed with antisense survivin oligodeoxynucleotide (asODN). The MNP then formed asODN-dendrimer-MNP composites, which we incubated with human tumor cell lines such as human breast cancer MCF-7, MDA-MB-435, and liver cancer HepG2 and then analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, quantitative reverse transcription-PCR, Western blotting, laser confocal microscopy, and high-resolution transmission electron microscopy. Results showed that the asODN-dendrimer-MNP composites were successfully synthesized, can enter into tumor cells within 15 min, caused marked down-regulation of the survivin gene and protein, and inhibited cell growth in dose- and time-dependent means. No.5 generation of asODN-dendrimer-MNP composites exhibits the highest efficiency for cellular transfection and inhibition. These results show that PAMAM dendrimer-modified MNPs may be a good gene delivery system and have potential applications in cancer therapy and molecular imaging diagnosis.

Synthesis and characterization of PAMAM dendrimer-based multifunctional nanodevices for targeting alphavbeta3 integrins.
Lesniak WG, Kariapper MS, Nair BM, Tan W, Hutson A, Balogh LP, Khan MK.
Bioconjug Chem. 2007 Jul-Aug;18(4):1148-54.
[ expand abstract ]

We have synthesized a stable and clinically relevant nanodevice (cRGD-BT-ND; ND for short) that exhibits superior binding to the biologic target alphavbeta3 integrins, when either compared to the same free cRGD peptide or to the biotinylated nanodevice without covalently attached peptides (BT-ND). Selective targeting of alphavbeta3 integrins was achieved by coupling cyclic cRGD peptides to the nanodevice (ND) surface, while biotin groups (BT) were used for amplified detection of bound cRGD-BT-ND by anti-biotin antibody or avidin linked to horseradish peroxidase after binding. The synthesis involved the following steps: the amino-terminated ethylenediamine core generation 5 poly(amidoamine) (PAMAM_E5.NH2) dendrimer was first partially acetylated and then biotinylated, and residual primary amine termini were converted to succinamic acid groups (SAH), some of which finally were conjugated with cRGD peptide residues through the amino group of the lysine side chain. The starting material and all derivatives were extensively characterized by polyacrylamide gel electrophoresis (PAGE), size exclusion chromatography (SEC), potentiometric acid-base titration, MALDI-TOF, and NMR. Cytotoxicity of all dendrimer derivatives was examined in B16F10 melanoma cell cultures using the XTT colorimetric assay for cellular viability. Binding of nanodevices to the biological target was determined using plates coated with human alphavbeta3 integrin and alphavbeta3 receptor expressing human dermal microvascular endothelial cells (HDMECs). The PAMAM_E5.(NHAc)72(NHBT)8(NHSAH)35(NHSA-cR GD)4 nanodevice is nontoxic within physiologic concentration ranges and specifically binds to the alphavbeta3 integrins, apparently much stronger than the cyclic cRGD peptide itself.

Paclitaxel-functionalized gold nanoparticles.
Gibson JD, Khanal BP, Zubarev ER.
J Am Chem Soc. 2007 Sep 19;129(37):11653-61.
[ expand abstract ]

Here we describe the first example of 2 nm gold nanoparticles (Au NPs) covalently functionalized with a chemotherapeutic drug, paclitaxel. The synthetic strategy involves the attachment of a flexible hexaethylene glycol linker at the C-7 position of paclitaxel followed by coupling of the resulting linear analogue to phenol-terminated gold nanocrystals. The reaction proceeds under mild esterification conditions and yields the product with a high molecular weight, while exhibiting an extremely low polydispersity index (1.02, relative to linear polystyrene standards). TGA analysis of the hybrid nanoparticles reveals the content of the covalently attached organic shell as nearly 67% by weight, which corresponds to approximately 70 molecules of paclitaxel per 1 nanoparticle. The presence of a paclitaxel shell with a high grafting density renders the product soluble in organic solvents and allows for detailed (1)H NMR analysis and, therefore, definitive confirmation of its chemical structure. High-resolution TEM was employed for direct visualization of the inorganic core of hybrid nanoparticles, which were found to retain their average size, shape, and high crystallinity after multiple synthetic steps and purifications. The interparticle distance substantially increases after the attachment of paclitaxel as revealed by low-magnification TEM, suggesting the presence of a larger organic shell. The method described here demonstrates that organic molecules with exceedingly complex structures can be covalently attached to gold nanocrystals in a controlled manner and fully characterized by traditional analytical techniques. In addition, this approach gives a rare opportunity to prepare hybrid particles with a well-defined amount of drug and offers a new alternative for the design of nanosized drug-delivery systems.

Differential tumor cell targeting of anti-HER2 (Herceptin) and anti-CD20 (Mabthera) coupled nanoparticles.
Cirstoiu-Hapca A, Bossy-Nobs L, Buchegger F, Gurny R, Delie F.
Int J Pharm. 2007 Mar 1;331(2):190-6.
[ expand abstract ]

Two types of antibody-labeled nanoparticles (mAb-NPs) were prepared with the aim to achieve specific tumor targeting. Anti-HER2 and anti-CD20 monoclonal antibodies (mAb) were used as model ligands. Small poly(dl-lactic acid) nanoparticles (PLA NPs) with a mean size of about 170 nm were prepared by the salting out method. Thereafter, the coating of PLA NPs with mAbs was performed in two steps. First, thiol groups (-SH) were introduced on the surface of PLA-NPs by a two-step carbodiimide reaction. The number of -SH groups on the surface of NPs increased from 150 to 400 mmol-SH/mol PLA when cystamine concentrations of 25-1518 mol cystamine/mol PLA were used during the thiolation reaction. In the second step, covalent coupling of antibodies to thiolated NPs (NPs-SH) was obtained via a bifunctional cross-linker, m-maleimidobenzoyl-N-hydroxy-sulfosuccinimide ester (sulfo-MBS). For both mAbs anti-HER2 and anti-CD20, respectively, the number of -SH functions on the NPs had no influence on the amount of mAb coupled to the NPs. Approximately, 295 anti-HER2 and 557 anti-CD20 molecules, respectively, were covalently coupled per nanoparticle. The NPs size after the coupling reactions was about 250 nm. The specific interaction between tumor cells and mAb-NPs was determined by confocal microscopy using two cell lines: SKOV-3 human ovarian cancer cells (overexpressing HER2) and Daudi lymphoma cells (overexpressing CD20). The results showed the selective targeting of mAb-NPs to tumor cells overexpressing the specific antigen. While anti-CD20 labeled NPs (anti-CD20 NPs) bound to and remained at the cellular surface, anti-HER2 labeled NPs (anti-HER2 NPs) were efficiently internalized. The mAb-NPs represent a promising approach to improve the efficacy of NPs in active targeting for cancer therapy while the choice of the antibody-target system defines the fate of the mAb-NPs after their binding to the cells.

Synthesis and antibody conjugation of magnetic nanoparticles with improved specific power absorption rates for alternating magnetic field cancer therapy
Grüttnera C, Müllera K, Tellera J, Westphala F, Foremanb A, Ivkovb R.
J Magn Magn Mater. 2007 Apr; 311(1):181-186.
[ expand abstract ]

Bionized nanoferrite (BNF) particles with high specific power absorption rates were synthesized in the size range of 20–100 nm by high-pressure homogenization for targeted cancer therapy with alternating magnetic fields. Several strategies were used to conjugate antibodies to the BNF particles. These strategies were compared using an immunoassay to find optimal conditions to reach a high immunoreactivity of the final antibody–particle conjugate.

Paclitaxel and ceramide co-administration in biodegradable polymeric nanoparticulate delivery system to overcome drug resistance in ovarian cancer.
Devalapally H, Duan Z, Seiden MV, Amiji MM.
Int J Cancer. 2007 Oct 15;121(8):1830-8.
[ expand abstract ]

The objective of this study was to overcome drug resistance upon systemic administration of combination paclitaxel (PTX) and the apoptotic signaling molecule C(6)-ceramide (CER) in biodegradable poly(ethylene oxide)-modified poly(epsilon-caprolactone (PEO-PCL) nanoparticles. Subcutaneous sensitive (wild-type) and multidrug resistant (MDR-1 positive) SKOV-3 human ovarian adenocarcinoma xenografts were established in female Nu/Nu mice. PTX and CER were administered intravenously either as a single agent or in combination in aqueous solution and in PEO-PCL nanoparticles to the tumor-bearing mice. There was significant (p< 0.05) tumor growth suppression in both wild-type SKOV-3 and multidrug resistant SKOV-3(TR) models upon single dose co-administration of PTX (20 mg/kg) and CER (100 mg/kg) in nanoparticle formulations as compared to the individual agents and administration in aqueous solutions. For instance, in SKOV-3 wild-type model, more than 4.3-fold increase (p < 0.05) in tumor growth delay and 3.6-fold (p < 0.05) increase in tumor volume doubling time (DT) were observed with the combination treatment in nanoparticles as compared to untreated animals. Similarly, 3-fold increase (p < 0.05) in tumor growth delay and tumor volume DT was observed in SKOV-3(TR) model. Body weight changes and blood cells counts were used as measures of safety and, except for an increase in platelet counts (p < 0.05) in PTX + CER treated animals, there was no difference between various treatment strategies. The results of this study show that combination of PTX and CER in biodegradable polymeric nanoparticles can serve as a very effective therapeutic strategy to overcome drug resistance in ovarian cancer.

Immunonanoparticles--an effective tool to impair harmful proteolysis in invasive breast tumor cells.
Obermajer N, Kocbek P, Repnik U, Kuznik A, Cegnar M, Kristl J, Kos J.
FEBS J. 2007 Sep;274(17):4416-27.
[ expand abstract ]

Breast cancer cells exhibit excessive proteolysis, which is responsible for extensive extracellular matrix degradation, invasion and metastasis. Besides other proteases, lysosomal cysteine protease cathepsin B has been implicated in these processes and the impairment of its intracellular activity was suggested to reduce harmful proteolysis and hence diminish progression of breast tumors. Here, we present an effective system composed of poly(D,L-lactide-coglycolide) nanoparticles, a specific anti-cytokeratin monoclonal IgG and cystatin, a potent protease inhibitor, that can neutralize the excessive intracellular proteolytic activity as well as invasive potential of breast tumor cells. The delivery system distinguishes between breast and other cells due to the monoclonal antibody specifically recognizing cytokeratines on the membrane of breast tumor cells. Bound nanoparticles are rapidly internalized by means of endocytosis releasing the inhibitor cargo within the lysosomes. This enables intracellular cathepsin B proteolytic activity to be inhibited, reducing the invasive and metastatic potential of tumor cells without affecting proteolytic functions in normal cells and processes. This approach may be applied for treatment of breast and other tumors in which intracellular proteolytic activity is a part of the process of malignant progression.

Synthesis, liposomal preparation, and in vitro toxicity of two novel dodecaborate cluster lipids for boron neutron capture therapy.
Justus E, Awad D, Hohnholt M, Schaffran T, Edwards K, Karlsson G, Damian L, Gabel D.
Bioconjug Chem. 2007 Jul-Aug;18(4):1287-93.
[ expand abstract ]

A new class of lipids, containing the closo-dodecaborate cluster, has been synthesized. Two lipids, S-(N, N-(2-dimyristoyloxyethyl)acetamido)thioundecahydro-closo-dodecaborate (2-) (B-6-14) and S-(N, N-(2-dipalmitoyloxyethyl)acetamido)thioundecahydro-closo-dodecaborate (2-) (B-6-16) are described. Both of them have a double-tailed lipophilic part and a headgroup carrying two negative charges. Differential scanning calorimetry shows that B-6-14 and B-6-16 bilayers have main phase transition temperatures of 18.8 and 37.9 degrees C, respectively. Above the transition temperature of 18.8 degrees C, B-6-14 can form liposomal vesicles, representing the first boron-containing lipid with this capability. Upon cooling below the transition temperature, stiff bilayers are formed. When incorporated into liposomal formulations with equimolar amounts of distearoyl phosphatidylcholine (DSPC) and cholesterol, stable liposomes are obtained. The zeta-potential measurements indicate that both B-6-14- and B-6-16-containing vesicles are negatively charged, with the most negative potential described of any liposome so far. The liposomes are of high potential value as transporters of boron to tumor cells in treatments based on boron neutron capture therapy (BNCT). Liposomes prepared from B-6-14 were slightly less toxic in V79 Chinese hamster cells (IC50 5.6 mM) than unformulated Na2B12H11SH (IC50 3.9 mM), while liposomes prepared from B-6-16 were not toxic even at 30 mM.

Preparation, characterisation and maintenance of drug efficacy of doxorubicin-loaded human serum albumin (HSA) nanoparticles.
Dreis S, Rothweiler F, Michaelis M, Cinatl J Jr, Kreuter J, Langer K.
Int J Pharm. 2007 Aug 16;341(1-2):207-14.
[ expand abstract ]

Human serum albumin (HSA) nanoparticles represent promising drug carrier systems. Binding of cytostatics to HSA nanoparticles may diminish their toxicity, optimise their body distribution and/or may overcome multidrug resistance. In the present study, doxorubicin-loaded HSA nanoparticle preparations were prepared. Doxorubicin was loaded to the HSA nanoparticles either by adsorption to the nanoparticles' surfaces or by incorporation into the particle matrix. Both loading strategies resulted in HSA nanoparticles of a size range between 150nm and 500nm with a loading efficiency of 70-95%. The influence on cell viability of the resulting nanoparticles was investigated in two different neuroblastoma cell lines. The anti-cancer effects of the drug-loaded nanoparticles were increased in comparison to doxorubicin solution. Based on these result a standard protocol for the preparation of doxorubicin-loaded HSA nanoparticles for further antitumoural studies was established.

Anticancer activity of cisplatin-loaded PLGA-mPEG nanoparticles on LNCaP prostate cancer cells.
Gryparis EC, Hatziapostolou M, Papadimitriou E, Avgoustakis K.
Eur J Pharm Biopharm. 2007 Aug;67(1):1-8.
[ expand abstract ]

The in vitro anticancer activity of cisplatin-loaded PLGA-mPEG nanoparticles on human prostate cancer LNCaP cells was investigated. The uptake of the PLGA-mPEG nanoparticles by the LNCaP cells was also studied. Blank PLGA-mPEG nanoparticles exhibited low cytotoxicity, which increased with increasing PLGA/PEG ratio in the PLGA-mPEG copolymer used to prepare the nanoparticles, possibly due to the increased cell uptake observed with increasing PLGA/PEG ratio. PLGA-mPEG nanoparticles loaded with cisplatin exerted in vitro anticancer activity against LNCaP cells that was comparable to the activity of free (non-entrapped in nanoparticles) cisplatin. Little differences in the in vitro anticancer activity of the different nanoparticle compositions were found, which may result from the differences observed between the different nanoparticles compositions in the uptake by the LNCaP cells and in the leakage of cisplatin from the nanoparticles during incubation with the cells. Visual evidence of nanoparticles' uptake by the LNCaP cells was obtained with nanoparticles labeled with PLGA(4165)-PyrBu(274) or dextran-rhodamine B isothiocyanate using fluorescence microscopy. Moreover, in some cases fluorescence around or inside cell nuclei was observed, which, if verified by further studies, would indicate that PLGA-PEG nanoparticles might prove to be useful in site-specific delivery of drugs whose site of pharmacological activity is cell nucleus.

Development of gelatin nanoparticles with biotinylated EGF conjugation for lung cancer targeting.
Tseng CL, Wang TW, Dong GC, Yueh-Hsiu Wu S, Young TH, Shieh MJ, Lou PJ, Lin FH.
Biomaterials. 2007 Sep;28(27):3996-4005.
[ expand abstract ]

Since lung cancer is the most malignant cancer today, a specific drug-delivery system has been developed for superior outcome. In this study, gelatin nanoparticles (GPs) employed as native carriers were grafted with NeutrAvidin(FITC) on the particle's surface (GP-Av). Next, the biotinylated epithelial growth factor (EGF) molecules were conjugated with NeutrAvidin(FITC), forming a core-shell-like structure (GP-Av-bEGF) to achieve the enhancement of targeting efficiency. These nanoparticles were applied as an EGF receptor (EGFR)-seeking agent to detect lung adenocarcinoma. The results showed that the modification process had no significant influence on particle size (220 nm) and zeta potential (-9.3 mV). By the in vitro cell culture test, GP-Av-bEGF resulted in higher entrance efficiency on adenocarcinoma cells (A549) than that on normal lung cells (HFL1) because A549 possessed greater amounts of EGFR. We also found that uptake of GP-Av-bEGF by A549 cells was time and dose dependent. Confocal microscopy confirmed the cellular internalization of GP-Av-bEGF, and more fluorescent spots of GP-Av-bEGF nanoparticles were obviously observed as well as lysosomal entrapment in A549. Finally, the delivery was demonstrated by in vivo aerosol administration to cancerous lung of the SCID mice model, and specific accumulation in cancerous lung was confirmed by image quantification. The targeting ability of GP-Av-bEGF was proved in vitro and in vivo, which holds promise for further anti-cancer drug applications.

Simplified preparation via streptavidin of antisense oligomers/carriers nanoparticles showing improved cellular delivery in culture.
Wang Y, Nakamura K, Liu X, Kitamura N, Kubo A, Hnatowich DJ.
Bioconjug Chem. 2007 Jul-Aug;18(4):1338-43.
[ expand abstract ]

OBJECTIVE: Carriers are increasingly now viewed as helpful or even essential to improve cellular uptake in connection with antisense tumor targeting and other applications requiring transmembrane delivery of oligomers. Evaluation of many of the large number of available and potentially useful carriers is limited only by the complexities of preparing the oligomer/carriers by covalent conjugation. However, using streptavidin as a linker between biotinylated carriers and biotinylated antisense oligomers would require only simple mixing for preparation. The goal of this study was to evaluate the preparation and cell accumulation in culture of carrier/streptavidin nanoparticle of an antisense phosphorodiamide morpholino (MORF) oligomer. METHODS: The model carriers were cholesterol, a 10 mer HIV-tat peptide, and a 10 mer polyarginine, each having been reported elsewhere to improve cellular delivery of oligomers. The model antisense oligomer was the 25 mer MORF targeting the survivin mRNA. The accumulations of the antisense MORF/carrier nanoparticle were compared to the sense MORF/carrier, to the carrier-free nanoparticles, and to the naked antisense MORF in the survivin-expressing MCF-7 cells. The MORFs and peptides were purchased biotinylated, while the cholesterol was biotinylated in-house. In all cases, the 99mTc radiolabel was placed on the oligomers. Cell studies were performed at low nM concentration as required for antisense imaging applications and at 37 degrees C primarily in 1% FBS. RESULTS: Each radiolabeled oligomer/streptavidin/carrier nanoparticle was successfully prepared by careful mixing at a 1:1 molar ratio. As evidence of carrier participation, the radiolabeled MORF showed increased accumulation in cells when incubated as the nanoparticle compared to the carrier-free nanoparticle and by as much as a factor of 11. Accumulation of the antisense MORF/streptavidin/tat nanoparticle was significantly higher than the sense MORF/streptavidin/tat nanoparticle as evidence of specific antisense targeting.
CONCLUSIONS: The preparation of oligomer/carrier nanoparticles was greatly simplified over covalent conjugations by using streptavidin as a linker. Furthermore, our results suggest that the addition of streptavidin did not interfere with the cellular delivery function of the tat, polyarginine, or cholesterol carriers nor with the specific antisense mRNA binding function of the MORF oligomer.

Targeting cancer cells using PLGA nanoparticles surface modified with monoclonal antibody.
Kocbek P, Obermajer N, Cegnar M, Kos J, Kristl J.
J Control Release. 2007 Jul 16;120(1-2):18-26.
[ expand abstract ]

Targeting drugs to their sites of action is still a major challenge in pharmaceutical research. In this study, polylactic-co-glycolic acid (PLGA) immuno-nanoparticles were prepared for targeting invasive epithelial breast tumour cells. Monoclonal antibody (mAb) was used as a homing ligand and was attached to the nanoparticle surface either covalently or non-covalently. The presence of mAb on the nanoparticle surface, its stability and recognition properties were tested. Protein assay, surface plasmon resonance, flow cytometry and fluorescence-immunostaining confirmed the presence of mAb on nanoparticles in both cases. However, a binding assay using cell lysate revealed that the recognition properties were preserved only for nanoparticles with adsorbed mAb. These nanoparticles were more likely to be bound to the targeted cells than non-coated nanoparticles. Both types of nanoparticles entered the target MCF-10A neoT cells in mono-culture. In co-culture of MCF-10A neoT and Caco-2 cells immuno-nanoparticles were localized solely to MCF-10A neoT cells, whereas non-coated nanoparticles were distributed randomly. Immuno-nanoparticles entered only MCF-10A neoT cells, while non-coated nanoparticles were taken up by both cell types, indicating specific targeting of the immuno-nanoparticles. In conclusion, we demonstrate a method by which mAbs can be bound to nanoparticles without detriment to their targeting ability. Furthermore, the results show the effectiveness of the new carrier system for targeted delivery of small or large active substances into cells or tissues of interest.

Mesoporous silica nanoparticles as a delivery system for hydrophobic anticancer drugs.
Lu J, Liong M, Zink JI, Tamanoi F.
Small. 2007 Aug;3(8):1341-6.
[ expand abstract ]

No abstract available. (Citation link )

The drug loading, cytotoxicty and tumor vascular targeting characteristics of magnetite in magnetic drug targeting.
Dandamudi S, Campbell RB.
Biomaterials. 2007 Nov;28(31):4673-83.
[ expand abstract ]

Chemotherapy is a popular treatment approach against cancer but significant uptake of drugs by normal tissues is still a major limitation. Magnetic drug targeting (MDT) has been used to improve localized drug delivery to interstitial tumor targets. MDT is now being developed to improve drug delivery to tumor vessels. We thus seek to understand the role of magnetite (MAG-C) in drug loading, influence on cytotoxicity and vascular targeting characteristics. The inclusion of MAG-C at lower concentrations (0.5 mg/ml) in cationic liposomes did not alter the efficiency of loading etoposide, but at higher concentrations (2.5 mg/ml) incorporation decreased from 80+/-3.4% to 44+/-4.26%. MAG-C reduced the incorporation of dacarbazine. The incorporation was significantly lower compared to liposomal etoposide, both in the presence and absence of MAG-C. The incorporation efficiency of vinblastine sulfate in cationic liposomes was similar for low and relatively high MAG-C content; values for incorporation were 21+/-0.11 and 23+/-2, respectively. Polyethylene-glycol improved the efficiency of loading chemotherapeutic agents regardless of drug type. Additionally, cytotoxicity and tumor vascular targeting characteristics of liposome therapeutics were not influenced by MAG-C. The components used to prepare magnetic liposomes for MDT should be optimized for maximum therapeutic benefit.

Targeted quantum dot conjugates for siRNA delivery.
Derfus AM, Chen AA, Min DH, Ruoslahti E, Bhatia SN.
Bioconjug Chem. 2007 Sept-Oct; 18(5):1391-6.
[ expand abstract ]

Treatment of human diseases such as cancer generally involves the sequential use of diagnostic tools and therapeutic modalities. Multifunctional platforms combining therapeutic and diagnostic imaging functions in a single vehicle promise to change this paradigm. in particular, nanoparticle-based multifunctional platforms offer the potential to improve the pharmacokinetics of drug formulations, while providing attachment sites for diagnostic imaging and disease targeting features. We have applied these principles to the delivery of small interfering RNA (siRNA) therapeutics, where systemic delivery is hampered by rapid excretion and nontargeted tissue distribution. Using a PEGlyated quantum dot (QD) core as a scaffold, siRNA and tumor-homing peptides (F3) were conjugated to functional groups on the particle's surface. We found that the homing peptide was required for targeted internalization by tumor cells, and that siRNA cargo could be coattached without affecting the function of the peptide. Using an EGFP model system, the role of conjugation chemistry was investigated, with siRNA attached to the particle by disulfide cross-linkers showing greater silencing efficiency than when attached by a nonreducible thioether linkage. Since each particle contains a limited number of attachment sites, we further explored the tradeoff between number of F3 peptides and the number of siRNA per particle, leading to an optimized formulation. Delivery of these F3/siRNA-QDs to EGFP-transfected HeLa cells and release from their endosomal entrapment led to significant knockdown of EGFP signal. By designing the siRNA sequence against a therapeutic target (e.g., oncogene) instead of EGFP, this technology may be ultimately adapted to simultaneously treat and image metastatic cancer.

Multifunctional nanoparticles for combining ultrasonic tumor imaging and targeted chemotherapy.
Rapoport N, Gao Z, Kennedy A.
J Natl Cancer Inst. 2007 Jul 18;99(14):1095-106.
[ expand abstract ]

BACKGROUND: Drug delivery in polymeric micelles combined with tumor irradiation by ultrasound results in effective drug targeting, but this technique requires prior tumor imaging. A technology that combined ultrasound imaging with ultrasound-mediated nanoparticle-based targeted chemotherapy could therefore have important applications in cancer treatment. METHODS: Mixtures of drug-loaded polymeric micelles and perfluoropentane (PFP) nano/microbubbles stabilized by the same biodegradable block copolymer were prepared. Size distribution of nanoparticles was measured by dynamic light scattering. Cavitation activity (oscillation, growth, and collapse of microbubbles) under ultrasound was assessed based on the changes in micelle/microbubble volume ratios. The effect of the nano/microbubbles on the ultrasound-mediated cellular uptake of doxorubicin (Dox) in MDA MB231 breast tumors in vitro and in vivo (in mice bearing xenograft tumors) was determined by flow cytometry. Statistical tests were two-sided. RESULTS: Phase state and nanoparticle sizes were sensitive to the copolymer/perfluorocarbon volume ratio. At physiologic temperatures, nanodroplets converted into nano/microbubbles. Doxorubicin was localized in the microbubble walls formed by the block copolymer. Upon intravenous injection into mice, Dox-loaded micelles and nanobubbles extravasated selectively into the tumor interstitium, where the nanobubbles coalesced to produce microbubbles with a strong, durable ultrasound contrast. Doxorubicin was strongly retained in the microbubbles but released in response to therapeutic ultrasound. Microbubbles cavitated under the action of tumor-directed ultrasound, which enhanced intracellular Dox uptake by tumor cells in vitro to a statistically significant extent relative to that observed with unsonicated microbubbles (drug uptake ratio = 4.60; 95% confidence interval [CI] = 1.70 to 12.47; P = .017) and unsonicated micelles (drug uptake ratio = 7.97; 95% CI = 3.72 to 17.08; P = .0032) and resulted in tumor regression in the mouse model. CONCLUSIONS: Multifunctional nanoparticles that are tumor-targeted drug carriers, long-lasting ultrasound contrast agents, and enhancers of ultrasound-mediated drug delivery have been developed and deserve further exploration as cancer therapeutics.

211AtCl@US-tube nanocapsules: a new concept in radiotherapeutic-agent design
Hartman KB, Hamlin DK, Wilbur S, Wilson LJ.
Small. 3(9):1496–1499.
[ expand abstract ]

No abstract available (Citation link)

Near-infrared resonant nanoshells for combined optical imaging and photothermal cancer therapy.
Gobin AM, Lee MH, Halas NJ, James WD, Drezek RA, West JL.
Nano Lett. 2007 Jul;7(7):1929-34.
[ expand abstract ]

Metal nanoshells are core/shell nanoparticles that can be designed to either strongly absorb or scatter within the near-infrared (NIR) wavelength region ( approximately 650-950 nm). Nanoshells were designed that possess both absorption and scattering properties in the NIR to provide optical contrast for improved diagnostic imaging and, at higher light intensity, rapid heating for photothermal therapy. Using these in a mouse model, we have demonstrated dramatic contrast enhancement for optical coherence tomography (OCT) and effective photothermal ablation of tumors.

Functionalized fullerenes mediate photodynamic killing of cancer cells: Type I versus Type II photochemical mechanism.
Mroz P, Pawlak A, Satti M, Lee H, Wharton T, Gali H, Sarna T, Hamblin MR.
Free Radic Biol Med. 2007 Sep 1;43(5):711-9.
[ expand abstract ]

Photodynamic therapy (PDT) employs the combination of nontoxic photosensitizers (PS) and harmless visible light to generate reactive oxygen species (ROS) and kill cells. Most clinically studied PS are based on the tetrapyrrole structure of porphyrins, chlorines, and related molecules, but new nontetrapyrrole PS are being sought. Fullerenes are soccer-ball shaped molecules composed of 60 or 70 carbon atoms and have attracted interest in connection with the search for biomedical applications of nanotechnology. Fullerenes are biologically inert unless derivatized with functional groups, whereupon they become soluble and can act as PS. We have compared the photodynamic activity of six functionalized fullerenes with 1, 2, or 3 hydrophilic or 1, 2, or 3 cationic groups. The octanol-water partition coefficients were determined and the relative contributions of Type I photochemistry (photogeneration of superoxide in the presence of NADH) and Type II photochemistry (photogeneration of singlet oxygen) were studied by measurement of oxygen consumption, 1270-nm luminescence and EPR spin trapping of the superoxide product. We studied three mouse cancer cell lines: (J774, LLC, and CT26) incubated for 24 h with fullerenes and illuminated with white light. The order of effectiveness as PS was inversely proportional to the degree of substitution of the fullerene nucleus for both the neutral and the cationic series. The monopyrrolidinium fullerene was the most active PS against all cell lines and induced apoptosis 4-6 h after illumination. It produced diffuse intracellular fluorescence when dichlorodihydrofluorescein was added as an ROS probe, suggesting a Type I mechanism for phototoxicity. We conclude that certain functionalized fullerenes have potential as novel PDT agents and phototoxicity may be mediated both by superoxide and by singlet oxygen.

Intracellular heating of living cells through Néel relaxation of magnetic nanoparticles.
Fortin JP, Gazeau F, Wilhelm C.
Eur Biophys J. 2008 Feb;37(2):223-8.
[ expand abstract ]

Maghemite and cobalt ferrite anionic magnetic nanoparticles enter tumor cells and can be used as heat sources when exposed to a high-frequency magnetic field. Comparative studies of the two particles enable to unravel the magnetic heating mechanisms (Néel relaxation vs. Brown relaxation) responsible for the cellular temperature rise, and also to establish a simple model, adjusted to the experimental results, allowing to predict the intracellular heating efficiency of iron oxide nanoparticles. Hence, we are able to derive the best nanoparticle design for a given material with a view to intracellular hyperthermia-based applications.

Tumor targeting with antibody-functionalized, radiolabeled carbon nanotubes.
McDevitt MR, Chattopadhyay D, Kappel BJ, Jaggi JS, Schiffman SR, Antczak C, Njardarson JT, Brentjens R, Scheinberg DA.
J Nucl Med. 2007 Jul;48(7):1180-9
[ expand abstract ]

Single-walled carbon nanotubes (CNT) are mechanically robust graphene cylinders with a high aspect ratio that are comprised of sp(2)-bonded carbon atoms and possessing highly regular structures with defined periodicity. CNT exhibit unique mechanochemical properties that can be exploited for the development of novel drug delivery platforms. We hypothesized that novel prototype nanostructures consisting of biologics, radionuclides, fluorochromes, and CNT could be synthesized and designed to target tumor cells. METHODS: Tumor-targeting CNT constructs were synthesized from sidewall-functionalized, water-soluble CNT platforms by covalently attaching multiple copies of tumor-specific monoclonal antibodies, radiometal-ion chelates, and fluorescent probes. The constructs were characterized spectroscopically, chromatographically, and electrophoretically. The specific reactivity of these constructs was evaluated in vitro by flow cytometry and cell-based immunoreactivity assays and in vivo using biodistribution in a murine xenograft model of lymphoma. RESULTS: A soluble, reactive CNT platform was used as the starting point to build multifunctional constructs with appended antibody, metal-ion chelate, and fluorescent chromophore moieties to effect specific targeting, to carry and deliver a radiometal-ion, and to report location, respectively. These nanoconstructs were found to be specifically reactive with the human cancer cells they were designed to target in vivo in a model of disseminated human lymphoma and in vitro by flow cytometry and cell-based immunoreactivity assays versus appropriate controls. CONCLUSION: The key achievement in these studies was the selective targeting of tumor in vitro and in vivo by the use of specific antibodies appended to a soluble, nanoscale CNT construct. The ability to specifically target tumor with prototype-radiolabeled or fluorescent-labeled, antibody-appended CNT constructs was encouraging and suggested further investigation of CNT as a novel delivery platform.

Multiplex targeting, tracking, and imaging of apoptosis by fluorescent surface enhanced Raman spectroscopic dots.
Yu KN, Lee SM, Han JY, Park H, Woo MA, Noh MS, Hwang SK, Kwon JT, Jin H, Kim YK, Hergenrother PJ, Jeong DH, Lee YS,Cho MH.
Bioconjug Chem. 2007 Jul-Aug;18(4):1155-62.
[ expand abstract ]

We have developed multifunctional fluorescent surface enhanced Raman spectroscopic tagging material (F-SERS dots) composed of silver nanoparticle-embedded silica spheres with fluorescent organic dye and specific Raman labels for multiplex targeting, tracking, and imaging of cellular/molecular events in the living organism. In this study, F-SERS dots fabricated with specific target antibodies (BAX and BAD) were employed for the detection of apoptosis. The F-SERS dots did not show any particular toxicity in several cell lines. The F-SERS dots could monitor the apoptosis effectively and simultaneously through fluorescent images as well as Raman signals in both cells and tissues with high selectivity. Our results clearly demonstrate that F-SERS dots can be easily applicable to multiplex analysis of diverse cellular/molecular events important for maintaining cellular homeostasis.

Internalization of MWCNTs by microglia: possible application in immunotherapy of brain tumors.
Kateb B, Van Handel M, Zhang L, Bronikowski MJ,Manohara H, Badie B.
Neuroimage. 2007;37 Suppl 1:S9-17.
[ expand abstract ]

There is a pressing need for new therapeutic, diagnostic, and drug delivery approaches for treating brain cancers. Nanotechnology offers a new method for targeted brain cancer therapy and could play a major role in gene and drug delivery. The goals of our study were to visualize in vitro ingestion, cytotoxicity, and loading capacity of Multi-Walled Carbon Nanotubes (MWCNTs) in microglia. Furthermore, we investigated internalization differences between microglia and glioma cells. BV2 microglia and GL261 glioma cells were incubated with MWCNTs, which were synthesized through catalytic chemical vapor deposition technique. Real-time RT-PCR, cell proliferation analysis, siRNA and DNA loading, electron microscopy, and flow cytometry were performed. We demonstrated that MWCNTs do not result in proliferative or cytokine changes in vitro, are capable of carrying DNA and siRNA and are internalized at higher levels in phagocytic cells as compared to tumor cells. This study suggests MWCNTs could be used as a novel, non-toxic, and biodegradable nano-vehicles for targeted therapy in brain cancers. Further studies are needed to demonstrate the full capacity of MWCNTs as nanovectors.

Methylene blue-containing silica-coated magnetic particles: a potential magnetic carrier for photodynamic therapy.
Tada DB, Vono LL, Duarte EL, Itri R, Kiyohara PK, Baptista MS, Rossi LM.
Langmuir. 2007 Jul 17;23(15):8194-9.
[ expand abstract ]

We present the preparation and characterization of methylene blue-containing silica-coated magnetic particles. The entrapment of methylene blue (MB), a photodynamic therapy drug under study in our group, in the silica matrix took place during the growth of a silica layer over a magnetic core composed of magnetite nanoparticles. The resulting material was characterized by transmission electron microscopy (TEM), light scattering, and X-ray diffraction. It is composed of approximately 30 nm silica spheres containing magnetic particles of 11 +/- 2 nm and methylene blue entrapped in the silica matrix. The immobilized drug can generate singlet oxygen, which was detected by its characteristic phosphorescence decay curve in the near-infrared and by a chemical method using 1,3-diphenylisobenzofuran to trap singlet oxygen. The lifetime of singlet oxygen was determined to be 52 micros (in acetonitrile) and 3 micros (in water), with both values being in good agreement with those in the literature. The release of singlet oxygen (etaDelta) was affected by the encapsulation of MB in the silica matrix, which caused a reduction to 6% of the quantum yield of MB free in solution. The magnetization curve confirmed the superparamagnetic behavior with a reduced saturation magnetization in respect to uncoated magnetic nanoparticles, which is consistent with the presence of a diamagnetic component over the magnetite surface. The result is a single particle platform that combines therapy (photosensitizer) and diagnostic (MRI contrast agent) possibilities at the same time, as well as drug targeting.

Co-Delivery of Hydrophobic and Hydrophilic Drugs from Nanoparticle-Aptamer Bioconjugates.
Zhang L, Radovic-Moreno AF, Alexis F, Gu FX, Basto PA, Bagalkot V, Jon S, Langer RS, Farokhzad OC.
ChemMedChem. 2(9):1268 – 1271.
[ expand abstract ]

No abstract available. (Citation link)

Modulation of intracellular ceramide using polymeric nanoparticles to overcome multidrug resistance in cancer.
van Vlerken LE, Duan Z, Seiden MV, Amiji MM.
Cancer Res. 2007 May 15;67(10):4843-50.
[ expand abstract ]

Although multidrug resistance (MDR) is known to develop through a variety of molecular mechanisms within the tumor cell, many tend to converge toward the alteration of apoptotic signaling. The enzyme glucosylceramide synthase (GCS), responsible for bioactivation of the proapoptotic mediator ceramide to a nonfunctional moiety glucosylceramide, is overexpressed in many MDR tumor types and has been implicated in cell survival in the presence of chemotherapy. The purpose of this study was to investigate the therapeutic strategy of coadministering ceramide with paclitaxel, a commonly used chemotherapeutic agent, in an attempt to restore apoptotic signaling and overcome MDR in the human ovarian cancer cell line SKOV3. Poly(ethylene oxide)-modified poly(epsilon-caprolactone) (PEO-PCL) nanoparticles were used to encapsulate and deliver the therapeutic agents for enhanced efficacy. Results show that indeed the cotherapy eradicates the complete population of MDR cancer cells when they are treated at their IC(50) dose of paclitaxel. More interestingly, when the cotherapy was combined with the properties of nanoparticle drug delivery, the MDR cells can be resensitized to a dose of paclitaxel near the IC(50) of non-MDR (drug sensitive) cells, indicating a 100-fold increase in chemosensitization via this approach. Molecular analysis of activity verified the hypothesis that the efficacy of this therapeutic approach is indeed due to a restoration in apoptotic signaling, although the beneficial properties of PEO-PCL nanoparticle delivery seemed to enhance the therapeutic success even further, showing the promising potential for the clinical use of this therapeutic strategy to overcome MDR.

Dendrimer-entrapped gold nanoparticles as a platform for cancer-cell targeting and imaging.
Shi X, Wang S, Meshinchi S, Van Antwerp ME, Bi X, Lee I, Baker JR Jr.
Small. 2007 Jul;3(7):1245-52.
[ expand abstract ]

We present a general approach for the targeting and imaging of cancer cells using dendrimer-entrapped gold nanoparticles (Au DENPs). Au DENPs were found to be able to covalently link with targeting and imaging ligands for subsequent cancer-cell targeting and imaging. The Au DENPs linked with defined numbers of folic acid (FA) and fluorescein isothiocyanate (FI) molecules are water soluble, stable, and biocompatible. In vitro studies show that the FA- and FI-modified Au DENPs can specifically bind to KB cells (a human epithelial carcinoma cell line) that overexpress high-affinity folate receptors and they are internalized dominantly into lysosomes of target cells within 2 h. These findings demonstrate that Au DENPs may serve as a general platform for cancer imaging and therapeutics.

Morbidity and quality of life during thermotherapy using magnetic nanoparticles in locally recurrent prostate cancer: results of a prospective phase I trial.
Johannsen M, Gneveckow U, Taymoorian K, Thiesen B, Waldöfner N, Scholz R, Jung K, Jordan A, Wust P, Loening SA.
Int J Hyperthermia. 2007 May;23(3):315-23.
[ expand abstract ]

PURPOSE: To investigate the treatment-related morbidity and quality of life (QoL) during thermotherapy using superparamagnetic nanoparticles in patients with locally recurrent prostate cancer. MATERIALS AND METHODS: Ten patients with biopsy-proven locally recurrent prostate cancer following primary therapy with curative intent and no detectable metastases were entered on a prospective phase I trial. Endpoints were feasibility, toxicity and QoL. Following intraprostatic injection of a nanoparticle dispersion, six thermal therapy sessions of 60 min duration were delivered at weekly intervals using an alternating magnetic field. National Cancer Institute (NCI) common toxicity criteria (CTC) and the European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30 and QLQ-PR25 questionnaires were used to evaluate toxicity and QoL, respectively. In addition, prostate specific antigen (PSA) measurements were carried out. RESULTS: Maximum temperatures up to 55 degrees C were achieved in the prostates at 25-30% of the available magnetic field strength. Nanoparticle deposits were detectable in the prostates one year after thermal therapy. At a median follow-up of 17.5 months (3-24), no systemic toxicity was observed. Acute urinary retention occurred in four patients with previous history of urethral stricture. Treatment-related morbidity was moderate and QoL was only temporarily impaired. Prostate-specific antigen (PSA) declines were observed in eight patients. CONCLUSIONS: Interstitial heating using magnetic nanoparticles was feasible and well tolerated in patients with locally recurrent prostate cancer. Deposition of nanoparticles in the prostate was highly durable. Further refinement of the technique is necessary to allow application of higher magnetic field strengths.

A p53-derived apoptotic peptide derepresses p73 to cause tumor regression in vivo.
Bell, HS, Dufes C, O'Prey J, Crighton D, Bergamaschi D, Lu X, Schatzlein AG, Vousden KH, Ryan KM.
J Clin Invest. 2007;117(4):1008-18.
[ expand abstract ]

The tumor suppressor p53 is a potent inducer of tumor cell death, and strategies exist to exploit p53 for therapeutic gain. However, because about half of human cancers contain mutant p53, application of these strategies is restricted. p53 family members, in particular p73, are in many ways functional paralogs of p53, but are rarely mutated in cancer. Methods for specific activation of p73, however, remain to be elucidated. We describe here a minimal p53-derived apoptotic peptide that induced death in multiple cell types regardless of p53 status. While unable to activate gene expression directly, this peptide retained the capacity to bind iASPP - a common negative regulator of p53 family members. Concordantly, in p53-null cells, this peptide derepressed p73, causing p73-mediated gene activation and death. Moreover, systemic nanoparticle delivery of a transgene expressing this peptide caused tumor regression in vivo via p73. This study therefore heralds what we believe to be the first strategy to directly and selectively activate p73 therapeutically and may lead to the development of broadly applicable agents for the treatment of malignant disease.

Ceramic nanoparticles deliver genes to the spleen, trigger antitumor immune response. Nanosized bioceramic particles could function as efficient gene delivery vehicles with target specificity for the spleen.
Tan K, Cheang P, Ho IA, Lam PY, Hui KM.
Gene Ther. 2007 May;14(10):828-35.
[ expand abstract ]

We have compared the ability of several nanosized bioceramic particles including negatively charged silica (SiO(2)), neutrally charged hydroxyapatite (HA) and positively charged zirconia (ZrO(2)) nanoparticles as non-viral vectors for efficient in vivo gene delivery. A mixture of highly monodispersed aqueous suspension of HA or SiO(2) nanoparticles, coated with protamine sulfate (PS), complexed efficiently with plasmid DNA and significantly enhanced transgene expression in vitro. In comparison, ZrO(2) nanoparticles gave poor transfection efficiency under similar conditions tested. It was also determined that, under the same conditions, PS-SiO(2)-DNA, but not PS-HA-DNA-nanoplexes, were able to mediate efficient transgene expression in vitro in the presence of 50% serum. Intraperitoneal injections of PS-SiO(2)-luciferase DNA nanoplexes targeted the highest level of transgene expression in the spleen of recipient mice that lasted for more than 48 h. Injection of PS-SiO(2)-pNGVL-hFLex-MUC-1 nanoplexes was able to mediate the production of Flt-3L in the sera of recipient mice. Simultaneously, the production of Flt-3L was accompanied by the stimulation of IL-2 and interferon-gamma (IFN-gamma). Most importantly, the injection of PS-SiO(2)-pNGVL-hFLex-MUC-1 nanoplexes could mount potent anti-tumour specific immune responses that led to the subsequent regression of parental tumor cells containing the muc-1 determinant.

Solid lipid nanoparticles: could they help to improve the efficacy of pharmacologic treatments for brain tumors?
Brioschi A, Zenga F, Zara GP, Gasco MR, Ducati A, Mauro A.
Neurol Res. 2007 Apr;29(3):324-30.
[ expand abstract ]

OBJECTIVES: Brain malignant neoplasms are still characterized by poor prognosis due to their peculiar hallmarks that severely limit aggressive multimodal therapeutic approaches. The optimization of the intratumoral drug delivery, directed to achieve effective concentrations and to reduce systemic undesired toxicity, is one of the primary goals of the brain tumors therapeutic strategies. Different passive and active delivery carriers allowing to a better control of drug distribution, metabolism, and elimination after parenteral administration have been developed. In the present review we will describe general characteristics and evaluate the efficacy of Solid Lipid Nanoparticles (SLN) as carriers of different drugs in experimental brain malignant tumor therapy. METHODS: SLN vehiculating different illustrative types of antineoplastic agents (conventional cytotoxic drugs such as doxorubicin and paclitaxel, the prodrug Cholesteryl butyrate, and anti VEGF antisense oligonucleotides) have been tested in experimental animal models of cerebral gliomas. RESULTS: SLN proved to successfully vehiculate into the brain different types of cytotoxic and gene therapeutical agents (otherwise unable to pass through the Blood-Brain Barrier) and to induce effective anti-tumoral therapeutical response. DISCUSSION: Compared to other vehicules, SLN seem to offer more advantages (such as higher physical stability, greater protection from degradation and better release profile of incorporated drugs, good tolerability and possibility of site-specific targeting) and could be regarded as an effective carrier for chemotherapeutic drugs, gene therapeutical agents, and diagnostic tools in neuro-oncology.

Preparation and characterization of radioactive dirhenium decacarbonyl-loaded PLLA nanoparticles for radionuclide intra-tumoral therapy.
Hamoudeh M, Salim H, Barbos D, Paunoiu C, Fessi H.
Eur J Pharm Biopharm. 2007 Nov;67(3):597-611.
[ expand abstract ]

This study describes the development of biocompatible radioactive rhenium-loaded nanoparticles for radionuclide anti-cancer therapy. To achieve this goal, dirhenium decacarbonyl [Re2(CO)10] has been encapsulated in poly(L-lactide) based nanoparticles by an oil-in-water emulsion-solvent evaporation method. A 3(3) factorial design method was applied to investigate the influence of both the proceeding and formulation parameters including the stirring speed and the concentration of both the PLLA polymer and the poly(vinyl alcohol) stabiliser on both nanoparticles size and the Re2(CO)10 encapsulation efficacy. The factorial design results attributed a clear negative effect for the stirring speed and the stabiliser concentration on the nanoparticles size while the polymer concentration exhibited a positive one. Regarding the Re2(CO)10 encapsulation efficacy, higher values were obtained when higher polymer concentrations, lower stabiliser concentrations or slower stirring speeds were applied in the preparation. Different tests were thereafter performed to characterize the Re2(CO)10-loaded nanoparticles. The nanoparticles size, being experimentally controlled by the above mentioned parameters, ranged between 330 and 1500 nm and the maximum rhenium loading was 24% by nanoparticles weight as determined by atomic emission assays and neutron activation analysis. Furthermore, the rhenium distribution within nanoparticles has been shown to be homogeneous as confirmed by the energy dispersive X-ray spectrometry. DSC assays demonstrated that Re2(CO)10 was encapsulated in its crystalline initial state. Other experiments including FT-IR and NMR did not show interactions between PLLA and Re2(CO)10. To render them radioactive, these nanoparticles have been bombarded with a neutron flux of 1.45x10(13) n/cm2/s during 1 h. The SEM micrographs of nanoparticles after neutron bombardment showed that the nanoparticles remained spherical and separated but slightly misshaped. These applied neutron activation conditions yielded a specific activity of about 32.5 GBq per gram of nanoparticles. Preliminary estimations allow us to think that a sole injection of 50 mg of these activated nanoparticles into a brain tumor model (4.2 cm diameter) would deliver a tumor absorbed dose of up to 47 Gy. In conclusion, these dirhenium decacarbonyl-loaded nanoparticles represent a novel promising tool for radionuclide anti-cancer therapy.

Encapsulation of paclitaxel in macromolecular nanoshells.
Zahr AS, Pishko MV.
Biomacromolecules. 2007 Jun;8(6):2004-10.
[ expand abstract ]

An electrostatic layer-by-layer self-assembly technique was used to encapsulate solid core paclitaxel nanoparticles within a polymeric nanometer-scale shell. This approach provides a new strategy for the development of polymeric vehicles that control drug release and target diseased tissues and cells specific to the ailment, such as breast cancer. Core paclitaxel nanoparticles, 153 +/- 28 nm in diameter, were prepared using a modified nanoprecipitation technique. A nanoshell composed of multilayered polyelectrolytes, poly(allylamine hydrochloride) and poly(styrene-4-sulfonate) was assembled stepwise onto core charged drug nanoparticles. In vitro studies were performed to determine the anticancer activity of paclitaxel core-shell nanoparticles. Paclitaxel core-shell nanoparticles induced cell cycle arrest in the G2/M phase after 24 and 48 h of incubation with a human breast carcinoma cell line, MCF-7. Changes in MCF-7 cell morphology, fragmentation of the nucleus, and loss of cell-cell contacts indicated that the cells responded to paclitaxel core nanoparticles upon treatment for 24 and 48 h. Cells arrested in G2/M phase illustrated abnormal microtubule and actin cytoskeleton morphology. The core-shell drug nanoparticles fabricated using this procedure provide a new approach in the delivery of paclitaxel devoid of Cremophor EL, a solvent that causes adverse side effects in patients undergoing chemotherapy for treatment of metastasized mammary cancers.

Nanoparticulate delivery of suicide DNA to murine prostate and prostate tumors.
Peng W, Anderson DG, Bao Y, Padera RF Jr, Langer R, Sawicki JA.
Prostate. 2007 Jun 1;67(8):855-62.
[ expand abstract ]

BACKGROUND: Currently available treatments for benign prostatic hyperplasia (BPH) and localized prostate cancer are generally effective but are often attended by serious side effects that impact on the quality of life. In particular, most current therapies are non-specific, with surgery, radiation, and chemical ablation having the potential to cause damage to surrounding tissue. Here, we demonstrate the effectiveness of a prostate-specific, locally delivered gene therapy for the targeted killing of prostate cells. METHODS: Using a degradable, poly(beta-amino ester) polymer, poly(butane diol diacrylate co amino pentanol) (C32), we developed a nanoparticulate system to deliver a diphtheria toxin suicide gene (DT-A) driven by a prostate specific promoter to cells. These C32/DT-A nanoparticles were directly injected to the normal prostate and to prostate tumors in mice. RESULTS: Nearly 50% of normal prostates showed a significant reduction in size, attributable to cellular apoptosis, whereas injection with naked DT-A-encoding DNA had little effect. Significant apoptosis was also observed in C32/DT-A injected prostate tumors. Importantly, no damage to surrounding tissue was observed. CONCLUSIONS: These results suggest that local delivery of poly(beta-amino ester) polymer/ DT-A nanoparticles may have application in the treatment of BPH and prostate cancer.

Nanosized bioceramic particles could function as efficient gene delivery vehicles with target specificity for the spleen.
Tan K, Cheang P, Ho IA, Lam PY, Hui KM.
Gene Ther. 2007 May;14(10):828-35.
[ expand abstract ]

We have compared the ability of several nanosized bioceramic particles including negatively charged silica (SiO(2)), neutrally charged hydroxyapatite (HA) and positively charged zirconia (ZrO(2)) nanoparticles as non-viral vectors for efficient in vivo gene delivery. A mixture of highly monodispersed aqueous suspension of HA or SiO(2) nanoparticles, coated with protamine sulfate (PS), complexed efficiently with plasmid DNA and significantly enhanced transgene expression in vitro. In comparison, ZrO(2) nanoparticles gave poor transfection efficiency under similar conditions tested. It was also determined that, under the same conditions, PS-SiO(2)-DNA, but not PS-HA-DNA-nanoplexes, were able to mediate efficient transgene expression in vitro in the presence of 50% serum. Intraperitoneal injections of PS-SiO(2)-luciferase DNA nanoplexes targeted the highest level of transgene expression in the spleen of recipient mice that lasted for more than 48 h. Injection of PS-SiO(2)-pNGVL-hFLex-MUC-1 nanoplexes was able to mediate the production of Flt-3L in the sera of recipient mice. Simultaneously, the production of Flt-3L was accompanied by the stimulation of IL-2 and interferon-gamma (IFN-gamma). Most importantly, the injection of PS-SiO(2)-pNGVL-hFLex-MUC-1 nanoplexes could mount potent anti-tumour specific immune responses that led to the subsequent regression of parental tumor cells containing the muc-1 determinant.

Quantum dots are phagocytized by macrophages and colocalize with experimental gliomas.
Jackson H, Muhammad O, Daneshvar H, Nelms J, Popescu A, Vogelbaum MA, Bruchez M, Toms SA.
Neurosurgery. 2007 Mar;60(3):524-9; discussion 529-30.
[ expand abstract ]

OBJECTIVE: The identification of neoplastic tissue within normal brain during biopsy and tumor resection remains a problem in the operative management of gliomas. A variety of nanoparticles are phagocytized by macrophages in vivo. This feature may allow optical nanoparticles, such as quantum dots, to colocalize with brain tumors and serve as an optical aid in the surgical resection or biopsy of brain tumors. METHODS: Male Fisher rats (Charles River Labs, Wilmington, MA) were implanted intracranially with C6 gliosarcoma cell lines to establish tumors. Two weeks after the implantation of tumors, 705-nm emission Qdot ITK Amino(PEG) Quantum Dots (Quantum Dot Corp., Hayward, CA) were injected via the tail vein at doses of 3 to 17 nmol. The animals were sacrificed 24 hours after the injection of quantum dots and their tissues were examined. RESULTS: Quantum dots are avidly phagocytized by macrophages and are taken up by the liver, spleen, and lymph nodes. A dose-response relationship was noted. At low doses, the majority of the quantum dots are sequestered in the liver, spleen, and lymph nodes. At higher doses, increasing quantities of quantum dots are noted within the experimental brain tumors. Macrophages and microglia colocalize with glioma cells, carrying the quantum dot and thereby optically outlining the tumor. Excitation with blue or ultraviolet wavelengths stimulates the quantum dots, which give off a deep red fluorescence detectable with charge-coupled device cameras, optical spectroscopy units, and in dark-field fluorescence microscopy. CONCLUSION: Quantum dots are optical nanoparticles that, when delivered in nanomole doses, are phagocytized by the macrophages and microglia that infiltrate experimental gliomas. The optical signal may be detected, allowing for improved identification and visualization of tumors, potentially augmenting brain tumor biopsy and resection.

Folic acid-conjugated protein cages of a plant virus: a novel delivery platform for doxorubicin.
Ren Y, Wong SM, Lim LY.
Bioconjug Chem. 2007 May-Jun;18(3):836-43.
[ expand abstract ]

The protein cage of a plant virus may provide a template for monodispersed nanosized systems for drug delivery. Using the Hibiscus chlorotic ringspot virus (HCRSV) as a model plant virus, we have prepared nanosized protein cages (30 nm) capable of encapsulating the anticancer drug, doxorubicin. The technique utilized the simultaneous encapsulation of a polyprotic acid of mw 200 kDa to produce an encapsulation efficiency for doxorubicin of about 7.5%. Folic acid was conjugated onto the capsids to impart cancer-targeting capability. The resultant nanosized systems improved the uptake and cytotoxicity of doxorubicin in the ovarian cancer cells, OVCAR-3, with statistical significance. Plant virus capsids may therefore provide viable templates for targeted drug delivery in cancer chemotherapy.

Polymeric nanoparticle-encapsulated curcumin ("nanocurcumin"): a novel strategy for human cancer therapy.
Bisht S, Feldmann G, Soni S, Ravi R, Karikar C, Maitra A, Maitra A.
J Nanobiotechnology. 2007 Apr 17;5:3.
[ expand abstract ]

BACKGROUND: Curcumin, a yellow polyphenol extracted from the rhizome of turmeric (Curcuma longa), has potent anti-cancer properties as demonstrated in a plethora of human cancer cell line and animal carcinogenesis models. Nevertheless, widespread clinical application of this relatively efficacious agent in cancer and other diseases has been limited due to poor aqueous solubility, and consequently, minimal systemic bioavailability. Nanoparticle-based drug delivery approaches have the potential for rendering hydrophobic agents like curcumin dispersible in aqueous media, thus circumventing the pitfalls of poor solubility. RESULTS: We have synthesized polymeric nanoparticle encapsulated formulation of curcumin - nanocurcumin - utilizing the micellar aggregates of cross-linked and random copolymers of N-isopropylacrylamide (NIPAAM), with N-vinyl-2-pyrrolidone (VP) and poly(ethyleneglycol)monoacrylate (PEG-A). Physico-chemical characterization of the polymeric nanoparticles by dynamic laser light scattering and transmission electron microscopy confirms a narrow size distribution in the 50 nm range. Nanocurcumin, unlike free curcumin, is readily dispersed in aqueous media. Nanocurcumin demonstrates comparable in vitro therapeutic efficacy to free curcumin against a panel of human pancreatic cancer cell lines, as assessed by cell viability and clonogenicity assays in soft agar. Further, nanocurcumin's mechanisms of action on pancreatic cancer cells mirror that of free curcumin, including induction of cellular apoptosis, blockade of nuclear factor kappa B (NFkappaB) activation, and downregulation of steady state levels of multiple pro-inflammatory cytokines (IL-6, IL-8, and TNFalpha). CONCLUSION: Nanocurcumin provides an opportunity to expand the clinical repertoire of this efficacious agent by enabling ready aqueous dispersion. Future studies utilizing nanocurcumin are warranted in pre-clinical in vivo models of cancer and other diseases that might benefit from the effects of curcumin.

Purpose: To determine if pulsed-high intensity focused ultrasound (HIFU) could effectively serve as a source of hyperthermia with thermosensitive liposomes to enhance delivery and efficacy of doxorubicin in tumors.
Experimental Design: Comparisons in vitro and in vivo were carried out between non–thermosensitive liposomes (NTSL) and low temperature–sensitive liposomes (LTSL). Liposomes were incubated in vitro over a range of temperatures and durations, and the amount of doxorubicin released was measured. For in vivo experiments, liposomes and free doxorubicin were injected i.v. in mice followed by pulsed-HIFU exposures in s.c. murine adenocarcinoma tumors at 0 and 24 h after administration. Combinations of the exposures and drug formulations were evaluated for doxorubicin concentration and growth inhibition in the tumors.
Results: In vitro incubations simulating the pulsed-HIFU thermal dose (42°C for 2 min) triggered release of 50% of doxorubicin from the LTSLs; however, no detectable release from the NTSLs was observed. Similarly, in vivo experiments showed that pulsed-HIFU exposures combined with the LTSLs resulted in more rapid delivery of doxorubicin as well as significantly higher i.t. concentration when compared with LTSLs alone or NTSLs, with or without exposures. Combining the exposures with the LTSLs also significantly reduced tumor growth compared with all other groups.
Conclusions: Combining low-temperature heat-sensitive liposomes with noninvasive and nondestructive pulsed-HIFU exposures enhanced the delivery of doxorubicin and, consequently, its antitumor effects. This combination therapy could potentially produce viable clinical strategies for improved targeting and delivery of drugs for treatment of cancer and other diseases.

Antiangiogenic gene therapy with systemically administered sFlt-1 plasmid DNA in engineered gelatin-based nanovectors.
Kommareddy S, Amiji M.
Cancer Gene Ther. 2007 May;14(5):488-98.
[ expand abstract ]

This study examined the potential of engineered gelatin-based nanoparticulate vectors for systemic delivery of therapeutic genes to human solid tumor xenografts in vivo. Plasmid DNA encoding for the soluble form of the extracellular domain of vascular endothelial growth factor receptor-1 (VEGF-R1 or sFlt-1) was encapsulated in the control and poly(ethylene glycol) (PEG)-modified gelatin-based nanoparticles. When the plasmid DNA was delivered in PEG-modified thiolated gelatin nanoparticles, highest levels of sFlt-1 expression was observed in vitro in MDA-MB-435 human breast adenocarcinoma cell line. In addition, upon intravenous administration in female Nu/Nu mice bearing orthotopic MDA-MB-435 breast adenocarcinoma xenografts, efficient in vivo expression of sFlt-1 plasmid DNA was confirmed quantitatively by enzyme-linked immunosorbent assay and qualitatively by Western blot analysis. The expressed sFlt-1 was therapeutically active as shown by suppression of tumor growth and microvessel density measurements. The results of this study show that PEG-modified gelatin-based nanovectors can serve as a safe and effective systemically administered gene delivery vehicle for solid tumor.

Modulation of Intracellular Ceramide Using Polymeric Nanoparticles to Overcome Multidrug Resistance in Cancer
van Vlerken LE, Duan Z, Seiden MV, Amiji MM.
Cancer Res. May 15; 67 (10):4843-50.
[ expand abstract ]

Although multidrug resistance (MDR) is known to develop through a variety of molecular mechanisms within the tumor cell, many tend to converge toward the alteration of apoptotic signaling. The enzyme glucosylceramide synthase (GCS), responsible for bioactivation of the proapoptotic mediator ceramide to a nonfunctional moiety glucosylceramide, is overexpressed in many MDR tumor types and has been implicated in cell survival in the presence of chemotherapy. The purpose of this study was to investigate the therapeutic strategy of coadministering ceramide with paclitaxel, a commonly used chemotherapeutic agent, in an attempt to restore apoptotic signaling and overcome MDR in the human ovarian cancer cell line SKOV3. Poly(ethylene oxide)-modified poly(epsilon-caprolactone) (PEO-PCL) nanoparticles were used to encapsulate and deliver the therapeutic agents for enhanced efficacy. Results show that indeed the cotherapy eradicates the complete population of MDR cancer cells when they are treated at their IC50 dose of paclitaxel. More interestingly, when the cotherapy was combined with the properties of nanoparticle drug delivery, the MDR cells can be resensitized to a dose of paclitaxel near the IC50 of non-MDR (drug sensitive) cells, indicating a 100-fold increase in chemosensitization via this approach. Molecular analysis of activity verified the hypothesis that the efficacy of this therapeutic approach is indeed due to a restoration in apoptotic signaling, although the beneficial properties of PEO-PCL nanoparticle delivery seemed to enhance the therapeutic success even further, showing the promising potential for the clinical use of this therapeutic strategy to overcome MDR.

Ultrafine hydrogel nanoparticles: synthetic approach and therapeutic application in living cells.
Gao D, Xu H, Philbert MA, Kopelman R.
Angew Chem Int Ed Engl. 2007;46(13):2224-7.
[ expand abstract ]

No abstract available. (Citation link)

On the suitability of nanocrystalline ferrites as a magnetic carrier for drug delivery: functionalization, conjugation and drug release kinetics.
Rana S, Gallo A, Srivastava RS, Misra RD.
Acta Biomater. 2007 Mar;3(2):233-42.
[ expand abstract ]

Superparamagnetic nickel ferrite nanoparticles functionalized with polyvinyl alcohol, polyethylene oxide and polymethacrylic acid (PMAA) polymers and subsequently conjugated with doxorubicin anti-cancer drug are studied for their use as a magnetic carrier for drug delivery. Fourier transform infrared spectroscopy enabled examination of the ability of the nanoparticles to be functionalized with polymers and conjugated with doxorubicin drug. The functionalized polymer-coated nanocrystalline nickel ferrites retain the magnetic characteristics of non-functionalized nanocrystalline nickel ferrites (superparamagnetism, absence of hysteresis, remanence and coercivity at room temperature), encouraging their application as a magnetic carrier for drug delivery. The PMAA-coated nanoferrites are demonstrated as being a potentially superior magnetically targeted drug carrier based on FTIR results and drug release kinetics in the absence and presence of an external magnetic field.

Improvement of cancer-targeting therapy, using nanocarriers for intractable solid tumors by inhibition of TGF-Β signaling.
Kano MR, Bae Y, Iwata C, Morishita Y,Yashiro M, Oka M, Fujii T, Komuro A, Kiyono K, Kaminishi M, Hirakawa K, Ouchi Y, Nishiyama N, Kataoka K, Miyazono K.
Proc Natl Acad Sci U S A. 2007 Feb 27;104 (9):3460-65.
[ expand abstract ]

Transforming growth factor (TGF)-Β plays a pivotal role in regulation of progression of cancer through effects on tumor microenvironment as well as on cancer cells. TGF-Β inhibitors have recently been shown to prevent the growth and metastasis of certain cancers. However, there may be adverse effects caused by TGF-Β signaling inhibition, including the induction of cancers by the repression of TGF-Β-mediated growth inhibition. Here, we present an application of a short-acting, small-molecule TGF-Β type I receptor (TΒR-I) inhibitor at a low dose in treating several experimental intractable solid tumors, including pancreatic adenocarcinoma and diffuse-type gastric cancer, characterized by hypovascularity and thick fibrosis in tumor microenvironments. Low-dose TΒR-I inhibitor altered neither TGF-Β signaling in cancer cells nor the amount of fibrotic components. However, it decreased pericyte coverage of the endothelium without reducing endothelial area specifically in tumor neovasculature and promoted accumulation of macromolecules, including anticancer nanocarriers, in the tumors. Compared with the absence of TΒR-I inhibitor, anticancer nanocarriers exhibited potent growth-inhibitory effects on these cancers in the presence of TΒR-I inhibitor. The use of TΒR-I inhibitor combined with nanocarriers may thus be of significant clinical and practical importance in treating intractable solid cancers.

Immunological properties of engineered nanomaterials.
Dobrovolskaia M, McNeil SE.
Nature Nanotechnology. 2007 Jul 29
[ view article ]

Nanoparticles of poly(lactide)-tocopheryl polyethylene glycol succinate (PLA-TPGS) copolymers for protein drug delivery.
Lee SH, Zhang Z, Feng SS.
Biomaterials. 2007 Apr;28(11):2041-50.
[ expand abstract ]

Nanoparticles (NPs) of poly(lactide)-tocopheryl polyethylene glycol succinate (PLA-TPGS) copolymers with various PLA:TPGS component ratios were prepared by the double emulsion technique for protein drug formulation with bovine serum albumin (BSA) as a model protein. Influence of the PLA:TPGS component ratio and the BSA loading level on the drug encapsulation efficiency (EE) and in vitro drug release behavior was investigated. The PLA-TPGS NPs achieved 16.7% protein drug loading and 75.6% EE, which exhibited a biphasic pattern of controlled protein release with higher initial burst for those NPs of more TPGS content. Furthermore, the released proteins retained good structural integrity for at least 35 days at 37 degrees C as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and circular dichroism (CD) spectroscopy. Compared with other biodegradable polymeric NPs such as poly(d,l-lactide-co-glycolide) (PLGA) NPs, PLA-TPGS NPs could provide the encapsulated proteins a milder environment. Confocal laser scanning microscopy (CLSM) observation demonstrated the intracellular uptake of the PLA-TPGS NPs by NIH-3T3 fibroblast cells and Caco-2 cancer cells. This research suggests that PLA-TPGS NPs could be of great potential for clinical formulation of proteins and peptides.

The Antioxidant Tempamine: In Vitro Antitumor and Neuroprotective Effects and Optimization of Liposomal Encapsulation and Release.
Wasserman V, Kizelsztein P, Garbuzenko O, Kohen R, Ovadia H, Tabakman R, Barenholz Y.
Langmuir. 2007 Feb 13;23(4):1937-1947.
[ expand abstract ]

The piperidine nitroxide tempamine (TMN) is a cell-permeable, stable radical having antioxidant, anticancer, and proapoptotic and/or pronecrotic activities, as was demonstrated by us in cell cultures. We also demonstrated synergism between TMN and doxorubicin in doxorubicin-sensitive and doxorubicin-resistant cell lines. Treatment of the C26 mouse colon carcinoma model in vivo also demonstrated synergism between TMN and doxorubicin in sterically stabilized liposomes (SSLs) containing TMN (SSL-TMN) and those containing doxorubicin. The above effects of TMN and SSL-TMN motivated us to develop and optimize the SSL-TMN formulation so that it will be able to reach the disease site with a sufficiently high TMN level and a release rate needed to achieve a therapeutic effect. Because TMN is an amphipathic weak base, it was remote loaded by an intraliposome high/extraliposome low transmembrane ammonium sulfate gradient. The kinetics and level of TMN loading were monitored by cyclic voltammetry (CV) and electron paramagnetic resonance (EPR); the latter also indicates TMN precipitation in the intraliposomal aqueous phase. The regeneration of the original CV and EPR signals by the ionophore nigericin indicates that TMN remained fully intact during loading and release. The cardinal role of the transmembrane ammonium ion gradient in the loading process was proven by the use of the selective ionophores nonactin (for NH4+) and nigericin (for H+). The anion of the ammonium salts affects loading stability and the rate of TMN release, both mediated through the TMN state of aggregation in the intraliposomal aqueous phase. The greater the TMN salt precipitation, the slower the TMN release rate. This was supported by measurement of osmolality, which is inversely related to TMN salt precipitate. Precipitation is in the order SO4-2 > Cl-1 > glucuronate-1. Liposome lipid composition, magnitude of the transmembrane ammonium ion gradient, and type of anion of the ammonium salt determine the amount of TMN loaded and its release rate.

Organically Modified Silica Nanoparticles Co-encapsulating Photosensitizing Drug and Aggregation-Enhanced Two-Photon Absorbing Fluorescent Dye Aggregates for Two-Photon Photodynamic Therapy.
Kim S, Ohulchanskyy TY, Pudavar HE, Pandey RK, Prasad PN.
J Am Chem Soc. 2007 Feb 9; [Epub ahead of print].
[ expand abstract ]

We report energy-transferring organically modified silica nanoparticles for two-photon photodynamic therapy. These nanoparticles co-encapsulate two-photon fluorescent dye nanoaggregates as an energy up-converting donor and a photosensitizing PDT drug as an acceptor. They combine two features: (i) aggregation-enhanced two-photon absorption and emission properties of a novel two-photon dye and (ii) nanoscopic fluorescence resonance energy transfer between this nanoaggregate and a photosensitizer, 2-devinyl-2-(1-hexyloxyethyl)pyropheophorbide. Stable aqueous dispersions of the co-encapsulating nanoparticles (diameter </= 30 nm) have been prepared in the nonpolar interior of micelles by coprecipitating an organically modified silica sol with the photosensitizer and an excess amount of the two-photon dye which forms fluorescent aggregates by phase separation from the particle matrix. Using a multidisciplinary nanophotonic approach, we show: (i) indirect excitation of the photosensitizer through efficient two-photon excited intraparticle energy transfer from the dye aggregates in the intracellular environment of tumor cells and (ii) generation of singlet oxygen and in vitro cytotoxic effect in tumor cells by photosensitization under two-photon irradiation.

siRNA Delivery into Human T Cells and Primary Cells with Carbon-Nanotube Transporters.
Liu Z, Winters M, Holodniy M, Dai H.
Angew Chem Int Ed Engl. 2007 Feb 9; [Epub ahead of print].
[ expand abstract ]

No abstract available

Bifunctional compounds for targeted hepatic gene delivery.
Kim KS, Lei Y, Stolz DB, Liu D.
Gene Ther. 2007 Feb 8; [Epub ahead of print].
[ expand abstract ]

A series of bifunctional compounds with galactosyl residues as targeting ligand for asialoglycoprotein receptors on hepatocytes and various dendrimers as the DNA-binding domain was synthesized. When mixed with plasmid DNA, these compounds self assembled into particles that exhibited high transfection activity both in vitro and in vivo. Optimal activity in liver cells was observed with compounds containing three galactosyl residues and 16 dendrimer arms. These results suggest that domain-based design is an effective strategy for development of a new generation of synthetic gene carriers.

Dendritic Molecular Transporters Provide Control of Delivery to Intracellular Compartments.
Huang K, Voss B, Kumar D, Hamm HE, Harth E.
Bioconjug Chem. 2007 Feb 7; [Epub ahead of print].
[ expand abstract ]

Novel biocompatible macromolecular vectors were developed that not only enable transport of bioactive cargo across the cell membrane but also control the delivery into defined intracellular compartments. This work describes the synthesis and design of two non-peptidic fluorescently labeled Newkome-type dendrimers, differentiated over a varied alkyl spacer with guanidine end moieties. The internalization of the fluorescein-labeled molecular transporter into mammalian cells showed strong subcellular localizations, evident with both live cells and fixed cells costained with DAPI, a nuclear stain. We observed that the subcellular distribution of these vectors varied significantly, as one of the vectors concentrates in the nucleus (FD-1) while the other (FD-2) concentrates in the cytosol. All experiments performed with NIH-3T3 fibroblasts and human microvascular endothelial cells (HMEC) showed similar results. The differential localization patterns of the two molecular transporters can be controlled through the variation of alkyl spacer length at the terminal generation of the dendrimer. Intracellular delivery of bioactive entities into specific subcellular locations, utilizing this practical approach, might overcome limitations in drug delivery and pioneer future technologies in drug transport.

Folate Receptor Targeted Delivery of Polyelectrolyte Complex Micelles Prepared from ODN-PEG-Folate Conjugate and Cationic Lipids.
Kim SH, Jeong JH, Mok H, Lee SH, Kim SW, Park TG.
Biotechnol Prog. 2007 Feb 2;23(1):232-237.
[ expand abstract ]

A polyelectrolyte complex micelle (PECM)-based delivery system for targeting folate (FOL) receptor overexpressing tumor cells is demonstrated using poly(ethylene glycol) (PEG)-conjugated oligonucleotide (ODN). The tumor targeting property was conferred to the PECM by tethering a folate moiety to the distal end of the PEG segment in an anti-sense green fluorescent protein (GFP) ODN-PEG conjugate. Nanoscale PECMs were spontaneously produced from ionic interactions between the ODN-PEG-FOL conjugate and a cationic lipid, lipofectamine (Lf). When treated with FOL receptor overexpressing cells (KB), the PCEMs caused a significant reduction in GFP expression in a dose-dependent manner. This effect was not observed in FOL receptor deficient cells (A549). The enhanced transfection of ODN-PEG-FOL/Lf PECMs to KB cells was caused by FOL receptor mediated endocytosis. The efficiency of target-specific gene suppression by ODN-PEG-FOL/Lf PECMs was maintained even in the presence of 10% fetal bovine serum in the transfection medium.

New Method for Delivering a Hydrophobic Drug for Photodynamic Therapy Using Pure Nanocrystal Form of the Drug.
Baba K, Pudavar HE, Roy I, Ohulchanskyy TY, Chen Y, Pandey RK, Prasad PN.
Mol Pharm. 2007 Feb 1; [Epub ahead of print].
[ expand abstract ]

A carrier-free method for delivery of a hydrophobic drug in its pure form, using nanocrystals (nanosized crystals), is proposed. To demonstrate this technique, nanocrystals of a hydrophobic photosensitizing anticancer drug, 2-devinyl-2-(1-hexyloxyethyl)pyropheophorbide (HPPH), have been synthesized using the reprecipitation method. The resulting drug nanocrystals were monodispersed and stable in aqueous dispersion, without the necessity of an additional stabilizer (surfactant). As shown by confocal microscopy, these pure drug nanocrystals were taken up by the cancer cells with high avidity. Though the fluorescence and photodynamic activity of the drug were substantially quenched in the form of nanocrystals in aqueous suspension, both these characteristics were recovered under in vitro and in vivo conditions. This recovery of drug activity and fluorescence is possibly due to the interaction of nanocrystals with serum albumin, resulting in conversion of the drug nanocrystals into the molecular form. This was confirmed by demonstrating similar recovery in presence of fetal bovine serum (FBS) or bovine serum albumin (BSA). Under similar treatment conditions, the HPPH in nanocrystal form or in 1% Tween-80/water formulation showed comparable in vitro and in vivo efficacy. Keywords: Nanocrystals; reprecipitation method; photosensitizers; photodynamic therapy; singlet oxygen; drug delivery.

Effect of coupling of albumin onto surface of PEG liposome on its in vivo disposition.
Furumoto K, Yokoe J, Ogawara K, Amano S, Takaguchi M, Higaki K, Kai T, Kimura T.
Int J Pharm. 2007 Feb 1;329(1-2):110-6.
[ expand abstract ]

To evaluate the effect of coupling of albumin onto the surface of poly(ethylene glycol)-modified liposome (PEG liposome) on the in vivo disposition of liposome, pharmacokinetics and tissue distribution were examined after intravenous administration of rat serum albumin-modified PEG (RSA/PEG) liposome into rats. RSA/PEG liposome showed longer blood-circulating property than PEG liposome and the hepatic clearance for RSA/PEG liposome was significantly smaller than that for PEG liposome. Single-pass liver perfusion experiments also showed that the hepatic disposition of RSA/PEG liposome was much less than that of PEG liposome and that pre-treatment of liver with trypsin did not significantly reduce the hepatic disposition of RSA/PEG liposome, suggesting that RSA/PEG liposome could avoid the hepatic uptake via the receptor-mediated endocytosis. To unravel the mechanism behind the less affinity of RSA/PEG liposome to the liver, serum proteins associated on their surface were quantitatively and qualitatively assessed. The results showed that the coupling of albumin onto PEG liposome significantly reduced the total amount of serum proteins associated onto the surface, and SDS-PAGE revealed that the decrease in the association with liposomes for several serum proteins, which might have opsonic activity. From these findings, introduction of serum albumin onto PEG liposome could be useful to develop a new nanoparticulate formulation with a better pharmacokinetic property.

Octaarginine-modified multifunctional envelope-type nanoparticles for gene delivery.
Khalil IA, Kogure K, Futaki S, Hama S, Akita H, Ueno M, Kishida H, Kudoh M, Mishina Y, Kataoka K, Yamada M, Harashima H.
Gene Ther. 2007 Feb 1; [Epub ahead of print].
[ expand abstract ]

This study describes a multifunctional envelope-type nano device (MEND) that mimics an envelope-type virus based on a novel packaging strategy. MEND particles contain a DNA core packaged into a lipid envelope modified with an octaarginine peptide. The peptide mediates internalization via macropinocytosis, which avoids lysosomal degradation. MEND-mediated transfection of a luciferase expression plasmid achieved comparable efficiency to adenovirus-mediated transfection, with lower associated cytotoxicity. Furthermore, topical application of MEND particles containing constitutively active bone morphogenetic protein (BMP) type IA receptor (caBmpr1a) gene had a significant impact on hair growth in vivo. These data demonstrate that MEND is a promising non-viral gene delivery system that may provide superior results to existing non-viral gene delivery technologies.

Effect of transferrin receptor-targeted liposomal doxorubicin in P-glycoprotein-mediated drug resistant tumor cells.
Kobayashi T, Ishida T, Okada Y, Ise S, Harashima H, Kiwada H.
Int J Pharm. 2007 Feb 1;329(1-2):94-102.
[ expand abstract ]

The over-expression of P-glycoprotein (P-gp) has been associated with the development of multidrug resistance (MDR) in cancer cells. In this study, we examined whether transferrin receptor (Tf-R) targeted liposomes can efficiently deliver encapsulated doxorubicin (DXR) into MDR cells (SBC-3/ADM) via Tf-R-mediated endocytosis thus overcoming MDR by by-passing P-gp-mediated drug efflux. We prepared four types of liposome, i.e. untargeted and Tf-R-targeted, made of either egg-PC/cholesterol or hydrogenated egg PC/cholesterol. Only with the targeted EPC-liposome we achieved significant delivery of encapsulated DXR and increased cytotoxicity of encapsulated DXR on the MDR cells (3.5-fold higher than free DXR). Confocal microscopy and an intracellular drug-accumulation assay indicated that the targeted liposomes efficiently delivered DXR into cells where it readily accumulated in the nucleus, in both drug-sensitive and MDR cells. These findings suggest that the targeted liposomes are rapidly internalized via Tf-R-mediated endocytosis followed by release of their contents into the cytoplasm. The rapid internalization and content release, most likely facilitated by the higher fluidity of the EPC-based liposomes, may explain why only targeted EPC-liposomes were able to prevent drug efflux by P-gp and to consequently circumvent MDR. Our results indicate that in order to achieve MDR circumvention by means of liposomal encapsulation of DXR the liposomes not only need to be targeted, but also to have the proper physicochemical properties for adequate release of the drug. Furthermore, these in vitro results suggest that Tf-R targeted EPC-liposomes are a potentially useful drug delivery system to circumvent P-gp-mediated MDR of tumors.

Oil-Encapsulating PEO-PPO-PEO/PEG Shell Cross-Linked Nanocapsules for Target-Specific Delivery of Paclitaxel.
Bae KH, Lee Y, Park TG.
Biomacromolecules. 2007 Feb;8(2):650-6.
[ expand abstract ]

PEO-PPO-PEO/PEG shell cross-linked nanocapsules encapsulating an oil phase in their nanoreservoir structure was developed as a target-specific carrier for a water-insoluble drug, paclitaxel. Oil-encapsulating PEO-PPO-PEO/PEG composite nanocapsules were synthesized by dissolving an oil (Lipiodol) and an amine-reactive PEO-PPO-PEO derivative in dichloromethane and subsequently dispersing in an aqueous solution containing amine-functionalized six-arm-branched poly(ethylene glycol) by ultrasonication. The resultant shell cross-linked nanocapsules had a unique core/shell architecture with an average size of 110.7 +/- 9.9 nm at 37 degrees C, as determined by dynamic light scattering and transmission electron microscopy. Paclitaxel could be effectively solubilized in the inner Lipiodol phase surrounded by a cross-linked PEO-PPO-PEO/PEG shell layer. The paclitaxel-loaded nanocapsules were further conjugated with folic acid to achieve folate receptor targeted delivery. Confocal microscopy and flow cytometric analysis revealed that folate-mediated targeting significantly enhanced the cellular uptake and apoptotic effect against folate receptor overexpressing cancer cells. The present study suggested that these novel nanomaterials encapsulating an oil reservoir could be potentially applied for cancer cell targeted delivery of various water-insoluble therapeutic and diagnostic agents.

Formulation of functionalized PLGA-PEG nanoparticles for in vivo targeted drug delivery.
Cheng J, Teply BA, Sherifi I, Sung J, Luther G, Gu FX, Levy-Nissenbaum E, Radovic-Moreno AF, Langer R, Farokhzad OC.
Biomaterials. 2007 Feb;28(5):869-76.
[ expand abstract ]

Nanoparticle (NP) size has been shown to significantly affect the biodistribution of targeted and non-targeted NPs in an organ specific manner. Herein we have developed NPs from carboxy-terminated poly(d,L-lactide-co-glycolide)-block-poly(ethylene glycol) (PLGA-b-PEG-COOH) polymer and studied the effects of altering the following formulation parameters on the size of NPs: (1) polymer concentration, (2) drug loading, (3) water miscibility of solvent, and (4) the ratio of water to solvent. We found that NP mean volumetric size correlates linearly with polymer concentration for NPs between 70 and 250 nm in diameter (linear coefficient=0.99 for NPs formulated with solvents studied). NPs with desirable size, drug loading, and polydispersity were conjugated to the A10 RNA aptamer (Apt) that binds to the prostate specific membrane antigen (PSMA), and NP and NP-Apt biodistribution was evaluated in a LNCaP (PSMA+) xenograft mouse model of prostate cancer. The surface functionalization of NPs with the A10 PSMA Apt significantly enhanced delivery of NPs to tumors vs. equivalent NPs lacking the A10 PSMA Apt (a 3.77-fold increase at 24h; NP-Apt 0.83%+/-0.21% vs. NP 0.22%+/-0.07% of injected dose per gram of tissue; mean+/-SD, n=4, p=0.002). The ability to control NP size together with targeted delivery may result in favorable biodistribution and development of clinically relevant targeted therapies.

The effect of paclitaxel-loaded nanoparticles with radiation on hypoxic MCF-7 cells.
Jin C, Wu H, Liu J, Bai L, Guo G.
J Clin Pharm Ther. 2007 Feb;32(1):41-7.
[ expand abstract ]

Background and Objective: The inability of radiotherapy to eradicate completely certain human tumours may be due to the presence of resistant hypoxic cells. Several studies have confirmed the radiosensitizing effect of paclitaxel, a microtubular inhibitor. The object of this study was to evaluate the physicochemical characteristics of paclitaxel-loaded nanoparticles, and determine the ability of the released paclitaxel to radiosensitize hypoxic human breast carcinoma cells (MCF-7) with respect to radiation dose. Methods: The poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles containing paclitaxel were prepared by o/w emulsification-solvent evaporation method. The morphology of the paclitaxel-loaded nanoparticles was investigated by scanning electron microscopy. The drug encapsulation efficiency (EE) and in vitro release profile were measured by high-performance liquid chromatography. Cell cycle was evaluated by flow cytometry. Cell viability was measured by the ability of single cells to form colonies in vitro. Results: The prepared nanoparticles were spherical with diameter between 200 and 800 nm. The EE was 85.5%. The drug release pattern was biphasic with a fast release rate followed by a slow one. Co-culture of human breast carcinoma cells (MCF-7) with paclitaxel-loaded nanoparticles demonstrated that released paclitaxel retained its bioactivity to block cells in the G2/M phase of the cell cycle and effectively sensitized hypoxic MCF-7 cells to radiation with radiosensitivity shown to be dependent of radiation dose at levels of dosages studied. The sensitizer enhancement ratio for paclitaxe-loaded nanoparticles at 10% survival is approximately 1.4. Conclusion: This work has demonstrated that paclitaxel can be effectively released from a biodegradable PLGA nanoparticle delivery system while maintaining potent combined cytotoxic and radiosensitizing abilities for hypoxic tumour cells.

Challenges to macromolecular drug delivery.
Juliano R.
Biochem Soc Trans. 2007 Feb;35(Pt 1):41-3.
[ expand abstract ]

The use of macromolecules, particularly monoclonal antibodies, as therapeutic agents has come to the forefront in recent years. The biodistribution and delivery issues for protein drugs are shared to a substantial degree with other emerging therapeutic approaches including pharmacologically active nucleic acids and nanoparticles. A generalized approach to these issues involves consideration of the multiple biological barriers that stand between the macromolecular drug or nanoparticle at its site of administration and its ultimate biological target. Considerations of size, stability, non-specific versus specific associations and potency versus toxicity all play a role. The creation of delivery approaches that combine high specificity for the target cell or tissue, high therapeutic payload and modest toxicity remains a challenge, although some very promising examples have emerged recently. A variety of sophisticated targeting strategies, based primarily on combinatorial library methods, when used in combination with new technologies to identify cell-surface receptor 'signatures' of specific tissues, will facilitate advances in targeted delivery of macromolecules and nanoparticles. The challenges to contemporary macromolecule drug delivery are complex, thus new research paradigms are emerging that combine the talents of physical and biological scientists to address this key issue for modern pharmacology and therapeutics.

Low-density lipoprotein receptor-mediated endocytosis of PEGylated nanoparticles in rat brain endothelial cells.
Kim HR, Gil S, Andrieux K, Nicolas V, Appel M, Chacun H, Desmaele D, Taran F, Georgin D, Couvreur P.
Cell Mol Life Sci. 2007 Feb;64(3):356-64.
[ expand abstract ]

Poly(methoxypolyethyleneglycol cyanoacrylate-co-hexadecylcyanoacrylate) (PEG-PHDCA) nanoparticles have demonstrated their capacity to diffuse through the blood-brain barrier after intravenous administration. However, the mechanism of transport of these nanoparticles into brain has not yet been clearly elucidated. The development of a model of rat brain endothelial cells (RBEC) in culture has allowed investigations into this mechanism. A study of the intracellular trafficking of nanoparticles by cell fractionation and confocal microscopy showed that nanoparticles are internalized by the endocytic pathway. Inhibition of the caveolae-mediated pathway by preincubation with filipin and nystatin did not modify the cellular uptake of the nanoparticles. In contrast, chlorpromazine and NaN(3) pretreatment, which interferes with clathrin and energy-dependent endocytosis, caused a significant decrease of nanoparticle internalization. Furthermore, cellular uptake experiments with nanoparticles preincubated with apolipoprotein E and blocking of low-density lipoprotein receptors (LDLR) clearly suggested that the LDLR-mediated pathway was involved in the endocytosis of PEGPHDCA nanoparticles by RBEC.

Biodistribution and pharmacokinetic analysis of long-circulating thiolated gelatin nanoparticles following systemic administration in breast cancer-bearing mice.
Kommareddy S, Amiji M.
J Pharm Sci. 2007 Feb;96(2):397-407.
[ expand abstract ]

The objective of the present study was to modify thiolated gelatin nanoparticles with poly(ethylene glycol) (PEG) chains and examine their long circulating and tumor-targeting properties in vivo in an orthotopic a human breast adenocarcinoma xenograft model. The crosslinked nanoparticle systems were characterized to have a size of 150-250 nm with rapid payload release properties in a highly reducing environment. Upon PEG modification, the nanoparticle size increased to 300-350 nm in diameter. The presence of PEG chains on the surface was confirmed by characterization with electron spectroscopy for chemical analysis. The in vivo long-circulating potential, biodistribution and passive tumor targeting of the controls, and PEG-modified thiolated gelatin nanoparticles were evaluated by injecting indium-111 ((111)In)-labeled nanoparticles into breast tumor (MDA-MB-435)-bearing nude mice. Upon modification with PEG, the nanoparticles were found to have longer circulation times, with the plasma and tumor half-lives of 15.3 and 37.8 h, respectively. The results also showed preferential localization of thiolated nanoparticles in the tumor mass. The resulting nanoparticulate systems with long circulation properties could be used to target encapsulated drugs and genes to tumors passively by utilizing the enhanced permeability and retention effect of the tumor vasculature.

The influence of polymeric properties on chitosan/siRNA nanoparticle formulation and gene silencing.
Liu X, Howard KA, Dong M, Andersen MO, Rahbek UL, Johnsen MG, Hansen OC, Besenbacher F, Kjems J.
Biomaterials. 2007 Feb;28(6):1280-8.
[ expand abstract ]

We have previously introduced the use of the biomaterial chitosan to form chitosan/siRNA nanoparticles for gene silencing protocols. This present study shows that the physicochemical properties (size, zeta potential, morphology and complex stability) and in vitro gene silencing of chitosan/siRNA nanoparticles are strongly dependent on chitosan molecular weight (Mw) and degree of deacetylation (DD). High Mw and DD chitosan resulted in the formation of discrete stable nanoparticles approximately 200 nm in size. Chitosan/siRNA formulations (N:P 50) prepared with low Mw (approximately 10 kDa) showed almost no knockdown of endogenous enhanced green fluorescent protein (EGFP) in H1299 human lung carcinoma cells, whereas those prepared from higher Mw (64.8-170 kDa) and DD (approximately 80%) showed greater gene silencing ranging between 45% and 65%. The highest gene silencing efficiency (80%) was achieved using chitosan/siRNA nanoparticles at N:P 150 using higher Mw (114 and 170 kDa) and DD (84%) that correlated with formation of stable nanoparticles of approximately 200 nm. In conclusion, this work confirms the application of chitosan as a non-viral carrier for siRNA and the importance of polymeric properties for the optimisation of gene silencing using chitosan/siRNA nanoparticles.

Paclitaxel nanoparticle inhibits growth of ovarian cancer xenografts and enhances lymphatic targeting.
Lu H, Li B, Kang Y, Jiang W, Huang Q, Chen Q, Li L, Xu C.
Cancer Chemother Pharmacol. 2007 Feb;59(2):175-81.
[ expand abstract ]

OBJECTIVES: Ovarian cancer has the highest mortality of all the gynecologic cancers. The antitumor agent paclitaxel has been proved to be efficient in the treatment of ovarian cancer. Our study is to develop a polymeric drug delivery system for paclitaxel and determine whether paclitaxel nanoparticle can inhibit growth of ovarian carcinoma xenografts in Fisher344 (F344) rats by intraperitoneal administration. The mechanism of paclitaxel nanoparticles in rats bearing ovarian cancer has been investigated in this study. METHODS: Synthesize paclitaxel loading nanoparticle (PLA) by ultrasonic emulsification; MTT analysis identified cytotoxic activity of paclitaxel nanoparticle in vitro; rat ovarian carcinoma cells were injected into the peritoneal cavity of F344 rats. The antitumor effect of paclitaxel nanoparticle in vivo has been evaluated by measuring tumor weight and ascite volume. At the end of the procedure rats were sacrificed; tumors were excised and processed for PCNA staining, tissue terminal deoxynucleotide transferase-mediated dUTP nick and labeling assay and RT-PCR to evaluate the proliferative and apoptotic changes and cancer transfer-related gene expression induced by PLA. Paclitaxel concentration in plasma, pelvic lymph nodes, liver, and heart were determined by high-performance liquid chromatography. RESULTS: Paclitaxel nanoparticle and PTX (Cremophor) showed equivalent cytotoxic activity in vitro. In rats implanted carcinoma cells, paclitaxel nanoparticles significantly reduced tumor weight and ascites volume, and induced apoptosis of tumor cells. PLA also inhibited cell proliferation and matrix metalloproteinase 9 mRNA expression. The paclitaxel concentration of pelvic lymph nodes in PLA treated animals was 20-fold higher than that of free PTX treated animals at 48 h after intraperitoneal administration. CONCLUSION: The intraperitoneal administration of paclitaxel nanoparticle can significantly inhibit the progression of ovarian carcinoma in peritoneal cavity of female F344 rat. The paclitaxel nanoparticle is safe and lymphatic targeting.

Nanostructured calcium phosphates (NanoCaPs) for non-viral gene delivery: influence of the synthesis parameters on transfection efficiency.
Olton D, Li J, Wilson ME, Rogers T, Close J, Huang L, Kumta PN, Sfeir C.
Biomaterials. 2007 Feb;28(6):1267-79.
[ expand abstract ]

Calcium phosphate (CaP) based approaches remain an attractive option for delivering plasmid DNA (pDNA) into cultured cells. However, despite their appeal, current synthesis methodologies typically yield lower, less consistent transfection efficiencies when compared to viral approaches. Therefore, we report here a novel method to consistently synthesize efficient, nano-sized, mono-dispersed CaP-pDNA particles; accomplished by optimizing both the stoichiometry (Ca/P ratio) of the CaP particles as well as the mode in which the calcium and phosphate precursor solutions are mixed. Our results indicate that calcium and phosphate precursors when mixed in a controlled and regulated manner reproducibly result in nano-sized particles that consistently yield higher transfection efficiencies when compared to particles synthesized via manual mixing (a two-fold increase was observed). Also, maximum transfection efficiencies in both HeLa and MC3T3-E1 cells lines were obtained when a Ca/P ratio between 100 and 300 was used. Particles synthesized within this optimum Ca/P ratio range were between 25 and 50 nm. Our data suggests that these maximized transfection efficiencies were obtained because these particles not only effectively condensed (70% efficient) but also efficiently bound (90% efficient) the pDNA. In addition, X-ray diffraction and Fourier transform infrared spectroscopy analyses confirmed that all of the synthesized CaP structures exhibited the hydroxyapatite phase.

Nanotechnology approaches for drug and small molecule delivery across the blood brain barrier.
Silva GA.
Surg Neurol. 2007 Feb;67(2):113-6.
[ expand abstract ]

Nanotechnology involves the design, synthesis, and characterization of materials and devices that have a functional organization in at least one dimension on the nanometer (ie, one billionth of a meter) scale. One area in which nanotechnology may have a significant clinical impact in neuroscience is the selective transport and delivery of drugs and other small molecules across the blood brain barrier that cannot cross otherwise. Using a variety of nanoparticles composed of different chemical compositions, different groups are exploring proof-of-concept approaches for the delivery of different antineoplastic drugs, oligonucleotides, genes, and magnetic resonance imaging contrast agents. This review discusses some of the main technical challenges associated with the development of nanotechnologies for delivery across the blood brain barrier and summarizes ongoing work.

Interaction of photosensitizers with liposomes containing unsaturated lipid.
Voszka I, Budai M, Szabo Z, Maillard P, Csik G, Grof P.
Chem Phys Lipids. 2007 Feb;145(2):63-71.
[ expand abstract ]

Small unilamellar liposomes were made of dipalmitoyl-phosphatidylcholine and dioleoyl-phosphatidylcholine, and photosensitized by a symmetrically or an asymmetrically substituted glycosilated tetraphenyl-porphyrin derivative. As differential scanning calorimetry and electron paramagnetic resonance spectroscopy (EPR) revealed these porphyrin derivatives were localized in different depth within the lipid bilayer. Both porphyrin derivatives were able to induce photoreaction and consequent structural changes in the membrane. 5-, 12-, or 16-doxyl stearic acid labeled lipid bilayers were applied and the efficiency of photoinduced reaction was followed by the decay of their EPR signal amplitude. Light dose-dependent destruction of nitroxide radical proved to be dependent on the position of spin label. In this process the porphyrin localized in closer connection with the double bond of unsaturated fatty acid was more effective. EPR signal decay was also dependent on the unsaturated fatty acid content of the liposome and the oxygen saturation of the solvent.

Examination of Nonendocytotic Bulk Transport of Nanoparticles Across Phospholipid Membranes.
Banerji SK, Hayes MA.
Langmuir. 2007 Jan 30; [Epub ahead of print].
[ expand abstract ]

Nonendocytotic transport is believed to play a role in the transmigration of particles less than 100 nm within biological systems. Determining the fundamental mechanism of this transport across cell membranes is essential if nanotechnology is to be utilized in general medical practice and may lead to methods of treating the deleterious internalization of ambient, possibly pollutant, nanoparticles. In order to gain a broader understanding of nonendocytotic transmembrane transport, it becomes essential to devise a method which allows the isolation of fundamental modes of transport such as passive Brownian diffusion through a membrane, as opposed to effusion-like transport of particles through transmembrane channels. The passive Brownian diffusion contribution was investigated using gold nanoparticles and mimetic biomembranes. Specifically, gold nanoparticle dispersions consisting of 7, 10, and 15 nm diameter particles were captured in giant unilamelar vesicles composed of phosphatidylcholine, phosphatidic acid, and cholesterol. Nonendocytotic transmembrane transport was modeled as the time derivative of the appearance of nanoparticles in the phosphate buffer outside the vesicles at 37 degrees C. The results show the transport rate to be zero; hence, a simple diffusive process of transmembrane transport is not supported.

Studies on the oridonin-loaded poly(D,L-lactic acid) nanoparticles in vitro and in vivo.
Xing J, Zhang D, Tan T.
Int J Biol Macromol. 2007 Jan 30;40(2):153-8.
[ expand abstract ]

The purpose of this paper was to investigate the possibility of developing a polymeric nanoparticle delivery system for ORI to increase its solubility, blood circulation time and tissue targeting. Oridonin-loaded poly(D,L-lactic acid) nanoparticles (ORI-PLA-NP) were prepared by the further modified spontaneous emulsion solvent diffusion (MSESD) method. Studies were carried out to characterize and evaluate the produced ORI-PLA-NP both in vitro and in vivo. The experimental results showed that the mean size of the nanoparticles were 137.3 nm, with 87.2% of the nanoparticles distributed between the range of 107 and 195 nm. The entrapment efficiency and actual drug loading of the nanoparticles were 91.88+/-1.83 and 2.32+/-0.05%, respectively. It was demonstrated by differential scanning calorimetry (DSC) and X-ray diffractometry (XRD) that ORI existed in the form of amorphous in the nanoparticles. The in vitro release profile of ORI-PLA-NP could be expressed well by the Higuchi equation: Q=8.944t(1/2)+11.246. The results of pharmacokinetics demonstrated that being encapsulated in PLA nanoparticles was remarkably effective for ORI to prolong its blood circulation time. After the i.v. administration of ORI-PLA-NP, we could observe a stable and high concentration of ORI in liver, lung and spleen, while its distribution in heart and kidney decreased.

Cisplatin encapsulated in phosphatidylethanolamine liposomes enhances the in vitro cytotoxicity and in vivo intratumor drug accumulation against melanomas.
Hwang TL, Lee WR, Hua SC, Fang JY.
J Dermatol Sci. 2007 Jan 29; [Epub ahead of print].
[ expand abstract ]

BACKGROUND: Cisplatin is a potent anticancer drug for treating melanoma. OBJECTIVE: The aim of this study was to evaluate the possibility of using liposomes, for intratumoral distribution in a melanoma, composed of phosphatidylethanolamine (PE), for its cytotoxicity. METHOD: The in vitro drug release, in vitro cytotoxicity against melanoma, and in vivo residence time in the tumor of liposome-encapsulated cisplatin were investigated. The liposomes were prepared and characterized in terms of their morphology, size, zeta potential, and drug loading. RESULT: The size of the PE liposomes attained a level of approximately 100nm. The concentration of cisplatin encapsulated in PE liposomes was 50-70% dependent on the presence or absence of polyethylene glycol (PEG) derivatives. On the other hand, no or negligible cisplatin molecules were encapsulated in egg phosphatidylcholine (EPC) liposomes. PE liposomes had higher cytotoxicity than classic liposomes or free cisplatin. Images of confocal laser scanning microscopy confirmed the great potency of PE liposomes to deliver cisplatin into cells. The incorporation of PEG derivatives completely inhibited the proliferation of melanoma cells. With in vivo intratumoral administration, the cisplatin concentration in the tumor tissue was maintained at a high level for 72h after application of the PE liposomes. The PE liposomes delivered cisplatin into the tumor approximately 3.6 times more efficiently than the free drug. CONCLUSION: These results demonstrate that PE liposomes represent a potentially useful strategy for targeting cisplatin delivery into melanomas.

Lipid-based Nanoparticles for Nucleic Acid Delivery.
Li W, Szoka FC Jr.
Pharm Res. 2007 Jan 25; [Epub ahead of print].
[ expand abstract ]

Lipid-based colloidal particles have been extensively studied as systemic gene delivery carriers. The topic that we would like to emphasize is the formulation/assembly of lipid-based nanoparticles (NP) with diameter under 100 nm for delivering nucleic acid in vivo. NP are different from cationic lipid-nucleic acid complexes (lipoplexes) and are vesicles composed of lipids and encapsulated nucleic acids with a diameter less than 100 nm. The diameter of the NP is an important attribute to enable NP to overcome the various in vivo barriers for systemic gene delivery such as: the blood components, reticuloendothelial system (RES) uptake, tumor access, extracellular matrix components, and intracellular barriers. The major formulation factors that impact the diameter and encapsulation efficiency of DNA-containing NP include the lipid composition, nucleic acid to lipid ratio and formulation method. The particle assembly step is a critical one to make NP suitable for in vivo gene delivery. NP are often prepared using a dialysis method either from an aqueous-detergent or aqueous-organic solvent mixture. The resulting particles have diameters about 100 nm and nucleic acid encapsulation ratios are >80%. Additional components can then be added to the particle after it is formed. This ordered assembly strategy enables one to optimize the particle physico-chemical attributes to devise a biocompatible particle with increased gene transfer efficacy in vivo. The components included in the sequentially assembled NP include: poly(ethylene glycol) (PEG)-shielding to improve the particle pharmacokinetic behavior, a targeting ligand to facilitate the particle-cell recognition and in some case a bioresponsive lipid or pH-triggered polymer to enhance nucleic acid release and intracellular trafficking. A number of groups have observed that a PEG-shielded NP is a robust and modestly effective system for systemic gene or small interfering RNA (siRNA) delivery.

Employment of Cationic Solid-Lipid Nanoparticles as RNA Carriers.
Montana G, Bondi ML, Carrotta R, Picone P, Craparo EF, Biagio PL, Giammona G, Carlo MD.
Bioconjug Chem. 2007 Jan 25; [Epub ahead of print].
[ expand abstract ]

Gene transfer represents an important advance in the treatment of both genetic and acquired diseases. In this article, the suitability of cationically modified solid-lipid nanoparticles (SLN) as a nonviral vector for gene delivery was investigated, in order to obtain stable materials able to condense RNA. Cationic SLN were produced by microemulsion using Compritol ATO 888 as matrix lipid, Pluronic F68 as tenside, and dimethyldioctadecylammonium bromide (DDAB) as cationic lipid. The resulting particles were approximately 100 nm in size and showed a highly positive surface charge (+41 mV) in water. Size and shape were further characterized by scanning electron microscopy (SEM) measurements. Moreover, we utilized the sea urchin as a model system to test their applicability on a living organism. To evaluate cationic SLN ability to complex the in vitro transcribed Paracentrotus lividus bep3 RNA, we utilized both light scattering and gel mobility experiments, and protection by nuclease degradation was also investigated. By microinjection experiment, we demonstrated that the nanoparticles do not inference with the viability of the P. lividus embryo and the complex nanoparticles-bep3 permits movement of the RNA during its localization in the egg, suggesting that it could be a suitable system for gene delivery. Taken together, all these results indicate that the cationic SNL are a good RNA carrier for gene transfer system and the sea urchin a simple and versatile candidate to test biological properties of nanotechnology devices.

A Parallel Approach for Subwavelength Molecular Surgery Using Gene-Specific Positioned Metal Nanoparticles as Laser Light Antennas.
Csaki A, Garwe F, Steinbruck A, Maubach G, Festag G, Weise A, Riemann I, Konig K, Fritzsche W.
Nano Lett. 2007 Jan 24; [Epub ahead of print].
[ expand abstract ]

An optical technique for the parallel manipulation of nanoscale structures with molecular resolution is presented. Bioconjugated metal nanoparticles are thereby positioned at the location of interest, such as, e.g., certain DNA sequences along metaphase chromosomes, prior to pulsed laser light irradiation of the whole sample. The nanoparticles are designed to absorb the introduced energy highly efficiently, in that way acting as nanoantenna. As result of the interaction, structural changes of the sample with subwavelength dimensions and nanoscale precision are observed at the location of the particles. The process leading to the nanolocalized destruction is caused by particle ablation as well as thermal damage of the surrounding material.

Nanosized Paclitaxel Particles from Supercritical Carbon Dioxide Processing and Their Biological Evaluation.
Pathak P, Prasad GL, Meziani MJ, Joudeh AA, Sun YP.
Langmuir. 2007 Jan 23; [Epub ahead of print].
[ expand abstract ]

The rapid expansion of a supercritical solution into a liquid solvent (RESOLV) technique with benign supercritical carbon dioxide was applied to obtain aqueous suspended nanoparticles of the highly potent anticancer drug paclitaxel. The paclitaxel nanoparticles were protected from agglomeration by using a known nontoxic stabilization agent. The aqueous suspended paclitaxel nanoparticles of different average particle sizes were evaluated in vitro against human breast cancer cells. The results suggest that the nanosized paclitaxel particles are effective, with an antineoplastic activity comparable to that of the commercial paclitaxel formulation. The technique should be generally applicable to the processing of nanoparticles from other important drugs with aqueous solubility problems.

Coating of negatively charged liposomes by polylysine: Drug release study.
Volodkin D, Mohwald H, Voegel JC, Ball V.
J Control Release. 2007 Jan 22;117(1):111-20.
[ expand abstract ]

The present work describes surface coating of carboxyfluorescein(CF)-loaded liposomes with poly-l-lysine (PLL) and liposome membrane permeability. The vesicles were prepared from synthetic or natural lipids. Interaction between PLL and the liposomes leads to the formation of complexes - either single PLL-coated vesicles or vesicle aggregates. Formation of the complexes is strongly affected by PLL/lipid molar ratio and the molecular mass of the PLL chains. Liposome permeability depends strongly on the lipid phase state - vesicles in the solid state retained the entrapped dye for a long time, but continuous CF release was registered for "fluid" vesicles. Crossing the transition temperature leads to intensive dye leakage because of the appearance of leaky interfacial domains between the coexisting solid and liquid phases and also because of a reversible change in the vesicle size upon the solid-liquid state phase transition. PLL coverage does not cause permeabilization of "solid" liposomes, but increases the permeability of "fluid" vesicles. At the same time, the results of differential scanning calorimetry and vesicle fusion suggest that PLL adsorption occurs exclusively on the vesicular surface and that the lipidic organization is not significantly disturbed. Moreover, PLL does not prevent lipid exchange between vesicles induced by temperature change.

The use of fullerene substituted phenylalanine amino acid as a passport for peptides through cell membranes.
Yang J, Wang K, Driver J, Yang J, Barron AR.
Org Biomol Chem. 2007 Jan 21;5(2):260-6.
[ expand abstract ]

We report the formation of a fullerene-peptide conjugate via the incorporation of a fullerene substituted phenylalanine derivative, "Bucky amino acid" (Baa), to a cationic peptide, which acts as a passport for intracellular delivery, enabling transport of a range of sequences into HEK-293, HepG2, and neuroblastoma cells where the peptides in the absence of the fullerene amino acid cannot enter the cell. Delivery of the fullerene species to either the cytoplasm or nucleus of the cell is demonstrated. Fullerene peptides based on the nuclear localization sequence (NLS), H-Baa-Lys(FITC)-Lys-Lys-Arg-Lys-Val-OH, can actively cross over the cell membrane and accumulate significantly around the nucleus of HEK-293 and neuroblastoma cells, while H-Baa-Lys(FITC)-Lys8-OH accumulates in the cytoplasm. Cellular studies show that the uptake for the anionic peptide Baa-Lys(FITC)Glu4Gly3Ser-OH is greatly reduced in comparison with the cationic fullerene peptides of the same concentration. The hydrophobic nature of the fullerene assisting peptide transport is suggested by the effect of gamma-cyclodextrin (CD) in lowering the efficacy of transport. These data suggest that the incorporation of a fullerene-based amino acid provides a route for the intracellular delivery of peptides and as a consequence the creation of a new class of cell penetrating peptides.

Biomimetic amplification of nanoparticle homing to tumors.
Simberg D, Duza T, Park JH, Essler M, Pilch J, Zhang L, Derfus AM, Yang M, Hoffman RM, Bhatia S, Sailor MJ, Ruoslahti E.
Proc Natl Acad Sci USA. 2007 Jan 16;104(3):932-6.
[ expand abstract ]

Nanoparticle-based diagnostics and therapeutics hold great promise because multiple functions can be built into the particles. One such function is an ability to home to specific sites in the body. We describe here biomimetic particles that not only home to tumors, but also amplify their own homing. The system is based on a peptide that recognizes clotted plasma proteins and selectively homes to tumors, where it binds to vessel walls and tumor stroma. Iron oxide nanoparticles and liposomes coated with this tumor-homing peptide accumulate in tumor vessels, where they induce additional local clotting, thereby producing new binding sites for more particles. The system mimics platelets, which also circulate freely but accumulate at a diseased site and amplify their own accumulation at that site. The self-amplifying homing is a novel function for nanoparticles. The clotting-based amplification greatly enhances tumor imaging, and the addition of a drug carrier function to the particles is envisioned.

Aclarubicin-loaded cationic albumin-conjugated pegylated nanoparticle for glioma chemotherapy in rats.
Lu W, Wan J, Zhang Q, She Z, Jiang X.
Int J Cancer. 2007 Jan 15;120(2):420-31.
[ expand abstract ]

Traditional glioma chemotherapy with those second-line drugs such as anthracyclines usually failed because they are inaccessible to blood-brain barrier (BBB) in tumor. In our study, we incorporated aclarubicin (ACL) into cationic albumin-conjugated pegylated nanoparticle (CBSA-NP-ACL) to determine its therapeutic potential of rats with intracranially implanted C6 glioma cells. When labeled with fluorescent probe, 6-coumarin, CBSA-NP was shown to accumulate much more in tumor mass than nanoparticle without conjugated CBSA (NP) 1 hr post intravenous injection, as well as better retention after 24 hr. Tumor drug concentration of CBSA-NP-ACL displayed 2.6- and 3.3-fold higher than that of NP-ACL and ACL solution 1 hr post injection, while 2.7 and 6.6-fold higher after 24 hr, respectively. Moreover, using tumor microdialysis sampling, AUC(0-24 hr) of free drug amount in tumor interstitium delivered by CBSA-NP-ACL was about 2.0- and 2.7-fold higher than that of NP-ACL and ACL solutions, respectively. When the tumor rat model was subjected to 4 cycles of 2 mg/kg of ACL in different formulations, a significant increase of median survival time was found in the group of CBSA-NP-ACL compared with that of saline control animals, animals treated with NP-ACL and ACL solution. By terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling, CBSA-NP-ACL can extensively make the tumor cell apoptosis. Histochemical evaluation by periodic acid Shiff staining and biochemical analysis depicted that the incorporation of ACL into CBSA-NP reduced its toxicity to liver, kidney and heart. Besides, CBSA-NP-ACL was not shown to open tight junction evaluated by BBB coculture. It was concluded that CBSA-NP-ACL could have a therapeutic potential for treatment of glioma.

Biomimetic amplification of nanoparticle homing to tumors.
Simberg D, Duza T, Park JH, Essler M, Pilch J, Zhang L, Derfus AM, Yang M, Hoffman RM, Bhatia S, Sailor MJ, Ruoslahti E.
Proc Natl Acad Sci USA. 2007 Jan 10; [Epub ahead of print].
[ expand abstract ]

Nanoparticle-based diagnostics and therapeutics hold great promise because multiple functions can be built into the particles. One such function is an ability to home to specific sites in the body. We describe here biomimetic particles that not only home to tumors, but also amplify their own homing. The system is based on a peptide that recognizes clotted plasma proteins and selectively homes to tumors, where it binds to vessel walls and tumor stroma. Iron oxide nanoparticles and liposomes coated with this tumor-homing peptide accumulate in tumor vessels, where they induce additional local clotting, thereby producing new binding sites for more particles. The system mimics platelets, which also circulate freely but accumulate at a diseased site and amplify their own accumulation at that site. The self-amplifying homing is a novel function for nanoparticles. The clotting-based amplification greatly enhances tumor imaging, and the addition of a drug carrier function to the particles is envisioned.

Hydrotropic Polymeric Micelles for Enhanced Paclitaxel Solubility: In Vitro and In Vivo Characterization.
Lee SC, Huh KM, Lee J, Cho YW, Galinsky RE, Park K.
Biomacromolecules. 2007 Jan 8;8(1):202-208.
[ expand abstract ]

The purpose of this investigation was to characterize the in vitro stability and in vivo disposition of paclitaxel in rats after solubilization of paclitaxel into hydrotropic polymeric micelles. The amphiphilic block copolymers consisted of a micellar shell-forming poly(ethylene glycol) (PEG) block and a core-forming poly(2-(4-vinylbenzyloxy)-N,N-diethylnicotinamide) (P(VBODENA)) block. N,N-Diethylnicotinamide (DENA) in the micellar inner core resulted in effective paclitaxel solubilization and stabilization. Solubilization of paclitaxel using polymeric micelles of poly(ethylene glycol)-b-P(d,l-lactide) (PEG-b-PLA) served as a control for the stability study. Up to 37.4 wt % paclitaxel could be loaded in PEG-b-P(VBODENA) micelles, whereas the maximum loading amount for PEG-b-PLA micelles was 27.6 wt %. Thermal analysis showed that paclitaxel in the polymeric micelles existed in the molecularly dispersed amorphous state even at loadings over 30 wt %. Paclitaxel-loaded hydrotropic polymeric micelles retained their stability in water for weeks, whereas paclitaxel-loaded PEG-b-PLA micelles precipitated in a few days. Hydrotropic polymer micelles were more effective than PEG-PLA micelle formulations in inhibiting the proliferation of human cancer cells. Paclitaxel in hydrotropic polymer micelles was administered orally (3.8 mg/kg), intravenously (2.5 mg/kg), or via the portal vein (2.5 mg/kg) to rats. The oral bioavailability was 12.4% of the intravenous administration. Our data suggest that polymeric micelles with a hydrotropic structure are superior as a carrier of paclitaxel due to a high solubilizing capacity combined with long-term stability, which has not been accomplished by other existing polymeric micelle systems.

Synthetic nano-low density lipoprotein as targeted drug delivery vehicle for glioblastoma multiforme.
Nikanjam M, Blakely EA, Bjornstad KA, Shu X, Budinger TF, Forte TM.
Int J Pharm. 2007 Jan 2;328(1):86-94.
[ expand abstract ]

The low density lipoprotein (LDL) receptor has been shown to be upregulated in GBM tumor cells and is therefore a potential molecular target for the delivery of therapeutic agents. A synthetic nano-LDL (nLDL) particle was developed and tested to determine its utility as a drug delivery vehicle targeted to GBM tumors. nLDL particles were constructed by combining a synthetic peptide containing a lipid binding motif and the LDL receptor (LDLR) binding domain of apolipoprotein B-100 with a lipid emulsion consisting of phosphatidyl choline, triolein, and cholesteryl oleate. Composition analysis, fast protein liquid chromatography, and electron microscopy revealed that nLDL was highly reproducible and intermediate in size between high density lipoprotein and LDL particles (10.5+/-2.8nm diameter). The binding and uptake of fluorescently labeled nLDL particles was assessed using fluorescence microscopy. Uptake of nLDL was time dependent, exhibiting saturation at approximately 3h, and concentration dependent, exhibiting saturation at concentrations greater than 5muM peptide. Using Lysotracker as a cellular marker, nLDL co-localized with lysosomes. nLDL binding was eliminated by blocking LDLRs with suramin and nLDL inhibited binding of plasma LDL to LDLRs. Collectively these data strongly suggest that the synthetic nano-LDLs described here are taken up by LDLR and can serve as a drug delivery vehicle for targeting GBM tumors via the LDLR.

Synthesis and characterization of chitosan-g-poly(ethylene glycol)-folate as a non-viral carrier for tumor-targeted gene delivery.
Chan P, Kurisawa M, Chung JE, Yang YY.
Biomaterials. 2007 Jan;28(3):540-9.
[ expand abstract ]

Poor water solubility and low transfection efficiency of chitosan are major drawbacks for its use as a gene delivery carrier. PEGylation can increase its solubility, and folate conjugation may improve gene transfection efficiency due to promoted uptake of folate receptor-bearing tumor cells. The aim of this study was to synthesize and characterize folate-poly(ethylene glycol)-grafted chitosan (FA-PEG-Chi) for targeted plasmid DNA delivery to tumor cells. Gel electrophoresis study showed strong DNA binding ability of modified chitosan. The pH(50) values, defined as the pH when the transmittance of a polymer solution at 600 nm has reached 50% of the original value, suggested that the water solubility of PEGylated chitosan had improved significantly. Regression analysis of pH(50) value as a function of substitution degree of PEG yielded an almost linear correlation for PEG-Chi and FA-PEG-Chi. The solubility of PEGylated chitosan decreased slightly by further conjugation of folic acid due to the relatively more hydrophobic nature of folic acid when compared to PEG. In addition, the chitosan-based DNA complexes did not induce remarkable cytotoxicity against HEK 293 cells. FA-PEG-Chi can be a promising gene carrier due to its solubility in physiological pH, efficiency in condensing DNA, low cytotoxicity and targeting ability.

Amine-containing core-shell nanoparticles as potential drug carriers for intracellular delivery.
Feng M, Li P.
J Biomed Mater Res A. 2007 Jan;80(1):184-93.
[ expand abstract ]

The present study aimed at exploring the use of amine-containing core-shell nanoparticles as potential drug carriers for intracellular delivery. Stable nanoparticles (100-200 nm in diameter) that consisted of poly(methyl methacrylate) (PMMA) cores with hydrophilic poly(ethyleneimine) (PEI) shells were synthesized and used to study their complexation with model drug, ibuprofen (IB), and release it under various electrolyte concentrations. The complexed IB/PEI-PMMA nanoparticles were characterized with FTIR, photon correlation spectroscopy, zeta-potential, and transmission electron microscopy (TEM). Results suggested that the PEI-PMMA nanoparticles could effectively complex with the IB via electrostatic interaction. The thick PEI shells (approximately 30 nm) significantly enhanced the drug loading capacity up to 23% (w/w) of the complexed nanopartricle. In vitro release of the drug from the complexed nanoparticles was sensitive to the ionic strength of the media. Study of cellular entry of fluorescently labeled IB/nanoparticle complexes using a confocal laser scanning microscopy demonstrated that the entry of the complexed nanoparticles strongly depended on the complexing ratio between IB and PEI-PMMA nanoparticles.

Conjugates of poly(DL-lactide-co-glycolide) on amino cyclodextrins and their nanoparticles as protein delivery system.
Gao H, Wang YN, Fan YG, Ma JB.
J Biomed Mater Res A. 2007 Jan;80(1):111-22.
[ expand abstract ]

Poly(DL-lactide-co-glycolide) (PLG) was chemically conjugated on two amino cyclodextrins, mono(6-(2-aminoethyl)amino-6-deoxy)-beta-cyclodextrin and ethylenediamino bridged bis(beta-cyclodextrin), to afford novel amphiphilic conjugates. Those conjugates were then characterized with infrared spectrometry (IR), proton nuclear magnetic resonance ((1)H NMR) and gel permeation chromatography (GPC). A repeat-nanoprecipitation (RP-NP) method was also developed to fabricate the nanoparticles of the conjugates with a water-soluble model protein, bovine serum albumin (BSA). At the end of RP-NP process, the availability of BSA was over 80% while the entrapment efficiency was 40-50% for each nanoprecipitation. The nanoparticles were rigid and spherical with diameters of 110-180 nm determined by transmission electron microscope (TEM), atomic force microscopy (AFM) and particle size analyzer. Nanoparticles possessed good steric stability during freeze-drying and resuspensions due to the existence of cyclodextrins corona. Interactions between BSA and the conjugates in the nanoparticles were then elucidated with IR experiments. About 25% BSA adsorbed on the surface of nanoparticles due to the interaction and was easy to release in the first day. The release of BSA from the nanoparticles was in three phases: a burst effect in the first day, a followed plateau in about a week, and a sustained release of the protein over 14 days. By changing the lactide/glycolide ratio, the degradation time of the conjugates and the release rate of BSA could be controlled. The loss of CDs content was faster than that of overall Mw during degradation since CDs formed outer corona of the nanoparticles. Both the novel biomaterials and the nanosphere fabrication technique contributed to the maintenance of protein structure.

Development of a novel systemic gene delivery system for cancer therapy with a tumor-specific cleavable PEG-lipid.
Hatakeyama H, Akita H, Kogure K, Oishi M, Nagasaki Y, Kihira Y, Ueno M, Kobayashi H, Kikuchi H, Harashima H.
Gene Ther. 2007 Jan;14(1):68-77.
[ expand abstract ]

For successful cancer gene therapy via intravenous (i.v.) administration, it is essential to optimize the stability of carriers in the systemic circulation and the cellular association after the accumulation of the carrier in tumor tissue. However, a dilemma exists regarding the use of poly(ethylene glycol) (PEG), which is useful for conferring stability in the systemic circulation, but is undesirable for the cellular uptake and the following processes. We report the development of a PEG-peptide-lipid ternary conjugate (PEG-Peptide-DOPE conjugate (PPD)). In this strategy, the PEG is removed from the carriers via cleavage by a matrix metalloproteinase (MMP), which is specifically expressed in tumor tissues. An in vitro study revealed that the PPD-modified gene carrier (Multifunctional Envelope-type Nano Device: MEND) exhibited pDNA expression activity that was dependent on the MMP expression level in the host cells. In vivo studies further revealed that the PPD was potent in stabilizing MEND in the systemic circulation and facilitating tumor accumulation. Moreover, the i.v. administration of PPD or PEG/PPD dually-modified MEND resulted in the stimulation of pDNA expression in tumor tissue, as compared with a conventional PEG-modified MEND. Thus, MEND modified with PPD is a promising device, which has the potential to make in vivo cancer gene therapy achievable.Gene Therapy (2007) 14, 68-77. doi:10.1038/sj.gt.3302843; published online 17 August 2006.

Dendrimer-based targeted delivery of an apoptotic sensor in cancer cells.
Myc A, Majoros IJ, Thomas TP, Baker JR Jr.
Biomacromolecules. 2007 Jan;8(1):13-8.
[ expand abstract ]

Our previous studies have demonstrated the applicability of poly(amidoamine) (PAMAM) dendrimers as a platform for the targeted delivery of chemotherapeutic drugs both in vitro and in vivo. To monitor the rate and extent of cell-killing caused by the delivered chemotherapeutic drug, we wished to analyze the degree of apoptosis in targeted cells on a real-time basis. As the apoptosis-regulating caspases are activated during the apoptotic process, several caspase-hydrolyzable, fluorescence resonance energy transfer (FRET)-based substrates have been marketed for the detection of apoptosis. However, the applicability of these agents is limited because of their nonspecificity and the consequent high background fluorescence in tissues. Here we show the synthesis, characterization, and in vitro targeting of an engineered PAMAM nanodevice in which folic acid (FA) is conjugated as the targeting molecule and a caspase-specific FRET-based agent (PhiPhiLux G1D2) is conjugated as the apoptosis-detecting agent. This conjugate specifically targets FA-receptor-positive, KB cells. In these cells, the apoptosis-inducing agent staurosporine caused a 5-fold increase in the cellular fluorescence. These results show, for the first time, the potential applicability of a targeted apoptosis-measuring nanodevice, which could be used for simultaneously monitoring the apoptotic potential of a delivered drug.

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2006

Polymer genomics: An insight into pharmacology and toxicology of nanomedicines.
Kabanov AV.
Adv Drug Deliv Rev. 2006 Dec 30;58(15):1597-621.
[ expand abstract ]

Synthetic polymers and nanomaterials display selective phenotypic effects in cells and in the body signal transduction mechanisms involved in inflammation, differentiation, proliferation, and apoptosis. When physically mixed or covalently conjugated with cytotoxic agents, bacterial DNA or antigens, polymers can drastically alter specific genetically controlled responses to these agents. These effects, in part, result from cooperative interactions of polymers and nanomaterials with plasma cell membranes and trafficking of polymers and nanomaterials to intracellular organelles. Cells and whole organism responses to these materials can be phenotype or genotype dependent. In selected cases, polymer agents can bypass limitations to biological responses imposed by the genotype, for example, phenotypic correction of immune response by polyelectrolytes. Overall, these effects are relatively benign as they do not result in cytotoxicity or major toxicities in the body. Collectively, however, these studies support the need for assessing pharmacogenomic effects of polymer materials to maximize clinical outcomes and understand the pharmacological and toxicological effects of polymer formulations of biological agents, i.e. polymer genomics.

Intraperitoneal Delivery of Liposomal siRNA for Therapy of Advanced Ovarian Cancer.
Landen CN, Merritt WM, Mangala LS, Sanguino AM, Bucana C, Lu C, Lin YG, Han LY, Kamat AA, Schmandt R, Coleman RL, Gershenson DM, Lopez-Berestein G, Sood AK.
Cancer Biol Ther. 2006 Dec 30;5(12) [Epub ahead of print].
[ expand abstract ]

Purpose: Intravenous (IV) delivery of siRNA incorporated into neutral liposomes allows efficient delivery to tumor tissue, and has therapeutic efficacy in preclinical proof-of-concept studies using EphA2-targeting siRNA. We sought to determine whether intraperitoneal (IP) delivery of these siRNA complexes was as effective at delivery and therapy as IV delivery. Experimental design: SiRNA was incorporated into the neutral liposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). Alexa555-siRNA-DOPC was injected IP into nude mice bearing established ovarian tumors, and organs were collected for microscopic fluorescent examination. Subsequently, therapeutic efficacy of the IP versus IV routes was directly compared. Results: Alexa555-siRNA in DOPC liposomes injected IP was diffusely distributed into intraperitoneal ovarian tumors. Delivery was also seen deeply into the liver and kidney parenchyma, suggesting that the predominant means of distribution was through the vasculature, rather than direct diffusion from the peritoneal cavity. In mice with orthotopic ovarian tumors, treatment with combined paclitaxel and IP EphA2-targeting siRNA-DOPC reduced tumor growth by 48-81% compared to paclitaxel/control siRNA-DOPC IP (HeyA8: 0.34 g v 0.66 g; SKOV3ip1: 0.04 v 0.21, p < 0.01). This reduction was comparable to concurrently-treated mice with paclitaxel and EphA2 siRNA-DOPC injected IV, which showed a reduction in growth by 45-69% compared to paclitaxel/control siRNA-DOPC injected IV (HeyA8: 0.23g v. 0.42g; SKOV3ip1: 0.04 v. 0.13 g). Conclusions: IP injection of siRNA incorporated in DOPC allows intra-tumoral delivery and has therapeutic efficacy in orthotopic ovarian tumors. These findings may have therapeutic implications for siRNA-based strategies.

Arginine-conjugated polypropylenimine dendrimer as a non-toxic and efficient gene delivery carrier.
Kim TI, Baek JU, Zhe Bai C, Park JS.
Biomaterials. 2006 Dec 28; [Epub ahead of print].
[ expand abstract ]

We synthesized arginine-conjugated polypropylenimine dendrimer G2 (DAB-8), PPI2-R for gene delivery systems. Synthesized PPI2-R could retard plasmid DNA at a weight ratio of 4 completely and PPI2-R polyplexes showed a fluorescence of less than 10% over a charge ratio of 2 by PicoGreen reagent assay, suggesting its good DNA condensing ability. The size of PPI2-R polyplex was measured to about 200nm at a charge ratio of 150. PPI2-R displayed 80-90% cell viability at even a 150mug/mL concentration. Transfection efficiency of PPI2-R was found to be high comparable to that of PEI25kD and to be 8-214 times higher than that of unmodified PPI2 on HeLa and 293 cells. Moreover, PPI2-R showed 4 times higher transfection efficiency than PEI25kD, treating with 10mug pDNA because of its low cytotoxicity on HeLa cells. Finally, PPI2-R showed a transfection efficiency 2-3 times higher than PEI25kD on HUVECs, showing its potency as a gene delivery carrier for primary cells. These results demonstrate that arginine-conjugation of PPI2 is successful in developing a low toxic and highly transfection efficient gene delivery carrier.

Targeted and intracellular delivery of paclitaxel using multi-functional polymeric micelles.
Seow WY, Xue JM, Yang YY.
Biomaterials. 2006 Dec 18; [Epub ahead of print].
[ expand abstract ]

Natural paclitaxel (Taxol((R)) is an effective anti-cancer drug, although a critical disadvantage is its non-targeting nature. To address this issue, cholesterol-grafted poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide-co-undecenoic acid) was synthesized with different starting monomer ratios via a free radical copolymerization route. Folate was subsequently attached to the hydrophilic segment of the polymer in order to target folate receptors-overexpressing cancer cells. The success of synthesis was confirmed with (1)H-NMR carried out in CDCl(3)/D(2)O. Using a membrane dialysis method, the polymer was then self-assembled into micelles whose hydrophobic cores could be utilized to encapsulate paclitaxel, an extremely hydrophobic compound. The polymer had a low CMC of approximately 20mg/L in water. Dynamic light scattering further showed that the sizes of blank micelles formed from the polymer were below 180nm at different pH values tested and approximately 220nm upon drug incorporation. More importantly, it was demonstrated that the micelles exhibited a useful pH-induced thermo-sensitivity, such that drug was released more rapidly at pH 5.0 (acidic endosomal/lysosomal environment) than at pH 7.4 (normal extracellular pH). In vitro cytotoxicity assays performed against KB cells then provided concluding evidences that the cellular uptake of micelles surface-functionalised with folate was indeed enhanced due to a receptor-assisted endocytosis process. This novel polymeric design thus has the potential to be a useful paclitaxel vehicle for the treatment of folate-receptor positive cancers.

Differential tumor cell targeting of anti-HER2 (Herceptin((R))) and anti-CD20 (Mabthera((R))) coupled nanoparticles.
Cirstoiu-Hapca A, Bossy-Nobs L, Buchegger F, Gurny R, Delie F.
Int J Pharm. 2006 Dec 15; [Epub ahead of print].
[ expand abstract ]

Two types of antibody-labeled nanoparticles (mAb-NPs) were prepared with the aim to achieve specific tumor targeting. Anti-HER2 and anti-CD20 monoclonal antibodies (mAb) were used as model ligands. Small poly(dl-lactic acid) nanoparticles (PLA NPs) with a mean size of about 170nm were prepared by the salting out method. Thereafter, the coating of PLA NPs with mAbs was performed in two steps. First, thiol groups (-SH) were introduced on the surface of PLA-NPs by a two-step carbodiimide reaction. The number of -SH groups on the surface of NPs increased from 150 to 400mmol-SH/molPLA when cystamine concentrations of 25-1518molcystamine/molPLA were used during the thiolation reaction. In the second step, covalent coupling of antibodies to thiolated NPs (NPs-SH) was obtained via a bifunctional cross-linker, m-maleimidobenzoyl-N-hydroxy-sulfosuccinimide ester (sulfo-MBS). For both mAbs anti-HER2 and anti-CD20, respectively, the number of -SH functions on the NPs had no influence on the amount of mAb coupled to the NPs. Approximately, 295 anti-HER2 and 557 anti-CD20 molecules, respectively, were covalently coupled per nanoparticle. The NPs size after the coupling reactions was about 250nm. The specific interaction between tumor cells and mAb-NPs was determined by confocal microscopy using two cell lines: SKOV-3 human ovarian cancer cells (overexpressing HER2) and Daudi lymphoma cells (overexpressing CD20). The results showed the selective targeting of mAb-NPs to tumor cells overexpressing the specific antigen. While anti-CD20 labeled NPs (anti-CD20 NPs) bound to and remained at the cellular surface, anti-HER2 labeled NPs (anti-HER2 NPs) were efficiently internalized. The mAb-NPs represent a promising approach to improve the efficacy of NPs in active targeting for cancer therapy while the choice of the antibody-target system defines the fate of the mAb-NPs after their binding to the cells.

HER2-specific T-cell immune responses in patients vaccinated with truncated HER2 protein complexed with nanogels of cholesteryl pullulan.
Kitano S, Kageyama S, Nagata Y, Miyahara Y, Hiasa A, Naota H, Okumura S, Imai H, Shiraishi T, Masuya M, Nishikawa M, Sunamoto J, Akiyoshi K, Kanematsu T, Scott AM, Murphy R, Hoffman EW, Old LJ, Shiku H.
Clin Cancer Res. 2006 Dec 15;12(24):7397-405.
[ expand abstract ]

PURPOSE: We developed a complex of tumor antigen protein with a novel nanoparticle antigen delivery system of cholesteryl pullulan (CHP). To target HER2 antigen, we prepared truncated HER2 protein 1-146 (146HER2) complexed with CHP, the CHP-HER2 vaccine. We designed a clinical study to assess the safety of the vaccine and HER2-specific T-cell immune responses measured by the newly developed enzyme-linked immunospot assay with mRNA-transduced phytohemagglutinin-stimulated CD4(+) T cells in HLA-A2402-positive patients with therapy-refractory HER2-expressing cancers. EXPERIMENTAL DESIGN: Nine patients with various types of solid tumors were enrolled. Each patient was s.c. vaccinated biweekly with 300 microg of CHP-HER2 vaccine for three times followed by booster doses. HER2-specific T-cell responses were evaluated by enzyme-linked immunospot assay by targeting autologous phytohemagglutinin-stimulated CD4(+) T cells transduced with 146HER2-encoding mRNA to cover both identified peptides and unknown epitopes for MHC class I and class II that might exist in the sequence of the vaccine protein. RESULTS: CHP-HER2 vaccine was well tolerated; the only adverse effect was grade 1 transient skin reaction at the sites of vaccination. HER2-specific CD8(+) and/or CD4(+) T-cell immune responses were detected in five patients who received four to eight vaccinations, among whom both T-cell responses were detected in these patients. In four patients with CD8(+) T-cell responses, two patients reacted to previously identified HER2(63-71) peptide and the other two reacted only to 146HER2 mRNA-transduced cells. CONCLUSIONS: CHP-HER2 vaccine was safe and induced HER2-specific CD8(+) and/or CD4(+) T-cell immune responses.

Dendrimer-encapsulated camptothecins: increased solubility, cellular uptake, and cellular retention affords enhanced anticancer activity in vitro.
Morgan MT, Nakanishi Y, Kroll DJ, Griset AP, Carnahan MA, Wathier M, Oberlies NH, Manikumar G, Wani MC, Grinstaff MW.
Cancer Res. 2006 Dec 15;66(24):11913-21.
[ expand abstract ]

A biocompatible polyester dendrimer composed of the natural metabolites, glycerol and succinic acid, is described for the encapsulation of the antitumor camptothecins, 10-hydroxycamptothecin and 7-butyl-10-aminocamptothecin. The cytotoxicity of the dendrimer-drug complex toward four different human cancer cell lines [human breast adenocarcinoma (MCF-7), colorectal adenocarcinoma (HT-29), non-small cell lung carcinoma (NCI-H460), and glioblastoma (SF-268)] is also reported, and low nmol/L IC(50) values are measured. Cellular uptake and efflux measurements in MCF-7 cells show an increase of 16-fold for cellular uptake and an increase in drug retention within the cell when using the dendrimer vehicle.

The Anionic Boron Cluster (B(12)H(11)SH)(2-) as a Means To Trigger Release of Liposome Contents.
Gabel D, Awad D, Schaffran T, Radovan D, Daraban D, Damian L, Winterhalter M, Karlsson G, Edwards K.
ChemMedChem. 2006 Dec 12; [Epub ahead of print].
[ expand abstract ]

No abstract available

Characterization of (Aminoethyl)chitin/DNA Nanoparticle for Gene Delivery.
Je JY, Cho YS, Kim SK.
Biomacromolecules. 2006 Dec 11;7(12):3448-3451.
[ expand abstract ]

Nonviral gene delivery systems have been increasingly proposed as a safer alternative to viral vehicles. In the present study, we synthesized water-soluble chitin by aminoalkylating onto chitin at the C-6 position, and its transfection efficiency was investigated. Aminoethyl-chitin (AEC) was complexed with DNA, and AEC/DNA nanoparticles were characterized. AEC/DNA nanoparticles showed good DNA binding ability, high protection of DNA from nuclease and serum, and low cytotoxicity. Mean particle size decreased from 367 to 290 nm and &zgr; potential increased from -4.58 to 22.87 mV when the AEC/DNA charge ratio (N/P) increased from 1.15 to 18.5. The transfection efficiency of AEC/DNA nanoparticles was investigated in a human embryonic kidney cell line (HEK293), and the results showed that AEC/DNA nanoparticles were much enhanced compare with naked DNA.

Quantum-dot based nanoparticles for targeted silencing of HER2/neu gene via RNA interference.
Tan WB, Jiang S, Zhang Y.
Biomaterials. 2006 Dec 9; [Epub ahead of print].
[ expand abstract ]

Gene silencing using short interfering RNA (siRNA) is fast becoming an attractive approach to probe gene function in mammalian cells. Although there have been some success in the delivery of siRNA using various methods, tracking their delivery and monitoring their transfection efficiency prove to be hard without a suitable tracking agent. Therefore, a challenge lies with the design of an efficient and at the same time, self-tracking, transfection agent for RNA interference. In this paper, chitosan nanoparticles (NPs) with encapsulated quantum dots (QDs) were synthesized and used to deliver HER2/neu siRNA. Using such a construct, the delivery and transfection of the siRNA can be monitored by the presence of fluorescent QDs in the chitosan NPs. Targeted delivery of HER2 siRNA to HER2-overexpressing SKBR3 breast cancer cells was shown to be specific with chitosan/QD NP surface labeled with HER2 antibody targeting the HER2 receptors on SKBR3 cells. Gene-silencing effects of the conjugated siRNA was also established using the luciferase and HER2 ELISA assays. These self-tracking siRNA delivery NPs will also aid in the monitoring of future gene silencing studies in vivo.

Fluorocarbon nanoparticles as multifunctional drug delivery vehicles.
Dielectrophoresis microsystem with integrated flow cytometers for on-line monitoring of sorting efficiency.
Wang Z, Hansen O, Petersen PK, Rogeberg A, Kutter JP, Bang DD, Wolff A.
Electrophoresis. 2006 Dec 7;27(24):5081-5092 [Epub ahead of print].
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Dielectrophoresis (DEP) and flow cytometry are powerful technologies and widely applied in microfluidic systems for handling and measuring cells and particles. Here, we present a novel microchip with a DEP selective filter integrated with two microchip flow cytometers (FCs) for on-line monitoring of cell sorting processes. On the microchip, the DEP filter is integrated in a microfluidic channel network to sort yeast cells by positive DEP. The two FCs detection windows are set upstream and downstream of the DEP filter. When a cell passes through the detection windows, the light scattered by the cell is measured by integrated polymer optical elements (waveguide, lens, and fiber coupler). By comparing the cell counting rates measured by the two FCs, the collection efficiency of the DEP filter can be determined. The chips were used for quantitative determination of the effect of flow rate, applied voltage, conductivity of the sample, and frequency of the electric field on the sorting efficiency. A theoretical model for the capture efficiency was developed and a reasonable agreement with the experimental results observed. Viable and non-viable yeast cells showed different frequency dependencies and were sorted with high efficiency. At 2 MHz, more than 90% of the viable and less than 10% of the non-viable cells were captured on the DEP filter. The presented approach provides quantitative real-time data for sorting a large number of cells and will allow optimization of the conditions for, e.g., collecting cancer cells on a DEP filter while normal cells pass through the system. Furthermore, the microstructure is simple to fabricate and can easily be integrated with other microstructures for lab-on-a-chip applications.

A novel PEGylation of chitosan nanoparticle for gene delivery.
Zhang Y, Chen J, Zhang Y, Pan Y, Zhao J, Ren L, Liao M, Hu Z, Kong L, Wang J.
Biotechnol Appl Biochem. 2006 Dec 6; [Epub ahead of print].
[ expand abstract ]

Chitosan (CS) has emerged as a promising non-viral vector for gene delivery because of its capability to form complexes with plasmid DNA (pDNA) and enhance its transport across cellular membranes through endocytosis. Complexes of CS and pDNA may improve transfection efficiency; however, they are not capable of sustained DNA release and prolonging gene transfer. In order to achieve prolonged delivery of CS/DNA complexes, we had prepared CS nanoparticle (NP) and CS/DNA complexes; then, alpha-methoxy-omega-succinimidyl-polyethylene glycol (MSS-PEG) was conjugated to the surface of CS/DNA complexes using an active ester scheme; finally, the potential of PEGylation of CS NP as a non-viral gene delivery vector to transfer exogenous gene in vitro and in vivo were examined. Electrophoretic analysis suggested that CS NPs could protect the DNA from nuclease degradation. The pDNA carried by CS NPs could enter and express in HepG2 cells. However, its transfection efficiency was very low and the better dose of DNA transferred was 1.6microgram. The transfection activities of CS/DNA-PEG still remained and the better dose of pDNA transferred was 2.4microgram. It indicated that the transfection activity of PEGylation of complexes was improved efficiently. In vivo experiments also showed that CS/DNA-PEG complexes mediated higher gene expression in tissues than CS/DNA complexes did and the gene expression in tumor induced by CS/DNA-PEG complexes was highest of all. These results suggested that PEGylation of CS/DNA complexes have favorable properties for non-viral gene delivery in vitro or in vivo and have the potential to deliver therapeutic genes directly into hepatoma tissues.

Polyarginine segments in block copolypeptides drive both vesicular assembly and intracellular delivery.
Holowka EP, Sun VZ, Kamei DT, Deming TJ.
Nat Mater. 2006 Dec 3; [Epub ahead of print].
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Polymeric vesicles are a relatively new class of nanoscale self-assembled materials that show great promise as robust encapsulants. Compared with liposomes, use of polymeric building blocks for membrane formation allows increased stability, stimuli responsiveness and chemical diversity, which may prove advantageous for drug-delivery applications . A major drawback of most polymeric vesicles is the lack of biofunctionality, which restricts their ability to interact with cells and tissues. We have prepared vesicles composed of polyarginine and polyleucine segments that are stable in media, can entrap water soluble species, and can be processed to different sizes and prepared in large quantities. The remarkable feature of these materials is that the polyarginine segments both direct structure for vesicle formation and provide functionality for efficient intracellular delivery of the vesicles. This unique synergy between nanoscale self-assembly and inherent peptide functionality provides a new approach for design of multifunctional materials for drug delivery.

In vivo Radioprotection by the Fullerene Nanoparticle DF-1 as Assessed in a Zebrafish Model.
Daroczi B, Kari G, McAleer MF, Wolf JC, Rodeck U, Dicker AP.
Clin Cancer Res. 2006 Dec 1;12(23):7086-91.
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PURPOSE: We have previously shown that zebrafish (Danio rerio) embryos can be used as an in vivo model to validate modifiers of the radiation response. Here, we evaluated the radioprotective effect of the nanoparticle DF-1, a fullerene with antioxidant properties, in zebrafish embryos. EXPERIMENTAL DESIGN: Zebrafish embryos were exposed to different doses of ionizing radiation ranging from 20 to 80 Gy in the presence and absence of DF-1. Toxicity and radioprotective effects were assessed by monitoring overall survival and morphology as well as organ functions by employing assays to measure kidney excretory function and development of sensory nerve cells (neuromasts). Antioxidant properties of DF-1 were assessed in whole fish. RESULTS: DF-1 had no apparent adverse effects on normal zebrafish morphology or viability throughout the concentration range tested (1-1,000 mumol/L). Ionizing radiation (10-40 Gy) caused time-dependent and dose-dependent perturbations of normal zebrafish morphology and physiology, notably defective midline development resulting in dorsal curvature of the body axis ("curly-up"), neurotoxicity, impaired excretory function, and decreased survival of the exposed embryos. DF-1 (100 mumol/L) markedly attenuated overall and organ-specific radiation-induced toxicity when given within 3 hours before or up to 15 minutes after radiation exposure. By contrast, DF-1 afforded no protection when given 30 minutes after ionizing radiation. The degree of radioprotection provided by DF-1 was comparable with that provided by the Food and Drug Administration-approved radioprotector amifostine (4 mmol/L). Protection against radiation-associated toxicity using DF-1 in zebrafish embryos was associated with marked reduction of radiation-induced reactive oxygen species. CONCLUSION: The fullerene DF-1 protects zebrafish embryos against deleterious effects of ionizing radiation due, in part, to its antioxidant properties.

Nanomedicine: Developing smarter therapeutic and diagnostic modalities.
Farokhzad OC, Langer R.
Adv Drug Deliv Rev. 2006 Dec 1;58(14):1456-9.
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The early impact of nanotechnology on medicine is beginning to get realized, with novel nanoscale therapeutic and diagnostic modalities under development or in clinical practice today. In this commentary the field of "nanomedicine" is briefly reviewed form the perspective of where we were; where we are today; and where we are likely to go tomorrow.

Determination of the Minimum Temperature Required for Selective Photothermal Destruction of Cancer Cells Using Immunotargeted Gold Nanoparticles.
Huang X, Jain PK, El-Sayed IH, El-Sayed MA.
Photochem Photobiol. 2006 Dec 1; [Epub ahead of print].
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Laser photothermal therapy of cancer using gold nanoparticles immunotargeted to molecular markers on the cell surface has been shown to be an effective modality to selectively kill cancer cells at much lower laser powers than those needed for healthy cells. To elucidate the minimum light dosimetry required to induce cell death, photothermal destruction of two cancerous cell lines and a noncancerous cell line treated with anti-epidermal growth factor receptor (anti-EGFR) antibody conjugated gold nanoparticles is studied and a numerical heat transport model is used to estimate the local temperature rise within the cells as a result of the laser heating of the gold nanoparticles. It is found that cell samples with higher nanoparticle loading require a lower incident laser power to achieve a certain temperature rise. Numerically estimated temperatures of 70-80 degrees C achieved by heating the gold particles agree well with the measured threshold temperature for destruction of the cell lines by oven-heating and those measured in an earlier nanoshell method. Specific binding of anti-EGFR antibody to cancerous cells overexpressing EGFR selectively increases the gold nanoparticle loading within cancerous cells, thus allowing the cancerous cells to be destroyed at lower laser power thresholds needed for the noncancerous cells. In addition, photothermal therapy using gold nanoparticles requires lower laser power thresholds than that using conventional dyes due to the much higher absorption coefficient of the gold nanoparticles.

PEGylated lysine dendrimers for tumor-selective targeting after intravenous injection in tumor-bearing mice.
Okuda T, Kawakami S, Akimoto N, Niidome T, Yamashita F, Hashida M.
J Control Release. 2006 Dec 1;116(3):330-336.
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In this study, we synthesized a sixth generation lysine dendrimer (KG6) and two PEGylated derivatives thereof and evaluated their biodistribution characteristics in both normal and tumor-bearing mice. The intact KG6 showed a rapid clearance from the blood stream and non-specific accumulation in the liver and kidney. In contrast, the PEGylated derivatives showed a better retention in blood and low accumulativeness in organs dependent of the rate of PEGylation. In addition, PEGylated KG6 with a high modification rate was accumulated effectively in tumor tissue via the enhanced permeability and retention (EPR) effect. Moreover, we clarified that multiple administrations did not affect the biodistribution characteristics of a second dose of PEGylated KG6. PEGylated lysine dendrimer would be a useful material for a clinically applicable tumor-targeting carrier.

Simultaneous delivery of doxorubicin and GG918 (Elacridar) by new Polymer-Lipid Hybrid Nanoparticles (PLN) for enhanced treatment of multidrug-resistant breast cancer.
Wong HL, Bendayan R, Rauth AM, Wu XY.
J Control Release. 2006 Dec 1;116(3):275-84.
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Multidrug-resistant (MDR) cancer may be treated using combinations of encapsulated cytotoxic drugs and chemosensitizers. To optimize for the effectiveness of this combinational approach, novel polymer-lipid hybrid nanoparticle (PLN) formulations capable of delivering a cytotoxic drug, doxorubicin (Dox), a chemosensitizer, GG918, or their combination were prepared. Both acute and long-term anticancer activities of various combinations of Dox and GG918 in solution or PLN form were evaluated in a human MDR breast cancer cell line (MDA435/LCC6/MDR1) using trypan blue exclusion and clonogenic assays. Cellular Dox uptake and drug distribution within the cells were determined by fluoremetry and fluorescence microscopy. The results showed that the encapsulation efficiencies of Dox and GG918 in PLN were up to 89% and were not compromised by co-encapsulation of the two agents. Of various combinational treatment approaches, the Dox and GG918 co-encapsulated PLN formulation ((DG)n) demonstrated the greatest Dox uptake and anticancer activity to the MDR cells, while co-administration of two single-agent loaded PLN was least effective. Fluorescence microscopy indicated cellular internalization of (DG)n. These findings suggest that in addition to the total drug concentrations, the simultaneous delivery of Dox and GG918 to the same cellular location is critical in determining the therapeutic effectiveness of this anticancer drug-chemosensitizer combination.

In vitro assessment of transferrin-conjugated liposomes as drug delivery systems for inhalation therapy of lung cancer.
Anabousi S, Bakowsky U, Schneider M, Huwer H, Lehr CM, Ehrhardt C.
Eur J Pharm Sci. 2006 Dec;29(5):367-74.
[ expand abstract ]

Most human tumours over-express receptors for growth factors and peptide hormones, which are being increasingly studied as a means to selectively deliver cytotoxic agents. An example being the transferrin receptor (TfR, CD71). Here, we studied expression levels and location of TfR in different lung epithelial cell types (i.e., bronchial and alveolar epithelial cells) by flow-cytometry and confocal laser scanning microscopy (CLSM). Furthermore, we assessed uptake levels and cytotoxicity of transferrin (Tf)-conjugated liposomes in vitro. TfR was found to be expressed at a significantly higher level in bronchial epithelial cells compared with their alveolar counterparts. Cells of cancerous origin (i.e., A549 cell line) showed a higher TfR expression level than healthy alveolar epithelial type II cells in primary culture. CLSM revealed TfR to be located primarily at the basolateral aspect of cells, with the exception of cells undergoing mitotic proliferation, which also showed TfR at their apical membranes, due to their loss of cell polarity. Higher expression levels of TfR correlated well with enhanced uptake of Tf-liposomes and increased levels of cytotoxicity. Liposome uptake was temperature-dependent and inhibitable by excess free Tf. Tf-conjugated liposomes appear as good candidates for an approach to deliver cytostatic drugs to sites of lung cancer by inhalation.

Thermotropic phase behavior of DPPC liposome systems in the presence of the anti-cancer agent 'Ellipticine'.
Cavalcanti LP, Torriani IL.
Eur Biophys J. 2006 Dec;36(1):67-71.
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This letter presents our first results on the structural changes in DPPC multilamellar vesicles dispersed in water in the presence of the anti-cancer agent Ellipticine. The thermotropic phase transitions of the lamellar packing inside lipid vesicles were characterized in situ by small angle X ray diffraction. The results lead to the determination of a critical concentration value for drug loading on the vesicle system around 4% molar fraction of Ellipticine, an indication of the localization of the drug in the alkyl chains and the influence of the drug on the decreasing rate of the bilayer period after the main phase transition.

Inhibitory effect of the polyinosinic-polycytidylic acid/cationic liposome on the progression of murine B16F10 melanoma.
Fujimura T, Nakagawa S, Ohtani T, Ito Y, Aiba S.
Eur J Immunol. 2006 Dec;36(12):3371-80.
[ expand abstract ]

Cellular proteins, retinoic acid inducible gene-I and Toll-like receptor 3, sense dsRNA including polyinosinic-polycytidylic acid (PIC) to stimulate innate immune response. The local administration of PIC has been demonstrated to be effective in anti-tumor immunotherapy. However, the effects of PIC delivered cross the cell membrane have not yet been examined. To address this issue, we used a complex of PIC and cationic liposome (PIC liposome) and examined its anti-tumor effects in vitro and in vivo. PIC liposome could directly suppress the growth of B16F10 melanoma in vitro and repeated peritumoral injections of PIC liposome inhibited melanoma growth in a dose-dependent manner. This treatment induced tyrosinase-related protein-2 (TRP-2)-tetramer(+) CD8(+) cells in the lymph nodes. As the mechanism for its anti-tumor immune response, we showed that the intradermal injection of PIC liposome induced the maturation of dendritic cells (DC). Moreover, the intratumoral injection of immature DC after treatment with PIC liposome significantly increased the number of TRP-2-specific IFN-gamma-producing cells in the lymph nodes as well as spleen, which resulted in an augmentation of the anti-tumor immune response. These studies demonstrate the potential of peritumoral injection of PIC liposome as immunotherapy for malignant melanoma.

[Magnetically based enhancement of nanoparticle uptake in tumor cells: combination of magnetically induced cell labeling and magnetic heating.]
Kettering M, Winter J, Zeisberger M, Alexiou C, Bremer-Streck S, Bergemann C, Kaiser WA, Hilger I.
Rofo. 2006 Dec;178(12):1255-60.
[ expand abstract ]

PURPOSE: Magnetic nanoparticles (MNP) are known to be versatile tools in diagnostic and interventional radiology. The goal of the present study was to assess whether MNP can be selectively accumulated on human adenocarcinoma cells in vitro using an external magnetic field (magnetically induced cell labeling) and whether these labeled tumor cells can then be destroyed after being exposed to an alternating magnetic field (magnetically induced heating). In this context, a long-term goal is to combine these two developing methods to achieve an additive effect in tumor therapy. MATERIALS AND METHODS: BT-474 cells were incubated until confluence. Magnetic nanoparticles (0.32 mg Fe/ml culture medium) were then added and the flask was exposed to an external magnetic field gradient (magnetically induced cell labeling, 56 or 83 mT magnets) for 24 hours in order to label the tumor cells with nanoparticles. Cells without both MNP and magnetic labeling as well as cells with MNP incubation but without magnetic labeling served as controls. After MNP incubation, the magnetically labeled cells (5 x 10 (7) cells/ml) were exposed to an alternating magnetic field for 5.45 minutes (frequency 400 kHz, amplitude 24.6 kA/m). The combination effect of both magnetic labeling and magnetic heating was assessed by determining the temperature increase. The amount of MNP accumulated within the cells was determined by measuring the iron content via atomic absorption spectrometry. For statistical analysis mean values and standard deviations of temperature increases and iron contents were calculated and the differences were analyzed using the Student's t-test. RESULTS: A significant temperature increase (p < 0.01) during magnetic heating of 41.76 +/- 4.60 K was detected after magnetic labeling of the cells (5 x 10 (7) cells/ml, 83 mT) incubated with MNP. In comparison, the cells incubated with MNP but without magnetic labeling revealed a temperature increase of 32.03 +/- 3.33 K, naked cells of only 2.69 +/- 0.34 K. CONCLUSION: The results demonstrated the magnetically based enhancement of cellular uptake of nanoparticles by tumor cells, resulting in the intensification of the generated temperature increase during magnetic heating. Consequently, magnetic nanoparticles are shown to be valuable tools for the combination of magnetically based therapy modalities.

Current state, achievements, and future prospects of polymeric micelles as nanocarriers for drug and gene delivery.
Nishiyama N, Kataoka K.
Pharmacol Ther. 2006 Dec;112(3):630-48.
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Polymeric micelles, self-assemblies of block copolymers, are promising nanocarrier systems for drug and gene delivery. Until now, several micellar formulations of antitumor drugs have been intensively studied in preclinical and clinical trials, and their utility has been demonstrated. Even compared with long-circulating liposomes, polymeric micelles might have several advantages, such as controlled drug release, tissue-penetrating ability and reduced toxicity such as hand-foot syndrome and hypersensitivity reaction. Importantly, critical features of the polymeric micelles as drug carriers, including particle size, stability, and loading capacity and release kinetics of drugs, can be modulated by the structures and physicochemical properties of the constituent block copolymers. Also, nano-engineering of block copolymers might allow the preparation of polymeric micelles with integrated smart functions, such as specific-tissue targetability, as well as chemical or physical stimuli-sensitivity. Thus, polymeric micelles are nanotechnology-based carrier systems that might exert the activity of potent bioactive compounds in a site-directed manner, ensuring their effectiveness and safety in the clinical use.

Transition Metal-Mediated Liposomal Encapsulation of Irinotecan (CPT-11) Stabilizes the Drug in the Therapeutically Active Lactone Conformation.
Ramsay E, Alnajim J, Anantha M, Taggar A, Thomas A, Edwards K, Karlsson G, Webb M, Bally M.
Pharm Res. 2006 Dec;23(12):2799-808.
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PURPOSE: To determine whether entrapped transition metals could mediate the active encapsulation of the anticancer drug irinotecan into preformed liposomes. Further, to establish that metal complexation could stabilize liposomal irinotecan in the therapeutically active lactone conformation. MATERIALS AND METHODS: Irinotecan was added to preformed 1,2-distearoyl-sn-glycero-phosphocholine/cholesterol (DSPC/chol) liposomes prepared in CuSO(4), ZnSO(4), MnSO(4), or CoSO(4) solutions, and drug encapsulation was determined over time. The roles of the transmembrane pH gradient and internal pH were evaluated. TLC and HPLC were used to monitor drug stability and liposome morphology was assessed by cryo-TEM. RESULTS: Irinotecan was rapidly and efficiently loaded into preformed liposomes prepared in unbuffered ( approximately pH 3.5) 300 mM CuSO(4) or ZnSO(4). For Cu-containing liposomes, results suggested that irinotecan loading occurred when the interior pH and the exterior pH were matched; however, addition of nigericin to collapse any residual transmembrane pH gradient inhibited irinotecan loading. Greater than 90% of the encapsulated drug was in its active lactone form and cryo-TEM analysis indicated dark intravesicular electron-dense spots. CONCLUSION: Irinotecan is stably entrapped in the active lactone conformation within preformed copper-containing liposomes as a result of metal-drug complexation.

Fluorocarbon nanoparticles as multifunctional drug delivery vehicles.
Yu YB.
J Drug Target. 2006 Dec;14(10):663-9.
[ expand abstract ]

The history and current status of fluorocarbon nanoparticles in biomedicine is briefly reviewed. The deficiencies of current fluorocarbon nanoparticle formulations are highlighted. Strategies to remedy such deficiencies and to functionalize fluorocarbon nanoparticles are presented. Potential applications of fluorocarbon nanoparticles as multifunctional drug delivery vehicles are discussed. The strength of fluorocarbon nanoparticles as drug delivery vehicles is that they integrate drug delivery with non-invasive MR imaging so that the biodistribution of the pharmaceutical entity (drug+delivery vehicle) can be monitored in real time. This, in turn, permits the physician to adjust treatment plan for each patient based on his/her actual response to the ongoing treatment.

Comparing transfection efficiency and safety for antisense oligodeoxyribonucleotide between phospholipids-based microbubbles and liposomes.
Zhao YZ, Luo YK, Liang HD, Mei XG, Tang J, Lu CT, Zhang Y, Lin Q.
J Drug Target. 2006 Dec;14(10):687-93.
[ expand abstract ]

Objective: To compare transfection efficiency and safety for antisense oligodeoxynucleotides (AS-ODNs) between two type of phospholipids-based vectors.Methods: An AS-ODNs sequence HA824 combined with luciferase reporter plasmid was used. Under low intensity ultrasound (US), a breast cancer cell line SK-BR-3 was exposed to different concentration of microbubbles and liposomes. Transfection efficiency was detected by fluorescence microscopy. Cell viability was verified by propidium iodide assay. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the inhibitory effect of HA824 on HER-2 expression at mRNA level. Atomic force microscopy (AFM) scanning techniques was employed to observe the change of membrane pore size.Results: AS-ODNs transfection efficiency showed an increasing tend with microbubble concentration, but not with liposome concentration. Maximum transfection efficiency with minimum cell viability was achieved under 2% microbubble concentration. Too strong sonoporation activity would enlarge membrane pores significantly and cause low cell viability.Conclusion: US-mediated AS-ODNs transfection enhanced by phospholipids-based microbubbles represents an effective and safe avenue.

Mathematical modeling and simulation of drug release from microspheres: Implications to drug delivery systems.
Arifin DY, Lee LY, Wang CH.
Adv Drug Deliv Rev. 2006 Nov 30;58(12-13):1274-325.
[ expand abstract ]

This article aims to provide a comprehensive review of existing mathematical models and simulations of drug release from polymeric microspheres and of drug transport in adjacent tissues. In drug delivery systems, mathematical modeling plays an important role in elucidating the important drug release mechanisms, thus facilitating the development of new pharmaceutical products by a systematic, rather than trial-and-error, approach. The mathematical models correspond to the known release mechanisms, which are classified as diffusion-, swelling-, and erosion-controlled systems. Various practical applications of these models which explain experimental data are illustrated. The effect of gamma-irradiation sterilization on drug release mechanism from erosion-controlled systems will be discussed. The application of existing models to nanoscale drug delivery systems specifically for hydrophobic and hydrophilic molecules is evaluated. The current development of drug transport modeling in tissues utilizing computational fluid dynamics (CFD) will also be described.

Bio-functional micelles self-assembled from a folate-conjugated block copolymer for targeted intracellular delivery of anticancer drugs.
Liu SQ, Wiradharma N, Gao SJ, Tong YW, Yang YY.
Biomaterials. 2006 Nov 30; [Epub ahead of print].
[ expand abstract ]

In this study, a block copolymer, poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide-co-2-aminoethyl methacrylate)-b-poly(10-undecenoic acid) (P(NIPAAm-co-DMAAm-co-AMA)-b-PUA) was synthesized, and folic acid was conjugated to the hydrophilic block through the amine group in AMA. This polymer was self-assembled into micelles, which exhibited pH-induced temperature sensitivity. They were smaller in size, and possessed a better-defined core-shell structure as well as more stable hydrophobic core than the random copolymer P(NIPAAm-co-DMAAm-co-UA), and provided a shell with folate molecules. An anti-cancer drug, doxorubicin (DOX) was encapsulated into the micelles. The mean diameter of the blank and DOX-loaded micelles was less than 100nm. DOX release was pH-dependent, being faster at low pH (endosomes/lysosomes). Therefore, DOX was readily released from the micelles into the nucleus after being taken up. More importantly, IC50 of DOX-loaded micelles with folate against folate receptor-expressing 4T1 and KB cells was much lower than that of the DOX-loaded micelles without folate (3.8 vs. 7.6mg/L for 4T1 cells and 1.2 vs. 3.0mg/L for KB cells). In vivo experiments conducted in a 4T1 mouse breast cancer model demonstrated that DOX-loaded micelles had a longer blood circulation time than free DOX (t(1/2): 30min and 140min, respectively). In addition, the micelles delivered an increased amount of DOX to the tumor when compared to free DOX. These bio-functional micelles may make a promising carrier to transport anticancer drugs specifically to tumor cells and release the drug molecules inside the cells to the cytosols for improved chemotherapy.

A bacterial protein enhances the release and efficacy of liposomal cancer drugs.
Cheong I, Huang X, Bettegowda C, Diaz LA Jr, Kinzler KW, Zhou S, Vogelstein B.
Science. 2006 Nov 24;314(5803):1308-11.
[ expand abstract ]

Clostridium novyi-NT is an anaerobic bacterium that can infect hypoxic regions within experimental tumors. Because C. novyi-NT lyses red blood cells, we hypothesized that its membrane-disrupting properties could be exploited to enhance the release of liposome-encapsulated drugs within tumors. Here, we show that treatment of mice bearing large, established tumors with C. novyi-NT plus a single dose of liposomal doxorubicin often led to eradication of the tumors. The bacterial factor responsible for the enhanced drug release was identified as a previously unrecognized protein termed liposomase. This protein could potentially be incorporated into diverse experimental approaches for the specific delivery of chemotherapeutic agents to tumors.

Thermotherapy of Prostate Cancer Using Magnetic Nanoparticles: Feasibility, Imaging, and Three-Dimensional Temperature Distribution.
Johannsen M, Gneveckow U, Thiesen B, Taymoorian K, Cho CH, Waldofner N, Scholz R, Jordan A, Loening SA, Wust P.
Eur Urol. 2006 Nov 17; [Epub ahead of print].
[ expand abstract ]

OBJECTIVES: To investigate the feasibility of thermotherapy using biocompatible superparamagnetic nanoparticles in patients with locally recurrent prostate cancer and to evaluate an imaging-based approach for noninvasive calculations of the three-dimensional temperature distribution. METHODS: Ten patients with locally recurrent prostate cancer following primary therapy with curative intent were entered into a prospective phase 1 trial. The magnetic fluid was injected transperineally into the prostates according to a preplan. Patients received six thermal therapies of 60-min duration at weekly intervals using an alternating magnetic field applicator. A method of three-dimensional thermal analysis based on computed tomography (CT) of the prostates was developed and correlated with invasive and intraluminal temperature measurements. The sensitivity of nanoparticle detection by means of CT was investigated in phantoms. RESULTS: The median detection rate of iron oxide nanoparticles in tissue specimens using CT was 89.5% (range: 70-98%). Maximum temperatures up to 55 degrees C were achieved in the prostates. Median temperatures in 20%, 50%, and 90% of the prostates were 41.1 degrees C (range: 40.0-47.4 degrees C), 40.8 degrees C (range: 39.5-45.4 degrees C), and 40.1 degrees C (range: 38.8-43.4 degrees C), respectively. Median urethral and rectal temperatures were 40.5 degrees C (range: 38.4-43.6 degrees C) and 39.8 degrees C (range: 38.2-43.4 degrees C). The median thermal dose was 7.8 (range: 3.5-136.4) cumulative equivalent minutes at 43 degrees C in 90% of the prostates. CONCLUSION: The heating technique using magnetic nanoparticles was feasible. Hyperthermic to thermoablative temperatures were achieved in the prostates at 25% of the available magnetic field strength, indicating a significant potential for higher temperatures. A noninvasive thermometry method specific for this approach could be developed, which may be used for thermal dosimetry in future studies.

PLA/PLGA nanoparticles for sustained release of docetaxel.
Musumeci T, Ventura CA, Giannone I, Ruozi B, Montenegro L, Pignatello R, Puglisi G.
Int J Pharm. 2006 Nov 15;325(1-2):172-9.
[ expand abstract ]

This study investigates the potentiality of nanosphere colloidal suspensions as sustained release systems for intravenous administration of docetaxel (DTX). Nanospheres were prepared by solvent displacement method using polylactic acids (PLA) at different molecular weight and polylactic-co-glycolic (PLGA) as biodegradable matrices. The systems were characterized by light scattering analysis for their mean size, size distribution and zeta potential and by scanning electron microscopy (SEM) for surface morphology. The average diameters of the nanoparticles ranged from 100 to 200 nm. Negative zeta potential values were observed for all systems, particularly the nanospheres produced with the lowest molecular weight PLA showed a zeta potential value of -28mV. Differential scanning calorimetry analysis (DSC) suggested that DTX was molecularly dispersed in the polymeric matrices. A biphasic release of DTX was observed for all colloidal suspensions, after a burst effect in which about 50% (w/w) of the loaded drug was released a sustained release profile for about 10 days was observed. To evaluate the influence of the polymeric carrier on the interaction of DTX with biological membranes, we performed an in vitro study using lipid vesicles made of dipalmitoylphosphatidylcholine (DPPC) as a biomembrane model. DSC was used as a simple and not invasive technique of analysis. DTX produced a depression of DPPC pretransition peak, no variation of the main phase transition temperature and a significative increase of DeltaH value, showing a superficial penetration of the drug into DPPC bilayer. Kinetic experiments demonstrated that the release process of DTX form nanospheres is affected by the molecular weight of the employed polymers.

PEGylated Nanoparticles Based on a Polyaspartamide. Preparation, Physico-Chemical Characterization, and Intracellular Uptake.
Craparo EF, Cavallaro G, Bondi ML, Mandracchia D, Giammona G.
Biomacromolecules. 2006 Nov 13; 7(11): 3083-3092.
[ expand abstract ]

Nanoparticles with different surface PEGylation degree were prepared by using as starting material alpha,beta-poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA). PHEA was functionalized with a PEG amino-derivative for obtaining PHEA-PEG(2000) copolymer. Both PHEA and PHEA-PEG(2000) were derivatized with methacrylic anhydride (MA) for obtaining poly(hydroxyethylaspartamide methacrylated) (PHM) and poly(hydroxyethylaspartamide methacrylated)-PEGylated (PHM-PEG(2000)), respectively. Nanoparticles were obtained by UV irradiation of an inverse microemulsion, using as internal phase an aqueous solution of PHM alone or of the PHM/PHM-PEG(2000) mixture at different weight ratio and as external phase a mixture of propylene carbonate and ethyl acetate. Obtained nanoparticles were characterized by FT-IR analysis, dimensional analysis, and TEM micrography. XPS analysis and zeta potential measurements demonstrated the presence of PEG onto the nanoparticle surface. Moreover, the partial degradation of nanoparticles in the presence of esterase as a function of time was demonstrated. Finally, nanoparticles did not possess any cytotoxic activity against K-562 cells and were able to escape from phagocytosis depending on the surface PEGylation degree.

Rapid and Precise Release from Nano-Tracted Poly(N-isopropylacrylamide) Hydrogels Containing Linear Poly(acrylic acid).
Asoh TA, Kaneko T, Matsusaki M, Akashi M.
Macromol Biosci. 2006 Nov 10;6(11):959-965 [Epub ahead of print].
[ expand abstract ]

We investigated the rapid and precise molecular release from hydrogels in response to dual stimuli. To achieve precise on/off drug release using thermoresponsive poly(N-isopropylacrylamide) hydrogels, we prepared nano-structured semi-IPNs, which consisted of thermosensitive PNIPAAm networks penetrated by pH-responsive poly(acrylic acid) (PAAc) linear chains and perforated to create nano-tracts as a molecular pathway. The present nano-tracted semi-IPNs show a rapid deswelling response to both temperature and pH. Model drug releases were investigated when simultaneous changes in temperature and pH were applied. We observed that the cationic drug was rapidly released and then abruptly discontinued from the nano-tracted semi-IPNs in response to the dual stimuli, and clear release and stopping cycles were repeatedly observed on successive steps. Moreover, the release rates and amount of drug released were controllable by the deswelling speed of the gels and the PAAc content inside the gels. This novel release system using the nano-tracted semi-IPNs may be useful for the high performance, pulsed release of molecules.Release profiles of MB from semi-IPNs at pH = 5.5, 20 degrees C (white region) and pH = 2, 40 degrees C (gray region).

Nanoparticles for Two-Photon Photodynamic Therapy in Living Cells.
Gao D, Agayan RR, Xu H, Philbert MA, Kopelman R.
Nano Lett. 2006 Nov 8;6(11):2383-2386.
[ expand abstract ]

We describe here a nontoxic two-photon photodynamic nanoparticle platform and its cellular application. We demonstrate that the dye's potential toxicity can be circumvented by its permanent encapsulation into a biocompatible nanoparticle polymer matrix; this was examined by dye leaching experiments and confirmed by cell uptake experiments. Infrared two-photon nanoplatform phototoxicity was demonstrated for rat C6 glioma cells, while the controls showed no dark toxicity for these living cells.

A single dose of doxorubicin-functionalized bow-tie dendrimer cures mice bearing C-26 colon carcinomas.
Lee CC, Gillies ER, Fox ME, Guillaudeu SJ, Frechet JM, Dy EE, Szoka FC.
Proc Natl Acad Sci USA. 2006 Nov 7;103(45):16649-54.
[ expand abstract ]

The antitumor effect of doxorubicin (DOX) conjugated to a biodegradable dendrimer was evaluated in mice bearing C-26 colon carcinomas. An asymmetric biodegradable polyester dendrimer containing 8-10 wt % DOX was prepared. The design of the dendrimer carrier optimized blood circulation time through size and molecular architecture, drug loading through multiple attachment sites, solubility through PEGylation, and drug release through the use of pH-sensitive hydrazone linkages. In culture, dendrimer-DOX was >10 times less toxic than free DOX toward C-26 colon carcinoma cells after exposure for 72 h. Upon i.v. administration to BALB/c mice with s.c. C-26 tumors, dendrimer-DOX was eliminated from the serum with a half-life of 16 +/- 1 h, and its tumor uptake was ninefold higher than i.v. administered free DOX at 48 h. In efficacy studies performed with BALB/c mice bearing s.c. C-26 tumors, a single i.v. injection of dendrimer-DOX at 20 mg/kg DOX equivalents 8 days after tumor implantation caused complete tumor regression and 100% survival of the mice over the 60-day experiment. No cures were achieved in tumor-implanted mice treated with free DOX at its maximum tolerated dose (6 mg/kg), drug-free dendrimer, or dendrimer-DOX in which the DOX was attached by means of a stable carbamate bond. The antitumor effect of dendrimer-DOX was similar to that of an equimolar dose of liposomal DOX (Doxil). The remarkable antitumor activity of dendrimer-DOX results from the ability of the dendrimer to favorably modulate the pharmacokinetics of attached DOX.

Preparation of poly varepsilon-caprolactone nanoparticles containing magnetite for magnetic drug carrier.
Yang J, Park SB, Yoon HG, Huh YM, Haam S.
Int J Pharm. 2006 Nov 6;324(2):185-90.
[ expand abstract ]

Magnetic poly varepsilon-caprolactone (PCL) nanoparticles were prepared in a well shaped spherical form by the o/w emulsion method. The influence of some preparative variables on the size and surface property was investigated. Nanoparticles were smooth, well individualized and homogeneous in size. The presence of magnetite and its superparamagnetic characteristic were confirmed by transmission electron microscope (TEM), Fourier transform infrared spectroscopy (FT-IR) and vibrating sample magnetometer (VSM), respectively. The anti-cancer drug was encapsulated in the magnetic nanoparticle during preparation. A typical release behavior was observed for 30 days. In vitro experiment of magnetic susceptibility under external magnetic field demonstrated that the magnetic PCL nanoparticles have sufficient magnetic susceptibility for a potential magnetic drug carrier for targeted delivery.

Thermally associating polypeptides designed for drug delivery produced by genetically engineered cells.
Hart DS, Gehrke SH.
J Pharm Sci. 2006 Nov 1; [Epub ahead of print].
[ expand abstract ]

Thermally associating polymers, including gelatin, cellulose ethers (e.g., Methocels(R) and poloxamers (e.g., Pluronics(R)) have a long history of use in pharmacy. Over the past 20 years, significant advances in genetic engineering and the understanding of protein secondary and tertiary structures have been made. This has led to the development of a variety of polypeptides that do not occur naturally but can be expressed in recombinant cells and have useful properties that lend themselves to novel applications where current materials cannot perform. The most intensively studied motifs are derived from the consensus repeats of elastin and silk, as well as coiled-coil helices. Many of these designed polypeptides or 'artificial proteins' are thermally associating materials. This property can be exploited to develop solid dosage forms, injectable drug delivery systems, micro- or nanoparticle drug carriers, triggered or targeted release systems, or as a means of simplifying the purification process and thus reducing costs of production of these materials. This review focuses on the development and characterization of this novel class of biomaterials and examines their potential for pharmaceutical applications.

A Parenteral Econazole Formulation Using a Novel Micelle-to-Liposome Transfer Method: In Vitro Characterization and Tumor Growth Delay in a Breast Cancer Xenograft Model.
Cogswell S, Berger S, Waterhouse D, Bally MB, Wasan EK.
Pharm Res. 2006 Nov;23(11):2575-85.
[ expand abstract ]

PURPOSE: The purpose of this study was to develop a parenteral liposomal formulation of econazole, a poorly water-soluble compound not previously available in an intravenous form. We are investigating econazole as an anticancer agent based on its unique mechanism of action to which cancer cells are preferentially sensitive. An intravenous formulation of econazole was desired for preclinical toxicity and efficacy studies of econazole. METHODS: Liposomal econazole was prepared using a novel micelle exchange technique to incorporate the drug into the lipid bilayer of pre-formed liposomes using a poly(ethylene) glycol-linked phospholipid, distearoyl phosphatidylethanolamine (DSPE-PEG). This method allowed for stable and efficient drug incorporation into DPPC and DMPC liposomes at a final drug:lipid ratio of 0.05 (w/w) and increased solubility in saline from <0.1 to 5 mg/ml. RESULTS: Stability over 14 days at 4 degrees C in buffer was demonstrated as well as in vitro plasma stability at 37 degrees C. Plasma elimination studies of micelle-loaded liposomal econazole showed a half-life of approximately 35 min and plasma AUC of 281 mug/ml min. In MCF-7 human breast cancer xenografts in Rag2M mice. Liposomal econazole did not induce significant hepatoxicity, renal toxity or weight loss compared to empty liposomes. Tumor growth was slightly delayed in liposomal econazole-treated mice, with approximately 10-day lag time to reach 300 mm(3) compared to vehicle controls. CONCLUSIONS: The micelle transfer method provided an efficient means of preparing liposomal econazole suitable for intravenous administration. Liposomal econazole was successfully administered to tumor bearing mice at 50 mg/kg, and no significant toxicities attributable to econazole were observed.

Pathotropic nanoparticles for cancer gene therapy Rexin-G IV: three-year clinical experience.
Gordon EM, Lopez FF, Cornelio GH, Lorenzo CC 3rd, Levy JP, Reed RA, Liu L, Bruckner HW, Hall FL.
Int J Oncol. 2006 Nov;29(5):1053-64.
[ expand abstract ]

Metastatic cancer is a life-threatening illness with a predictably fatal outcome, thereby representing a major unmet medical need. In 2003, Rexin-G became the world's first targeted injectable vector approved for clinical trials in the treatment of intractable metastatic disease. Uniquely suited, by design, to function within the context of the human circulatory system, Rexin-G is a pathotropic (disease-seeking) gene delivery system bearing a designer killer gene; in essence, a targeted nanoparticle that seeks out and selectively accumulates in metastatic sites upon intravenous infusion. The targeted delivery of the cytocidal gene to primary tumors and metastatic foci, in effective local concentrations, compels both cancer cells and tumor-associated neovasculature to self-destruct, without causing untoward collateral damage to non-target organs. In this study: i) we report the results of three distinctive clinical studies which demonstrate the initial proofs of concept, safety, and efficacy of Rexin-G when used as a single agent for advanced or metastatic cancer, ii) we introduce the quantitative foundations of an innovative personalized treatment regimen, designated the 'Calculus of Parity', based on a patient's calculated tumor burden, iii) we propose a refinement of surrogate end-points commonly used for defining success in cancer therapy, and iv) we map out a strategic plan for the accelerated approval of Rexin-G based on the oncologic Threshold of Credibility paradigm being developed by the Food and Drug Administration.

Busulfan loading into poly(alkyl cyanoacrylate) nanoparticles: physico-chemistry and molecular modeling.
Layre AM, Couvreur P, Chacun H, Aymes-Chodur C, Ghermani NE, Poupaert J, Richard J, Requier D, Gref R.
J Biomed Mater Res B Appl Biomater. 2006 Nov;79(2):254-62.
[ expand abstract ]

The busulfan is an alkylating agent widely used for the treatment of haematological malignancies and nonmalignant disorders. For a long time, it has been available only in an oral form. This treatment leads to a wide variability in bioavailability and side effects such as the veino-occlusive disease. Thus, an intravenous formulation of busulfan-loaded nanoparticles may be considered as a major progress. This study deals with busulfan entrapment by nanoprecipitation into five different types of poly(alkyl cyanoacrylate) polymers. The polymers leading to the highest busulfan loading efficiencies were poly(isobutyl cyanoacrylate) (PIBCA) and poly(ethyl cyanoacrylate). Molecular modeling along with energy minimization process was employed to identify the nature of the interactions occurring between busulfan and PIBCA. Further, optimization studies enabled to obtain PIBCA nanoparticles displaying busulfan loading ratios equal to 5.9% (w/w) together with nanoparticle yields of 71% (w/w). Since busulfan is a highly reactive molecule, we performed (1)H-NMR spectroscopy experiments showing that chemical integrity of the drug was preserved after loading into nanoparticles. The in vitro release studies under sink conditions, in water, or in rat plasma showed a fast release in the first 10 min followed by a slower one over 6 h. This phenomenon could be explained by the semi-polar characteristics of busulfan.

Vaccination with liposome--DNA complexes elicits enhanced antitumor immunity.
U'Ren L, Kedl R, Dow S.
Cancer Gene Ther. 2006 Nov;13(11):1033-44.
[ expand abstract ]

Cationic liposomes have been shown to potentiate markedly the ability of plasmid DNA to activate innate immune responses. We reasoned therefore that liposome-DNA complexes (LDC) could be used to produce more effective plasmid DNA vaccines for cancer. To test this hypothesis, tumor-bearing mice were vaccinated with conventional plasmid DNA vaccines or with LDC vaccines encoding model tumor antigens and CD8(+) T-cell responses and antitumor activity were assessed. We found that although plasmid DNA vaccines generated large increases in antigen-specific CD8(+) T cells, they failed to elicit significant antitumor immunity. In contrast, LDC vaccines elicited large numbers of antigen-specific CD8(+) T cells and also generated significant antitumor activity against established tumors. The antitumor activity elicited by immunization with LDC vaccines was mediated primarily by CD8(+) T cells. Studies of the interaction of LDC with antigen-presenting cells found that LDC triggered dendritic cell production of interleukin-12 and interferon (IFN)-gamma production by natural killer cells in vivo. Activation by LDC was also accompanied by upregulation of costimulatory molecule expression. These findings suggest that by concurrently activating strong systemic innate immune responses and generating cytotoxic T-lymphocyte responses, LDC may be used to increase the effectiveness of therapeutic plasmid DNA vaccination for cancer.

Cytotoxicity of doxorubicin bound to poly(butyl cyanoacrylate) nanoparticles in rat glioma cell lines using different assays.
Sanchez De Juan B, Von Briesen H, Gelperina SE, Kreuter J.
J Drug Target. 2006 Nov;14(9):614-22.
[ expand abstract ]

The cytotoxicity of doxorubicin bound to poly(butyl cyanoacrylate) nanoparticles (Dox-PBCA-NP) was investigated in the rat glioma cell lines GS-9L, F-98 and RG-2. MTT and LDH assays were used as cytotoxic assays. In general, the cytotoxicity of nanoparticle-bound doxorubicin (Dox) was enhanced compared to the free drug in solution. However, responses of the cell lines towards the drug effects were different. In the case of free Dox in solution, this difference correlated with different intracellular concentrations of Dox, which in turn, depended on the level of P-glycoprotein (P-gp) expression in these cell lines. Accordingly, the 9L gliosarcoma (GS-9L) cells, which appeared to be most resistant towards Dox, were characterized by the highest P-gp expression.Additionally, the influence of surfactants on the cytotoxic effect was investigated at different Dox concentrations. It was shown that the presence of polysorbate 80 (Tween(R) 80) in the nanoparticle formulation significantly enhanced the cytotoxicity, whereas poloxamer 188 (Pluronic(R) F68) and poloxamine 908 (Tetronic(R) 908) had a negligible influence.

Biodistribution and pharmacokinetic analysis of long-circulating thiolated gelatin nanoparticles following systemic administration in breast cancer-bearing mice.
Kommareddy S, Amiji M.
J Pharm Sci. 2006 Oct 30; [Epub ahead of print].
[ expand abstract ]

The objective of the present study was to modify thiolated gelatin nanoparticles with poly(ethylene glycol) (PEG) chains and examine their long circulating and tumor-targeting properties in vivo in an orthotopic a human breast adenocarcinoma xenograft model. The crosslinked nanoparticle systems were characterized to have a size of 150-250 nm with rapid payload release properties in a highly reducing environment. Upon PEG modification, the nanoparticle size increased to 300-350 nm in diameter. The presence of PEG chains on the surface was confirmed by characterization with electron spectroscopy for chemical analysis. The in vivo long-circulating potential, biodistribution and passive tumor targeting of the controls, and PEG-modified thiolated gelatin nanoparticles were evaluated by injecting indium-111 ((111)In)-labeled nanoparticles into breast tumor (MDA-MB-435)-bearing nude mice. Upon modification with PEG, the nanoparticles were found to have longer circulation times, with the plasma and tumor half-lives of 15.3 and 37.8 h, respectively. The results also showed preferential localization of thiolated nanoparticles in the tumor mass. The resulting nanoparticulate systems with long circulation properties could be used to target encapsulated drugs and genes to tumors passively by utilizing the enhanced permeability and retention effect of the tumor vasculature.

Nanomedicine: Developing smarter therapeutic and diagnostic modalities.
Farokhzad OC, Langer R.
Adv Drug Deliv Rev. 2006 Oct 27; [Epub ahead of print].
[ expand abstract ]

The early impact of nanotechnology on medicine is beginning to get realized, with novel nanoscale therapeutic and diagnostic modalities under development or in clinical practice today. In this commentary the field of "nanomedicine" is briefly reviewed form the perspective of where we were; where we are today; and where we are likely to go tomorrow.

Aclarubicin-loaded cationic albumin-conjugated pegylated nanoparticle for glioma chemotherapy in rats.
Lu W, Wan J, Zhang Q, She Z, Jiang X.
Int J Cancer. 2006 Oct 25; [Epub ahead of print].
[ expand abstract ]

Traditional glioma chemotherapy with those second-line drugs such as anthracyclines usually failed because they are inaccessible to blood-brain barrier (BBB) in tumor. In our study, we incorporated aclarubicin (ACL) into cationic albumin-conjugated pegylated nanoparticle (CBSA-NP-ACL) to determine its therapeutic potential of rats with intracranially implanted C6 glioma cells. When labeled with fluorescent probe, 6-coumarin, CBSA-NP was shown to accumulate much more in tumor mass than nanoparticle without conjugated CBSA (NP) 1 hr post intravenous injection, as well as better retention after 24 hr. Tumor drug concentration of CBSA-NP-ACL displayed 2.6- and 3.3-fold higher than that of NP-ACL and ACL solution 1 hr post injection, while 2.7 and 6.6-fold higher after 24 hr, respectively. Moreover, using tumor microdialysis sampling, AUC(0-24 hr) of free drug amount in tumor interstitium delivered by CBSA-NP-ACL was about 2.0- and 2.7-fold higher than that of NP-ACL and ACL solutions, respectively. When the tumor rat model was subjected to 4 cycles of 2 mg/kg of ACL in different formulations, a significant increase of median survival time was found in the group of CBSA-NP-ACL compared with that of saline control animals, animals treated with NP-ACL and ACL solution. By terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling, CBSA-NP-ACL can extensively make the tumor cell apoptosis. Histochemical evaluation by periodic acid Shiff staining and biochemical analysis depicted that the incorporation of ACL into CBSA-NP reduced its toxicity to liver, kidney and heart. Besides, CBSA-NP-ACL was not shown to open tight junction evaluated by BBB coculture. It was concluded that CBSA-NP-ACL could have a therapeutic potential for treatment of glioma.

Transition Metal-Mediated Liposomal Encapsulation of Irinotecan (CPT-11) Stabilizes the Drug in the Therapeutically Active Lactone Conformation.
Ramsay E, Alnajim J, Anantha M, Taggar A, Thomas A, Edwards K, Karlsson G, Webb M, Bally M.
Pharm Res. 2006 Oct 25; [Epub ahead of print].
[ expand abstract ]

PURPOSE: To determine whether entrapped transition metals could mediate the active encapsulation of the anticancer drug irinotecan into preformed liposomes. Further, to establish that metal complexation could stabilize liposomal irinotecan in the therapeutically active lactone conformation. MATERIALS AND METHODS: Irinotecan was added to preformed 1,2-distearoyl-sn-glycero-phosphocholine/cholesterol (DSPC/chol) liposomes prepared in CuSO(4), ZnSO(4), MnSO(4), or CoSO(4) solutions, and drug encapsulation was determined over time. The roles of the transmembrane pH gradient and internal pH were evaluated. TLC and HPLC were used to monitor drug stability and liposome morphology was assessed by cryo-TEM. RESULTS: Irinotecan was rapidly and efficiently loaded into preformed liposomes prepared in unbuffered ( approximately pH 3.5) 300 mM CuSO(4) or ZnSO(4). For Cu-containing liposomes, results suggested that irinotecan loading occurred when the interior pH and the exterior pH were matched; however, addition of nigericin to collapse any residual transmembrane pH gradient inhibited irinotecan loading. Greater than 90% of the encapsulated drug was in its active lactone form and cryo-TEM analysis indicated dark intravesicular electron-dense spots. CONCLUSION: Irinotecan is stably entrapped in the active lactone conformation within preformed copper-containing liposomes as a result of metal-drug complexation.

In vitro and in vivo suppression of hepatocellular carcinoma growth by chitosan nanoparticles.
Qi L, Xu Z, Chen M.
Eur J Cancer. 2006 Oct 16; [Epub ahead of print].
[ expand abstract ]

Chitosan nanoparticles (CNP), a kind of widely used drug carrier, have shown potent cytotoxic effects on various tumour cell lines in vitro and in vivo. This study sought to evaluate the antitumour effect of CNP on growth of human hepatocellular carcinoma (BEL7402) and the possible mechanisms involved. Cells were grown in the absence and presence of various concentrations of CNP with mean particle size of about 40nm. Cell viability, ultrastructural changes, surface charge, mitochondrial membrane potential, reactive oxygen species (ROS) generation, lipid peroxidation, DNA fragmentation and fatty acid composition were analysed by MTT assay, electron microscopy, zetasizer analysis, flow cytometry, spectrophotometric thiobarbituric (TBA) assays, DNA agarose gel electrophoresis and GC/MS respectively. For in vivo experiments, male BABL/c nude mice were implanted with BEL7402 cells subcutaneously to establish human hepatoma model. Chitosan, saline, and CNP with different mean particle size (40, 70 and 100nm) were administrated by oral administration (1mg/kg body weight). Tumour and body weight were measured, morphologic changes of tumour and liver tissues were studied under electron microscope. In vitro, CNP exhibited high antitumour activities with an IC(50) value of 15.01mug/ml, 6.19mug/ml and 0.94mug/ml after treatment for 24h, 48h and 72h respectively. CNP could induce cell necrosis observed by electron microscope and DNA fragmentation. The antitumour mechanism was mediated by neutralisation of cell surface charge, decrease of mitochondrial membrane potential and induction of lipid peroxidation. The tumour growth inhibitory rates on BEL7402 cells in nude mice treated with chitosan and CNP with different mean particle size (40, 70 and 100nm) were 24.07%, 61.69%, 58.98% and 34.91% respectively. Typical necrotic morphological changes of tumour tissues and no liver abnormalities were found under electron microscope. In this paper, results show a strong antitumour effect of CNP on human hepatoma cell line BEL7402 in vitro and in vivo. These findings suggest that CNP could be a kind of promising agent for further evaluations in the treatment of hepatocellular carcinoma.

Multivalent effects of RGD peptides obtained by nanoparticle display.
Montet X, Funovics M, Montet-Abou K, Weissleder R, Josephson L.
J Med Chem. 2006 Oct 5;49(20):6087-93.
[ expand abstract ]

The binding of RGD peptides to integrins offers an excellent system to study the multivalent mediated changes in affinity that arise when peptides, displayed on the surface of a nanoparticle carrier, bind to integrins displayed on the cell membrane. The IC50 of an RGD nanoparticle for endothelial adhesion was 1.0 nM nanoparticle or 20 nM peptide (20 peptide/nanoparticle) and was associated with strong multivalent effects, defined as a multivalent enhancement factor (MVE) of 38 (MVE=IC50 (peptide)/IC50 (peptide when displayed by nanoparticle)). The attachment of RGD peptides to nanoparticles resulted in an extension of the peptide blood half-life from 13 to 180 min. Based on the multivalent enhancement of affinity and extension of blood half-life, multivalent RGD nanoparticle-sized materials should be potent inhibitors of the alpha(V)beta(3) function on endothelial cells in vivo.

Amine-containing core-shell nanoparticles as potential drug carriers for intracellular delivery.
Feng M, Li P.
J Biomed Mater Res A. 2006 Oct 3; [Epub ahead of print] .
[ expand abstract ]

The present study aimed at exploring the use of amine-containing core-shell nanoparticles as potential drug carriers for intracellular delivery. Stable nanoparticles (100-200 nm in diameter) that consisted of poly(methyl methacrylate) (PMMA) cores with hydrophilic poly(ethyleneimine) (PEI) shells were synthesized and used to study their complexation with model drug, ibuprofen (IB), and release it under various electrolyte concentrations. The complexed IB/PEI-PMMA nanoparticles were characterized with FTIR, photon correlation spectroscopy, zeta-potential, and transmission electron microscopy (TEM). Results suggested that the PEI-PMMA nanoparticles could effectively complex with the IB via electrostatic interaction. The thick PEI shells ( approximately 30 nm) significantly enhanced the drug loading capacity up to 23% (w/w) of the complexed nanopartricle. In vitro release of the drug from the complexed nanoparticles was sensitive to the ionic strength of the media. Study of cellular entry of fluorescently labeled IB/nanoparticle complexes using a confocal laser scanning microscopy demonstrated that the entry of the complexed nanoparticles strongly depended on the complexing ratio between IB and PEI-PMMA nanoparticles.

Liposome-mediated RNA transfection should be used with caution.
Barreau C, Dutertre S, Paillard L, Osborne HB.
RNA. 2006 Oct;12(10):1790-3.
[ expand abstract ]

Liposome-mediated RNA transfection appears to present a number of advantages for studying the metabolism of reporter mRNAs in mammalian cells. This method is also widely used to transfect siRNAs. Here we describe results indicating that reporter mRNAs introduced into HeLa cells by liposomes do not present the expected behaviors. Namely, the stability of reporter mRNAs was independent of the presence or absence of an AUUUA instability element, a poly(A) tail, or even a 5' methylated cap. Confocal microscopy showed that fluorescent RNAs introduced by liposome-mediated transfection were present in discrete particles. These observations imply that a number of control experiments are required when using liposome to mediated RNA transfection, and the possible consequences are discussed.

Liposomal plasmid DNA encoding human thymosin alpha and interferon omega potently inhibits liver tumor growth in ICR mice.
Chen PF, Fu GF, Zhang HY, Xu GX, Hou YY.
J Gastroenterol Hepatol. 2006 Oct;21(10):1538-43.
[ expand abstract ]

Aim: To evaluate the potential therapeutic effect of liposomal gene delivery, genes encoding for human thymosin alpha1 (Talpha(1)) and interferon omega(1) were injected via the tail vein into mice bearing a Hep-A-22 liver tumor. The cDNA of human Talpha(1) and interferon omega(1) were obtained by synthesis or reverse transcription-polymerase chain reaction (RT-PCR), respectively. Eukaryotic expressing vectors pIRES2, encoding Talpha(1) and/or interferon omega(1), were constructed and injected with liposome via the tail vein into ICR mice bearing a Hep-A-22 tumor. The potency of tumor inhibition was evaluated when three treated groups were compared with the group receiving the empty vector. Apoptosis of tumor cells was investigated by analyzing DNA fragmentation. Only the group treated with dual-gene plasmid reached an eligible level of tumor inhibition (43%). The difference in tumor weight was statistically significant between the Talpha(1) gene or the interferon omega(1) gene treated groups and the control (P < 0.05), and highly significant between the dual-gene treated group and the control (P < 0.01). DNA ladder was observed in the tumor cells from the purpose gene treated groups but not from the control. The dual-gene plasmid-liposome complex showed more potent inhibition than the single gene constructs on the growth of Hep-A-22 tumor cells in mice, which may be attributed to indirect and additive induction of apoptosis in tumor cells by increased expression of Talpha(1) and interferon omega(1).

Optimized ultrasound-mediated gene transfection in cancer cells.
Feril LB Jr, Ogawa R, Tachibana K, Kondo T.
Cancer Sci. 2006 Oct;97(10):1111-4.
[ expand abstract ]

Ultrasound-mediated gene transfection (sonotransfection) is a promising physical method for gene therapy, especially for cancer gene therapy. To investigate the optimal sonotransfection conditions and to determine whether the optimal transfection rate using sonotransfection is comparable to that of electrotransfection or liposome-mediated transfection, we sonicated different cancer cell lines (U937, HeLa, PC-3, Meth A and T-24) using a 1-MHz unfocused ultrasound at different intensities, pulse repetition frequencies and exposure times. The ideal ultrasound conditions were noted to be at 1.5 Watt/cm(2) pulsed at 0.5 Hz with a duty factor of 50%. The results showed that transfection rate increased with the number of pulses, and peaked between 10 and 15 pulses before it started to decline. Using such optimal conditions, we have shown that sonotransfection is superior to electrotransfection and liposome-mediated transfection at the fixed conditions used in the present study. These findings suggest that sonotransfection could be a better alternative to other non-viral methods (e.g. electroporation and liposome-mediated transfection) of gene transfection, particularly in cancer gene therapy.

Combining cell therapy and nanotechnology.
Halberstadt C, Emerich DF, Gonsalves K.
Expert Opin Biol Ther. 2006 Oct;6(10):971-81.
[ expand abstract ]

Cell transplantation to treat diseases characterised by tissue and cell dysfunction, ranging from diabetes to spinal cord injury, has made great strides preclinically and towards clinical efficacy. In order to enhance clinical outcomes, research needs to continue in areas including the development of a universal cell source that can be differentiated into specific cellular phenotypes, methods to protect the transplanted allogeneic or xenogeneic cells from rejection by the host immune system, techniques to enhance cellular integration of the transplant within the host tissue, strategies for in vivo detection and monitoring of the cellular implants, and new techniques to deliver genes to cells without eliciting a host immune response. Overcoming these obstacles will be of considerable benefit, as it allows understanding, visualising and controlling cellular interactions at a submicron level. Nanotechnology is a multidisciplinary field that allows us to manipulate materials, tissues, cells and DNA at the level of and within the individual cell. As such, nanotechnology may be well suited to optimise the generally encouraging results already achieved in cell transplantation. This review presents some of the ways that nanotechnology can directly contribute to cell transplantation.

RNA interference in vitro and in vivo using a novel chitosan/siRNA nanoparticle system.
Howard KA, Rahbek UL, Liu X, Damgaard CK, Glud SZ, Andersen MO, Hovgaard MB, Schmitz A, Nyengaard JR, Besenbacher F, Kjems J.
Mol Ther. 2006 Oct;14(4):476-84.
[ expand abstract ]

This work introduces a novel chitosan-based siRNA nanoparticle delivery system for RNA interference in vitro and in vivo. The formation of interpolyelectrolyte complexes between siRNA duplexes (21-mers) and chitosan polymer into nanoparticles, ranging from 40 to 600 nm, was shown using atomic force microscopy and photon correlation spectroscopy. Rapid uptake (1 h) of Cy5-labeled nanoparticles into NIH 3T3 cells, followed by accumulation over a 24 h period, was visualized using fluorescence microscopy. Nanoparticle-mediated knockdown of endogenous enhanced green fluorescent protein (EGFP) was demonstrated in both H1299 human lung carcinoma cells and murine peritoneal macrophages (77.9% and 89.3% reduction in EGFP fluorescence, respectively). In addition, Western analysis showed approximately 90% reduced expression of BCR/ABL-1 leukemia fusion protein while BCR expression was unaffected in K562 (Ph(+)) cells after transfection using nanoparticles containing siRNA specific to the BCR/ABL-1 junction sequence. Effective in vivo RNA interference was achieved in bronchiole epithelial cells of transgenic EGFP mice after nasal administration of chitosan/siRNA formulations (37% and 43% reduction compared to mismatch and untreated control, respectively). These findings highlight the potential application of this novel chitosan-based system in RNA-mediated therapy of systemic and mucosal disease.

Targeting gene therapy for prostate cancer cells by liposomes complexed with anti-prostate-specific membrane antigen monoclonal antibody.
Ikegami S, Yamakami K, Ono T, Sato M, Suzuki S, Yoshimura I, Asano T, Hayakawa M, Tadakuma T.
Hum Gene Ther. 2006 Oct;17(10):997-1005.
[ expand abstract ]

Prostate-specific membrane antigen (PSMA) is a membrane-bound antigen expressed on the surface of prostate cancer cells, and this paper describes the use of an antibody against PSMA for targeting gene therapy. We coupled anti-PSMA monoclonal antibody with poly-L-lysine and then incubated it with plasmids. These complexes were then transfected with cationic liposomes into cells. The transfection efficiency of anti-PSMA- liposome complex was higher than that of normal IgG-liposome complex in PSMA-positive LNCaP cells. Furthermore, anti-PSMA-liposome complex containing a suicide gene, thymidine kinase, demonstrated a selective growth-inhibitory effect on LNCaP cells in vitro, but did not exert a significant effect on PSMA-negative cells. In an in vivo xenograft model of LNCaP cells in nu/nu mice, we administered the complexes via the tail vein. Judging on the basis of both 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining and luciferase assay findings, a significant enrichment of plasmid DNA was observed in LNCaP xenografts with anti-PSMA-liposome complex in comparison with normal IgG-liposome complex. However, the distribution of plasmid DNA did not change substantially in any other organs including the liver, kidney, lung, and spleen. Moreover, in suicide gene therapy, anti-PSMA-liposome complex exerted a significant inhibitory effect on the growth of LNCaP xenograft, in contrast to normal IgG-liposome complex.

Crossing cellular barriers using dendrimer nanotechnologies.
Najlah M, D'Emanuele A.
Curr Opin Pharmacol. 2006 Oct;6(5):522-7.
[ expand abstract ]

Dendrimers represent a class of polymers characterised by their well-defined structure, with a high degree of molecular uniformity and low polydispersity. They have found a wide-range of pharmaceutical applications; however, more recently, they have been shown to function as effective intracellular carriers for drugs. In addition, dendrimers have been shown to be capable of bypassing efflux transporters. A new generation of dendrimer-based delivery systems will enable the efficient transport of drugs across cellular barriers.

Co-delivery of drugs and DNA from cationic core-shell nanoparticles self-assembled from a biodegradable copolymer.
Wang Y, Gao S, Ye WH, Yoon HS, Yang YY.
Nat Mater. 2006 Oct;5(10):791-6.
[ expand abstract ]

Non-viral gene-delivery systems are safer to use and easier to produce than viral vectors, but their comparatively low transfection efficiency has limited their applications. Co-delivery of drugs and DNA has been proposed to enhance gene expression or to achieve the synergistic/combined effect of drug and gene therapies. Attempts have been made to deliver drugs and DNA simultaneously using liposomes. Here we report cationic core-shell nanoparticles that were self-assembled from a biodegradable amphiphilic copolymer. These nanoparticles offer advantages over liposomes, as they are easier to fabricate, and are more readily subject to modulation of their size and degree of positive charge. More importantly, they achieve high gene-transfection efficiency and the possibility of co-delivering drugs and genes to the same cells. Enhanced gene transfection with the co-delivery of paclitaxel has been demonstrated by in vitro and in vivo studies. In particular, the co-delivery of paclitaxel with an interleukin-12-encoded plasmid using these nanoparticles suppressed cancer growth more efficiently than the delivery of either paclitaxel or the plasmid in a 4T1 mouse breast cancer model. Moreover, the co-delivery of paclitaxel with Bcl-2-targeted small interfering RNA (siRNA) increased cytotoxicity in MDA-MB-231 human breast cancer cells.

Blood progenitor cell separation from clinical leukapheresis product by magnetic nanoparticle binding and magnetophoresis.
Jing Y, Moore LR, Williams PS, Chalmers JJ, Farag SS, Bolwell B, Zborowski M.
Biotechnol Bioeng. 2006 Sep 28; [Epub ahead of print] .
[ expand abstract ]

Positive selection of CD34+ blood progenitor cells from circulation has been reported to improve patient recovery in applications of autologous transplantation. Current magnetic separation methods rely on cell capture and release on solid supports rather than sorting from flowing suspensions, which limits the range of therapeutic applications and the process scale up. We tested CD34+ cell immunomagnetic labeling and isolation from fresh leukocyte fraction of peripheral blood (leukapheresis) using the continuous quadrupole magnetic flow sorter (QMS), consisting of a flow channel (SHOT, Greenville, IN) and a quadrupole magnet with a maximum field intensity (B(o)) of 1.42 T and a mean force field strength (S(m)) of 1.45 x 10(8)TA/m(2). Both the sample magnetophoretic mobility (m) and the inlet and outlet flow patterns highly affect the QMS performance. Seven commercial progenitor cell labeling reagent combinations were quantitatively evaluated by measuring magnetophoretic mobility of a high CD34 expression cell line, KG-1a, using the cell tracking velocimeter (CTV). The CD34 Progenitor Cell Isolation Kittrade mark (Miltenyi Biotec, Bergisch Gladbach, Germany) showed the strongest labeling of KG-1a cells and was selected for progenitor cell enrichment from 11 fresh and 11 cryopreserved clinical leukapheresis samples derived from different donors. The CD34+ cells were isolated with a purity of 60%-96%, a recovery of 18%- 60%, an enrichment rate of 12-169 and a throughput of (1.7-9.3) x 10(4) cells/s. The results also showed a highly regular dependence of the QMS performance on the flow conditions that agreed with the theoretical predictions based on the CD34+ cell magnetophoretic mobility.

N-acetyl histidine-conjugated glycol chitosan self-assembled nanoparticles for intracytoplasmic delivery of drugs: Endocytosis, exocytosis and drug release.
Park JS, Han TH, Lee KY, Han SS, Hwang JJ, Moon DH, Kim SY, Cho YW.
J Control Release. 2006 Sep 28;115(1):37-45.
[ expand abstract ]

Nano-sized vesicular systems (nanoparticles), ranging from 10 nm to 1000 nm in size, have potential applications as drug delivery systems. Successful clinical applications require the efficient intracellular delivery of drug-loaded nanoparticles. Here we describe N-acetyl histidine-conjugated glycol chitosan (NAcHis-GC) self-assembled nanoparticles as a promising system for intracytoplasmic delivery of drugs. Because N-acetyl histidine (NAcHis) is hydrophobic at neutral pH, the conjugates formed self-assembled nanoparticles with mean diameters of 150-250 nm. In slightly acidic environments, such as those in endosomes, the nanoparticles were disassembled due to breakdown of the hydrophilic/hydrophobic balance by the protonation of the imidazole group of NAcHis. Cellular internalization and drug release of the pH-sensitive self-assembled nanoparticles were investigated by flow cytometry and confocal microscopy. NAcHis-GC nanoparticles internalized by adsorptive endocytosis were exocytosed or localized in endosomes. The amount of exocytosed nanoparticles was dependent on the pre-incubation time prior to removal of free nanoparticles from the culture media. Flow cytometry and confocal microscopy showed that NAcHis-GC nanoparticles released drugs into the cytosol and cell cycle analysis demonstrated that paclitaxel-incorporated NAcHis-GC nanoparticles were effective in inducing arrest of cell growth.

Supramolecular assembly of cyclodextrin-based nanoparticles on solid surfaces for gene delivery.
Park IK, von Recum HA, Jiang S, Pun SH.
Langmuir. 2006 Sep 26;22(20):8478-84.
[ expand abstract ]

In this work, a new approach for surface-mediated gene delivery based on inclusion complex formation between the solid surface and delivery vehicles is presented. beta-Cyclodextrin (CD) molecules form high-affinity inclusion complexes with adamantane. This complexation ability was used to specifically immobilize beta-CD-modified poly(ethylenimine) (CD-PEI) nanoparticles on adamantane- (AD-) modified self-assembled monolayers. To investigate the nanoparticle/surface interaction, CD-PEI-based and PEI-based nanoparticles were passed through a surface plasmon resonance flow cell containing the monolayers. CD-PEI nanoparticles are specifically immobilized on the chip surface by cyclodextrin-adamantane inclusion complex formation. Minimal nanoparticle adsorption was detected with PEI-based nanoparticles or on control surfaces. Competition studies with free cyclodextrins reveal that the multivalent interactions between CD-PEI nanoparticles and the adamantane-modified surface results in significantly higher binding affinity than single cyclodextrin-adamantane complexes. Immobilized nanoparticles were characterized by atomic force microscopy and quantified by fluorescence assay. Thus, the ability of CD-PEI nanoparticles to form inclusion complexes can be exploited to attain specific, high-affinity loading of delivery vehicles onto solid surfaces.

Surface functionalization of inorganic nano-crystals with fibronectin and E-cadherin chimera synergistically accelerates trans-gene delivery into embryonic stem cells.
Kutsuzawa K, Chowdhury EH, Nagaoka M, Maruyama K, Akiyama Y, Akaike T.
Biochem Biophys Res Commun. 2006 Sep 25; [Epub ahead of print] .
[ expand abstract ]

Stem cells holding great promises in regenerative medicine have the potential to be differentiated to a specific cell type through genetic manipulation. However, conventional ways of gene transfer to such progenitor cells suffer from a number of disadvantages particularly involving safety and efficacy issues. Here, we report on the development of a bio-functionalized inorganic nano-carrier of DNA by embedding fibronectin and E-cadherin chimera on the carrier, leading to its high affinity interactions with embryonic stem cell surface and accelerated trans-gene delivery for subsequent expression. While only apatite nano-particles were very inefficient in transfecting embryonic stem cells, fibronectin-anchored particles and to a more significant extent, fibronectin and E-cadherin-Fc-associated particles dramatically enhanced trans-gene delivery with a value notably higher than that of commercially available lipofection system. The involvement of both cell surface integrin and E-cadherin in mediating intracellular localization of the hybrid carrier was verified by blocking integrin binding site with excess free fibronectin and up-regulating both integrin and E-cadherin through PKC activation. Thus, the new establishment of a bio-functional hybrid gene-carrier would promote and facilitate development of stem cell-based therapy in regenerative medicine.

Synthesis and characterization of chitosan-g-poly(ethylene glycol)-folate as a non-viral carrier for tumor-targeted gene delivery.
Chan P, Kurisawa M, Chung JE, Yang YY.
Biomaterials. 2006 Sep 22; [Epub ahead of print] .
[ expand abstract ]

Poor water solubility and low transfection efficiency of chitosan are major drawbacks for its use as a gene delivery carrier. PEGylation can increase its solubility, and folate conjugation may improve gene transfection efficiency due to promoted uptake of folate receptor-bearing tumor cells. The aim of this study was to synthesize and characterize folate-poly(ethylene glycol)-grafted chitosan (FA-PEG-Chi) for targeted plasmid DNA delivery to tumor cells. Gel electrophoresis study showed strong DNA binding ability of modified chitosan. The pH(50) values, defined as the pH when the transmittance of a polymer solution at 600nm has reached 50% of the original value, suggested that the water solubility of PEGylated chitosan had improved significantly. Regression analysis of pH(50) value as a function of substitution degree of PEG yielded an almost linear correlation for PEG-Chi and FA-PEG-Chi. The solubility of PEGylated chitosan decreased slightly by further conjugation of folic acid due to the relatively more hydrophobic nature of folic acid when compared to PEG. In addition, the chitosan-based DNA complexes did not induce remarkable cytotoxicity against HEK 293 cells. FA-PEG-Chi can be a promising gene carrier due to its solubility in physiological pH, efficiency in condensing DNA, low cytotoxicity and targeting ability.

Cell Biological and Biophysical Aspects of Lipid-mediated Gene Delivery.
Rao NM, Gopal V.
Biosci Rep. 2006 Sep 22; [Epub ahead of print].
[ expand abstract ]

Cationic lipids are conceptually and methodologically simple tools to deliver nucleic acids into the cells. Strategies based on cationic lipids are viable alternatives to viral vectors and are becoming increasingly popular owing to their minimal toxicity. The first-generation cationic lipids were built around the quaternary nitrogen primarily for binding and condensing DNA. A large number of lipids with variations in the hydrophobic and hydrophilic region were generated with excellent transfection efficiencies in vitro. These cationic lipids had reduced efficiencies when tested for gene delivery in vivo. Efforts in the last decade delineated the cell biological basis of the cationic lipid gene delivery to a significant detail. The application of techniques such as small angle X-ray spectroscopy (SAXS) and fluorescence microscopy, helped in linking the physical properties of lipid:DNA complex (lipoplex) with its intracellular fate. This biological knowledge has been incorporated in the design of the second-generation cationic lipids. Lipid-peptide conjugates (peptoids) are effective strategies to overcome the various cellular barriers along with the lipoplex formulations methodologies. In this context, cationic lipid-mediated gene delivery is considerably benefited by the methodologies of liposome-mediated drug delivery. Lipid mediated gene delivery has an intrinsic advantage of being a biomimetic platform on which considerable variations could be built to develop efficient in vivo gene delivery protocols.

Controlled release from a nanocarrier entrapped within a microcarrier.
Rojas EC, Sahiner N, Lawson LB, John VT, Papadopoulos KD.
J Colloid Interface Sci. 2006 Sep 15;301(2):617-23.
[ expand abstract ]

This study illustrates the entrapment of the dye molecule fluorescein sodium salt (FSS) by hydrogel nanoparticles, which are in turn confined inside a water-in-oil-in-water double-emulsion globule, and its subsequent release by the action of the competing agent hydrochloric acid (HCl). Thus, a "double carrier" concept is being introduced in which a nanoscale delivery vehicle is being transported by a microscale delivery vehicle in order to simultaneously take advantage of both systems. This may facilitate storage and handling while protecting the active substance and improving its action upon application.

PEG-modified gold nanorods with a stealth character for in vivo applications.
Niidome T, Yamagata M, Okamoto Y, Akiyama Y, Takahashi H, Kawano T, Katayama Y, Niidome Y.
J Control Release. 2006 Sep 12;114(3):343-7.
[ expand abstract ]

Gold nanorods prepared in hexadecyltrimethylammonium bromide (CTAB) solution are expected to provide novel materials for photothermal therapy and photo-controlled drug delivery systems. Since gold nanorods stabilized with CTAB show strong cytotoxicity, we developed a technique to modify these with polyethyleneglycol (PEG) for medical applications. PEG-modification was achieved by adding mPEG-SH in the CTAB solution, then, excess CTAB was removed by dialysis. PEG-modified gold nanoparticles showed a nearly neutral surface, and had little cytotoxicity in vitro. Following intravenous injection into mice, 54% of injected PEG-modified gold nanoparticles were found in blood at 0.5 h after intravenous injection, whereas most of gold was detected in the liver in the case of original gold nanorods stabilized with CTAB.

Heparin-deoxycholic acid chemical conjugate as an anticancer drug carrier and its antitumor activity.
Park K, Lee GY, Kim YS, Yu M, Park RW, Kim IS, Kim SY, Byun Y.
J Control Release. 2006 Sep 12;114(3):300-6.
[ expand abstract ]

A chemically modified heparin-DOCA (HD) conjugate was developed as a drug carrier for cancer therapy. HD conjugate was found to have markedly low anticoagulant activity and to form self-assembled nanoparticles in aqueous condition. We observed that HD conjugate prevented squamous cell carcinoma (SCC) and human umbilical vascular endothelial cell (HUVEC) proliferation during BrdU incorporation assays. Here, we prepared doxorubicin-loaded heparin nanoparticles by entrapping doxorubicin into the amphiphilic HD conjugate by physical interaction and characterized the properties of these nanoparticles using Dynamic Light Scattering (DLS) and Atomic Force Microscope (AFM). In this study, doxorubicin-loaded heparin nanoparticles were designed to improve the antitumor effects of nano-sized particles (range of 180 to 210 nm) at high drug-loading efficiencies in the range 64% to 96%. These doxorubicin-loaded heparin nanoparticles displayed sustained drug release patterns. It was confirmed in vivo toxicity studies that HD conjugate did not induce unexpected side effects and that DHN 20 was safer than free DOX. An in vivo study showed that HD conjugate, doxorubicin and DHN 20 (one of doxorubicin-loaded heparin nanoparticles) induced tumor volume reductions of 43%, 56% and 74%, respectively, relative to the saline treated control. These results suggest that the drug-entrapped with heparin nanoparticles might provide a novel therapy for SCC.

Cisplatin Nanoliposomes for Cancer Therapy: AFM and Fluorescence Imaging of Cisplatin Encapsulation, Stability, Cellular Uptake, and Toxicity.
Ramachandran S, Quist AP, Kumar S, Lal R.
Langmuir. 2006 Sep 12;22(19):8156-62.
[ expand abstract ]

Cisplatin is the most effective cytotoxic agent against many cancers. Its usage, however, is limited due to inefficient uptake by the target cells. A liposomal formulation of cisplatin is reported to partly overcome this limitation. Physicochemical characteristics of the liposome-cisplatin preparation, including its size, stability, encapsulation efficiency, and cytoplasmic internalization efficiency, play a significant role in an effective usage of liposomal formulations. We have used atomic force microscopy (AFM) to determine physicochemical characteristics of cisplatin-encapsulated liposomes, AFM and fluorescence microscopy to examine their cytoplasmic internalization, and Live/Dead assay to examine their cell toxicity. Nonencapsulated cisplatin is globular and 10-50 nm in size. AFM force-dissection and stiffness measurements show that cisplatin-encapsulated liposomes are significantly stiffer ( approximately 100%) and more stable than liposomes without encapsulated cisplatin. Cisplatin-encapsulated liposomes of approximately 250 nm diameter (nanoliposomes) are most efficiently internalized and induce cell toxicity in a time-dependent manner. Liposomes without cisplatin of similar dimensions, although internalized in the cell cytoplasm, do not induce cell toxicity.

A dual-ligand approach for enhancing targeting selectivity of therapeutic nanocarriers.
Saul JM, Annapragada AV, Bellamkonda RV.
J Control Release. 2006 Sep 12;114(3):277-287.
[ expand abstract ]

Conjugation of ligands to nano-scale drug carriers targeting over-expressed cell surface receptors is a promising approach for delivery of therapeutic agents to tumor cells. However, most commonly utilized ligands are directed at receptors expressed not only on target cells but also on other cells in the body, leading to unintended uptake in these off-target cells. In this study, a novel, dual-ligand approach is reported, which targets tumor cells while sparing off-target cells by exploiting the fact that tumor cells typically over-express multiple types of surface receptors. This approach was tested in the human KB cell line, which over-expresses both folate receptor (FR) and the epidermal growth factor receptor (EGFR). Liposomal nanocarriers loaded with doxorubicin and bearing controlled numbers of both folic acid and a monoclonal antibody against the EGFR were designed. Cytotoxicity was used to determine targeting selectivity of the designed carriers in vitro by utilizing KB cells expressing both FR and EGFR and off-target control cells in which one or both receptors were blocked. The data demonstrates that nanocarriers can be designed to achieve toxicity only when all targeted receptors are available, providing an approach to improve selectivity over current single-ligand approaches.

Macrophage Uptake of Core-Shell Nanoparticles Surface Modified with
Poly(ethylene glycol).
Zahr AS, Davis CA, Pishko MV.
Langmuir. 2006 Sep 12;22(19):8178-8185.
[ expand abstract ]

The in vitro uptake of core-shell nanoparticles encapsulated in a bio-macromolecular nanoshell assembled from multilayered polyelectrolytes was studied. Sulfate modified fluorescent polystyrene nanobeads (diameter 200 nm) were used as a solid core upon which charged multilayers of poly-l-lysine, chitosan, and heparin sulfate are electrostatically deposited utilizing a layer-by-layer (LbL) self-assembly process. The nanoshell composed of the multilayered polyelectrolytes was modified with poly(ethylene glycol) (PEG) of varying molecular weights (either MW 2000, 5000, or 20 000 Da) to form a hydrophilic and long-circulating nanoparticle. The assembly of the nanoshell was confirmed by zeta potential, transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS). The reversal in charge upon the deposition of alternating polyelectrolytes was observed by zeta potential measurements. The nanometer thickness of the nanoshell was confirmed by TEM. The presence of the (C-C-O)(n)() backbone in PEG at the surface of the nanoshell was confirmed by the increase in (C-O,N) peak area concentrations compared to (C-C) peak area, and these results were gathered from XPS. In vitro studies between suspension macrophages and core-shell nanoparticles were performed to determine how the hydrophilicity and the charge on the nanoshell can promote or reduce uptake. Results showed that after 24 h uptake was decreased 3-fold when PEGs of 2000 and 20 000 Da were chemisorbed to the nanoshell, as opposed to a nanoshell with either a positive or highly negative charge. Confocal microscopy aided in verifying that core-shell nanoparticles were internalized within the cell cytoplasm and were not attached to the cell surface. Protein adhesion studies with bovine serum albumin were performed to determine the relationship between surface charge and opsonization of core-shell nanoparticles. It was found that a hydrophilic surface with a low negative charge reduced protein adsorption and uptake. The in vitro uptake of macrophages and protein adsorption onto core-shell nanoparticles formed using layer-by-layer assembly has not been previously studied.

Conjugates of poly(DL-lactide-co-glycolide) on amino cyclodextrins and their nanoparticles as protein delivery system.
Gao H, Wang YN, Fan YG, Ma JB.
J Biomed Mater Res A. 2006 Sep 7; [Epub ahead of print] .
[ expand abstract ]

Poly(DL-lactide-co-glycolide) (PLG) was chemically conjugated on two amino cyclodextrins, mono(6-(2-aminoethyl)amino-6-deoxy)-beta-cyclodextrin and ethylenediamino bridged bis(beta-cyclodextrin), to afford novel amphiphilic conjugates. Those conjugates were then characterized with infrared spectrometry (IR), proton nuclear magnetic resonance ((1)H NMR) and gel permeation chromatography (GPC). A repeat-nanoprecipitation (RP-NP) method was also developed to fabricate the nanoparticles of the conjugates with a water-soluble model protein, bovine serum albumin (BSA). At the end of RP-NP process, the availability of BSA was over 80% while the entrapment efficiency was 40-50% for each nanoprecipitation. The nanoparticles were rigid and spherical with diameters of 110-180 nm determined by transmission electron microscope (TEM), atomic force microscopy (AFM) and particle size analyzer. Nanoparticles possessed good steric stability during freeze-drying and resuspensions due to the existence of cyclodextrins corona. Interactions between BSA and the conjugates in the nanoparticles were then elucidated with IR experiments. About 25% BSA adsorbed on the surface of nanoparticles due to the interaction and was easy to release in the first day. The release of BSA from the nanoparticles was in three phases: a burst effect in the first day, a followed plateau in about a week, and a sustained release of the protein over 14 days. By changing the lactide/glycolide ratio, the degradation time of the conjugates and the release rate of BSA could be controlled. The loss of CDs content was faster than that of overall Mw during degradation since CDs formed outer corona of the nanoparticles. Both the novel biomaterials and the nanosphere fabrication technique contributed to the maintenance of protein structure.

NK105, a paclitaxel-incorporating micellar nanoparticle, is a more potent radiosensitising agent compared to free paclitaxel.
Negishi T, Koizumi F, Uchino H, Kuroda J, Kawaguchi T, Naito S, Matsumura Y.
Br J Cancer. 2006 Sep 4;95(5):601-6.
[ expand abstract ]

NK105 is a micellar nanoparticle formulation designed to enhance the delivery of paclitaxel (PTX) to solid tumours. It has been reported to exert antitumour activity in vivo and to have reduced neurotoxicity as compared to that of free PTX. The purpose of this study was to investigate the radiosensitising effect of NK105 in comparison with that of PTX. Lewis lung carcinoma (LLC)-bearing mice were administered a single intravenous (i.v.) injection of PTX or NK105; 24 h after the drug administration, a proportion of the mice received radiation to the tumour site or lung fields. Then, the antitumour activity and lung toxicity were evaluated. In one subset of mice, the tumours were excised and specimens were prepared for analysis of the cell cycle distribution by flow cytometry. Combined NK105 treatment with radiation yielded significant superior antitumour activity as compared to combined PTX treatment with radiation (P=0.0277). On the other hand, a histopathological study of lung sections revealed no significant difference in histopathological changes between mice treated with PTX and radiation and those treated with NK105 and radiation. Flow-cytometric analysis showed that NK105-treated LLC tumour cells showed more severe arrest at the G2/M phase as compared to PTX-treated tumour cells. The superior radiosensitising activity of NK105 was thus considered to be attributable to the more severe cell cycle arrest at the G2/M phase induced by NK105 as compared to that induced by free PTX. The present study results suggest that further clinical trials are warranted to determine the efficacy and feasibility of combined NK105 therapy with radiation.

Biodegradable nanoparticles of amphiphilic triblock copolymers based on
poly(3-hydroxybutyrate) and poly(ethylene glycol) as drug carriers.
Chen C, Yu CH, Cheng YC, Yu PH, Cheung MK.
Biomaterials. 2006 Sep;27(27):4804-14.
[ expand abstract ]

New amorphous amphiphilic triblock copolymers of poly(3-hydroxybutyrate)-poly(ethylene glycol)-poly(3-hydroxybutyrate) (PHB-PEG-PHB) were synthesized using the ring-opening copolymerization of beta-butyrolactone monomer. They were characterized by fluorescence, SEM and (1)H NMR. These triblock copolymers can form biodegradable nanoparticles with core-shell structure in aqueous solution. Comparing to the poly(ethylene oxide)-PHB-poly(ethylene oxide) (PEO-PHB-PEO) copolymers, these nanoparticles exhibited much smaller critical micelle concentrations and better drug loading properties, which indicated that the nanoparticles were very suitable for delivery carriers of hydrophobic drugs. The drug release profile monitored by fluorescence showed that the release of pyrene from the PHB-PEG-PHB nanoparticles exhibited the second-order exponential decay behavior. The initial biodegradation rate of the PHB-PEG-PHB nanoparticles was related to the enzyme amount, the initial concentrations of nanoparticle dispersions and the PHB block length. The biodegraded products detected by (1)H NMR contained 3HB monomer, dimer and minor trimer, which were safe to the body.

Nanoparticulate drug carriers based on hybrid poly(d,l-lactide-co-glycolide)-dendron structures.
Costantino L, Gandolfi F, Bossy-Nobs L, Tosi G, Gurny R, Rivasi F, Angela Vandelli M, Forni F.
Biomaterials. 2006 Sep;27(26):4635-45; Epub 2006 May 22.
[ expand abstract ]

We describe a general method for incorporating target moieties in a well-defined arrangement into the surface of biocompatible polyester poly(d,l-lactic-co-glycolic acid) (PLGA) materials using dendrons. In this way it is possible to obtain nanoparticles (NPs) with a high degree of surface coverage. This new strategy was successfully applied to the preparation of peptide- and beta-d-glucose-covered NPs. The first application is based on the discovery of NPs made of conjugates between PLGA and short peptidic sequences able to cross the blood-brain barrier (BBB) after systemic administration. In this paper, we used a branched structure (dendron) in order to prepare a derivative of PLGA able to form, by simple nanoprecipitation, NPs with a higher degree of surface coverage than previously reported by us, characteristic that could influence the uptake by the liver and spleen. The NPs thus obtained retain the ability to cross the BBB and possess a core-shell structure, as evidenced from zeta-potential, X-ray photoelectron (ESCA) spectroscopy and elemental analyses. These results are comparable with the NPs obtained by the derivatization of preformed NPs. The same strategy, namely the use of a branched spacer (a dendron or a G1 dendrimer) inserted between one end of the PLGA chain and a derivatizing molecule, was also successfully applied to obtain beta-d-glucose-covered NPs; in this case, the surface analysis of the NPs was performed by using high resolution magic angle spinning (HRMAS) NMR spectroscopy and zeta-potential measurements.

The pinpoint promise of nanoparticle-based drug delivery and molecular diagnosis.
Emerich DF, Thanos CG.
Biomol Eng. 2006 Sep;23(4):171-84.
[ expand abstract ]

Nanotechnology, or systems/device manufacture at the molecular level, is a multidisciplinary scientific field undergoing explosive development. The genesis of nanotechnology can be traced to the promise of revolutionary advances across medicine, communications, genomics and robotics. Without doubt one of the greatest values of nanotechnology will be in the development of new and effective medical treatments (i.e., nanomedicine). This review focuses on the potential of nanomedicine as it specifically relates to (1) the development of nanoparticles for enabling and improving the targeted delivery of therapeutic agents; (2) developing novel and more effective diagnostic and screening techniques to extend the limits of molecular diagnostics providing point-of-care diagnosis and more personalized medicine.

Liposome formulation of a novel hydrophobic aryl-imidazole compound for anti-cancer therapy.
Liu J, Lee H, Huesca M, Young A, Allen C.
Cancer Chemother Pharmacol. 2006 Sep;58(3):306-18.
[ expand abstract ]

PURPOSE: A cholesterol-free liposome formulation formed from mixtures of egg phosphatidylcholine (ePC) and poly (ethylene glycol) conjugated distearoylphosphatidylethanolamine (DSPE-PEG 2000) was optimized and evaluated for delivery of a novel anti-cancer agent ML220 (2-(5-bromo-1H-indol-3-yl)-1H-phenanthro [9,10-d] imidazole). RESULTS AND DISCUSSION: ML220 is highly lipophilic with a water solubility of 0.14 mug/ml and calculated log P of 5.69. The ML220-loaded liposomes had a unimodal size-distribution and a mean diameter of 89 nm. The drug to lipid ratio in the formulation was 1:3.5 (mol:mol) and the drug loading efficiency was 83% providing a more than 50,000-fold increase in the water solubility of ML220. The formulation was demonstrated to be stable in vitro at 37 degrees C for over 2 weeks with a delayed drug release profile. Evaluation of the subacute toxicity of the liposome formulated drug in C3H mice revealed no overt signs of toxicity. Also, a biexponential drug plasma concentration pattern was found upon evaluation of the pharmacokinetics in Balb/C mice. The in vivo evaluation of the anti-cancer activity in a human colon HT29 carcinoma model revealed a significant delay in tumor growth. CONCLUSION: Overall, the ePC/DSPE-PEG liposomes were demonstrated to be a suitable delivery system for ML220. These studies also highlight the potential of cholesterol-free liposomes as a formulation strategy for highly lipophilic drugs.

Enhancement of anticancer activity in antineovascular therapy is based on the intratumoral distribution of the active targeting carrier for anticancer drugs.
Maeda N, Miyazawa S, Shimizu K, Asai T, Yonezawa S, Kitazawa S, Namba Y, Tsukada H, Oku N.
Biol Pharm Bull. 2006 Sep;29(9):1936-40.
[ expand abstract ]

We previously observed the enhanced anticancer efficacy of anticancer drugs encapsulated in Ala-Pro-Arg-Pro-Gly-polyethyleneglycol-modified liposome (APRPG-PEG-Lip) in tumor-bearing mice, since APRPG peptide was used as an active targeting tool to angiogenic endothelium. This modality, antineovascular therapy (ANET), aims to eradicate tumor cells indirectly through damaging angiogenic vessels. In the present study, we examined the in vivo trafficking of APRPG-PEG-Lip labeled with [2-(18)F]2-fluoro-2-deoxy-D-glucose ([2-(18)F]FDG) by use of positron emission tomography (PET), and observed that the trafficking of this liposome was quite similar to that of non-targeted long-circulating liposome (PEG-Lip). Then, histochemical analysis of intratumoral distribution of both liposomes was performed by use of fluorescence-labeled liposomes. In contrast to in vivo trafficking, intratumoral distribution of both types of liposomes was quite different: APRPG-PEG-Lip was colocalized with angiogenic endothelial cells that were immunohistochemically stained for CD31, although PEG-Lip was localized around the angiogenic vessels. These results strongly suggest that intratumoral distribution of drug carrier is much more important for therapeutic efficacy than the total accumulation of the anticancer drug in the tumor, and that active delivery of anticancer drugs to angiogenic vessels is useful for cancer treatment.

Nanocarriers: promising vehicle for bioactive drugs.
Rawat M, Singh D, Saraf S, Saraf S.
Biol Pharm Bull. 2006 Sep;29(9):1790-8.
[ expand abstract ]

Development of new delivery systems that deliver the potential drug specifically to the target site in order to meet the therapeutic needs of the patients at the required time and level remains the key challenge in the field of pharmaceutical biotechnology. Developments in this context to achieve desired goal has led to the evolution of the multidisciplinary field nanobiotechnology which involves the combination of two most promising technologies of 21st century--biotechnology and nanotechnology. Nanobiotechnology encompasses a wide array of different techniques to improve the delivery of biotech drugs, and nanoparticles offer the most suitable form whose properties can be tailored by chemical methods. This review highlights the different types of nanoparticulate delivery systems employed for biotech drugs in the field of molecular medicine with a short overlook at its applications and the probable associated drawbacks.

Nanoparticles of 5-fluorouracil (5-FU) loaded N-succinyl-chitosan (Suc-Chi) for cancer chemotherapy: preparation, characterization--in-vitro drug release and anti-tumour activity.
Yan C, Chen D, Gu J, Qin J.
J Pharm Pharmacol. 2006 Sep;58(9):1177-81.
[ expand abstract ]

N-Succinyl-chitosan has favourable properties as a drug carrier, such as biocompatibility, low toxicity and long-term retention in the body. It is a good candidate for cancer chemotherapy as a polymeric drug carrier. This study describes the preparation and characterization of 5-fluorouracil-loaded N-succinyl-chitosan nanoparticles (5-FU-Suc-Chi/NP) by an emulsification solvent diffusion method. The influence of the initial 5-FU concentration on the nanoparticle characteristics and release behaviour in phosphate-buffered saline solution (PBS) was evaluated. The Suc-Chi nanoparticles had a particle diameter (Z-average) in the range 202 approximately 273 nm and a negative zeta-potential (approx. -27 to -18 mV). The formulation with an initial 5-FU concentration of 1000 microg mL-1 provided the highest loading capacity (19%) and the highest extent of release (61% at 24 h). The 5-FU-Suc-Chi/NP showed good anti-tumour activity against Sarcoma 180 solid tumour and mild toxicity. According to the data obtained, this Suc-Chi-based nanotechnology opens new and interesting perspectives for cancer chemotherapy.

Susceptibility of nanoparticle-encapsulated paclitaxel to P-glycoprotein-mediated drug efflux.
Chavanpatil MD, Patil Y, Panyam J.
Int J Pharm. 2006 Aug 31;320(1-2):150-6.
[ expand abstract ]

Overexpression of P-glycoprotein (P-gp) is a key factor contributing to the development of multidrug resistance (MDR) in cancer cells. The objective of the study is to investigate whether a P-gp substrate, paclitaxel, delivered to MDR tumor cells in poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles is susceptible to P-gp - mediated drug efflux. Paclitaxel-loaded nanoparticles were formulated by emulsion-solvent evaporation technique. Nanoparticles had a mean hydrodynamic diameter of about 195nm, and demonstrated sustained release of paclitaxel. In vitro cell culture studies indicated that paclitaxel nanoparticles result in sustained, dose-dependent and significant cytotoxicity in drug-sensitive MCF-7 tumor cells but not in drug-resistant NCI-ADR/RES cells. Resistance to nanoparticle-encapsulated paclitaxel was reversed by verapamil, a P-gp inhibitor. Further, sustained inhibition of P-gp was necessary for sustaining the cytotoxicity of nanoparticle-encapsulated paclitaxel in drug-resistant cells. Inhibition of P-gp by verapamil did not significantly affect the uptake or retention of nanoparticles in drug-resistant cells. In conclusion, our studies suggest that P-gp substrates, such as paclitaxel, delivered to MDR cells by PLGA nanoparticles, are susceptible to efflux by P-gp. Inhibition of P-gp restores sensitivity to paclitaxel; however, sustained inhibition of P-gp is required for sustained therapeutic efficacy of nanoparticle-encapsulated drug.

Template synthesis of multifunctional nanotubes for controlled release.
Son SJ, Bai X, Nan A, Ghandehari H, Lee SB.
J Control Release. 2006 Aug 28;114(2):143-52.
[ expand abstract ]

In the past few decades, nanoscale materials have been widely used for controlled release applications. Importantly, many researches have focused on multifunctional nanoparticles for targeted delivery of bioactive and imaging agents as therapeutics and diagnostics. Recent advances in nanotechnology have made possible the design and development of tubular nanoscale particles called nanotubes. The tubular shape of such particles is highly attractive since it is possible to differentially functionalize the inner and outer surfaces to facilitate drug loading, biocompatibility and biorecognition. Novel synthetic strategies allow the fabrication of tubular structures with well-defined diameters and lengths. This can have important implications in biodistribution, subcellular trafficking and drug release. In this article the biomedical applications of nanotubes will be discussed with emphasis on the template synthesis of composite nanotubes containing silica and iron oxide that have potential use in drug delivery, magnetic resonance imaging (MRI), and chemical and biochemical separations.

Multicentre phase II pharmacokinetic and pharmacodynamic study of OSI-7904L in previously untreated patients with advanced gastric or gastroesophageal junction adenocarcinoma.
Falk S, Anthoney A, Eatock M, Van Cutsem E, Chick J, Glen H, Valle JW, Drolet DW, Albert D, Ferry D, Ajani J.
Br J Cancer. 2006 Aug 21;95(4):450-6.
[ expand abstract ]

A two-stage Simon design was used to evaluate the response rate of OSI-7904L, a liposome encapsulated thymidylate synthase inhibitor, in advanced gastric and/or gastroesophageal adenocarcinoma (A-G/GEJA), administered intravenously at 12 mg m(-2) over 30 min every 21 days. Fifty patients were treated. Median age was 64 years (range 35-82), 62% were male and 89% had ECOG PS of 0/1. A total of 252 cycles were administered; median of 4 per patient (range 1-21). Twelve patients required dose reductions, mainly for skin toxicity. Investigator assessed response rate was 17.4% (95% CI 7.8-31.4) with one complete and seven partial responses in 46 evaluable patients. Twenty-one patients (42%) had stable disease. Median time to progression and survival were 12.4 and 36.9 weeks, respectively. NCI CTCAE Grade 3/4 neutropenia (14%) and thrombocytopenia (4%) were uncommon. The main G3/4 nonhaematological toxicities were skin-related 22%, stomatitis 14%, fatigue/lethargy 10%, and diarrhea 8%. Pharmacokinetic data showed high interpatient variability. Patients with higher AUC were more likely to experience G3/4 toxicity during cycle 1 while baseline homocysteine did not predict toxicity. Response did not correlate with AUC. Elevations in 2'-dU were observed indicating target inhibition. Analysis of TS genotype, TS protein and expression did not reveal any correlation with outcome. OSI-7904L has activity in A-G/GEJA similar to other active agents and an acceptable safety profile.

Antigen Chemically Coupled to the Surface of Liposomes Are Cross-Presented to CD8+ T Cells and Induce Potent Antitumor Immunity.
Taneichi M, Ishida H, Kajino K, Ogasawara K, Tanaka Y, Kasai M, Mori M, Nishida M, Yamamura H, Mizuguchi J, Uchida T.
J Immunol. 2006 Aug 15;177(4):2324-30.
[ expand abstract ]

We have previously demonstrated that liposomes with differential lipid components display differential adjuvant effects when Ags are chemically coupled to their surfaces. In the present study, Ag presentation of liposome-coupled OVA was investigated in vitro, and it was found that OVA coupled to liposomes made using unsaturated fatty acid was presented to both CD4(+) and CD8(+) T cells, whereas OVA coupled to liposomes made using saturated fatty acid was presented only to CD4(+) T cells. Confocal laser scanning microscopic analysis demonstrated that a portion of the OVA coupled to liposomes made using unsaturated, but not saturated fatty acid, received processing beyond the MHC class II compartment, suggesting that the degradation of OVA might occur in the cytosol, and that the peptides generated in this manner would be presented to CD8(+) T cells via MHC class I. The ability to induce cross-presentation of an Ag coupled to liposomes consisting of unsaturated fatty acid was further confirmed by in vivo induction of CTL and by the induction of tumor eradication in mice; E.G7 tumors in mice that received combined inoculation with OVA(257-264)-liposome conjugates, CpG, and anti-IL-10 mAbs were completely eradicated. In those mice, the frequency of CD8(+) T cells reactive with OVA(257-264) peptides in the context of H-2K(b) was significantly increased. These results suggested that, by choosing lipid components for liposomes, surface-coupled liposomal Ags might be applicable for the development of tumor vaccines to present tumor Ags to APCs and induce antitumor responses.

In vivo antitumor activity of chitosan nanoparticles.
Qi L, Xu Z.
Bioorg Med Chem Lett. 2006 Aug 15;16(16):4243-5.
[ expand abstract ]

Chitosan nanoparticles have been synthesized as potential anticancer agents, and evaluated, in vitro, against various cancer cell lines. In this study, in vivo antitumor activity of chitosan nanoparticles against Sarcoma-180 and mouse hepatoma H22 was investigated. Chitosan nanoparticles showed significant antitumor activity in vivo. The doses and particle size made a great effect on their efficacy.

Studies on the oridonin-loaded poly(d,l-lactic acid) nanoparticles in vitro and in vivo.
Xing J, Zhang D, Tan T.
Int J Biol Macromol. 2006 Aug 7; [Epub ahead of print] .
[ expand abstract ]

The purpose of this paper was to investigate the possibility of developing a polymeric nanoparticle delivery system for ORI to increase its solubility, blood circulation time and tissue targeting. Oridonin-loaded poly(d,l-lactic acid) nanoparticles (ORI-PLA-NP) were prepared by the further modified spontaneous emulsion solvent diffusion (MSESD) method. Studies were carried out to characterize and evaluate the produced ORI-PLA-NP both in vitro and in vivo. The experimental results showed that the mean size of the nanoparticles were 137.3nm, with 87.2% of the nanoparticles distributed between the range of 107 and 195nm. The entrapment efficiency and actual drug loading of the nanoparticles were 91.88+/-1.83 and 2.32+/-0.05%, respectively. It was demonstrated by differential scanning calorimetry (DSC) and X-ray diffractometry (XRD) that ORI existed in the form of amorphous in the nanoparticles. The in vitro release profile of ORI-PLA-NP could be expressed well by the Higuchi equation: Q=8.944t(1/2)+11.246. The results of pharmacokinetics demonstrated that being encapsulated in PLA nanoparticles was remarkably effective for ORI to prolong its blood circulation time. After the i.v. administration of ORI-PLA-NP, we could observe a stable and high concentration of ORI in liver, lung and spleen, while its distribution in heart and kidney decreased.

Clodronate-liposome-mediated depletion of tumour-associated macrophages: a new and highly effective antiangiogenic therapy approach.
Zeisberger SM, Odermatt B, Marty C, Zehnder-Fjallman AH, Ballmer-Hofer K, Schwendener RA.
Br J Cancer. 2006 Aug 7;95(3):272-81.
[ expand abstract ]

Tumour-associated macrophages, TAMs, play a pivotal role in tumour growth and metastasis by promoting tumour angiogenesis. Treatment with clodronate encapsulated in liposomes (clodrolip) efficiently depleted these phagocytic cells in the murine F9 teratocarcinoma and human A673 rhabdomyosarcoma mouse tumour models resulting in significant inhibition of tumour growth ranging from 75 to >92%, depending on therapy and schedule. Tumour inhibition was accompanied by a drastic reduction in blood vessel density in the tumour tissue. Vascular endothelial growth factor (VEGF) is one of the major inducers of tumour angiogenesis and is also required for macrophage recruitment. The strongest effects were observed with the combination therapy of clodrolip and a VEGF-neutralising antibody, whereas free clodronate was not significantly active. Immunohistologic evaluation of the tumours showed significant depletion of F4/80(+) and MOMA-1(+) and a less pronounced depletion of CD11b(+) TAMs. Blood vessel staining (CD31) and quantification of the vessels as well as TAMs and tumour-associated dendritic cells (TADCs) in the A673 model showed reduction rates of 85 to >94%, even 9 days after the end of therapy. In addition, CD11c(+) TADCs, which have been shown to potentially differentiate into endothelial-like cells upon stimulation by tumour released growth and differentiation factors, were similarly reduced by clodrolip or antibody treatment. These results validate clodrolip therapy in combination with angiogenesis inhibitors as a promising novel strategy for an indirect cancer therapy aimed at the haematopoietic precursor cells that stimulate tumour growth and dissemination and as a tool to study the role of macrophages and dendritic cells in tumorigenesis.

Crossing cellular barriers using dendrimer nanotechnologies.
Najlah M, D'Emanuele A.
Curr Opin Pharmacol. 2006 Aug 2; [Epub ahead of print] .
[ expand abstract ]

Dendrimers represent a class of polymers characterised by their well-defined structure, with a high degree of molecular uniformity and low polydispersity. They have found a wide-range of pharmaceutical applications; however, more recently, they have been shown to function as effective intracellular carriers for drugs. In addition, dendrimers have been shown to be capable of bypassing efflux transporters. A new generation of dendrimer-based delivery systems will enable the efficient transport of drugs across cellular barriers.

Parameters influencing the stealthiness of colloidal drug delivery systems.
Vonarbourg A, Passirani C, Saulnier P, Benoit JP.
Biomaterials. 2006 Aug;27(24):4356-73.
[ expand abstract ]

Over the last few decades, colloidal drug delivery systems (CDDS) such as nano-structures have been developed in order to improve the efficiency and the specificity of drug action. Their small size permits them to be injected intravenously in order to reach target tissues. However, it is known that they can be rapidly removed from blood circulation by the immune system. CDDS are removed via the complement system and via the cells of the mononuclear phagocyte system (MPS), after their recognition by opsonins and/or receptors present at the cell surface. This recognition is dependent on the physicochemical characteristics of the CDDS. In this study, we will focus on parameters influencing the interactions of opsonins and the macrophage plasma membrane with the surface of CDDS, whereby parameters of the polymer coating become necessary to provide good protection.

Intracellular photodynamic therapy with photosensitizer-nanoparticle conjugates: cancer therapy using a 'Trojan horse'.
Wieder ME, Hone DC, Cook MJ, Handsley MM, Gavrilovic J, Russell DA.
Photochem Photobiol Sci. 2006 Aug;5(8):727-34.
[ expand abstract ]

Phthalocyanine-nanoparticle conjugates have been designed and synthesised for the delivery of hydrophobic photosensitizers for photodynamic therapy (PDT) of cancer. The phthalocyanine photosensitizer stabilized gold nanoparticles have an average diameter of 2-4 nm. The synthetic strategy interdigitates a phase transfer reagent between phthalocyanine molecules on the particle surface that solubilises the hydrophobic photosensitizer in polar solvents enabling delivery of the nanoparticle conjugates to cells. The phthalocyanine is present in the monomeric form on the nanoparticle surface, absorbs radiation maximally at 695 nm and catalytically produces the cytotoxic species singlet oxygen with high efficiency. These properties suggest that the phthalocyanine-nanoparticle conjugates are ideally suited for PDT. In a process that can be considered as cancer therapy using a 'Trojan horse', when the nanoparticle conjugates are incubated with HeLa cells (a cervical cancer cell line), they are taken up thus delivering the phthalocyanine photosensitizer directly into the cell interior. Irradiation of the nanoparticle conjugates within the HeLa cells induced substantial cell mortality through the photodynamic production of singlet oxygen. The PDT efficiency of the nanoparticle conjugates, determined using colorimetric assay, was twice that obtained using the free phthalocyanine derivative. Following PDT with the nanoparticle conjugates, morphological changes to the HeLa cellular structure were indicative of cell mortality via apoptosis. Further evidence of apoptosis was provided through the bioluminescent assay detection of caspase 3/7. Our results suggest that gold nanoparticle conjugates are an excellent vehicle for the delivery of surface bound hydrophobic photosensitizers for efficacious photodynamic therapy of cultured tumour cells.

Emerging use of nanoparticles in diagnosis and treatment of breast cancer.
Yezhelyev MV, Gao X, Xing Y, Al-Hajj A, Nie S, O'Regan RM.
Lancet Oncol. 2006 Aug;7(8):657-67.
[ expand abstract ]

The biological application of nanoparticles is a rapidly developing area of nanotechnology that raises new possibilities in the diagnosis and treatment of human cancers. In cancer diagnostics, fluorescent nanoparticles can be used for multiplex simultaneous profiling of tumour biomarkers and for detection of multiple genes and matrix RNA with fluorescent in-situ hybridisation. In breast cancer, three crucial biomarkers can be detected and accurately quantified in single tumour sections by use of nanoparticles conjugated to antibodies. In the near future, the use of conjugated nanoparticles will allow at least ten cancer-related proteins to be detected on tiny tumour sections, providing a new method of analysing the proteome of an individual tumour. Supermagnetic nanoparticles have exciting possibilities as contrast agents for cancer detection in vivo, and for monitoring the response to treatment. Several chemotherapy agents are available as nanoparticle formulations, and have at least equivalent efficacy and fewer toxic effects compared with conventional formulations. Ultimately, the use of nanoparticles will allow simultaneous tumour targeting and drug delivery in a unique manner. In this review, we give an overview of the use of clinically applicable nanoparticles in oncology, with particular focus on the diagnosis and treatment of breast cancer.

Fullerene (C60) immunoconjugates: interaction of water-soluble C60 derivatives with the murine anti-gp240 melanoma antibody.
Ashcroft JM, Tsyboulski DA, Hartman KB, Zakharian TY, Marks JW, Weisman RB, Rosenblum MG, Wilson LJ.
Chem Commun (Camb). 2006 Jul 28;(28):3004-6.
[ expand abstract ]

The first fullerene (C60) immunoconjugates have been prepared and characterized as an initial step toward the development of fullerene immunotherapy (FIT).

Method of laser activated nano-thermolysis for elimination of tumor cells.
Lapotko D, Lukianova E, Potapnev M, Aleinikova O, Oraevsky A.
Cancer Lett. 2006 Jul 28;239(1):36-45.
[ expand abstract ]

We describe novel ex vivo method for elimination of tumor cells from cell suspension, Laser Activated Nanothermolysis and propose this method for purging of bone marrow and blood transplants. K562 and human lympholeukemia cells were eliminated in experiments by laser-induced micro-bubbles that emerge inside individual target cells around selectively formed clusters of light-absorbing gold nanoparticles. Pretreatment of tumor cells with specific monoclonal antibodies and Ig-conjugated 30-nm gold particles allowed the formation of clusters of 10-20 on the surface of cell membrane. Electron microscopy found the nanoparticulate clusters inside the cells. Total (100%) elimination of K562 cells targeted with specific antibodies was achieved with single laser pulses with optical fluence of 5J/cm(2) at the wavelength of 532 nm without damage to the same cells targeted without specific antibodies. Total elimination of human lymphoblasts from suspension of normal stem cells was achieved by a single laser pulse with the optical fluence of 1.7J/cm(2), while the damage level of normal cells was 16%.

Synthesis of branched antisense oligonucleotides having multiple specificities Treatment of hormone insensitive prostate cancer.
Rubenstein M, Anderson KM, Tsui P, Guinan P.
Med Hypotheses. 2006 Jul 24; [Epub ahead of print] .
[ expand abstract ]

Antisense oligonucleotides (oligos) directed against transforming growth factor-alpha (TGF-alpha) and its binding site, the epidermal growth factor receptor (EGFR), have demonstrated in vitro and in vivo efficacy against both the PC-3 and LNCaP prostate tumor models. In an attempt to increase the efficiency of these oligos a new type of antisense compound called a bispecific oligo has been evaluated in vitro both alone and in combination with traditional chemotherapeutic agents. These bispecifics, which were first proposed in this journal in 2004, include binding sites for both TGF-alpha and EGFR along the same stretch of complementary DNA. Such bispecifics are able to deliver essentially two antisense activities in an equal molar ratio and can be directed against mRNA encoding proteins of different biochemical pathways. The first bispecifics were developed against two proteins regulating a single autocrine loop. Subsequent bispecifics have been developed which target both EGFR and the apoptosis regulating protein bcl-2. Bispecific activity of a single linear sequence oligo has already been shown to have efficacy. To further develop this multispecific approach, we now propose a branched antisense compound, again, having multiple binding site activities (to complementary sequenced mRNA). Active oligos would be attached to a fat soluble backbone which might enhance targeting and also intracellular entry, release and activity. Such a structure would also permit the customization of these branched forms to include oligos targeting specific proteins related to the growth of various tumor types. Problems associated with the development of antisense oligos have included both membrane solubility and specific targeting. By designing this branched form of antisense structure, multiple activities can be retained (added), solubility improved and delivery enhanced. Such a new formulation would include several antisense oligos covalently bound to and branching off from a lipid-like backbone. An elongated hydrocarbon chain would increase fat solubility and would permit oligo incorporation into nanoparticles or liposome derived delivery vehicles. Specific delivery of oligos could also be enhanced by the tendency of these nanoparticle or liposomal microbubbles to be disrupted under the influence of ultrasonic waves beamed at the targeted tissue.

Poly(ethylene oxide)-modified poly(beta-amino ester) nanoparticles as a pH-sensitive system for tumor-targeted delivery of hydrophobic drugs: part 3. Therapeutic efficacy and safety studies in ovarian cancer xenograft model.
Devalapally H, Shenoy D, Little S, Langer R, Amiji M.
Cancer Chemother Pharmacol. 2006 Jul 22; [Epub ahead of print].
[ expand abstract ]

PURPOSE: The objective of this study was to evaluate the anti-tumor efficacy and lack of systemic toxicity of paclitaxel when administered in pH-sensitive poly(ethylene oxide) (PEO)-modified poly(beta-amino ester) (PbAE) nanoparticles in mice bearing human ovarian adenocarcinoma (SKOV-3) xenograft. METHODS: Paclitaxel-encapsulated PEO-modified PbAE (PEO-PbAE) nanoparticles were prepared by the solvent displacement method. PEO-modified poly(epsilon-caprolactone) (PCL) (PEO-PCL) nanoparticles were used as a non pH-responsive control formulation. Efficacy studies were conducted in SKOV-3 tumor-bearing athymic (Nu/Nu) mice at an equivalent paclitaxel dose of 20 mg/kg with the control and nanoparticle formulations. Safety of the drug when administered in the control and nanoparticle formulation was determined from blood cell counts and changes in body weight of the animals. RESULTS: The formulated paclitaxel-containing PEO-PbAE and PEO-PCL nanoparticles had a particle size in the range of 100-200 nm and a surface charge of + 39.0 and - 30.8 mV, respectively. After intravenous administration of paclitaxel in these formulations, the tumor growth was inhibited significantly. Both of the formulated nanoparticles tested have shown improved therapeutic efficacy as compared to the paclitaxel aqueous solution. Additionally, significantly lower toxicity profile of paclitaxel was observed with PEO-modified nanoparticles as compared to the aqueous solution formulation CONCLUSION: PEO-modified PbAE nanoparticles are a unique pH-sensitive drug delivery system that elicits enhanced efficacy and safety profile in solid tumor therapy.

Clodronate-liposome-mediated depletion of tumour-associated macrophages: a new and highly effective antiangiogenic therapy approach.
Zeisberger SM, Odermatt B, Marty C, Zehnder-Fjallman AH, Ballmer-Hofer K, Schwendener RA.
Br J Cancer. 2006 Jul 11; [Epub ahead of print] .
[ expand abstract ]

Tumour-associated macrophages, TAMs, play a pivotal role in tumour growth and metastasis by promoting tumour angiogenesis. Treatment with clodronate encapsulated in liposomes (clodrolip) efficiently depleted these phagocytic cells in the murine F9 teratocarcinoma and human A673 rhabdomyosarcoma mouse tumour models resulting in significant inhibition of tumour growth ranging from 75 to >92%, depending on therapy and schedule. Tumour inhibition was accompanied by a drastic reduction in blood vessel density in the tumour tissue. Vascular endothelial growth factor (VEGF) is one of the major inducers of tumour angiogenesis and is also required for macrophage recruitment. The strongest effects were observed with the combination therapy of clodrolip and a VEGF-neutralising antibody, whereas free clodronate was not significantly active. Immunohistologic evaluation of the tumours showed significant depletion of F4/80(+) and MOMA-1(+) and a less pronounced depletion of CD11b(+) TAMs. Blood vessel staining (CD31) and quantification of the vessels as well as TAMs and tumour-associated dendritic cells (TADCs) in the A673 model showed reduction rates of 85 to >94%, even 9 days after the end of therapy. In addition, CD11c(+) TADCs, which have been shown to potentially differentiate into endothelial-like cells upon stimulation by tumour released growth and differentiation factors, were similarly reduced by clodrolip or antibody treatment. These results validate clodrolip therapy in combination with angiogenesis inhibitors as a promising novel strategy for an indirect cancer therapy aimed at the haematopoietic precursor cells that stimulate tumour growth and dissemination and as a tool to study the role of macrophages and dendritic cells in tumorigenesis.

RNA Interference in Vitro and in Vivo Using a Novel Chitosan/siRNA Nanoparticle System.
Howard KA, Rahbek UL, Liu X, Damgaard CK, Glud SZ, Andersen MO, Hovgaard MB, Schmitz A, Nyengaard JR, Besenbacher F, Kjems J.
Mol Ther. 2006 Jul 7; [Epub ahead of print] .
[ expand abstract ]

This work introduces a novel chitosan-based siRNA nanoparticle delivery system for RNA interference in vitro and in vivo. The formation of interpolyelectrolyte complexes between siRNA duplexes (21-mers) and chitosan polymer into nanoparticles, ranging from 40 to 600 nm, was shown using atomic force microscopy and photon correlation spectroscopy. Rapid uptake (1 h) of Cy5-labeled nanoparticles into NIH 3T3 cells, followed by accumulation over a 24-h period, was visualized using fluorescence microscopy. Nanoparticle-mediated knockdown of endogenous enhanced green fluorescent protein (EGFP) was demonstrated in both H1299 human lung carcinoma cells and murine peritoneal macrophages (77.9% and 89.3% reduction in EGFP fluorescence, respectively). In addition, Western analysis showed approximately 90% reduced expression of BCR/ABL-1 leukemia fusion protein while BCR expression was unaffected in K562 (Ph(+)) cells after transfection using nanoparticles containing siRNA specific to the BCR/ABL-1 junction sequence. Effective in vivo RNA interference was achieved in bronchiole epithelial cells of transgenic EGFP mice after nasal administration of chitosan/siRNA formulations (37% and 43% reduction compared to mismatch and untreated control, respectively). These findings highlight the potential application of this novel chitosan-based system in RNA-mediated therapy of systemic and mucosal disease.

pH-sensing nano-crystals of carbonate apatite: Effects on intracellular delivery and release of DNA for efficient expression into mammalian cells.
Chowdhury EH, Maruyama A, Kano A, Nagaoka M, Kotaka M, Hirose S, Kunou M, Akaike T.
Gene. 2006 Jul 5;376(1):87-94; Epub 2006 Apr 5.
[ expand abstract ]

Two unique and fascinating properties of carbonate apatite which are well-known in hard tissue engineering, have been unveiled, for the first time, for the development of the simplest, but most efficient non-viral gene delivery device - ability of preventing the growth of crystals needed for high frequency DNA transfer across a plasma membrane and a fast dissolution rate for effective release of DNA during endosomal acidification, leading to a remarkably high transgene expression (5 to 100-fold) in mammalian cells compared to the widely used transfecting agents. Moreover, by modulating the crystal dissolution rate of carbonate apatite through incorporation of fluoride or strontium into it, transfection activity could be dramatically controlled, thus shedding light on a new barrier in the non-viral route, which was overlooked so far. Thus we have developed an innovative technology with significant insights, that would come as a promising tool for both basic research laboratories and clinical settings.

Phase I trial of Doxorubicin-containing low temperature sensitive liposomes in spontaneous canine tumors.
Hauck ML, Larue SM, Petros WP, Poulson JM, Yu D, Spasojevic I, Pruitt AF, Klein A, Case B, Thrall DE, Needham D, Dewhirst MW.
Clin Cancer Res. 2006 Jul 1;12(13):4004-10.
[ expand abstract ]

PURPOSE: To determine the maximum tolerated dose, dose-limiting toxicities, and pharmacokinetic characteristics of doxorubicin encapsulated in a low temperature sensitive liposome (LTSL) when given concurrently with local hyperthermia to canine solid tumors. EXPERIMENTAL DESIGN: Privately owned dogs with solid tumors (carcinomas or sarcomas) were treated. The tumors did not involve bone and were located at sites amenable to local hyperthermia. LTSL-doxorubicin was given (0.7-1.0 mg/kg i.v.) over 30 minutes during local tumor hyperthermia in a standard phase I dose escalation study. Three treatments, given 3 weeks apart, were scheduled. Toxicity was monitored for an additional month. Pharmacokinetics were evaluated during the first treatment cycle. RESULTS: Twenty-one patients were enrolled: 18 with sarcomas and 3 with carcinomas. Grade 4 neutropenia and acute death secondary to liver failure, possibly drug related, were the dose-limiting toxicities. The maximum tolerated dose was 0.93 mg/kg. Other toxicities, with the possible exception of renal damage, were consistent with those observed following free doxorubicin administration. Of the 20 dogs that received >/=2 doses of LTSL-doxorubicin, 12 had stable disease, and 6 had a partial response to treatment. Pharmacokinetic variables were more similar to those of free doxorubicin than the marketed liposomal product. Tumor drug concentrations at a dose of 1.0 mg/kg averaged 9.12 +/- 6.17 ng/mg tissue. CONCLUSION: LTSL-doxorubicin offers a novel approach to improving drug delivery to solid tumors. It was well tolerated and resulted in favorable response profiles in these patients. Additional evaluation in human patients is warranted.

Shell cross-linked stearic acid grafted chitosan oligosaccharide self-aggregated micelles for controlled release of paclitaxel.
Hu FQ, Ren GF, Yuan H, Du YZ, Zeng S.
Colloids Surf B Biointerfaces. 2006 Jul 1;50(2):97-103; Epub 2006 May 5.
[ expand abstract ]

Stearic acid grafted chitosan oligosaccharide (CSO-SA) with different degree of amino substitution (SD) was synthesized by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated coupling reaction. The critical micelle concentration (CMC) of CSO-SA with different SD was about 0.06, 0.04, 0.01mg/ml, respectively. With the increase of micelle concentration, the micelle size decreased, and the zeta potential increased. On the other hand, with the increase of SD of CSO-SA, the micelle size and zeta potential decreased due to the increased hydrophobic interaction of SA and the reduced free amino groups. To increase the stability of the micelle in vivo and controll drug release, the shells of micelles were cross-linked by glutaraldehyde. By controlling the molar ratio of CSO-SA to glutaraldehyde, the cross-linking of intra-micelle could be reached, and the nanoparticle with smaller size than that of its initial micelle was obtained. Paclitaxel was then used as model drug to incorporate into the micelles, and the surfaces of the micelles were further cross-linked by glutaraldehyde to form drug loaded and shell cross-linked nanoparticles. The effects of drug loading, SD of CSO-SA and cross-link degree on the size, zeta potential, drug entrapment efficiency and in vitro drug release behavior of micelles and its cross-linked nanoparticles were investigated. The higher drug entrapment efficiencies (above 94%) were observed in all case. The charged amounts of drug did not affect the drug release behavior. The drug release rate decreased with the increase of SD of CSO-SA and cross-link degree.

Antibody targeting of long-circulating lipidic nanoparticles does not increase tumor localization but does increase internalization in animal models.
Kirpotin DB, Drummond DC, Shao Y, Shalaby MR, Hong K, Nielsen UB, Marks JD, Benz CC, Park JW.
Cancer Res. 2006 Jul 1;66(13):6732-40.
[ expand abstract ]

We describe evidence for a novel mechanism of monoclonal antibody (MAb)-directed nanoparticle (immunoliposome) targeting to solid tumors in vivo. Long-circulating immunoliposomes targeted to HER2 (ErbB2, Neu) were prepared by the conjugation of anti-HER2 MAb fragments (Fab' or single chain Fv) to liposome-grafted polyethylene glycol chains. MAb fragment conjugation did not affect the biodistribution or long-circulating properties of i.v.-administered liposomes. However, antibody-directed targeting also did not increase the tumor localization of immunoliposomes, as both targeted and nontargeted liposomes achieved similarly high levels (7-8% injected dose/g tumor tissue) of tumor tissue accumulation in HER2-overexpressing breast cancer xenografts (BT-474). Studies using colloidal gold-labeled liposomes showed the accumulation of anti-HER2 immunoliposomes within cancer cells, whereas matched nontargeted liposomes were located predominantly in extracellular stroma or within macrophages. A similar pattern of stromal accumulation without cancer cell internalization was observed for anti-HER2 immunoliposomes in non-HER2-overexpressing breast cancer xenografts (MCF-7). Flow cytometry of disaggregated tumors posttreatment with either liposomes or immunoliposomes showed up to 6-fold greater intracellular uptake in cancer cells due to targeting. Thus, in contrast to nontargeted liposomes, anti-HER2 immunoliposomes achieved intracellular drug delivery via MAb-mediated endocytosis, and this, rather than increased uptake in tumor tissue, was correlated with superior antitumor activity. Immunoliposomes capable of selective internalization in cancer cells in vivo may provide new opportunities for drug delivery.

Drug delivery system based on covalently bonded poly[N-isopropylacrylamide-co-2-hydroxyethylacrylate]-based nanoparticle networks.
Mathews AS, Ha CS, Cho WJ, Kim I.
Drug Deliv. 2006 Jul-Aug;13(4):245-51.
[ expand abstract ]

The present work is focused on investigating the controlled drug release behavior of Poly [N-isopropylacrylamide-co-2-Hydroxyethylacrylate] (P [NIPAm-HEAc])-based hydrogel. The synthesis process includes the preparation of monodispersed hydrogel nanoparticles containing specific functional groups, followed by cross-linking to neighboring spheres to stabilize the entire network. The color and volume of these crystalline hydrogel networks can reversibly change in response to external stimuli such as temperature, pH, and other environmental conditions. The feasibility of the hydrogel as a controlled release vehicle for 5-fluorouracil is evaluated.

Evaluation of firefly luciferase bioluminescence mediated photodynamic toxicity in cancer cells.
Schipper ML, Patel MR, Gambhir SS.
Mol Imaging Biol. 2006 Jul-Aug;8(4):218-25.
[ expand abstract ]

PURPOSE: This work investigated whether fLuc-catalyzed oxidation of D: -luciferin generates sufficient light to induce photodynamic toxicity in cancer cells. PROCEDURES: Light emission was assessed via cooled CCD (charge-coupled device) camera. Parental and fLuc expressing cancer cells were exposed to subtoxic concentrations of photosensitizers (Rose Bengal or hypericin) and D: -luciferin, sunlight, or lamplight. Toxicity was assessed by MTT assay. RESULTS: fLuc expressing cells emitted up to 500-fold higher levels of photons than parental cell lines. Although exposure to photosensitizer and sunlight reduced survival of various cell lines, survival of fLuc expressing cells incubated with photosensitizer and D: -luciferin, or photosensitizer and lamplight, did not differ significantly from parental or untreated cells. CONCLUSIONS: Contesting recent reports, fLuc bioluminescence does not generate sufficient photons to induce Rose Bengal or hypericin photodynamic toxicity in a range of malignant and nonmalignant cell lines, and is not suitable as a generalizable approach to antineoplastic therapy.

Effects of siRNAs in combination with Gleevec on K-562 cell proliferation and Bcr-Abl expression.
Baker BE, Kestler DP, Ichiki AT.
J Biomed Sci. 2006 Jul;13(4):499-507; Epub 2006 Mar 18.
[ expand abstract ]

RNA interference (RNAi) is the repression of gene expression through a cellular mechanism of transcript-specific mRNA degradation. RNAi has been observed in human cells and applied to the modulation of a variety of human transcripts. Our goals were to deliver small interfering RNA (siRNA) using a liposome-based method, and to show Bcr-Abl siRNA specificity against K-562 cells, alone or in combination with Gleevec. Both synthetic (syn) siRNA, consisting of homogeneous 21-nucleotide-long RNA duplexes specific for the Bcr-Abl fusion site, and recombinant (r)-generated Bcr-Abl siRNA were employed. siRNA was transfected into K-562 cells with greater than 90% efficiency using RNAiFecttrade mark, as judged by fluorescence analysis. The Bcr-Abl transcript was inhibited using either siRNA preparation as measured by RT-PCR or real-time PCR. The IC(50) of Gleevec in the K-562 subline F(1) was lowered over 3-fold from 0.2 to 0.06 muM in cells transfected with either syn or rBcr-Abl siRNA. No effect was observed in cells after transfection with an irrelevant control siRNA. Therefore, K-562 cells transfected with RNAifect deliver Bcr-Abl siRNA efficiently and the Bcr-Abl siRNA decreased the IC(50) of Gleevec required to inhibit the high levels of Bcr-Abl protein found in K-562 cells.

Nanotechnology: intelligent design to treat complex disease.
Couvreur P, Vauthier C.
Pharm Res. 2006 Jul;23(7):1417-50 ; Epub 2006 Jun 21.
[ expand abstract ]

The purpose of this expert review is to discuss the impact of nanotechnology in the treatment of the major health threats including cancer, infections, metabolic diseases, autoimmune diseases, and inflammations. Indeed, during the past 30 years, the explosive growth of nanotechnology has burst into challenging innovations in pharmacology, the main input being the ability to perform temporal and spatial site-specific delivery. This has led to some marketed compounds through the last decade. Although the introduction of nanotechnology obviously permitted to step over numerous milestones toward the development of the "magic bullet" proposed a century ago by the immunologist Paul Ehrlich, there are, however, unresolved delivery problems to be still addressed. These scientific and technological locks are discussed along this review together with an analysis of the current situation concerning the industrial development.

Plasmid size up to 20 kbp does not limit effective in vivo lung gene transfer using compacted DNA nanoparticles.
Fink TL, Klepcyk PJ, Oette SM, Gedeon CR, Hyatt SL, Kowalczyk TH, Moen RC, Cooper MJ.
Gene Ther. 2006 Jul;13(13):1048-51; Epub 2006 Mar 9.
[ expand abstract ]

Nanoparticles consisting of single molecules of DNA condensed with polyethylene glycol-substituted lysine 30-mers efficiently transfect lung epithelium following intrapulmonary administration. Nanoparticles formulated with lysine polymers having different counterions at the time of DNA mixing have distinct geometric shapes: trifluoroacetate or acetate counterions produce ellipsoids or rods, respectively. Based on intracytoplasmic microinjection studies, nanoparticle ellipsoids having a minimum diameter less than the 25 nm nuclear membrane pore efficiently transfect non-dividing cells. This 25 nm size restriction corresponds to a 5.8 kbp plasmid when compacted into spheroids, whereas the 8-11 nm diameter of rod-like particles is smaller than the nuclear pore diameter. In mice, up to 50% of lung cells are transfected after dosing with a rod-like compacted 6.9 kbp lacZ expression plasmid, and correction of the CFTR chloride channel was observed in humans following intranasal administration of a rod-like compacted 8.3 kbp plasmid. To further investigate the potential size and shape limitations of DNA nanoparticles for in vivo lung delivery, reporter gene activity of ellipsoidal and rod-like compacted luciferase plasmids ranging in size between 5.3 and 20.2 kbp was investigated. Equivalent molar reporter gene activities were observed for each formulation, indicating that microinjection size limitations do not apply to the in vivo gene transfer setting.Gene Therapy (2006) 13, 1048-1051. doi:10.1038/sj.gt.3302761; published online 9 March 2006.

Microfabricated nanochannel implantable drug delivery devices: trends, limitations and possibilities.
Gardner P.
Expert Opin Drug Deliv. 2006 Jul;3(4):479-87.
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This is a review of the application of microfabrication technologies, borrowed from the semiconductor industry, to drug delivery implants incorporating structures in the nanometer dimension. In the futuristic ideal, these systems would involve the implantation of precisely microfabricated drug delivery systems with nanopores, nanochannels and/or nanoreservoirs fabricated from silicon, coupled with electronic sensing and actuator systems, for the precise, timed and/or targeted delivery of drugs. After more than a decade in conceptualisation and experimentation, four systems that have commercial potential are discussed: i) implantable microchips with on-demand microdosage for one or more therapeutic agents under internal control or external control using a wireless link; ii) nanopore pumps, implantable titanium pumps, consisting of a drug reservoir with a nanopore-release membrane, capable of delivering potent small or macromolecules at constant serum levels for sustained periods of time; iii) nanocages, microfabricated nanopore immunoisolation chambers for cellular implants, capable of natural feedback-controlled delivery of proteins and peptides; and iv) nanobuckets, micromachined silicon porous particles with drug-loading capacity and targeting ligands for localised delivery. Each of the systems, along with future trends in microfabrication manufacturing, limitations and possibilities, are discussed.

A pilot study of the liposomal MUC1 vaccine BLP25 in prostate specific antigen failures after radical prostatectomy.
North SA, Graham K, Bodnar D, Venner P.
J Urol. 2006 Jul;176(1):91-5.
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PURPOSE: Men with biochemical failure after radical prostatectomy have few therapeutic options other than androgen deprivation therapy. Targeted therapies in this group are appropriate because the optimal timing of the initiation of hormonal therapy in this patient population is unknown. A single institution pilot trial was performed using BLP25 liposome vaccine in hormone naive patients with prostate specific antigen failure after radical prostatectomy to determine if prostate specific antigen progression could be halted. MATERIALS AND METHODS: Men with biochemical failure after radical prostatectomy were enrolled. Primary end points were efficacy and safety of the MUC1 BLP25 liposomal vaccine. Changes in prostate specific antigen doubling time were also evaluated. Patients received a single intravenous dose of cyclophosphamide, followed by vaccinations with BLP25 liposome vaccine for up to 1 year. Prostate specific antigen was measured at baseline and during treatment, and prostate specific antigen doubling time was calculated for these intervals. RESULTS: A total of 16 patients with a median age of 60 years were enrolled. All patients received cyclophosphamide and 15 of 16 completed the primary treatment period. Ten patients completed the maintenance period. After the 8-week primary treatment period 8 of 16 patients had stable or decreased prostate specific antigen. At the last on-study prostate specific antigen measurement 1 patient maintained stable prostate specific antigen but all others had progression. However, 6 of the 16 patients had greater than 50% prolongation of prostate specific antigen doubling time compared to pre-study prostate specific antigen doubling time. CONCLUSIONS: BLP25 liposome vaccine shows promise for prolonging prostate specific antigen doubling time in hormone naive men with biochemical failure after prostatectomy and little morbidity. This could potentially translate into the deferral of hormonal therapy. Further testing in this population of patients is warranted.

Convection-enhanced delivery of Ls-TPT enables an effective, continuous, low-dose chemotherapy against malignant glioma xenograft model.
Saito R, Krauze MT, Noble CO, Drummond DC, Kirpotin DB, Berger MS, Park JW, Bankiewicz KS.
Neuro-oncol. 2006 Jul;8(3):205-14; Epub 2006 May 24.
[ expand abstract ]

Treatment of malignant gliomas represents one of the most formidable challenges in oncology. The combination of surgery, radiation, and chemotherapy yields median survivals of less than one year. Here we demonstrate the use of a minimally invasive surgical technique, convection-enhanced delivery (CED), for local administration of a novel nanoparticle liposome containing topotecan. CED of this liposomal topotecan (Ls-TPT) resulted in extended brain tissue retention (t((1/2)) = 1.5 days), whereas free topotecan was rapidly cleared (t((1/2)) = 0.1 days) after CED. The favorable pharmacokinetic profile of extended topotecan release for about seven days, along with biodistribution featuring perivascular accumulation of the nanoparticles, provided, in addition to the known topoisomerase I inhibition, an effective antiangiogenic therapy. In the rat intracranial U87MG tumor model, vascular targeting of Ls-TPT with CED was associated with reductions in laminin expression and vascular density compared to free topotecan or control treatments. A single CED treatment on day 7 showed that free topotecan conferred no survival benefit versus control. However, Ls-TPT produced a significant (P = 0.0002) survival benefit, with six of seven complete cures. Larger U87MG tumors, where CED of Ls-TPT on day 12 resulted in one of six cures, indicated the necessity to cover the entire tumor with the infused therapeutic agent. CED of Ls-TPT was also efficacious in the intracranial U251MG tumor model (P = 0.0005 versus control). We conclude that the combination of a novel nanoparticle Ls-TPT and CED administration was very effective in treating experimental brain tumors.

A phase I study with oral SU5416 in patients with advanced solid tumors: a drug inducing its clearance.
Salzberg M, Pless M, Rochlitz C, Ambrus K, Scigalla P, Herrmann R.
Invest New Drugs. 2006 Jul;24(4):299-304.
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Vascular endothelial growth factor (VEGF) is a potent stimulant of angiogenesis. SU5416, is a small molecule tyrosine kinase inhibitor, and a potent inhibitor of VEGF-mediated Flk-1 receptor signaling. Intravenous agent SU5416 has shown evidence of biological activity against a variety of tumor types. The current intravenous dosing regimen is not optimal for long-term administration, which is needed for optimal efficacy. The aim of this study was to evaluate the safety profile and pharmacokinetics of a Nanocrystal Colloidal Dispersion (NCD) SU5416 formulation in humans. Patients with advanced and/or metastatic solid organ tumors were included in the trial; various SU5416 regimens were tested for tolerability, safety and were evaluated concerning pharmacokinetics. The results of this study indicate that induction of clearance after oral dosing of NCD SU5416 in humans occurs and is greater than following i.v. administration. It has been confirmed that SU5416 is a high clearance compound, also as an oral NCD formulation. The NCD formulation was well tolerated, but no effective drug serum levels could be achieved. These data help to understand the ADME (Absorption, Distribution, Metabolism, Excretion) properties of indoline chemical class compounds. The lessons learned should be applied in the development of next generation indoline anti-angiogenic and anti-tumor compounds.

A new polymer-lipid hybrid nanoparticle system increases cytotoxicity of Doxorubicin against multidrug-resistant human breast cancer cells.
Wong HL, Rauth AM, Bendayan R, Manias JL, Ramaswamy M, Liu Z, Erhan SZ, Wu XY. Pharm Res. 2006 Jul;23(7):1574-85; Epub 2006 Jun 24.
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PURPOSE: This work is intended to develop and evaluate a new polymer-lipid hybrid nanoparticle system that can efficiently load and release water-soluble anticancer drug doxorubicin hydrochloride (Dox) and enhance Dox toxicity against multidrug-resistant (MDR) cancer cells. METHODS: Cationic Dox was complexed with a new soybean-oil-based anionic polymer and dispersed together with a lipid in water to form Dox-loaded solid lipid nanoparticles (Dox-SLNs). Drug loading and release properties were measured spectrophotometrically. The in vitro cytotoxicity of Dox-SLN and the excipients in an MDR human breast cancer cell line (MDA435/LCC6/MDR1) and its wild-type line were evaluated by trypan blue exclusion and clonogenic assays. Cellular uptake and retention of Dox were determined with a microplate fluorometer. RESULTS: Dox-SLNs were prepared with a drug encapsulation efficiency of 60-80% and a particle size range of 80-350 nm. About 50% of the loaded drug was released in the first few hours and an additional 10-20% in 2 weeks. Treatment of the MDR cells with Dox-SLN resulted in over 8-fold increase in cell kill when compared to Dox solution treatment at equivalent doses. The blank SLN and the excipients exhibited little cytotoxicity. The biological activity of the released Dox remained unchanged from fresh, free Dox. Cellular Dox uptake and retention by the MDR cells were both significantly enhanced (p < 0.05) when Dox was delivered in Dox-SLN form. CONCLUSIONS: The new polymer-lipid hybrid nanoparticle system is effective for delivery of Dox and enhances its efficacy against MDR breast cancer cells.

Poly(ethylene oxide)-modified poly(beta-amino ester) nanoparticles as a pH-sensitive system for tumor-targeted delivery of hydrophobic drugs: part 3. Therapeutic efficacy and safety studies in ovarian cancer xenograft model.
Devalapally H, Shenoy D, Little S, Langer R, Amiji M.
Cancer Chemother Pharmacol. 2006 Jul 22; [Epub ahead of print].
[ expand abstract ]

PURPOSE: The objective of this study was to evaluate the anti-tumor efficacy and lack of systemic toxicity of paclitaxel when administered in pH-sensitive poly(ethylene oxide) (PEO)-modified poly(beta-amino ester) (PbAE) nanoparticles in mice bearing human ovarian adenocarcinoma (SKOV-3) xenograft. METHODS: Paclitaxel-encapsulated PEO-modified PbAE (PEO-PbAE) nanoparticles were prepared by the solvent displacement method. PEO-modified poly(epsilon-caprolactone) (PCL) (PEO-PCL) nanoparticles were used as a non pH-responsive control formulation. Efficacy studies were conducted in SKOV-3 tumor-bearing athymic (Nu/Nu) mice at an equivalent paclitaxel dose of 20 mg/kg with the control and nanoparticle formulations. Safety of the drug when administered in the control and nanoparticle formulation was determined from blood cell counts and changes in body weight of the animals. RESULTS: The formulated paclitaxel-containing PEO-PbAE and PEO-PCL nanoparticles had a particle size in the range of 100-200 nm and a surface charge of + 39.0 and - 30.8 mV, respectively. After intravenous administration of paclitaxel in these formulations, the tumor growth was inhibited significantly. Both of the formulated nanoparticles tested have shown improved therapeutic efficacy as compared to the paclitaxel aqueous solution. Additionally, significantly lower toxicity profile of paclitaxel was observed with PEO-modified nanoparticles as compared to the aqueous solution formulation CONCLUSION: PEO-modified PbAE nanoparticles are a unique pH-sensitive drug delivery system that elicits enhanced efficacy and safety profile in solid tumor therapy.

The drug encapsulation efficiency, in vitro drug release, cellular uptake and cytotoxicity of paclitaxel-loaded poly(lactide)-tocopheryl polyethylene glycol succinate nanoparticles.
Zhang Z, Feng SS.
Biomaterials. 2006 Jul;27(21):4025-33; Epub 2006 Mar 27.
[ expand abstract ]

Paclitaxel is one of the most effective antineoplastic drugs. Its current clinical administration is formulated in Cremophor EL, which causes serious side effects. Nanoparticle (NP) technology may provide a solution for such poisonous adjuvant problems and promote a sustained chemotherapy, in which biodegradable polymers play a key role. Our group has successfully synthesized novel poly(lactide)-tocopheryl polyethylene glycol succinate (TPGS) (PLA-TPGS) copolymers of desired hydrophobic-hydrophilic balance for NP formulation of anticancer drugs. The present work is focused on effects of the PLA:TPGS composition ratio on drug encapsulation efficiency, in vitro drug release, in vitro cellular uptake and viability of the PLA-TPGS NP formulation of paclitaxel. The PLA-TPGS copolymers of various PLA:TPGS ratios were synthesized by the ring-opening polymerization method and characterized by GPC and (1)H NMR for their molecular structure. Paclitaxel-loaded PLA-TPGS NPs were prepared by a modified solvent extraction/evaporation method and characterized by laser light scattering for size and size distribution, scanning electron microscopy for surface morphology and zeta potential for surface charge. High performance liquid chromatography was used to measure the drug encapsulation efficiency and in vitro drug release profile. Cancer cell lines HT-29 and Caco-2 were used to image and measure the cellular uptake of fluorescent PLA-TPGS NPs. Cancer cell viability of the drug-loaded PLA-TPGS was measured by MTT assay. It was found that the PLA:TPGS composition ratio has little effects on the particle size and size distribution. However, the PLA-TPGS NPs of 89:11 PLA:TPGS ratio achieved the best effects on the drug encapsulation efficiency, the cellular uptake and the cancer cell mortality of the drug-loaded PLA-TPGS NPs. This research was also carried out in close comparison with the drug-loaded PLGA NPs.

Current state, achievements, and future prospects of polymeric micelles as nanocarriers for drug and gene delivery.
Nishiyama N, Kataoka K.
Pharmacol Ther. 2006 Jun 29; [Epub ahead of print] .
[ expand abstract ]

Polymeric micelles, self-assemblies of block copolymers, are promising nanocarrier systems for drug and gene delivery. Until now, several micellar formulations of antitumor drugs have been intensively studied in preclinical and clinical trials, and their utility has been demonstrated. Even compared with long-circulating liposomes, polymeric micelles might have several advantages, such as controlled drug release, tissue-penetrating ability and reduced toxicity such as hand-foot syndrome and hypersensitivity reaction. Importantly, critical features of the polymeric micelles as drug carriers, including particle size, stability, and loading capacity and release kinetics of drugs, can be modulated by the structures and physicochemical properties of the constituent block copolymers. Also, nano-engineering of block copolymers might allow the preparation of polymeric micelles with integrated smart functions, such as specific-tissue targetability, as well as chemical or physical stimuli-sensitivity. Thus, polymeric micelles are nanotechnology-based carrier systems that might exert the activity of potent bioactive compounds in a site-directed manner, ensuring their effectiveness and safety in the clinical use.

Poly(ethylene glycol)/poly(epsilon-caprolactone) diblock copolymeric nanoparticles for non-viral gene delivery: The role of charge group and molecular weight in particle formation, cytotoxicity and transfection.
Jeong Soon Jang, So Yeon Kim, Sang Bong Lee, Kyung Ok Kim, Joong Soo Han, Young Moo Lee.
J Control Release. 2006 Jun 28;113(2):173-82; Epub 2006 Apr 26.
[ expand abstract ]

Two types of nanoparticles containing pGL3-Control (plasmid DNA) were prepared using nonionic amphiphlic block copolymers and ionic amphiphilic block copolymers containing a terminal cationic group to investigate the effect of charge on the vehicle properties for systemic gene delivery. Methoxy poly(ethylene glycol) (MPEG)/poly(epsilon-caprolactone) (PCL) diblock copolymers were synthesized by the ring-opening polymerizatrion of epsilon-caprolactone in the presence of a catalyst-free MPEG homopolymer. The hydroxy groups of MPEG/PCL block copolymer were then modified into an amine group to synthesize an amine-terminated MPEG/PCL diblock copolymer (AMPEG/PCL). DNA was incorporated into the polymeric nanoparticles by physical entrapment and electrostatic interaction. All nanoparticle samples exhibited spherical structures and although their sizes increased slightly after DNA-loading, they remained less than 160 nm. The AMPEG/PCL nanoparticles exhibited smaller particle sizes than the MPEG/PCL nanoparticles of the same molecular weight after DNA-loading. The optimum mixing ratio of MPEG/PCL and AMPEG/PCL copolymers to DNA ranged from 4:1 to 1:2 depending on the molecular weight of the block copolymer, the composition of MPEG and PCL and terminal amine group. Based on in vitro cytotoxicity tests, the DNA-loaded MPEG/PCL and AMPEG/PCL nanoparticles did not induce any remarkable cytotoxicity against normal human fibroblasts. Transfection efficiencies of DNA-loaded nanoparticles were improved about 3.4 approximately 12.9 times under serum conditions.

Trehalose click polymers inhibit nanoparticle aggregation and promote pDNA delivery in serum.
Srinivasachari S, Liu Y, Zhang G, Prevette L, Reineke TM.
J Am Chem Soc. 2006 Jun 28;128(25):8176-84.
[ expand abstract ]

Herein, three new glycopolymers have been synthesized via "click polymerization" to promote nucleic acid delivery in the presence of biological media containing serum. These structures were designed to contain a trehalose moiety to promote biocompatibility, water solubility, and stability against aggregation, amide-triazole groups to enhance DNA binding affinity, and an oligoamine unit to facilitate DNA encapsulation, phosphate neutralization, and interactions with cell surfaces. A 2,3,4,2',3',4'-hexa-O-acetyl-6,6'-diazido-6,6'-dideoxy-D-trehalose (4) monomer was polymerized via copper(I)-catalyzed azide-alkyne cycloaddition with a series of dialkyne-amide comonomers that contain either one, two, or three Boc-protected secondary amines (7a, 7b, or 7c, respectively). After deprotection, three water-soluble polycations (9a, 9b, or 9c) were obtained with similar degrees of polymerization (n = 56-61) to elucidate the role of amine number on nucleic acid binding, complex formation, stability, and cellular delivery. Gel electrophoresis and ethidium bromide experiments showed that 9a-9c associated with plasmid DNA (pDNA) and formed complexes (polyplexes) at N/P ratios dependent on the amine number. TEM experiments revealed that 9a-9c polyplexes were small (50-120 nm) and had morphologies (spherical and rodlike) associated with the polymer chain stiffness. Dynamic light scattering studies in the presence of media containing serum demonstrated that 9c polyplexes had a low degree of flocculation, whereas 9a and 9b polyplexesd aggregate rapidly. Further biological studies revealed that these structures were biocompatible and deliver pDNA into HeLa cells. Particularly, 9c polyplexes promoted high delivery efficacy and gene expression profiles in the presence of serum.

Adenoviral Gene Vector Tethering to Nanoparticle Surfaces Results in Receptor-Independent Cell Entry and Increased Transgene Expression.
Chorny M, Fishbein I, Alferiev IS, Nyanguile O, Gaster R, Levy RJ.
Mol Ther. 2006 Jun 23; [Epub ahead of print] .
[ expand abstract ]

The present studies investigated the hypothesis that affinity immobilization of replication-defective adenoviruses (Ad) on the surfaces of biodegradable nanoparticles (NP) can improve transduction through uncoupling cellular uptake from the coxsackie-adenovirus receptor (CAR). Ad was tethered to the surfaces of polylactide-based NP that were surface-activated using a photoreactive polyallylamine-benzophenone-pyridyldithiocarboxylate polymer, which enabled (via thiol chemistry) the covalent attachment of Ad-binding proteins, either the recombinant D1 domain of CAR or an adenoviral knob-specific monoclonal antibody. Gene transfer by NP-Ad complexes was studied in relation to cellular uptake as a function of cell type and the character of NP-Ad binding. NP-Ad complexes, but not Ad applied with or without control nonimmune IgG-modified NP, significantly increased green fluorescent protein reporter expression in endothelioma and endothelial and arterial smooth muscle cells (SMC) in direct correlation to the extent of NP-Ad internalization. CAR-independent uptake of NP-Ad was confirmed by demonstrating inhibition of free Ad- but not NP-Ad complex-mediated transduction by knob protein. Complexes formulated with an Ad encoding inducible nitric oxide synthase inhibited growth of cultured SMC to a significantly greater extent than those with (GFP)Ad or (NULL)Ad or free vector. It is concluded that Ad-specific affinity tethering to biodegradable NP can significantly increase the level of gene expression via a CAR-independent uptake mechanism.

Potential use of drug carried-liposomes for cancer therapy via direct intratumoral injection.
Bao A, Phillips WT, Goins B, Zheng X, Sabour S, Natarajan M, Ross Woolley F, Zavaleta C, Otto RA.
Int J Pharm. 2006 Jun 19;316(1-2):162-9; Epub 2006 Mar 31.
[ expand abstract ]

Liposomes have recognized advantages as nano-particle drug carriers for tumor therapy. In this study, the pharmacokinetics and distribution of intratumorally administered liposomes were investigated as drug carriers for treating solid tumors via direct intratumoral administration. 99mTc-liposomes were administered intratumorally to nude rats bearing human head and neck squamous cell carcinoma xenografts. Planar gamma camera images were analyzed to evaluate the local retention of the intratumorally administered liposomes. Co-registered pinhole micro-single photon emission computed tomography (SPECT)/computed tomography (CT) images were acquired of the whole animal as well as the dissected tumors to determine intratumoral distribution of the 99mTc-liposomes. For 99mTc-liposomes, there was an initial retention of 47.4 +/- 11.0% (n = 4) in tumors and surrounding tissues. At 20 h, 39.2 +/- 10.6% (n = 4) of 99mTc-activity still remained in the tumor. In contrast, only 18.7 +/- 3.3% (n = 3) of the intratumoral 99mTc-activity remained for unencapsulated 99mTc-complex at 20 h. Pinhole micro-SPECT images demonstrated that 99mTc-liposomes also have a superior intratumoral 99mTc-activity diffusion compared with unencapsulated 99mTc-complex. Higher intratumoral retention of 99mTc-liposomes accompanied by an improved intratumoral diffusion suggests that intratumorally administered liposomal drugs are potentially promising agents for solid tumor local therapy.

A folate receptor-targeted liposomal formulation for paclitaxel.
Wu J, Liu Q, Lee RJ.
Int J Pharm. 2006 Jun 19;316(1-2):148-53; Epub 2006 Mar 6.
[ expand abstract ]

A novel liposomal formulation of paclitaxel targeting the folate receptor (FR) was synthesized and characterized. This formulation was designed to overcome vehicle toxicity associated with the traditional Cremophor EL-based formulation and to provide the added advantages of prolonged systemic circulation time and selective targeting of the FR, which is frequently overexpressed on epithelial cancer cells. The formulation had the composition of dipalmitoyl phosphatidylcholine/dimyristoyl phosphatidylglycerol/monomethoxy-polyethylene glycol (PEG)2000-distearoyl phosphatidylethanolamine/folate-PEG3350-distearoyl phosphatidylethanolamine (DPPC/DMPG/mPEG-DSPE/folate-PEG-DSPE) at molar ratios of (85.5:9.5:4.5:0.5) and a drug-to-lipid molar ratio of 1:33. The liposomes were prepared by polycarbonate membrane extrusion. The mean particle size of the liposomes was 97.1 nm and remained stable for at least 72 h at 4 degrees C. FR-targeted liposomes of the same lipid composition entrapping calcein were shown to be efficiently taken up by KB oral carcinoma cells, which are highly FR+. FR-targeted liposomes containing paclitaxel showed 3.8-fold greater cytotoxicity compared to non-targeted control liposomes in KB cells. Plasma clearance profiles of paclitaxel in the liposomal formulations were then compared to paclitaxel in Cremophor EL formulation. The liposomal formulations showed much longer terminal half-lives (12.33 and 14.23 h for FR-targeted and non-targeted liposomes, respectively) than paclitaxel in Cremophor EL (1.78 h). In conclusion, the paclitaxel formulation described in this study has substantial stability and favorable pharmacokinetic properties. The FR-targeted paclitaxel formulation is potentially useful for treatment of FR+ tumors and warrants further investigation.

PAMAM dendrimers for efficient siRNA delivery and potent gene silencing.
Zhou J, Wu J, Hafdi N, Behr JP, Erbacher P, Peng L.
Chem Commun (Camb). 2006 Jun 14;(22):2362-4; Epub 2006 May 10.
[ expand abstract ]

Genuine, nondegraded PAMAM dendrimers self-assemble with siRNA into nanoscale particles that are efficient for siRNA delivery and induce potent endogenous gene silencing.

Anti-angiogenic effects of liposomal prednisolone phosphate on B16 melanoma in mice.
Banciu M, Schiffelers RM, Fens MH, Metselaar JM, Storm G.
J Control Release. 2006 Jun 12;113(1):1-8; Epub 2006 Apr 7.
[ expand abstract ]

Prednisolone phosphate (PLP) encapsulated in long-circulating liposomes can inhibit tumor growth after intravenous administration (i.v.). These antitumor effects of liposomal PLP are the result of the tumor-targeting property of the liposome formulation. The mechanism by which liposomal PLP inhibits tumor growth is unclear. We investigated the effects of liposome-encapsulated PLP versus free PLP on angiogenic protein production in tumor tissue in vivo and on viability and proliferation of tumor and endothelial cells in vitro. In vivo, liposomal PLP had a stronger reducing effect on pro-angiogenic protein levels than free PLP, whereas levels of anti-angiogenic proteins were hardly affected. Cell viability was only slightly affected with either treatment. Liposomal PLP had strong anti-proliferative effects on human umbilical vein endothelial cells, whereas free PLP had hardly any effect. Taken together, the present study points to a strong inhibitory effect of liposomal PLP on tumor angiogenesis by reduction of the intratumoral production of the majority of pro-angiogenic factors studied and direct inhibition of endothelial cell proliferation, which is the result of high prolonged levels of prednisolone in the tumor by liposomal delivery.

In vivo antitumor activity of chitosan nanoparticles.
Qi L, Xu Z.
Bioorg Med Chem Lett. 2006 Jun 5; [Epub ahead of print] .
[ expand abstract ]

Chitosan nanoparticles have been synthesized as potential anticancer agents, and evaluated, in vitro, against various cancer cell lines. In this study, in vivo antitumor activity of chitosan nanoparticles against Sarcoma-180 and mouse hepatoma H22 was investigated. Chitosan nanoparticles showed significant antitumor activity in vivo. The doses and particle size made a great effect on their efficacy.

Lipoplexes prepared from cationic liposomes and mammalian DNA induce CpG-independent, direct cytotoxic effects in cell cultures and in mice.
Khazanov E, Simberg D, Barenholz Y.
J Gene Med. 2006 Jun 2; [Epub ahead of print] .
[ expand abstract ]

BACKGROUND: Recent studies demonstrated the cytotoxic activity of bacterial DNA (pDNA) complexed with cationic lipids. This cytotoxicity is related to the ability of pDNA to induce potently the immune system, which is associated with release of inflamatory cytokines. Both activities seem to be related to the nonmethylated CpG sequences present in the pDNA. Here we study the cytotoxic activity of nonbacterial DNA complexed with cationic lipids against various tumor cell lines. METHODS: Various nucleic acids complexed with cationic liposomes were prepared and their cytotoxic activity was studied in cell cultures and in tumor-bearing mice. Cell uptake of lipoplexes was evaluated, and mechanism of DNA cytotoxic activity was studied. RESULTS: We found that nonbacterial (vertebrate) genomic DNA when complexed with cationic lipids is highly cytotoxic against C-26 and M-109 tumor cells. Cationic lipids alone were not toxic to these cells. The cytotoxic activity does not result from nonspecific acidification of the intracellular milieu, as substitution of DNA by poly-L-glutamate did not result in cytotoxicity, although the level of uptake of anionic charges per cell was similar to that of the nucleic acids, suggesting that this cytotoxic effect is specific to nucleic acids. By studying the nucleic acid fate using confocal microscopy, we found that cytotoxicity correlated with the release of DNA into the cytoplasm following uptake of lipoplexes. Injection of calf thymus DNA-based lipoplexes to mice with peritoneal C-26 metastases resulted in doubling of median survival time and long-term survival in 20% of the tumor-bearing mice. Judging by low levels of IFN-gamma, TNF-alpha and IL-6 in the treated mice, this effect cannot be ascribed to Th-1 inflammation, but rather to a direct cytotoxic effect on the tumor cells. CONCLUSIONS: The above data provide a new insight into the mechanisms of lipoplex-mediated antitumor effects in vitro and in vivo and new perspectives in cancer therapy.

Protein transduction by lipidic peptide dendrimers.
Bayele HK, Ramaswamy C, Wilderspin AF, Srai KS, Toth I, Florence AT.
J Pharm Sci. 2006 Jun;95(6):1227-37.
[ expand abstract ]

We investigated the potential of a new family of lipidic peptide dendrimers in protein transduction into cultured cells. Dendrimer-protein interaction was determined by gel retardation assays using purified recombinant protein. To assess intracellular protein delivery, two marker proteins were used: recombinant firefly luciferase and a Cy3-labeled monoclonal antibody to the c-myc proto-oncogene. Protein delivery was determined by luciferase assays and fluorescence microscopy, respectively. While there was minimal delivery of luciferase or antibody in the absence of the dendrimers, the latter increased protein delivery substantially. Luciferase delivery was concentration and cell type-dependent; the efficiency of delivery also varied with the number of terminal amino groups on the dendrimers. In previous reports, we showed that these dendrimers could be used for gene and drug delivery; the data we report herein suggest that they may also be capable of intracellular protein delivery. This finding has important implications for the use of these dendrimers in protein therapeutics and vaccinology. (c) 2006 Wiley-Liss, Inc. and the American Pharmacists Association.

Pegylated liposomal doxorubicin (Lipo-Dox(R)) for platinum-resistant or refractory epithelial ovarian carcinoma: A Taiwanese gynecologic oncology group study with long-term follow-up.
Chou HH, Wang KL, Chen CA, Wei LH, Lai CH, Hsieh CY, Yang YC, Twu NF, Chang TC, Yen MS.
Gynecol Oncol. 2006 Jun;101(3):423-8; Epub 2005 Dec 1.
[ expand abstract ]

OBJECTIVES.: To evaluate the efficacy and safety of a distearoylphosphatidylcholine pegylated liposomal doxorubicin, Lipo-Dox(R), in platinum-resistant or refractory epithelial ovarian cancer. METHODS.: A multicenter phase II trial enrolled women with platinum-resistant or refractory epithelial ovarian carcinoma and naive to anthracycline. Eligible patients had either measurable tumor(s) or elevated serum CA 125 titer. Lipodox was initiated with a dose of 45 mg/m(2) at a 4-week interval with subsequent escalation or reduction. A total of six cycles were scheduled. RESULTS.: 29 patients, 20 with platinum-resistant and 9 with platinum refractory tumors, were enrolled. Lipo-Dox was given for an average of 4.6 cycles per patient with a total of 134 cycles. Among the 26 evaluable patients, one achieved CR, 5 PR and 9 SD. The overall response rate was 23.1% (95% CI, 6.8%-39.3%) with a median response duration of 11.6 weeks. 5 of the 6 responses were in patients with resistant disease. The median progression-free duration in the SD patients was 25.7 weeks. With a median follow-up of 13.8 months, the median progression-free and median overall survivals in the 26 patients were 5.4 months and 13.8 months, respectively. Hand-foot skin reaction occurred in 4.5% and skin pigmentation in 11.2% of all treatment cycles, all were Grade 1/2. Nausea and vomiting occurred in 14.2%, while anemia, leukopenia and thrombocytopenia occurred in 20.9%, 32.8% and 9% of cycle, respectively, and were mostly Grade 1 or 2. CONCLUSION.: Lipo-Dox, the third liposome encapsulated doxorubicin, at 45 mg/m(2) every 4 weeks, is effective against recurrent, platinum-resistant epithelial ovarian cancers.

Nano- and micro-particulate formulations of poorly water-soluble drugs by using a novel optimized technique.
Douroumis D, Fahr A.
Eur J Pharm Biopharm. 2006 Jun;63(2):173-5; Epub 2006 Mar 6.
[ expand abstract ]

A novel technique for the production of nano- and micro-particulate formulations of poorly water-soluble drugs has been developed. This technique involves the use of static mixer elements to provide fast precipitation by continuous turbulent mixing of two liquid flows, an aqueous phase and an organic phase, respectively. The objective of this study was to develop the mixer technique by investigating the influence of the element number on the particle size of the resulting dispersions. Four model active pharmaceutical ingredients (APIs) with a variety of polymers, lipids or surfactants underwent intensive mixing and the final suspensions showed a narrow size distribution. Parameters such as the flow rate and the temperature of the precipitated organic-aqueous phases were also significant in the reduction of particle size. Further development of the mixing technique led to reproducible and stable formulations with minimal excipient amounts. These formulations were spray- or freeze-dried to improve stability.

Optimization of preparation of DHAQ-loaded PEG-PLGA-PEG nonaparticles using central composite design.
Duan Y, Xu S, Wang Q, Liu J, Zhang Z.
J Mater Sci Mater Med. 2006 Jun;17(6):559-63.
[ expand abstract ]

Mitoxantrone (DHAQ)-loaded poly (ethylene glycol)-poly (lactic acid-co-glycolic acid) -poly (ethyleneglycol) (PELGE) nanoparticles (NP) were fabricated using an emulsification/solvent evaporation technique. A central composite design (CCD) was applied to evaluate the joint influence of three formulation variables: the amounts of polymer, concentration of the DHAQ, and the ratio of the organic phase (inner-phase) and the aqueous phase (outer-phase). In this study, we optimize the preparation technology on the basis of the single factor evaluation. The optimal conditions for the preparation of DHAQ-loaded nanoparticle were found to be: the concentration of PELGE was 9 mg/mL, the concentration of inner-phase of DHAQ was 27.5 mg/L, and the ratio of inner-phase/outer-phase was 8.5/1. The results showed that CCD is an ideal technique for formulation studies. The entrapment efficiency ratio (ER) was 90% and particle sizes are less than 500 nm. The nanoparticles, as examined by transmission electron microscopy (TEM), have a smooth and spherical surface. The DHAQ could be loaded into PELGE copolymers. In this study, the DHAQ nanoparticle-polymer delivery system was established by using PELGE polymers as carrier material.

Toward the emergence of nanoneurosurgery: part III--nanomedicine: targeted nanotherapy, nanosurgery, and progress toward the realization of nanoneurosurgery.
Leary SP, Liu CY, Apuzzo ML.
Neurosurgery. 2006 Jun;58(6):1009-26; discussion 1009-26.
[ expand abstract ]

The notion of nanotechnology has evolved since its inception as a fantastic conceptual idea to its current position as a mainstream research initiative with broad applications among all divisions of science. In the first part of this series, we reviewed the structures and principles that comprise the main body of knowledge of nanoscience and nanotechnology. In the second part, we discussed applications of nanotechnology to the emerging field of nanomedicine, with specific attention on medical diagnostics and imaging. This article further explores the applications of nanotechnology to nanomedicine. Specific attention is given to developments in therapeutic modalities, including advanced drug delivery systems and targeted nanotherapy, which will form the basis for the treatment arm of mature nanomedicine. A variety of modalities are discussed, including polymeric nanoparticles, micelles, liposomes, dendrimers, fullerenes, hydrogels, nanoshells, and smart surfaces. Applications of nanotechnology to nanosurgery and nanoneurosurgery are presented. Femtosecond laser systems, nanoneedles, and nanotweezers are presented as technologies that are operational at the nanoscale level and have the potential to revolutionize the practice of neurosurgery in a profound and momentous way.

Mannosylated liposomes as antigen delivery vehicles for targeting to dendritic cells.
White KL, Rades T, Furneaux RH, Tyler PC, Hook S.
J Pharm Pharmacol. 2006 Jun;58(6):729-37.
[ expand abstract ]

The immune stimulating ability of mannosylated liposomes containing FITC-ovalbumin as a model antigen and displaying either a branched tri-mannose or a mono-mannose ligand on the liposome surface was investigated in human monocyte-derived dendritic cells (MoDCs) and murine bone-marrow-derived dendritic cells (BMDCs). Uptake of liposomes, dendritic cell activation and proliferation of CD8(+) T cells from OT-I transgenic mice were determined by flow cytometry. Uptake of liposomes displaying the tri-mannose ligand was enhanced in human MoDCs compared with both non-mannosylated liposomes and liposomes displaying mono-mannose ligands. However, this increased uptake did not result in an increase in expression of CD80 or CD86 on the surface of the MoDCs. In contrast, neither tri-mannose- nor mono-mannose-containing liposomes were taken up by murine BMDCs to a greater extent than non-mannose-containing liposomes. The expression of CD86 and CD40 on the surface of BMDCs was not increased after exposure to mannosylated liposomes and BMDCs incubated with mannosylated liposomes were not able to stimulate proliferation of CD8(+) T cells to any greater extent than BMDCs incubated with non-mannosylated liposomes. These findings suggest that while mannose-containing ligands can enhance the uptake of antigen-containing liposomes by some dendritic cells, important differences in the affinity of carbohydrate-binding receptors for mannose-containing ligands do exist between species. In addition, the increase in uptake of antigen by dendritic cells using mannosylated liposomes does not necessarily result in enhanced dendritic cell activation.

A mechanistic study of enhanced doxorubicin uptake and retention in multidrug resistant breast cancer cells using a polymer-lipid hybrid nanoparticle system.
Wong HL, Bendayan R, Rauth AM, Xue HY, Babakhanian K, Wu XY.
J Pharmacol Exp Ther. 2006 Jun;317(3):1372-81; Epub 2006 Mar 17.
[ expand abstract ]

The objectives of this study were to evaluate the potential of a polymer-lipid hybrid nanoparticle (PLN) system to enhance cellular accumulation and retention of doxorubicin (Dox), a widely used anticancer drug and an established P-glycoprotein (Pgp) substrate, in Pgp-overexpressing cancer cell lines and to explore the underlying mechanisms. Nanoparticles containing Dox complexed with a novel anionic polymer (Dox-PLN) were prepared using an ultrasound method. Two Pgp-overexpressing breast cancer cell lines (a human cell line, MDA435/LCC6/MDR1, and a mouse cell line, EMT6/AR1) were used to investigate the effect of nanoparticles on cellular uptake and retention of Dox. Endocytosis inhibition studies and fluorescence microscopic imaging were performed to elucidate the mechanisms of cellular drug uptake. Treatment of Pgp-overexpressing cell lines with Dox-PLNs resulted in significantly enhanced Dox uptake and more substantial increases in drug retention after the end of treatment compared with free Dox solutions (p < 0.05). Fluorescence microscopic images showed improved nuclear localization of Dox and uptake of lipid when the drug was delivered in the Dox-PLN form to MDA435/LCC6/MDR1 cells. Endocytosis inhibition studies revealed that phagocytosis is an important pathway in the membrane permeability of the nanoparticles. These findings suggest that some of the Dox physically associated with the nanoparticles bypass the membrane-associated Pgp when delivered as Dox-PLNs, and in this form, the drug is better retained within the Pgp-overexpressing cells than the free drug. The present study suggests a new mechanism for overcoming drug resistance in Pgp-overexpressing tumor cells using lipid-based nanoparticle formulations.

Selective laser nano-thermolysis of human leukemia cells with microbubbles generated around clusters of gold nanoparticles.
Lapotko DO, Lukianova E, Oraevsky AA.
Lasers Surg Med. 2006 May 30; [Epub ahead of print] .
[ expand abstract ]

BACKGROUND AND OBJECTIVE: Previously reported studies on laser nano-thermolysis of cancerous cells demonstrated insufficient efficacy and specificity of malignant cell damage. Safety, that is, absence of damage to normal cells in the course of the laser thermolysis was also low due to less than optimal protocol of cancer cell targeting with nanoparticles (NP). The objective of this study was two-fold: to optimize NP targeting to real tumor (human) cells and to better understand physical mechanisms of cell damage for improved control of the laser ablation. STUDY DESIGN/MATERIALS AND METHODS: We have suggested (1) two-stage targeting method to form clusters of light-absorbing gold NPs selectively in target cells, and (2) the cell damage mechanism through laser-induced generation of vapor bubbles around NP clusters. Experimental investigation of laser nano-thermolysis of leukemia cells was performed using 30 nm spherical gold nanoparticles as a light absorbing agent, and photothermal and fluorescent microscopies as well as flow cytometry as methods to monitor microbubble formation and resulting damage of leukemia cells in human bone marrow specimens. RESULTS: NP clusters were formed and visualized using fluorescence microscopy at cell membranes and in cytoplasm of B-lymphoblasts. Laser irradiation of cells (532 nm, 10 nanoseconds, 0.6 J/cm(2)) induced microbubbles selectively in leukemia cells with large clusters, but not in cells with single NPs or small clusters. Quantitative analysis demonstrated that only 0.1%-1.5% of tumor cells and 77%-84% of normal bone marrow cells survived laser pulse. CONCLUSIONS: Two-stage cell targeting method permits formation of NP clusters selectively in diagnosis-specific tumor cells. The clusters serve as effective sources of photothermally-induced microbubbles, which kill individual target cells after a single laser pulse. The laser fluence threshold for generation of microbubbles is inversely proportional to the volume of NP clusters.

Construction, gene delivery, and expression of DNA tethered nanoparticles.
Prow T, Smith JN, Grebe R, Salazar JH, Wang N, Kotov N, Lutty G, Leary J.
Mol Vis. 2006 May 26;12:606-15.
[ expand abstract ]

PURPOSE: Layered nanoparticles have the potential to deliver any number of substances to cells both in vitro and in vivo. The purpose of this study was to develop and test a relatively simple alternative to custom synthesized nanoparticles for use in multiple biological systems, with special focus on the eye. METHODS: The biotin-labeled transcriptionally active PCR products (TAP) were conjugated to gold, semiconductor nanocrystals, and magnetic nanoparticles (MNP) coated with streptavidin. The process of nanoparticle construction was monitored with gel electrophoresis. Fluorescence microscopy followed by image analysis was used to examine gene expression levels from DNA alone and tethered MNP in human hepatoma derived Huh-7 cells. Adult retinal endothelial cells from both dog (ADREC) and human (HREC) sources were transfected with nanoparticles and reporter gene expression evaluated with confocal and fluorescent microscopy. Transmission electron microscopy was used to quantify the concentration of nanoparticles in a stock solution. Nanoparticles were evaluated for transfection efficiency, determined by fluorescence microscopy cell counts. Cells treated with MNP were evaluated for increased reactive oxygen species (ROS) and necrosis with flow cytometry. RESULTS: Both 5' and 3' biotin-labeled TAP bound equally to MNP and there were no differences in functionality between the two tethering orientations. Free DNA was easily removed by the use of magnetic columns. These particles were also able to deliver genes to a human hepatoma cell line, Huh-7, but transfection efficiency was greater than TAP. The semiconductor nanocrystals and MNP had the highest transfection efficiencies. The MNP did not induce ROS formation or necrosis after 48 h of incubation. CONCLUSIONS: Once transfected, the MNP had reporter gene expression levels equivalent to TAP. The nanoparticles, however, had better transfection efficiencies than TAP. The magnetic nanoparticles were the most easily purified of all the nanoparticles tested. This strategy for bioconjugating TAP to nanoparticles is valuable because nanoparticle composition can be changed and the system optimized quickly. Since endothelial cells take up MNP, this strategy could be used to target neovascularization as occurs in proliferative retinopathies. Multiple cell types were used to test this technology and in each the nanoparticles were capable of transfection. In adult endothelial cells the MNP appeared innocuous, even at the highest doses tested with respect to ROS and necrosis. This technology has the potential to be used as more than just a vector for gene transfer, because each layer has the potential to perform its own unique function and then degrade to expose the next functional layer.

Convection-enhanced delivery of Ls-TPT enables an effective, continuous, low-dose chemotherapy against malignant glioma xenograft model.
Saito R, Krauze MT, Noble CO, Drummond DC, Kirpotin DB, Berger MS, Park JW, Bankiewicz KS.
Neuro-oncol. 2006 May 24; [Epub ahead of print] .
[ expand abstract ]

Treatment of malignant gliomas represents one of the most formidable challenges in oncology. The combination of surgery, radiation, and chemotherapy yields median survivals of less than one year. Here we demonstrate the use of a minimally invasive surgical technique, convection-enhanced delivery (CED), for local administration of a novel nanoparticle liposome containing topotecan. CED of this liposomal topotecan (Ls-TPT) resulted in extended brain tissue retention (t1/2 = 1.5 days), whereas free topotecan was rapidly cleared (t1/2 = 0.1 days) after CED. The favorable pharmacokinetic profile of extended topotecan release for about seven days, along with biodistribution featuring perivascular accumulation of the nanoparticles, provided, in addition to the known topoisomerase I inhibition, an effective antiangiogenic therapy. In the rat intracranial U87MG tumor model, vascular targeting of Ls-TPT with CED was associated with reductions in laminin expression and vascular density compared to free topotecan or control treatments. A single CED treatment on day 7 showed that free topotecan conferred no survival benefit versus control. However, Ls-TPT produced a significant (P = 0.0002) survival benefit, with six of seven complete cures. Larger U87MG tumors, where CED of Ls-TPT on day 12 resulted in one of six cures, indicated the necessity to cover the entire tumor with the infused therapeutic agent. CED of Ls-TPT was also efficacious in the intracranial U251MG tumor model (P = 0.0005 versus control). We conclude that the combination of a novel nanoparticle Ls-TPT and CED administration was very effective in treating experimental brain tumors.

Paclitaxel nanoparticle inhibits growth of ovarian cancer xenografts and enhances lymphatic targeting.
Lu H, Li B, Kang Y, Jiang W, Huang Q, Chen Q, Li L, Xu C.
Cancer Chemother Pharmacol. 2006 May 23; [Epub ahead of print] .
[ expand abstract ]

Objectives: Ovarian cancer has the highest mortality of all the gynecologic cancers. The antitumor agent paclitaxel has been proved to be efficient in the treatment of ovarian cancer. Our study is to develop a polymeric drug delivery system for paclitaxel and determine whether paclitaxel nanoparticle can inhibit growth of ovarian carcinoma xenografts in Fisher344 (F344) rats by intraperitoneal administration. The mechanism of paclitaxel nanoparticles in rats bearing ovarian cancer has been investigated in this study. Methods: Synthesize paclitaxel loading nanoparticle (PLA) by ultrasonic emulsification; MTT analysis identified cytotoxic activity of paclitaxel nanoparticle in vitro; rat ovarian carcinoma cells were injected into the peritoneal cavity of F344 rats. The antitumor effect of paclitaxel nanoparticle in vivo has been evaluated by measuring tumor weight and ascite volume. At the end of the procedure rats were sacrificed; tumors were excised and processed for PCNA staining, tissue terminal deoxynucleotide transferase-mediated dUTP nick and labeling assay and RT-PCR to evaluate the proliferative and apoptotic changes and cancer transfer-related gene expression induced by PLA. Paclitaxel concentration in plasma, pelvic lymph nodes, liver, and heart were determined by high-performance liquid chromatography. Results: Paclitaxel nanoparticle and PTX (Cremophor) showed equivalent cytotoxic activity in vitro. In rats implanted carcinoma cells, paclitaxel nanoparticles significantly reduced tumor weight and ascites volume, and induced apoptosis of tumor cells. PLA also inhibited cell proliferation and matrix metalloproteinase 9 mRNA expression. The paclitaxel concentration of pelvic lymph nodes in PLA treated animals was 20-fold higher than that of free PTX treated animals at 48 h after intraperitoneal administration. Conclusion: The intraperitoneal administration of paclitaxel nanoparticle can significantly inhibit the progression of ovarian carcinoma in peritoneal cavity of female F344 rat. The paclitaxel nanoparticle is safe and lymphatic targeting.

Oligonucleotide-modified gold nanoparticles for intracellular gene regulation.
Rosi NL, Giljohann DA, Thaxton CS, Lytton-Jean AK, Han MS, Mirkin CA.
Science. 2006 May 19;312(5776):1027-30.
[ expand abstract ]

We describe the use of gold nanoparticle-oligonucleotide complexes as intracellular gene regulation agents for the control of protein expression in cells. These oligonucleotide-modified nanoparticles have affinity constants for complementary nucleic acids that are higher than their unmodified oligonucleotide counterparts, are less susceptible to degradation by nuclease activity, exhibit greater than 99% cellular uptake, can introduce oligonucleotides at a higher effective concentration than conventional transfection agents, and are nontoxic to the cells under the conditions studied. By chemically tailoring the density of DNA bound to the surface of gold nanoparticles, we demonstrated a tunable gene knockdown.

Biodistribution and tumor-accumulation of gadolinium (Gd) encapsulated in long-circulating liposomes in tumor-bearing mice for potential neutron capture therapy.
Le UM, Cui Z.
Int J Pharm. 2006 May 17; [Epub ahead of print] .
[ expand abstract ]

To deliver and maintain a sufficient amount of Gd into tumors is required for a successful Gd neutron capture therapy (Gd-NCT), but it has been proven to be rather challenging to achieve. Previously, we have reported a Gd-encapsulated liposome formulation that has the potential to overcome this challenge. In the present study, we sought to systemically evaluate the biodistribution and the tumor-accumulation of the Gd in model tumor-bearing mice. The Gd-encapsulated liposomes were injected into mice pre-grafted with two different model tumors. The Gd content in the tumors and other organs were determined at various time after the injection. A sufficient amount of Gd was readily delivered into those two different model tumors. Increasing the dose of Gd by injecting the Gd-encapsulated liposomes multiple times tended to increase the uptake of the Gd by the tumors. Finally, the uptake of Gd by tumors was inversely correlated with the size of the tumors. The Gd-encapsulated liposomes hold great potentials as a Gd delivery system for NCT of small- and medium-size tumors. Alternative strategies may have to be adopted in order to use NCT to treat large, advanced solid tumors, although for which, Gd-NCT might be advantageous over boron-NCT.

Efficient delivery of a Bcl-2-specific antisense oligodeoxyribonucleotide (G3139) via transferrin receptor-targeted liposomes.
Chiu SJ, Liu S, Perrotti D, Marcucci G, Lee RJ.
J Control Release. 2006 May 15;112(2):199-207; Epub 2006 Mar 6.
[ expand abstract ]

A novel transferrin receptor (TfR)-targeted liposomal formulation was synthesized and evaluated for the delivery of a phosphorothioate antisense oligodeoxyribonucleotide (ODN) (G3139, oblimerson sodium, or Genasensetrade mark) to Bcl-2 in K562 leukemia cells. Liposomes composed of DC-Chol/egg PC/PEG-DSPE (25:73.5:1.5, mol/mol/mol) were loaded with G3139 with high efficiency (70-80%). To prepare targeted liposomes, transferrin was first coupled to PEG-DSPE and then incorporated into the bilayer by post-insertion. The liposomes had a mean diameter of 100 to 150 nm and exhibited colloidal stability for up to 8 weeks. Uptake of Tf-conjugated G3139-containing liposomes in TfR positive K562 cells was found to be more efficient than that of the non-targeted control formulation and could be blocked by excess free Tf. Treatment with Tf-conjugated liposomes resulted in Bcl-2 protein downregulation in K562 cells that was approximately 2-fold greater than with non-targeted liposomes (p<0.05) and 10-fold greater than with free G3139. Treatment with 2 muM G3139 in Tf-conjugated liposomes resulted in >80% reduction in Bcl-2 transcript. In addition, Tf-conjugated liposomal G3139-sensitized K562 cells to daunorubicin, lowering IC(50) from 1.8 muM to 0.18 muM. In conclusion, Tf-conjugated liposomes are effective delivery vehicles for G3139 antisense oligos in TfR positive K562 cells and warrant further investigation as an in vivo oligo delivery vehicle.

Development and in vitro validation of a targeted delivery vehicle for DNA vaccines.
Talsma SS, Babensee JE, Murthy N, Williams IR.
J Control Release. 2006 May 15;112(2):271-9; Epub 2006 Mar 6.
[ expand abstract ]

Usage of DNA vaccination has been limited by inefficient cellular expression of plasmid constructs used in DNA vaccines. We describe a novel system for enhancing delivery of DNA vaccine plasmids into cells and their nuclei. This delivery system uses recombinant reovirus type 3 sigma1 attachment protein genetically modified with a nuclear localization sequence (sigma1-NLS) as a targeting ligand. Purified sigma1-NLS was covalently conjugated to the polycation polyethyleneimine (PEI) using a carboxyl-reactive cross-linking agent and complexed with plasmid DNA. The benefit of the NLS in enhancement of protein delivery into the nucleus was demonstrated by liposome-mediated loading of cells with sigma1 or sigma1-NLS. In L929 fibroblasts loaded with sigma1-NLS, 69% of the internalized protein was recovered in the nuclear fraction after 6 h compared to just 10% when using unmodified sigma1. Transfection of L929 cells with sigma1-NLS-conjugated PEI complexed with a luciferase expression plasmid resulted in a mean 16-fold increase in luciferase activity over complexes made with unmodified PEI, compared to a mean 3-fold boost obtained using sigma1-conjugated PEI. These results suggest that sigma1-NLS is a useful bifunctional targeting ligand suitable for enhancing DNA delivery and subsequent gene expression for both DNA vaccine applications and nonviral gene therapy.

siRNA-containing liposomes modified with polyarginine effectively silence the targeted gene.
Zhang C, Tang N, Liu X, Liang W, Xu W, Torchilin VP.
J Control Release. 2006 May 15;112(2):229-39; Epub 2006 Mar 20.
[ expand abstract ]

Development of RNA interference (RNAi) technology utilizing the short interfering RNA sequences (siRNA) based 'targeted' therapeutics has focused on creating methods for delivering siRNAs to cells and for enhancing siRNA stability in vitro and in vivo. Here, we describe a novel approach for siRNA cellular delivery using siRNA encapsulated into liposomes additionally bearing arginine octamer (R8) molecules attached to their outer surface (R8-liposomes). The R8-liposomal human double minute gene 2 (HDM2)-siRNA demonstrated a significant stability against degradation in the blood serum (siRNA-loaded R8-liposomes remained intact even after 24-h incubation), and higher transfection efficiency into all three tested lung tumor cell lines. siRNA delivery successfully proceeds in the presence of plasma proteins, and R8-liposomes demonstrate low non-specific toxicity. The mechanism of action of R8-liposome-encapsulated siRNA is associated with the RNAi-mediated degradation of the target mRNA. siRNA in R8-liposomes effectively inhibited the targeted gene and significantly reduced the proliferation of cancer cells. The approach offers the potential for siRNA delivery for various in vitro and in vivo applications.

Polyelectrolyte Nanoparticles with High Drug Loading Enhance the Oral Uptake of Hydrophobic Compounds.
Cheng WP, Gray AI, Tetley L, Hang TL, Schatzlein AG, Uchegbu IF.
Biomacromolecules. 2006 May 8;7(5):1509-1520.
[ expand abstract ]

In the pharmaceutical industry, orally active compounds are required to have sufficient water solubility to enable dissolution within the gastrointestinal tract prior to absorption. Limited dissolution within the gastrointestinal tract often reduces the bioavailability of hydrophobic drugs. To improve gastrointestinal tract dissolution, nonaqueous solvents are often used in the form of emulsions and microemulsions. Here, we show that oil-free polyelectrolyte nanosystems (micellar dispersions and 100-300 nm particles) prepared from poly(ethylenimines) derivatized with cetyl chains and quaternary ammonium groups are able to encapsulate high levels of hydrophobic drug (0.20 g of drug per g of polymer) for over 9 months, as demonstrated using cyclosporine A (log P = 4.3). The polyelectrolytes facilitate the absorption of hydrophobic drugs within the gastrointestinal tract by promoting drug dissolution and by a hypothesized mechanism involving paracellular drug transport. Polyelectrolyte nanoparticle drug blood levels are similar to those obtained with commercial microemulsion formulations. The polyelectrolytes do not promote absorption by inhibition of the P-glycoprotein efflux pump.

Water Soluble Nanoparticles from PEG-Based Cationic Hyperbranched Polymer and RNA That Protect RNA from Enzymatic Degradation.
Khan JA, Kainthan RK, Ganguli M, Kizhakkedathu JN, Singh Y, Maiti S.
Biomacromolecules. 2006 May 8;7(5):1386-1388.
[ expand abstract ]

Recent advances in understanding biological systems have proven that RNA is not merely the carrier of genetic information, but also a key molecule in regulation of gene expression and other crucial metabolic processes. Therefore, it is being considered as an ideal therapeutic candidate both for metabolic and genetic disorders. However, research involving RNA molecules faces a practical limitation since RNA is highly labile. We have developed a novel method to protect RNA from cleavage by complexing it with a hyperbranched cationic polymer. It was found that total cellular RNA isolated from yeast spontaneously interacts with the positively charged polymer to form a spherical nanoparticle morphology. This interaction protects the RNA against enzymatic degradation. This methodology can be easily adapted for long-term storage of RNA, long distance transfer of RNA, and genetic engineering using RNA as a building block.

Application of Micro- and Nano-Electromechanical Devices to Drug Delivery.
Staples M, Daniel K, Cima MJ, Langer R.
Pharm Res. 2006 May 5; [Epub ahead of print] .
[ expand abstract ]

Micro- and nano-electromechanical systems (MEMS and NEMS)-based drug delivery devices have become commercially-feasible due to converging technologies and regulatory accommodation. The FDA Office of Combination Products coordinates review of innovative medical therapies that join elements from multiple established categories: drugs, devices, and biologics. Combination products constructed using MEMS or NEMS technology offer revolutionary opportunities to address unmet medical needs related to dosing. These products have the potential to completely control drug release, meeting requirements for on-demand pulsatile or adjustable continuous administration for extended periods. MEMS or NEMS technologies, materials science, data management, and biological science have all significantly developed in recent years, providing a multidisciplinary foundation for developing integrated therapeutic systems. If small-scale biosensor and drug reservoir units are combined and implanted, a wireless integrated system can regulate drug release, receive sensor feedback, and transmit updates. For example, an "artificial pancreas" implementation of an integrated therapeutic system would improve diabetes management. The tools of microfabrication technology, information science, and systems biology are being combined to design increasingly sophisticated drug delivery systems that promise to significantly improve medical care.

In vivo targeting of dendritic cells in lymph nodes with poly(propylene sulfide) nanoparticles.
Reddy ST, Rehor A, Schmoekel HG, Hubbell JA, Swartz MA.
J Control Release. 2006 May 1;112(1):26-34; Epub 2006 Mar 10.
[ expand abstract ]

Delivery of biodegradable nanoparticles to antigen-presenting cells (APCs), specifically dendritic cells (DCs), has potential for immunotherapy. This study investigates the delivery of 20, 45, and 100nm diameter poly(ethylene glycol)-stabilized poly(propylene sulfide) (PPS) nanoparticles to DCs in the lymph nodes. These nanoparticles consist of a cross-linked rubbery core of PPS surrounded by a hydrophilic corona of poly(ethylene glycol). The PPS domain is capable of carrying hydrophobic drugs and degrades within oxidative environments. 20 nm particles were most readily taken up into lymphatics following interstitial injection, while both 20 and 45nm nanoparticles showed significant retention in lymph nodes, displaying a consistent and strong presence at 24, 72, 96 and 120h post-injection. Nanoparticles were internalized by up to 40-50% of lymph node DCs (and APCs) without the use of a targeting ligand, and the site of internalization was in the lymph nodes rather than at the injection site. Finally, an increase in nanoparticle-containing DCs (and other APCs) was seen at 96h vs. 24h, suggesting an infiltration of these cells to lymph nodes. Thus, PPS nanoparticles of 20-45nm have the potential for immunotherapeutic applications that specifically target DCs in lymph nodes.

Quantum Dot-Based Energy Transfer: Perspectives and Potential for Applications in Photodynamic Therapy.
Samia AC, Dayal S, Burda C.
Photochem Photobiol. 2006 May 1; [Epub ahead of print] .
[ expand abstract ]

Quantum dots have emerged as an important class of material that offers great promise to a diverse range of applications ranging from energy conversion to biomedicine. Here, we review the potential of using quantum dots and quantum dot conjugates as sensitizers for photodynamic therapy (PDT). The photophysics of singlet oxygen generation in relation to quantum dot-based energy transfer is discussed and the possibility of using quantum dots as photosensitizer in PDT is assessed, including their current limitations to applications in biological systems. The biggest advantage of quantum dots over molecular photosensitizers that comes into perspective is their tunable optical properties and surface chemistries. Recent developments in the preparation and photophysical characterization of quantum dot energy transfer processes are also presented in this review, to provide insights on the future direction of quantum dot-based photosensitization studies from the viewpoint of our ongoing research.

Nano-fibrous scaffold for controlled delivery of recombinant human PDGF-BB.
Wei G, Jin Q, Giannobile WV, Ma PX.
J Control Release. 2006 May 1;112(1):103-10; Epub 2006 Mar 3.
[ expand abstract ]

The localized and temporally controlled delivery of growth factors is key to achieving optimal clinical efficacy. In sophisticated tissue engineering strategies, the biodegradable scaffold is preferred to serve as both a three-dimensional (3-D) substrate and a growth factor delivery vehicle to promote cellular activity and enhance tissue neogenesis. This study presents a novel approach to fabricate tissue engineering scaffolds capable of controlled growth factor delivery whereby growth factor containing microspheres were incorporated into 3-D scaffolds with good mechanical properties, well-interconnected macroporous and nano-fibrous structures. The microspheres were uniformly distributed throughout the nano-fibrous scaffold and their incorporation did not interfere the macro-, micro-, and nanostructures of the scaffold. The release kinetics of platelet-derived growth factor-BB (PDGF-BB) from microspheres and scaffolds was investigated using poly(lactic-co-glycolic acid) (PLGA50) microspheres with different molecular weights (6.5 and 64kDa, respectively) and microsphere-incorporated poly(l-lactic acid) (PLLA) nano-fibrous scaffolds. Incorporation of microspheres into scaffolds significantly reduced the initial burst release. Sustained release from several days to months was achieved through different microspheres in scaffolds. Released PDGF-BB was demonstrated to possess biological activity as evidenced by stimulation of human gingival fibroblast DNA synthesis in vitro. The successful generation of 3-D nano-fibrous scaffold incorporating controlled-release factors indicates significant potential for more complex tissue regeneration.

Antitumor activity of an Ets protein, PEA3, in breast cancer cell lines MDA-MB-361DYT2 and BT474M1.
Yu Z, Xia W, Wang HY, Wang SC, Pan Y, Kwong KY, Hortobagyi GN, Hung MC.
Mol Carcinog. 2006 May 1; [Epub ahead of print] .
[ expand abstract ]

Polyomavirus enhancer activator 3 (PEA3) is a member of the Ets family of transcription factors. We demonstrated in a previous study that, by downregulating the HER-2/neu oncogene at the transcriptional level, PEA3 can inhibit the growth and development into tumors of HER-2/neu-overexpressing ovarian cancer cells. Here, we establish stable clones of the human breast cancer cell line MDA-MB-361DYT2 that express PEA3 under the control of a tetracycline-inducible promoter. Ectopic expression of PEA3 in this cell line inhibited cell growth and resulted in cell cycle accumulation in the G1 phase. We demonstrate that expression of PEA3 in an orthotopic breast cancer model inhibited tumor growth and prolonged the survival of tumor-bearing mice. In a parallel experiment with another breast cancer cell line, BT474M1, we were unable to obtain stable PEA3-inducible transfectants, suggesting that PEA3 may exert a strong growth inhibition effect in this cell line. Indeed, PEA3 coupled with the liposome SN2 demonstrated therapeutic effects in mice bearing tumors induced by BT474M1. These results provide evidence for the antitumor activity of PEA3 in human breast cancers.

Nanoparticle-aptamer bioconjugates for cancer targeting.
Farokhzad OC, Karp JM, Langer R.
Expert Opin Drug Deliv. 2006 May;3(3):311-324.
[ expand abstract ]

The combination of targeted drug delivery and controlled-release technology may pave the road for more effective yet safer chemotherapeutic options for cancer therapy. Drug-encapsulated polymeric nanoparticle-aptamer bioconjugates represent an emerging technology that can facilitate the delivery of chemotherapeutics to primary and metastatic tumours. Aptamers are short nucleic acid molecules with binding properties and biochemical characteristics that may make them suitable for use as targeting molecules. The goal of this review is to summarise the key components that are required for creating effective cancer targeting nanoparticle-aptamer bioconjugates. The field of controlled release and the structure and properties of aptamers, as well as the criteria for constructing effective conjugates, will be discussed.

ICS-283: a system for targeted intravenous delivery of siRNA.
Schiffelers RM, Storm G.
Expert Opin Drug Deliv. 2006 May;3(3):445-454.
[ expand abstract ]

ICS-283 was developed within Intradigm Corporation as a system that is designed for the systemic delivery of therapeutic small interfering (siRNA) to sites of pathological angiogenesis. The non-viral siRNA delivery system is based on synthetic nanoparticles, known as TargeTrantrade mark (Intradigm Corporation), which functions as a broad-platform technology to deliver siRNA to specific target cells in diseased tissues. The system is constructed to incorporate different functionalities that address critical needs for successful nucleic acid delivery. The TargeTran synthetic vector is a self-assembling, layered nanoparticle that protects and targets siRNA to specific cell types in pathological tissues. At present, ICS-283 is the only antiangiogenic siRNA delivery system that is designed for intravenous administration to treat angiogenesis-driven diseases.

Pharmacokinetic and cytotoxic studies of pegylated liposomal daunorubicin.
Song H, Zhang J, Han Z, Zhang X, Li Z, Zhang L, Fu M, Lin C, Ma J.
Cancer Chemother Pharmacol. 2006 May;57(5):591-8; Epub 2005 Aug 30.
[ expand abstract ]

Pegylated liposomes have been studied for nearly two decades. However, fewer pharmacological studies about its application in daunorubicin (DNR) than those in doxorubicin have been reported. In order to conduct a complete pharmacokinetic study, radiolabeled DNR was encapsulated in pegylated liposomes. Its in vitro drug release kinetics was determined to be in a slow manner, which was reflected in its cytotoxic effect on four cell lines. The lethal dose, plasma pharmacokinetics as well as tissue distribution of the formulation were evaluated in comparison with free DNR. The results revealed that liposomal daunorubicin significantly reduced the toxicity of the drug, with a half lethal dose of 29.35 mg/kg, compared with 5.45 mg/kg for free drug. Pharmacokinetic study of liposomal DNR demonstrated a slower clearance rate, an elevated area under the concentration-time curve, as well as increased half-lives compared to free drug. In addition, an altered tissue distribution of liposomal DNR was observed, with lower cardiac accumulation. Taken together, pegylated liposome-loaded DNR may be a promising anticancer drug and worth further therapeutic study.

Pharmacokinetic and pharmacodynamic evaluation of a novel in situ forming poly(ethylene glycol)-based hydrogel for the controlled delivery of the camptothecins.
Lalloo A, Chao P, Hu P, Stein S, Sinko PJ.
J Control Release. 2006 Apr 29; [Epub ahead of print] .
[ expand abstract ]

Inadequate drug delivery, due to problems associated with achieving constant therapeutic blood levels, has hampered the use of anticancer agents of the camptothecin (CPT) class. The objective of the current studies was to develop a depot delivery system for the water-soluble analog of CPT, topotecan (TPT). In this study, a 2-phase drug depot consisting of TPT-loaded liposomes entrapped in a poly(ethylene glycol) hydrogel was designed. Physically entrapped unaltered TPT displayed a rapid release rate from the hydrogel. Controlled release was demonstrated in vitro and in vivo from the 2-phase system with constant blood levels being achieved for several days in rats. Cytotoxicity and antitumor activity were also evaluated in rats inoculated with syngeneic MAT B III breast cancer cells. Rats treated with the liposome-loaded hydrogel displayed significantly longer tumor growth suppression and did not exhibit body weight loss compared to those treated with other delivery modes. These experiments constitute a proof-of-principle of the 2-phase depot concept and its potential value for enhancing safety and efficacy in chemotherapy.

In vitro enhanced cytotoxicity of tumor-infiltrating lymphocytes transfected with tumor necrosis factor-related apoptosis-inducing ligand and/or interleukin-2 gene in human renal cell carcinoma.
Tian JQ, Wang ZP, Rodriguez R, Fu JS, Lu JZ, Ma BL.
Urology. 2006 Apr 22; [Epub ahead of print] .
[ expand abstract ]

OBJECTIVES: To investigate whether tumor-infiltrating lymphocytes (TILs) transfected with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and interleukin-2 (IL-2) genes are capable of improving the potency and efficacy of propagation and cytotoxicity against renal cell carcinoma (RCC) cells in vitro. METHODS: A mammal expression vector system was constructed. TILs were transfected by liposome-mediated gene transfection. The degree of cytokine mRNA expression was evaluated with Northern blot. Protein expression was determined with Western blot and enzyme-linked immunosorbent assay. Cytotoxicity of TILs against autologous RCC cells and the human RCC cell line (786-0) were examined by chromium release assay. Flow cytometric analyses were performed to determine the apoptosis of tumor cells. RESULTS: A high level of expression of the human TRAIL and IL-2 stable transfected TILs was observed. The mean IL-2 production was 22.6 +/- 5.2, 507.7 +/- 52.4, and 549.0 +/- 74.0 ng/10(6) cells/24 hours in the TIL/parental, TIL/IL-2, and TIL/TRAIL+IL-2 genes, respectively. The mean cytotoxicity (effector/target ratio 20:1) of TIL/parental, TIL/IL-2, TIL/TRAIL, and TIL/TRAIL+IL-2 against autologous RCC cells in the percentage of cytolysis was 21.2% +/- 4.8%, 32.1% +/- 5.5%, 63.5% +/- 6.6%, and 78.1% +/- 9.63%, respectively. These four groups showed cytotoxic activity against allogeneic 786-0 RCC cells; the corresponding values were 9.8% +/- 3.5%, 12.3% +/- 3.4%, 24.1% +/- 4.9%, and 30.4% +/- 6.2%. The number of apoptotic cells was significantly greater for autologous RCC cells than for 786-0 cells after TIL/TRAIL and TIL/TRAIL+IL-2 treatment. CONCLUSIONS: TIL/TRAIL+IL-2 and TIL/IL-2 were expanded by autocrine IL-2. TIL/TRAIL+IL-2 and TIL/TRAIL showed significant cytotoxicity that was induced by TRAIL. TILs, including parental TILs and transfected TILs, demonstrated a potent cytotoxicity against RCC cells with remarkable selectivity. Autologous RCC cells seemed more sensitive than allogeneic RCC cells.

Antiangiogenesis and damaging blood flow by antisense vascular endothelial growth factor oligodeoxynucleotides to suppress lung cancers.
Li C, Cheng X, Jiang H, Sun X.
Tumour Biol. 2006;27(3):158-65; Epub 2006 Apr 20.
[ expand abstract ]

Angiogenesis plays a key role in the growth and metastasis of lung cancers, and vascular endothelial growth factor (VEGF) is one of the major angiogenic factors. The study aims to investigate whether phosphoro thioate-modified antisense VEGF oligodeoxynucleo tides (ASODN) formulated in cationic liposome could inhibit the growth of Lewis lung carcinoma (LLC) tumors by antiangiogenesis. The study demonstrated that ASODN downregulated the expression of VEGF in LLC cells at levels of protein and mRNA in vitro and in vivo. The conditioned media obtained from LLC cells treated with ASODN significantly inhibited the proliferation of bovine aortic endothelial cells. The ASODN therapy significantly suppressed the growth of established subcutaneous LLC tumors in mice by inhibiting angiogenesis and damaging the blood flow of tumors. In conclusion, our results suggest that ASODN targeting VEGF presents a potent therapeutic strategy to combat lung cancers.

Eradication of established HPV 16-expressing tumors by a single administration of a vaccine composed of a liposome-encapsulated CTL-T helper fusion peptide in a water-in-oil emulsion.
Daftarian P, Mansour M, Benoit AC, Pohajdak B, Hoskin DW, Brown RG, Kast WM.
Vaccine. 2006 Apr 18; [Epub ahead of print] .
[ expand abstract ]

Human papillomavirus (HPV)-induced cervical cancer is the second most common cancer among women worldwide with half a million new cases per year. Despite the encouraging development of a preventive vaccine for HPV, a therapeutic vaccine for cervical cancer or pre-cancerous lesions remains a high priority. The preclinical study reported here used VacciMax((R)) (VM) to deliver a peptide-based vaccine composed of an HPV 16 E7-derived cytotoxic T lymphocyte (CTL) epitope fused to the T helper epitope PADRE (FP) and combined with CpG or lipopeptide adjuvant. In the study, C57BL/6 mice received 0.5million HPV 16-expressing C3 tumor cells. Mice were inoculated post-tumor challenge with a single s.c. injection of FP-CpG-VM on either day 4, 5, 6, 9, or 14. All mice that received the FP-CpG-VM vaccine were tumor-free to day 130 when the experiment was terminated. In contrast, only a minority of mice that received a control vaccine were tumor-free on day 60. Cytotoxicity assays, ELISPOT and intracellular staining for interferon (IFN)-gamma showed the immune response was specific for the selected CTL epitope. All mice that received the FP-CpG-VM vaccine remained tumor-free when re-challenged with 6million C3 cells. Cytotoxicity assays 4 months post-challenge showed that only splenocytes from mice inoculated with the FP-CpG-VM vaccine had high lysis activity. These results indicate that VacciMax((R)) causes a rapid, robust, durable and therapeutic CTL response to HPV 16 E7 protein expressing tumors.

Targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo.
Farokhzad OC, Cheng J, Teply BA, Sherifi I, Jon S, Kantoff PW, Richie JP, Langer R.
Proc Natl Acad Sci U S A. 2006 Apr 18;103(16):6315-20; Epub 2006 Apr 10.
[ expand abstract ]

Targeted uptake of therapeutic nanoparticles in a cell-, tissue-, or disease-specific manner represents a potentially powerful technology. Using prostate cancer as a model, we report docetaxel (Dtxl)-encapsulated nanoparticles formulated with biocompatible and biodegradable poly(D,L-lactic-co-glycolic acid)-block-poly(ethylene glycol) (PLGA-b-PEG) copolymer and surface functionalized with the A10 2'-fluoropyrimidine RNA aptamers that recognize the extracellular domain of the prostate-specific membrane antigen (PSMA), a well characterized antigen expressed on the surface of prostate cancer cells. These Dtxl-encapsulated nanoparticle-aptamer bioconjugates (Dtxl-NP-Apt) bind to the PSMA protein expressed on the surface of LNCaP prostate epithelial cells and get taken up by these cells resulting in significantly enhanced in vitro cellular toxicity as compared with nontargeted nanoparticles that lack the PSMA aptamer (Dtxl-NP) (P < 0.0004). The Dtxl-NP-Apt bioconjugates also exhibit remarkable efficacy and reduced toxicity as measured by mean body weight loss (BWL) in vivo [body weight loss of 7.7 +/- 4% vs. 18 +/- 5% for Dtxl-NP-Apt vs. Dtxl-NP at nadir, respectively (mean +/- SD); n = 7]. After a single intratumoral injection of Dtxl-NP-Apt bioconjugates, complete tumor reduction was observed in five of seven LNCaP xenograft nude mice (initial tumor volume of approximately 300 mm3), and 100% of these animals survived our 109-day study. In contrast, two of seven mice in the Dtxl-NP group had complete tumor reduction with 109-day survivability of only 57%. Dtxl alone had a survivability of only 14%. Saline and nanoparticles without drug were similarly nonefficacious. This report demonstrates the potential utility of nanoparticle-aptamer bioconjugates for a therapeutic application.

Targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo.
Farokhzad OC, Cheng J, Teply BA, Sherifi I, Jon S, Kantoff PW, Richie JP, Langer R.
Proc Natl Acad Sci USA. 2006 Apr 10; [Epub ahead of print] .
[ expand abstract ]

Targeted uptake of therapeutic nanoparticles in a cell-, tissue-, or disease-specific manner represents a potentially powerful technology. Using prostate cancer as a model, we report docetaxel (Dtxl)-encapsulated nanoparticles formulated with biocompatible and biodegradable poly(D,L-lactic-co-glycolic acid)-block-poly(ethylene glycol) (PLGA-b-PEG) copolymer and surface functionalized with the A10 2'-fluoropyrimidine RNA aptamers that recognize the extracellular domain of the prostate-specific membrane antigen (PSMA), a well characterized antigen expressed on the surface of prostate cancer cells. These Dtxl-encapsulated nanoparticle-aptamer bioconjugates (Dtxl-NP-Apt) bind to the PSMA protein expressed on the surface of LNCaP prostate epithelial cells and get taken up by these cells resulting in significantly enhanced in vitro cellular toxicity as compared with nontargeted nanoparticles that lack the PSMA aptamer (Dtxl-NP) (P < 0.0004). The Dtxl-NP-Apt bioconjugates also exhibit remarkable efficacy and reduced toxicity as measured by mean body weight loss (BWL) in vivo [body weight loss of 7.7 +/- 4% vs. 18 +/- 5% for Dtxl-NP-Apt vs. Dtxl-NP at nadir, respectively (mean +/- SD); n = 7]. After a single intratumoral injection of Dtxl-NP-Apt bioconjugates, complete tumor reduction was observed in five of seven LNCaP xenograft nude mice (initial tumor volume of approximately 300 mm(3)), and 100% of these animals survived our 109-day study. In contrast, two of seven mice in the Dtxl-NP group had complete tumor reduction with 109-day survivability of only 57%. Dtxl alone had a survivability of only 14%. Saline and nanoparticles without drug were similarly nonefficacious. This report demonstrates the potential utility of nanoparticle-aptamer bioconjugates for a therapeutic application.

Induction of cancer cell-specific apoptosis by folate-labeled cationic liposomes.
Yoshida T, Oide N, Sakamoto T, Yotsumoto S, Negishi Y, Tsuchiya S, Aramaki Y.
J Control Release. 2006 Apr 10;111(3):325-32; [Epub 2006 Feb 14].
[ expand abstract ]

We have previously reported that cationic liposomes themselves can induce apoptosis in macrophages and lymphocytes. In this paper, we attempted the cancer cell-specific delivery of cationic liposomes and the induction of apoptosis utilizing this characteristic. Cationic liposomes composed of stearylamine (SA-liposomes) induced apoptosis in human nasopharyngeal epidermoid carcinoma cells (KB cells) overexpressing the folate receptor and human fibroblasts (WI-38 cells) with no folate receptor, without showing selectivity. To recruit liposomes to cancer cells and induce apoptosis, we focused on the folate receptor and prepared folic acid-labeled liposomes using polyethyleneglycol (PEG) (folate-PEG-liposomes). Folate-PEG-liposomes showed selectivity and induced apoptosis in KB cells, but not WI-38 cells. The apoptosis occurred in a dose-dependent manner. Furthermore, folate-PEG-liposomes appear to associate with KB cells via the folate receptor, whereas SA-liposomes may associate with cells through electrostatic interactions. To confirm the contribution of the folate receptor to apoptosis of KB cells induced by folate-PEG-liposomes, the effect of folic acid on the apoptosis was examined. The addition of free folic acid drastically suppressed the apoptosis of KB cells and the percentage of cells with hypodiploid nuclei returned to the control level. Taken together, cationic liposomes labeled with folate bound to KB cells via folate receptors and, interestingly, the cationic liposomes themselves could cause apoptosis in cancer cells.

Long-circulating gadolinium-encapsulated liposomes for potential application in tumor neutron capture therapy.
Le UM, Cui Z.
Int J Pharm. 2006 Apr 7;312(1-2):105-12; [Epub 2006 Feb 2].
[ expand abstract ]

Gadolinium neutron capture therapy (Gd-NCT) is a promising cancer therapy modality. One of the key factors for a successful Gd-NCT is to deliver and maintain a sufficient amount of Gd in tumor tissues during neutron irradiation. We proposed to prepare a Gd delivery system by complexing a Gd-containing compound, diethylenetriaminepentaacetic acid (Gd-DTPA), with a polycationic peptide, poly-l-lysine (pLL), and then encapsulate the complexed Gd-DTPA into PEGylated liposomes. Complexation of Gd-DTPA with pLL not only enhanced the encapsulation efficiency of Gd-DTPA in liposomes, but also significantly limited the release of Gd-DTPA from the liposomes. A Gd-DTPA-encapsulated liposome formulation that contained 6.8+/-0.3mg/mL of pure encapsulated Gd was prepared. The blood half-life of the Gd encapsulated into the liposome formulation was estimated to be about 24h in healthy tumor-free mice. About 12h after the Gd-encapsulated liposomes were intravenously injected into mice with pre-established model tumors, the Gd content in the tumors reached an average of 159mug/g of wet tumor tissue. This Gd-DTPA encapsulated liposome may be used to deliver Gd into solid tumors for NCT and tumor imaging.

Characterization and cytotoxicity of mixed polyethyleneglycol modified liposomes containing doxorubicin.
Sadzuka Y, Sugiyama I, Tsuruda T, Sonobe T.
Int J Pharm. 2006 Apr 7;312(1-2):83-9; [Epub 2006 Feb 2].
[ expand abstract ]

Liposomes are recognized as one of the useful drug carriers, but have many problems to overcome before their clinical application. Liposomes, bonding peculiarly with serum protein (opsonization), are taken up by reticuloendothelial system (RES) cells in the liver and spleen. It is known that polyethyleneglycol (PEG) modification of the liposome surface induces the formation of a fixed aqueous layer around the liposomes due to the interaction between the PEG-polymer and water molecule, and thus prevents the attraction of opsonins. Namely, PEG-modified liposomes are able to escape trapping by the RES cells, and have a prolonged circulation time. In this study, the effects of different anchors with the same PEG molecular weight on the cell uptake and cytotoxicity of mixed PEG-modified liposomal doxorubicin (DOX) were examined. The fixed aqueous layer thickness (FALT) of liposomes covered with mixtures of PEG-molecules which differ in their chain length were increased, compared to that of the single PEG2000-modified liposome. Mixed PEG-modification of liposomes with different anchors (PEG2000-(1-monomethoxypolyethyleneglycol-2,3-distearoylglycerol (DSG): cholesterol (CHO)=1:1)-modified liposome) led to an increase in the FALT, compared to that of each single PEG-modification. The uptake of DOX into Ehrlich ascites carcinoma cells by the liposomes covered with PEG-CHO was higher than the other liposomes. Thus, liposomes covered with PEG-DSG and PEG-CHO have an enhanced cytotoxicity. In conclusion, it was confirmed that mix-modified liposomes using PEG-lipid with different anchors were superior.

The in vitro inhibition of multidrug resistance by combined nanoparticulate titanium dioxide and UV irradition.
Song M, Zhang R, Dai Y, Gao F, Chi H, Lv G, Chen B, Wang X.
Biomaterials. 2006 Apr 3; [Epub ahead of print] .
[ expand abstract ]

The appearance of drug-resistant (especially, multidrug-resistant (MDR)) tumor cells is a major obstacle to the success of chemotherapy; thus, the development of effective anti-MDR agents plays an important role in the tumor therapy. In this report, the considerable effect of nano-TiO(2) and UV illumination on the drug resistance of target cancer cells has been explored, and the fresh evidence from the fluorescence spectroscopy and microscopy as well as electrochemical studies demonstrates the significant enhancement effect of nano-TiO(2) to the drug uptake by drug-resistant leukemia cells. Besides, it is also observed that the combination of the nano-TiO(2) and UV irradiation with the accompanying anticancer drug daunorubicin could provoke some considerable changes of the cell membrane of the target leukemia cells, which indicates that nano-TiO(2) could not only increase the drug accumulation in target cancer cells, but also act as an effective anti-MDR agent to inhibit the relative drug resistance.

Immunogenic and structural properties of the Asn-Gly-Arg (NGR) tumor neovasculature-homing motif.
Di Matteo P, Curnis F, Longhi R, Colombo G, Sacchi A, Crippa L, Protti MP, Ponzoni M, Toma S, Corti A.
Mol Immunol. 2006 Apr;43(10):1509-18; [Epub 2005 Dec 7].
[ expand abstract ]

Tumor homing peptides containing the NGR motif, such as CNGRC and GNGRG, have been used for delivering cytokines, chemotherapeutic drugs, apoptotic peptides, and liposomes to a CD13 isoform expressed in tumor blood vessels. In view of the potential clinical applications of these drugs and considering the risk that NGR peptides could elicit blocking antibodies we have investigated the immunogenic properties of CNGRC and GNGRG in mice and rabbits, using various products containing these residues and different administration schedules. The results suggest that the immunogenicity of the NGR motif is very low, even when it is conjugated to tumor necrosis factor-alpha or to highly immunogenic carrier proteins. Molecular dynamics simulation experiments showed that both peptides have a strong propensity to populate a turn conformation. Superposition of predicted structures to the CTGNGRGEWKC loop of the 5th type I repeat of human fibronectin, a protein that contains four NGR motives, showed that the root mean square deviation of backbones was 0.7A for GNGRG and 0.5A for NGR. These results suggest that NGR peptides could mimic from an immunological point of view a "self" structure, likely the GNGRG loop of fibronectin, with important implications for the use of these targeting peptides in patients.

Delivery of liposomal Doxorubicin (doxil) in a breast cancer tumor model: investigation of potential enhancement by pulsed-high intensity focused ultrasound exposure.
Frenkel V, Etherington A, Greene M, Quijano J, Xie J, Hunter F, Dromi S, Li KC.
Acad Radiol. 2006 Apr;13(4):469-79.
[ expand abstract ]

RATIONALE AND OBJECTIVES: To investigate the potential of using pulsed high-intensity focused ultrasound (HIFU) exposures to enhance the delivery, and hence therapeutic effect of liposomal doxorubicin (Doxil) in a murine breast cancer tumor model. MATERIALS AND METHODS: Tumors were grown in the bilateral flanks of mice using a mammary adenocarcinoma cell line. Experiments consisted of exposing one of two tumors to pulsed-HIFU, followed by tail vein injections of Doxil. Tumor growth rates were monitored, and assays carried out for doxorubicin concentration in these tumors as well as in a second (squamous cell carcinoma) tumor model and in muscle. Laser scanning confocal microscopy was used with fluorescent probes to observe both the uptake of polystyrene nanoparticles and dilation of exposed blood vessels. Additional experiments involving histologic analysis and real-time temperature measurements were performed to determine the safety of the exposures. RESULTS: Pulsed-HIFU exposures were shown to be safe, producing no apparent deleterious effects in the tumors. The exposures, however, were not found to enhance the delivery of Doxil, and consequently did not allow for lower doses for obtaining tumor regression. Imaging with a fluorescent dextran showed blood vessels to be dilated as a result of the exposures. Experiments with polystyrene nanoparticles of similar size to the liposomes showed a greater abundance to be present in the treated tumors. CONCLUSION: Although past studies have shown the advantages of pulsed-HIFU exposures for enhancing delivery, this was not observed with the liposomes, apparently because of their inherent ability to preferentially accumulate into tumors on their own. Potential mechanisms for enhanced uptake of non-liposomal nanoparticles are discussed.

Interleukin-2/liposomes potentiate immune responses to a soluble protein cancer vaccine in mice.
Johnston D, Reynolds SR, Bystryn JC.
Cancer Immunol Immunother. 2006 Apr;55(4):412-9; [Epub 2005 Sep 6].
[ expand abstract ]

A critical element in improving the potency of cancer vaccines, especially pure protein or peptide antigens, is to develop procedures that can strongly but safely increase their ability to induce immune responses. Here, we describe that encapsulation of a pure protein antigen and interleukin-2 (IL-2) together into liposomes significantly improves immune responses and tumor protection. Groups of C57Bl/6 mice were immunized weekly x4 with -0.1 mg of ovalbumin (OVA) injected subcutaneously in PBS or encapsulated in liposomes with or without human recombinant IL-2. Control groups included mice immunized to irradiated E.G7-OVA cells (that express ovalbumin), or to PBS. Sera were collected and pooled by immunization group at baseline and at weeks 2 and 4 to measure antibody responses to OVA by ELISA. Splenocytes obtained at week 4 were tested for anti-OVA cellular responses by ELISPOT. Mice were then challenged to a lethal dose of E.G7-OVA cells to measure tumor-protective immunity. IL-2 liposomes caused no detectable toxicity. Antibody, CD8(+) T cell, and tumor-protective immune responses were markedly enhanced in mice immunized to OVA + IL-2 in liposomes compared to mice immunized to OVA, either alone or encapsulated into liposomes without IL-2. These results indicate that IL-2 liposomes enhance antibody, cellular, and tumor-protective immune responses to immunization with a soluble protein. This may provide a simple, safe, and effective way to enhance the immunogenicity of vaccines that consist of pure protein antigens.

Novel biodegradable HSAM nanoparticle for drug delivery.
Liu B, Jiang S, Zhang W, Ye F, Wang YH, Wu J, Zhang DY.
Oncol Rep. 2006 Apr;15(4):957-61.
[ expand abstract ]

The systemic pharmacological treatment of disease is limited by severe toxicity to normal organs/tissue. Therefore, various delivery vehicles have been designed to carry therapeutic drugs to their target tissues. We designed a novel vehicle formed by the interaction of biotins in a DNA (polymer) with avidins (crosslink), resulting in a porous particle. This self-assembled (HSAM) nanoparticle vehicle has been tested in our laboratory both in vitro and in vivo for its ability to carry doxorubicin, a widely used anticancer drug with a high toxicity to normal organs. Doxorubicin binds to the nanoparticle by intercalating into the DNA strands that are later degraded by nucleases released from cancer cells. Our results showed that 1.1 microg of HSAM DNA can carry 1 microg of doxorubicin, and the doxorubicin-bound HSAM nanoparticle can still be degraded by nucleases (BAL-31 and DNase I). The HSAM nanoparticle carrying doxorubicin can efficiently inhibit cancer cell growth in vitro and in a murine model. Furthermore, this nanoparticle is able to deliver up to 180 ng/mg of doxorubicin to the target tumor tissue, which is 15-fold above the systemic toxicity dose (12 mg/kg). These results suggest that the HSAM nanoparticle is both biocompatible and biodegradable, making it a valuable vehicle for drug delivery in cancer treatment.

Significant and prolonged antisense effect of a multifunctional envelope-type nano device encapsulating antisense oligodeoxynucleotide.
Nakamura Y, Kogure K, Yamada Y, Futaki S, Harashima H.
J Pharm Pharmacol. 2006 Apr;58(4):431-7.
[ expand abstract ]

A multifunctional envelope-type nano device (MEND) was developed for use as an efficient non-viral system for the delivery of plasmid DNA (pDNA) using octaarginine (R8) as an internalizing ligand. Three types of R8-MENDs were prepared, co-encapsulating luciferase-encoding pDNA and anti-luciferase oligodeoxynucleotide (ODN) condensed by three polycations, stearyl octaarginine (STRR8), poly-L-lysine (PLL) and protamine, and the antisense effects of the ODN-encapsulated R8-MENDs (ODN-MEND) were analysed in-vitro. The ODN-MEND packaged using protamine as a condenser showed a 90% antisense effect 16 h after the transfection, and a persistent antisense effect of over 75% for up to 48 h, which was much more effective than that of LipofectAmine2000. On the other hand, the ODN-MENDs prepared using PLL and STR-R8 as condensers did not show any significant inhibition of luciferase activity. Although there was no specific relation between the physicochemical characteristics of the ODN-MENDs and their antisense effect, the pattern of the antisense effect among the ODN-MENDs was similar to that of the silencing effect of R8-MEND encapsulating plasmid DNA encoding siRNA. These results suggest that R8-MENDs are able to deliver encapsulated DNA to the cytosol as well as to the nucleus, and that protamine can also function as an efficient decondenser, not only in the nucleus but also in the cytosol. In conclusion, we successfully developed an ODN-MEND with a high antisense effect using protamine as a DNA condensing as well as a decondensing agent.

Pharmacokinetics and phototoxicity of purpurin-18 in human colon carcinoma cells using liposomes as delivery vehicles.
Sharma S, Dube A, Bose B, Gupta PK.
Cancer Chemother Pharmacol. 2006 Apr;57(4):500-6; [Epub 2005 Aug 2].
[ expand abstract ]

Pharmacokinetics and phototoxicity of purpurin-18 (Pp18) in human colon carcinoma cells (Colo-205) was studied using liposomes as delivery vehicles. Cytotoxicity was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and neutral red uptake assay, and mode of cell death was assessed by the study of cell morphology and nuclear staining with Hoechst 33342-propidium iodide. Pp18 solubilized in dimethyl sulfoxide saline solution was observed to aggregate (Q-band absorption 740 nm), resulting in very poor cellular uptake. Pp18 incorporated in liposome remained in monomeric form (Q-band absorption 695 nm), but due to the presence of an anhydride ring in the molecule it readily yielded another photosensitizer, chlorin p6 (Q-band absorption 662 nm). Measurements at various pH showed that Pp18 in liposome was stable at acidic pH (6.5). Incubation of cells with 6.0 microM Pp18 in liposome at pH 6.5 showed a rapid cellular uptake. Spectrofluorometric measurements showed the presence of both Pp18 and chlorin p6, indicating conversion of some amount of Pp18 into chlorin p6 in the cells. Fluorescence microscopy revealed that the fluorescence was localized mainly in the cytoplasm, sparing the nucleus. Illumination of cells to white light after 4-h incubation with Pp18 liposome preparation was observed to lead to dose-dependent decrease in cell viability. At low irradiation time, cells displayed formation of plasma membrane blebs and micronuclei typical of apoptotic cell death. In contrast, at higher irradiation time, cell swelling and vacuolization in nucleus was observed, suggesting cell death due to necrosis. Irradiation with narrow bandwidth light showed that at low pH, the relative phototoxicity due to pp18 was higher than that due to chlorin p6. It is suggested that the pH-dependent conversion of pp18 to chlorin p6 can be exploited to increase PDT selectivity.

Potential use of drug carried-liposomes for cancer therapy via direct intratumoral injection.
Bao A, Phillips WT, Goins B, Zheng X, Sabour S, Natarajan M, Ross Woolley F, Zavaleta C, Otto RA.
Int J Pharm. 2006 Mar 29; [Epub ahead of print] .
[ expand abstract ]

Liposomes have recognized advantages as nano-particle drug carriers for tumor therapy. In this study, the pharmacokinetics and distribution of intratumorally administered liposomes were investigated as drug carriers for treating solid tumors via direct intratumoral administration. (99m)Tc-liposomes were administered intratumorally to nude rats bearing human head and neck squamous cell carcinoma xenografts. Planar gamma camera images were analyzed to evaluate the local retention of the intratumorally administered liposomes. Co-registered pinhole micro-single photon emission computed tomography (SPECT)/computed tomography (CT) images were acquired of the whole animal as well as the dissected tumors to determine intratumoral distribution of the (99m)Tc-liposomes. For (99m)Tc-liposomes, there was an initial retention of 47.4+/-11.0% (n=4) in tumors and surrounding tissues. At 20h, 39.2+/-10.6% (n=4) of (99m)Tc-activity still remained in the tumor. In contrast, only 18.7+/-3.3% (n=3) of the intratumoral (99m)Tc-activity remained for unencapsulated (99m)Tc-complex at 20h. Pinhole micro-SPECT images demonstrated that (99m)Tc-liposomes also have a superior intratumoral (99m)Tc-activity diffusion compared with unencapsulated (99m)Tc-complex. Higher intratumoral retention of (99m)Tc-liposomes accompanied by an improved intratumoral diffusion suggests that intratumorally administered liposomal drugs are potentially promising agents for solid tumor local therapy.

A columnar phase of dendritic lipid-based cationic liposome-DNA complexes for gene delivery: hexagonally ordered cylindrical micelles embedded in a DNA honeycomb lattice.
Ewert KK, Evans HM, Zidovska A, Bouxsein NF, Ahmad A, Safinya CR.
J Am Chem Soc. 2006 Mar 29;128(12):3998-4006.
[ expand abstract ]

Gene therapy holds great promise as a future approach to fighting disease and is explored in worldwide clinical trials. Cationic liposome (CL)-DNA complexes are a prevalent nonviral delivery vector, but their efficiency requires improvement and the understanding of their mechanism of action is incomplete. As part of our effort to investigate the structure-transfection efficiency relationships of self-assembled CL-DNA vectors, we have synthesized a new, highly charged (16+) multivalent cationic lipid, MVLBG2, with a dendritic headgroup. Our synthetic scheme allows facile variation of the headgroup charge and the spacer connecting hydrophobic and headgroup moieties as well as gram-scale synthesis. Complexes of DNA with mixtures of MVLBG2 and neutral 1,2-dioleoyl-sn-glycerophosphatidylcholine (DOPC) exhibit the well-known lamellar phase at 90 mol % DOPC. Starting at 20 mol % dendritic lipid, however, two novel nonlamellar phases are observed by synchrotron X-ray diffraction. The structure of one of these phases, present in a narrow range of composition around 25 mol % MVLBG2, has been solved. In this novel dual lattice structure, termed H(I)C, hexagonally arranged tubular lipid micelles are surrounded by DNA rods forming a three-dimensionally continuous substructure with honeycomb symmetry. Complexes in the H(I)C phase efficiently transfect mouse and human cells in culture. Their transfection efficiency, as well as that of the lamellar complexes containing only 10 mol% dendritic lipid, reaches and surpasses that of commercially available, optimized DOTAP-based complexes. In particular, complexes containing MVLBG2 are significantly more transfectant over the entire composition range in mouse embryonic fibroblasts, a cell line empirically known to be hard to transfect.

Intracellular uptake and release of poly(ethyleneimine)-co-poly(methyl methacrylate) nanoparticle/pDNA complexes for gene delivery.
Feng M, Lee D, Li P.
Int J Pharm. 2006 Mar 27;311(1-2):209-14; [Epub 2006 Jan 25].
[ expand abstract ]

Our previous studies demonstrated that cationic nanoparticles composed of well-defined poly(methyl methacrylate) (PMMA) cores surrounded by a hairly poly(ethyleneimine) (PEI) shells have comparative advantages over the PEI system for gene delivery. In this study, we focused on the intracellular uptake and release of PEI-PMMA nanoparticle/pDNA complexes. The behavior of the nanoparticle/pDNA complexes in recipient cells was monitored by using confocal laser scanning microscopy. We found that the nanoparticle/pDNA complexes were internalized very effectively by endocytosis. In the recipient cells the nanoparticles were found localized in the cytoplasm. At the same time, the pDNA carried by the nanoparticles successfully detached from the nanoparticles and localized in the nucleus of the HeLa cells.

The drug encapsulation efficiency, in vitro drug release, cellular uptake and cytotoxicity of paclitaxel-loaded poly(lactide)-tocopheryl polyethylene glycol succinate nanoparticles.
Zhang Z, Feng SS.
Biomaterials. 2006 Mar 23; [Epub ahead of print] .
[ expand abstract ]

Paclitaxel is one of the most effective antineoplastic drugs. Its current clinical administration is formulated in Cremophor EL, which causes serious side effects. Nanoparticle (NP) technology may provide a solution for such poisonous adjuvant problems and promote a sustained chemotherapy, in which biodegradable polymers play a key role. Our group has successfully synthesized novel poly(lactide)-tocopheryl polyethylene glycol succinate (TPGS) (PLA-TPGS) copolymers of desired hydrophobic-hydrophilic balance for NP formulation of anticancer drugs. The present work is focused on effects of the PLA:TPGS composition ratio on drug encapsulation efficiency, in vitro drug release, in vitro cellular uptake and viability of the PLA-TPGS NP formulation of paclitaxel. The PLA-TPGS copolymers of various PLA:TPGS ratios were synthesized by the ring-opening polymerization method and characterized by GPC and (1)H NMR for their molecular structure. Paclitaxel-loaded PLA-TPGS NPs were prepared by a modified solvent extraction/evaporation method and characterized by laser light scattering for size and size distribution, scanning electron microscopy for surface morphology and zeta potential for surface charge. High performance liquid chromatography was used to measure the drug encapsulation efficiency and in vitro drug release profile. Cancer cell lines HT-29 and Caco-2 were used to image and measure the cellular uptake of fluorescent PLA-TPGS NPs. Cancer cell viability of the drug-loaded PLA-TPGS was measured by MTT assay. It was found that the PLA:TPGS composition ratio has little effects on the particle size and size distribution. However, the PLA-TPGS NPs of 89:11 PLA:TPGS ratio achieved the best effects on the drug encapsulation efficiency, the cellular uptake and the cancer cell mortality of the drug-loaded PLA-TPGS NPs. This research was also carried out in close comparison with the drug-loaded PLGA NPs.

Effects of siRNAs in combination with Gleevec on K-562 cell proliferation and Bcr-Abl expression.
Baker BE, Kestler DP, Ichiki AT.
J Biomed Sci. 2006 Mar 18; [Epub ahead of print] .
[ expand abstract ]

RNA interference (RNAi) is the repression of gene expression through a cellular mechanism of transcript-specific mRNA degradation. RNAi has been observed in human cells and applied to the modulation of a variety of human transcripts. Our goals were to deliver small interfering RNA (siRNA) using a liposome-based method, and to show Bcr-Abl siRNA specificity against K-562 cells, alone or in combination with Gleevec. Both synthetic (syn) siRNA, consisting of homogeneous 21-nucleotide-long RNA duplexes specific for the Bcr-Abl fusion site, and recombinant (r)-generated Bcr-Abl siRNA were employed. siRNA was transfected into K-562 cells with greater than 90% efficiency using RNAiFecttrade mark, as judged by fluorescence analysis. The Bcr-Abl transcript was inhibited using either siRNA preparation as measured by RT-PCR or real-time PCR. The IC(50) of Gleevec in the K-562 subline F(1) was lowered over 3-fold from 0.2 to 0.06 muM in cells transfected with either syn or rBcr-Abl siRNA. No effect was observed in cells after transfection with an irrelevant control siRNA. Therefore, K-562 cells transfected with RNAifect deliver Bcr-Abl siRNA efficiently and the Bcr-Abl siRNA decreased the IC(50) of Gleevec required to inhibit the high levels of Bcr-Abl protein found in K-562 cells.

A Mechanistic Study of Enhanced Doxorubicin Uptake and Retention in Multidrug Resistant Breast Cancer Cells using a Polymer-Lipid Hybrid Nanoparticle (PLN) System.
Wong HL, Bendayan R, Rauth AM, Xue HY, Babakhanian K, Wu XY.
J Pharmacol Exp Ther. 2006 Mar 17; [Epub ahead of print] .
[ expand abstract ]

The objectives of this study are i) to evaluate the potential of a polymer-lipid hybrid nanoparticle (PLN) system to enhance cellular accumulation and retention of doxorubicin (Dox), a widely used anticancer drug and an established P-glycoprotein (Pgp) substrate, in Pgp-overexpressing cancer cell lines, and ii) to explore the underlying mechanisms. Nanoparticles containing Dox complexed with a novel anionic polymer (Dox-PLN) were prepared using ultrasound method. Two Pgp-overexpressing breast cancer cell lines (a human cell line MDA435/LCC/MDR1 and a mouse cell line EMT6/AR1) were used to investigate the effect of nanoparticles on cellular uptake and retention of Dox. Endocytosis inhibition studies and fluorescence microscopic imaging were performed to elucidate the mechanisms of cellular drug uptake. Treatment of Pgp-overexpressing cell lines with Dox-PLN resulted in significantly enhanced Dox uptake and more substantial increases in drug retention after the end of treatment compared to free Dox solutions (p < 0.05). Fluorescence microscopic images showed improved nuclear localization of Dox and uptake of lipid when the drug was delivered in the Dox-PLN form to MDA435/LCC/MDR1 cells. Endocytosis inhibition studies revealed that phagocytosis is an important pathway in the membrane permeability of the nanoparticles. These findings suggest that some of the Dox physically associated with the nanoparticles bypass the membrane associated Pgp when delivered as Dox-PLN, and in this form the drug is better retained within the Pgp-overexpressing cells than the free drug. The present study suggests a new mechanism for overcoming drug resistance in Pgp-overexpressing tumor cells using lipid-based nanoparticles formulations.

Development of a highly active nanoliposomal irinotecan using a novel intraliposomal stabilization strategy.
Drummond DC, Noble CO, Guo Z, Hong K, Park JW, Kirpotin DB.
Cancer Res. 2006 Mar 15;66(6):3271-7.
[ expand abstract ]

Liposome formulations of camptothecins have been actively pursued because of the potential for significant pharmacologic advantages from successful drug delivery of this important class of anticancer drugs. We describe nanoliposomal CPT-11, a novel nanoparticle/liposome construct containing CPT-11 (irinotecan) with unprecedented drug loading efficiency and in vivo drug retention. Using a modified gradient loading method featuring a sterically hindered amine with highly charged, multivalent anionic trapping agents, either polymeric (polyphosphate) or nonpolymeric (sucrose octasulfate), liposomes were capable of entrapping CPT-11 at extremely high drug-to-lipid ratios (>800 g CPT-11/mol phospholipid) and retaining encapsulated drug in vivo with a half-life of drug release in the circulation of 56.8 hours. CPT-11 was also protected from hydrolysis to the inactive carboxylate form and from metabolic conversion to SN-38 while circulating. The maximum tolerated dose in normal mice was determined to be 80 mg/kg for free CPT-11 and >320 mg/kg for nanoliposomal CPT-11. Nanoliposomal CPT-11 showed markedly superior efficacy when compared with free CPT-11 in human breast (BT474) and colon (HT29) cancer xenograft models. This study shows that intraliposomal stabilization of CPT-11 using a polymeric or highly charged, nonpolymeric polyanionic trapping agent results in a markedly active antitumor agent with low toxicity.

Reduced toxicity and superior therapeutic activity of a mitomycin C lipid-based prodrug incorporated in pegylated liposomes.
Gabizon AA, Tzemach D, Horowitz AT, Shmeeda H, Yeh J, Zalipsky S.
Clin Cancer Res. 2006 Mar 15;12(6):1913-20.
[ expand abstract ]

PURPOSE: A lipid-based prodrug of mitomycin C [MMC; 2,3-(distearoyloxy)propane-1-dithio-4'-benzyloxycarbonyl-MMC] was designed for liposome formulation. The purpose of this study was to examine the in vitro cytotoxicity, pharmacokinetics, in vivo toxicity, and in vivo antitumor activity of this new lipid-based prodrug formulated in polyethylene glycol-coated (pegylated) liposomes. EXPERIMENTAL DESIGN: MMC was released from the MMC lipid-based prodrug (MLP) by thiolytic-induced cleavage with a variety of thiol-containing reducing agents. MLP was incorporated with nearly 100% efficiency in cholesterol-free pegylated liposomes with hydrogenated phosphatidylcholine as the main component and a mean vesicle size of approximately 90 nm. This formulation was used for in vitro and in vivo tests in rodents. RESULTS: In vitro, the cytotoxic activity of pegylated liposomal MLP (PL-MLP) was drastically reduced compared with free MMC. However, in the presence of reducing agents, such as cysteine or N-acetyl-cysteine, its activity increased to nearly comparable levels to those of free MMC. Intravenous administration of PL-MLP in rats resulted in a slow clearance indicating stable prodrug retention in liposomes and long circulation time kinetics, with a pharmacokinetic profile substantially different from that of free MMC. In vivo, PL-MLP was approximately 3-fold less toxic than free MMC. The therapeutic index and absolute antitumor efficacy of PL-MLP were superior to that of free MMC in the three tumor models tested. In addition, PL-MLP was significantly more active than a formulation of doxorubicin in pegylated liposomes (DOXIL) in the M109R tumor model, a mouse tumor cell line with a multidrug-resistant phenotype. CONCLUSIONS: Delivery of MLP in pegylated liposomes is a potential approach for effective treatment of multidrug-resistant tumors while significantly buffering the toxicity of MMC.

Enhancement of the efficiency of non-viral gene delivery by application of pulsed magnetic field.
Kamau SW, Hassa PO, Steitz B, Petri-Fink A, Hofmann H, Hofmann-Amtenbrink M, von Rechenberg B, Hottiger MO.
Nucleic Acids Res. 2006 Mar 15;34(5):e40.
[ expand abstract ]

New approaches to increase the efficiency of non-viral gene delivery are still required. Here we report a simple approach that enhances gene delivery using permanent and pulsating magnetic fields. DNA plasmids and novel DNA fragments (PCR products) containing sequence encoding for green fluorescent protein were coupled to polyethylenimine coated superparamagnetic nanoparticles (SPIONs). The complexes were added to cells that were subsequently exposed to permanent and pulsating magnetic fields. Presence of these magnetic fields significantly increased the transfection efficiency 40 times more than in cells not exposed to the magnetic field. The transfection efficiency was highest when the nanoparticles were sedimented on the permanent magnet before the application of the pulsating field, both for small (50 nm) and large (200-250 nm) nanoparticles. The highly efficient gene transfer already within 5 min shows that this technique is a powerful tool for future in vivo studies, where rapid gene delivery is required before systemic clearance or filtration of the gene vectors occurs.

Antioxidative function and biodistribution of [Gd@C(82)(OH)(22)](n) nanoparticles in tumor-bearing mice.
Wang J, Chen C, Li B, Yu H, Zhao Y, Sun J, Li Y, Xing G, Yuan H, Tang J, Chen Z, Meng H, Gao Y, Ye C, Chai Z, Zhu C, Ma B, Fang X, Wan L.
Biochem Pharmacol. 2006 Mar 14;71(6):872-881.
[ expand abstract ]

Oxidative stress is considered to be one of the important mechanisms involved in carcinogenesis. In our previous study, gadolinium endohedral metallofullerenol ([Gd@C(82)(OH)(22)](n) nanoparticles) have shown high inhibitory activity on hepatoma cell (H22) growth in mice. To explore the antioxidative functions of nanoparticles, we investigated the biodistribution of [Gd@C(82)(OH)(22)](n) nanoparticles, the changes of blood coagulation profiles, the metabolism of reactive oxygen species (ROS) in the tumor-bearing mice as well as the possible relationships between nanoparticles treatment and ROS production in this paper. The activities of hepatic superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione S-transferase (GST) and catalase (CAT) as well as the levels of reduced glutathione (GSH), protein-bound thiols and malondialdehyde (MDA) were compared between the tumor-bearing mice and normal mice. Transplanted tumors were grown in mice by subcutaneous injection of murine hepatoma cells in the mice. The comparison of the above parameters between nanoparticles and cyclophosphamide (CTX) therapy were also investigated. [Gd@C(82)(OH)(22)](n) administration can efficiently restore the damaged liver and kidney of the tumor-bearing mice. All the activities of enzymes and other parameters related to oxidative stress were reduced after [Gd@C(82)(OH)(22)](n) treatment and tended closely to the normal levels. The results suggest that [Gd@C(82)(OH)(22)](n) nanoparticle treatment could regulate ROS production in vivo.

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Hydrophobically modified glycol chitosan nanoparticles as carriers for paclitaxel.
Kim JH, Kim YS, Kim S, Park JH, Kim K, Choi K, Chung H, Jeong SY, Park RW, Kim IS, Kwon IC.
J Control Release. 2006 Mar 10;111(1-2):228-234.
[ expand abstract ]

Self-assembled nanoparticles based on hydrophobically modified glycol chitosan (HGC) were prepared as a carrier for paclitaxel. HGC conjugates were prepared by chemically linking 5beta-cholanic acid to glycol chitosan chains using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide chemistry. In phosphate-buffered saline (PBS; pH 7.4), the synthesized HGC conjugates formed nano-sized particles with a diameter of 200 nm and exhibited high thermodynamic stability as reflected by their low critical aggregation concentration (0.03 mg/ml). Paclitaxel was efficiently loaded into HGC nanoparticles up to 10 wt.% using a dialysis method. The paclitaxel-loaded HGC (PTX-HGC) nanoparticles were 400 nm in diameter and were stable in PBS for 10 days. These PTX-HGC nanoparticles also showed sustained release of the incorporated of paclitaxel (80% of the loaded dose was released in 8 days at 37 degrees C in PBS). Owing to sustained release, the PTX-HGC nanoparticles were less cytotoxic to B16F10 melanoma cells than free paclitaxel formulated in Cremophor EL. Injection of PTX-HGC nanoparticles into the tail vein of tumor-bearing mice prevented increases in tumor volume for 8 days. Finally, PTX was less toxic to the tumor-bearing mice when formulated in HGC nanoparticles than when formulated with Cremophor EL.

In vivo targeting of dendritic cells in lymph nodes with poly(propylene sulfide) nanoparticles.
Reddy ST, Rehor A, Schmoekel HG, Hubbell JA, Swartz MA.
J Control Release. 2006 Mar 8; [Epub ahead of print].
[ expand abstract ]

Delivery of biodegradable nanoparticles to antigen-presenting cells (APCs), specifically dendritic cells (DCs), has potential for immunotherapy. This study investigates the delivery of 20, 45, and 100nm diameter poly(ethylene glycol)-stabilized poly(propylene sulfide) (PPS) nanoparticles to DCs in the lymph nodes. These nanoparticles consist of a cross-linked rubbery core of PPS surrounded by a hydrophilic corona of poly(ethylene glycol). The PPS domain is capable of carrying hydrophobic drugs and degrades within oxidative environments. 20 nm particles were most readily taken up into lymphatics following interstitial injection, while both 20 and 45nm nanoparticles showed significant retention in lymph nodes, displaying a consistent and strong presence at 24, 72, 96 and 120h post-injection. Nanoparticles were internalized by up to 40-50% of lymph node DCs (and APCs) without the use of a targeting ligand, and the site of internalization was in the lymph nodes rather than at the injection site. Finally, an increase in nanoparticle-containing DCs (and other APCs) was seen at 96h vs. 24h, suggesting an infiltration of these cells to lymph nodes. Thus, PPS nanoparticles of 20-45nm have the potential for immunotherapeutic applications that specifically target DCs in lymph nodes.

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A novel stealth liposomal topotecan with amlodipine: Apoptotic effect is associated with deletion of intracellular Ca(2+) by amlodipine thus leading to an enhanced antitumor activity in leukemia.
Li X, Ruan GR, Lu WL, Hong HY, Liang GW, Zhang YT, Liu Y, Long C, Ma X, Yuan L, Wang JC, Zhang X, Zhang Q.
J Control Release. 2006 Mar 1; [Epub ahead of print].
[ expand abstract ]

The objectives of the present study were to define whether amlodipine induces apoptosis and what mechanism is involved in the process in human resistant and non-resistant leukemia cells following co-administration of stealth liposomal topotecan with amlodipine, a novel antiresistant liposomes developed by our institution. In three leukemias, K562, HL-60, and multidrug resistant (MDR) HL-60, cytotoxicity of topotecan was potentiated by amolodipne, while topotecan alone was resistant to MDR HL-60 cells. In two selected K562 or MDR HL-60 cells, the apoptotic effects were increased by addition of amlodipine, showing a dose-dependent manner. The activities of caspase 3 and 7 (marked as caspase 3/7), and caspase 8 were significantly activated by topotecan with amlodipine co-treated as the stealth liposomes. The deletions of intracellular Ca(2+) stores induced by amlodipine correlated with the activated activities of caspase 3/7, or 8, respectively. In xenograft model with MDR HL-60 in nude mice, antitumor activity of stealth liposomal topotecan with amlodipine was significantly enhanced as compared to that of stealth liposomal topotecan or topotecan alone. In conclusion, apoptotic effect is associated with deletion of intracellular Ca(2+) by amlodipine through activation of caspase 8 and then 3/7 activities. The enhanced antitumor activities by stealth liposomal topotecan with amlodipine are mainly due to the potentiating apoptotic effect and reversing the resistance by amlodipine. Stealth liposomal encapsulation of anticancer agent with a modulator may provide a novel strategy for improving the chemotherapeutic effects.

Novel nanoliposomal CPT-11 infused by convection-enhanced delivery in intracranial tumors: pharmacology and efficacy.
Noble CO, Krauze MT, Drummond DC, Yamashita Y, Saito R, Berger MS, Kirpotin DB, Bankiewicz KS, Park JW.
Cancer Res. 2006 Mar 1;66(5):2801-6.
[ expand abstract ]

We hypothesized that combining convection-enhanced delivery (CED) with a novel, highly stable nanoparticle/liposome containing CPT-11 (nanoliposomal CPT-11) would provide a dual drug delivery strategy for brain tumor treatment. Following CED in rat brains, tissue retention of nanoliposomal CPT-11 was greatly prolonged, with >20% injected dose remaining at 12 days for all doses. Tissue residence was dose dependent, with doses of 60 microg (3 mg/mL), 0.8 mg (40 mg/mL), and 1.6 mg (80 mg/mL) resulting in tissue half-life (t(1/2)) of 6.7, 10.7, and 19.7 days, respectively. In contrast, CED of free CPT-11 resulted in rapid drug clearance (tissue t(1/2) = 0.3 day). At equivalent CED doses, nanoliposomal CPT-11 increased area under the time-concentration curve by 25-fold and tissue t(1/2) by 22-fold over free CPT-11; CED in intracranial U87 glioma xenografts showed even longer tumor retention (tissue t(1/2) = 43 days). Plasma levels were undetectable following CED of nanoliposomal CPT-11. Importantly, prolonged exposure to nanoliposomal CPT-11 resulted in no measurable central nervous system (CNS) toxicity at any dose tested (0.06-1.6 mg/rat), whereas CED of free CPT-11 induced severe CNS toxicity at 0.4 mg/rat. In the intracranial U87 glioma xenograft model, a single CED infusion of nanoliposomal CPT-11 at 1.6 mg resulted in significantly improved median survival (>100 days) compared with CED of control liposomes (19.5 days; P = 4.9 x 10(-5)) or free drug (28.5 days; P = 0.011). We conclude that CED of nanoliposomal CPT-11 greatly prolonged tissue residence while also substantially reducing toxicity, resulting in a highly effective treatment strategy in preclinical brain tumor models.

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Preparation and characterization of Boron carbide nanoparticles for use as a novel agent in T cell-guided boron neutron capture therapy.
Mortensen MW, Sorensen PG, Bjorkdahl O, Jensen MR, Gundersen HJ, Bjornholm T.
Appl Radiat Isot. 2006 Mar;64(3):315-24.
[ expand abstract ]

Boron carbide nanoparticles are proposed as a system for T cell-guided boron neutron capture therapy. Nanoparticles were produced by ball milling in various atmospheres of commercially available boron carbide. The physical and chemical properties of the particles were investigated using transmission electron microscopy, photon correlation spectroscopy, X-ray photoelectron spectroscopy, X-ray diffraction, vibrational spectroscopy, gel electrophoresis and chemical assays and reveal profound changes in surface chemistry and structural characteristics. In vitro thermal neutron irradiation of B16 melanoma cells incubated with sub-100nm nanoparticles (381.5mug/g (10)B) induces complete cell death. The nanoparticles alone induce no toxicity.

Boron containing macromolecules and nanovehicles as delivery agents for neutron capture therapy.
Wu G, Barth RF, Yang W, Lee RJ, Tjarks W, Backer MV, Backer JM.
Anticancer Agents Med Chem. 2006 Mar;6(2):167-84.
[ expand abstract ]

Boron neutron capture therapy (BNCT) is based on the nuclear capture and fission reactions that occur when non-radioactive boron-10 is irradiated with low energy thermal neutrons to yield high linear energy transfer (LET) alpha particles ((4)He) and recoiling lithium -7((7)Li) nuclei. For BNCT to be successful, a sufficient number of (10)B atoms ( approximately 10(9) atoms/cell) must be selectively delivered to the tumor and enough thermal neutrons must be absorbed by them to sustain a lethal (10)B(n, alpha) (7)Li capture reaction. BNCT primarily has been used to treat patients with brain tumors, and more recently those with head and neck cancer. Two low molecular weight (LMW) boron delivery agents currently are being used clinically, sodium borocaptate and boronophenylalanine. However, a variety of high molecular weight (HMW) agents consisting of macromolecules and nanovehicles have been developed. This review will focus on the latter which include: monoclonal antibodies, dendrimers, liposomes, dextrans, polylysine, avidin, folic acid, and epidermal and vascular endothelial growth factors (EGF and VEGF). Procedures for introducing boron atoms into these HMW agents and their chemical properties will be discussed. In vivo studies on their biodistribution will be described, and the efficacy of a subset of them, which have been used for BNCT of tumors in experimental animals, will be discussed. Since brain tumors currently are the primary candidates for treatment by BNCT, delivery of these HMW agents across the blood-brain barrier presents a special challenge. Various routes of administration will be discussed including receptor-facilitated transcytosis following intravenous administration, direct intratumoral injection and convection enhanced delivery by which a pump is used to apply a pressure gradient to establish bulk flow of the HMW agent during interstitial infusion. Finally, we will conclude with a discussion relating to issues that must be addressed if these HMW agents are to be used clinically.

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Anticancer Efficacies of Cisplatin-Releasing pH-Responsive Nanoparticles.
Xu P, Van Kirk EA, Murdoch WJ, Zhan Y, Isaak DD, Radosz M, Shen Y.
Biomacromolecules. 2006 Mar;7(3):829-35.
[ expand abstract ]

The objective of these investigations was to test the hypothesis that a rapid cytoplasmic release profile from nanoparticles would potentiate the anticancer activity of cisplatin. Cisplatin-loaded nanoparticles with pH-responsive poly[2-(N,N-diethylamino)ethyl methacrylate] (PDEA) cores were synthesized from PDEA-block-poly(ethylene glycol) (PDEA-PEG) copolymer by using a solvent-displacement (acetone-water) method. Nanoparticles with pH-nonresponsive poly(epsilon-caprolactone) (PCL) cores made from PCL-block-PEG (PCL-PEG) were used for comparison. Nanoparticle sizes, zeta potentials, drug-loading capacities, and pH responsiveness were characterized. The cellular uptakes and localization in lysosomes were visualized by using confocal fluorescence microscopy. Cytostatic effects of free and encapsulated cis-diammineplatinum(II) dichloride (cisplatin) toward human SKOV-3 epithelial ovarian cancer cells were estimated by using the MTT assay. Intraperitoneal tumor responses to cisplatin and cisplatin/PDEA-PEG were evaluated in athymic mice at 4-6 weeks postinoculation of SKOV-3 cells. PDEA-PEG nanoparticles dissolved at pH < 6 and rapidly internalized and transferred to lysosomes; it therefore was predicted that the PDEA nanoparticles would rapidly release cisplatin into cytoplasm upon integration into acidic lysosomes and thereby overwhelm the chemoresistant properties of SKOV-3 cells. Indeed, relative proportions of viable cells were diminished to a greater extent by exposure in vitro to fast-releasing nanoparticles compared to slow-releasing nanoparticles or an equivalent dose of free cisplatin. Incidences of cellular pyknosis (a morphological indicator of apoptosis) were most evident within intestinal/mesentery tumors of mice treated with cisplatin/PDEA-PEG; tumor burdens were correspondingly reduced.

Construction of folate-conjugated pRNA of bacteriophage phi29 DNA packaging motor for delivery of chimeric siRNA to nasopharyngeal carcinoma cells.
Guo S, Huang F, Guo P.
Gene Ther. 2006 Feb 16; [Epub ahead of print] .
[ expand abstract ]

Nasopharyngeal carcinoma is a poorly differentiated upper respiratory tract cancer that highly expresses human folate receptors (hFR). Binding of folate to hFR triggers endocytosis. The folate was conjugated into adenosine 5'-monophosphate (AMP) by 1,6-hexanediamine linkages. After reverse HPLC to reach 93% purity, the folate-AMP, which can only be used for transcription initiation but not for chain extension, was incorporated into the 5'-end of bacteriophage phi29 motor pRNA. A 16:1 ratio of folate-AMP to ATP in transcription resulted in more than 60% of the pRNA containing folate. A pRNA with a 5'-overhang is needed to enhance the accessibility of the 5' folate for specific receptor binding. Utilizing the engineered left/right interlocking loops, polyvalent dimeric pRNA nanoparticles were constructed using RNA nanotechnology to carry folate, a detection marker, and siRNA targeting at an antiapoptosis factor. The chimeric pRNAs were processed into ds-siRNA by Dicer. Incubation of nasopharyngeal epidermal carcinoma (KB) cells with the dimer resulted in its entry into cancer cells, and the subsequent silencing of the target gene. Such a protein-free RNA nanoparticle with undetectable antigenicity has a potential for repeated long-term administration for nasopharyngeal carcinoma as the effectiveness and specificity were confirmed by ex vivo delivery in the animal trial.

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Novel PEG-matrix metalloproteinase-2 cleavable peptide-lipid containing galactosylated liposomes for hepatocellular carcinoma-selective targeting.
Terada T, Iwai M, Kawakami S, Yamashita F, Hashida M.
J Control Release. 2006 Feb 16; [Epub ahead of print].
[ expand abstract ]

In order to obtain an HCC-selective drug delivery system, a novel functional lipid, which is cleaved by the protease activity of matrix metalloproteinase-2 (MMP-2), was developed. The amino group of dioleoylphosphatidylethanolamine (DOPE) was conjugated with PEGylated MMP-2 substrate peptide (Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln), and MMP-2-cleavable PEG-Peptide-DOPE (PEG-PD) was synthesized. When PEG-PD was incorporated in galactosylated liposomes (Gal-PEG-PD-liposomes), we expected that Gal-PEG-PD-liposomes would not be taken up by normal hepatocytes due to the steric hindrance effect, but would be activated around HCC cells by secreted MMPs. In the pretreatment by hMMP2 (1, 5, and 10mug/ml), an hMMP2 concentration-dependent higher uptake of Gal-PEG-PD-liposomes was observed in HepG2 cells, suggesting PEG-PD cleavage. In the presence of an excess of galactose, the uptake of Gal-PEG-PD-liposomes with hMMP2 was significantly inhibited, suggesting asialoglycoprotein receptor-mediated uptake of Gal-PEG-PD-liposomes following the PEG-PD cleavage. Pretreatment of Gal-PEG-PD-liposomes with the conditioned medium of B16BL6, which contained secreted MMPs, enhanced the binding to HepG2 cells, as in the case of hMMP-2 treatment. Moreover, the cytotoxicity of N(4)-octadecyl-1-beta-d-arabinofuranosylcytosine (NOAC) incorporated Gal-PEG-PD-liposomes was enhanced by hMMPs (5mug/ml) and its cytotoxicity was significantly reduced by the presence of an excess of galactose in HepG2 cells. In conclusion, Gal-PEG-PD-liposomes were successfully developed for novel HCC-selective targeting.

Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods.
Huang X, El-Sayed IH, Qian W, El-Sayed MA.
J Am Chem Soc. 2006 Feb 15;128(6):2115-20.
[ expand abstract ]

Due to strong electric fields at the surface, the absorption and scattering of electromagnetic radiation by noble metal nanoparticles are strongly enhanced. These unique properties provide the potential of designing novel optically active reagents for simultaneous molecular imaging and photothermal cancer therapy. It is desirable to use agents that are active in the near-infrared (NIR) region of the radiation spectrum to minimize the light extinction by intrinsic chromophores in native tissue. Gold nanorods with suitable aspect ratios (length divided by width) can absorb and scatter strongly in the NIR region (650-900 nm). In the present work, we provide an in vitro demonstration of gold nanorods as novel contrast agents for both molecular imaging and photothermal cancer therapy. Nanorods are synthesized and conjugated to anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibodies and incubated in cell cultures with a nonmalignant epithelial cell line (HaCat) and two malignant oral epithelial cell lines (HOC 313 clone 8 and HSC 3). The anti-EGFR antibody-conjugated nanorods bind specifically to the surface of the malignant-type cells with a much higher affinity due to the overexpressed EGFR on the cytoplasmic membrane of the malignant cells. As a result of the strongly scattered red light from gold nanorods in dark field, observed using a laboratory microscope, the malignant cells are clearly visualized and diagnosed from the nonmalignant cells. It is found that, after exposure to continuous red laser at 800 nm, malignant cells require about half the laser energy to be photothermally destroyed than the nonmalignant cells. Thus, both efficient cancer cell diagnostics and selective photothermal therapy are realized at the same time.

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Characterization of Superparamagnetic Nanoparticle Interactions with Extracellular Matrix in an in Vitro System.
Kuhn SJ, Hallahan DE, Giorgio TD.
Ann Biomed Eng. 2006 Feb 14;:1-8 .
[ expand abstract ]

Controlled dispersion of therapeutic agents within liquid- and gel-filled cavities represents a barrier to treatment of some cancers and other pathological states. Interstitial delivery is compromised by the poor mobility of macromolecules and larger nanoscale structures. We developed an in vitro system to quantify the suitability of superparamagnetic nanoparticles (SPM NPs) as a site-specific therapeutic vehicle for delivery through fluid- and gel-based systems. SPM NP motion was induced by an external magnetic field. NP migration was modulated by NP concentration and surface coating. 135 nanometer radius PEGylated NPs moved through the extracellular matrix with an average velocity of 1.5 mm h(-1), suitable for some clinical applications. Increasing the SPM NP radius to 400 nm while maintaining the same per NP magnetic susceptibility resulted in a greater than 1000-fold reduction in magnetic mobility, to less than 0.01 mm h(-1). The critical influence of NP size on gel permeation was also observed in silica-coated 135 nm SPM NPs that aggregated under the experimental conditions. Aggregation played a critical role in determining the behavior of the nanoparticles. SPM NPs allow significant free-solution mobility to specific sites within a cavity and generate sufficient force to penetrate common in vivo gels.

Hypersensitivity and loss of disease site targeting caused by antibody responses to PEGylated liposomes.
Judge A, McClintock K, Phelps JR, Maclachlan I.
Mol Ther. 2006 Feb;13(2):328-37.
[ expand abstract ]

The systemic application of nucleic acid drugs requires delivery systems that overcome the poor pharmacokinetics, limited biodistribution, and inefficient uptake of nucleic acids. PEGylated liposomes show considerable promise because of their intrinsic ability to accumulate at disease sites and facilitate transfection of target cells. Unlike many viral vectors, PEGylated liposomes are generally considered to be nonimmunogenic. We have developed a PEGylated liposome for the systemic administration of plasmid DNA that achieves high levels of selective gene expression at distal tumor sites. Here we report that the in vivo efficacy and safety of these systems can be severely compromised following repeat administration. This phenomenon is characterized by a loss of disease site targeting, accelerated clearance from the blood, and acute hypersensitivity. These effects are fully attributable to a surprisingly robust, long-lived antibody response generated against polyethylene glycol (PEG) that results from the strong adjuvant effect of the plasmid payload. Importantly, immunogenicity may be substantially reduced by modifying the alkyl chain of the PEG-lipid conjugate, thereby allowing successful repeat dosing of the modified plasmid formulations without adverse side effects. Immunogenicity is a relevant concern for a number of nonviral delivery systems given the potent immunostimulatory properties of many nucleic acid drugs.

Preparation, cellular transport, and activity of polyamidoamine-based dendritic nanodevices with a high drug payload.
Kolhe P, Khandare J, Pillai O, Kannan S, Lieh-Lai M, Kannan RM.
Biomaterials. 2006 Feb;27(4):660-9.
[ expand abstract ]

Dendrimers are emerging as a relatively new class of polymeric biomaterials with applications in drug delivery, and imaging. Achieving a high drug payload in dendrimers, and understanding the therapeutic effect of the dendrimer-drug conjugates are receiving increasing attention. A high drug payload nanodevice was obtained by covalent conjugation of ibuprofen to a polyamidoamine (PAMAM-G4-OH) dendrimer. Using DCC as a coupling agent, 58 molecules of ibuprofen were covalently conjugated to one molecule of generation 4 PAMAM-OH dendrimer. Cellular entry of the fluoroisothiocynate (FITC)-labeled dendrimer-drug conjugate was evaluated in vitro by using human lung epithelial carcinoma A549 cells by flow cytometry, confocal microscopy and UV/Visible spectroscopy. The pharmacological activity of the dendrimer-ibuprofen conjugate was compared to pure ibuprofen at various time points by measuring the suppression of prostaglandin E2. Significant amounts of the conjugate entered the cells rapidly within 15 min. Suppression of prostaglandin was noted within 30 min for the dendrimer-drug conjugates versus 1 h for the free ibuprofen. The results suggest that dendrimers with high drug payload improve the drug's efficacy by enhanced cellular delivery, and may produce a rapid pharmacological response. These dendrimer-drug conjugates can potentially be further modified by attaching antibodies and ligands for targeted drug delivery.

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Astatination of nanoparticles containing silver as possible carriers of 211At.
Kucka J, Hruby M, Konak C, Kozempel J, Lebeda O.
Appl Radiat Isot. 2006 Feb;64(2):201-6.
[ expand abstract ]

The alpha emitter 211At is a prospective radionuclide for the therapy of smaller tumours and metastases. However, the chemical properties of 211At together with the fact that it is available only in trace amounts, makes the labelling of prospective astatine carriers rather complicated. In this context we have studied a new class of possible astatine carriers--nanoparticle systems, which tend to concentrate themselves in some types of tumours by means of the EPR effect. Additionally, such nanoparticles have the advantage that they may be chemically modified by the attachment of a tumour-seeking agent, and also directly applied to the target site. In order to reach high labelling yields, and in order to protect the nanoparticles from rapid degradation by the immune system, silver-containing particles covalently coated by poly(ethylene oxide) were developed and tested. The effect of the different reducing and oxidizing agents on the labelling yield was also determined. It was found that labelling yields were almost quantitative and well reproducible under reducing conditions, while under oxidizing conditions they dropped to ca. 50%. In the absence of any reducing or oxidizing agent, the labelling yields were randomly distributed between a range of 50% and 97%. The labelled nanoparticles were stable even in a large surplus of competing chloride ions.

PAMAM Dendrimer-Based Multifunctional Conjugate for Cancer Therapy: Synthesis, Characterization, and Functionality.
Majoros IJ, Myc A, Thomas T, Mehta CB, Baker JR Jr.
Biomacromolecules. 2006 Feb;7(2):572-9.
[ expand abstract ]

Poly(amidoamine) (PAMAM) dendrimer-based multifunctional cancer therapeutic conjugates have been designed and synthesized. The primary amino groups on the surface of the generation 5 (G5) PAMAM dendrimer were neutralized through partial acetylation, providing enhanced solubility of the dendrimer (in conjugation of FITC (fluorescein isothiocyanate)) and preventing nonspecific targeting interactions (in vitro and in vivo) during delivery. The functional molecules fluorescein isothiocyanate (FITC, an imaging agent), folic acid (FA, targets overexpressed folate receptors on specific cancer cells), and paclitaxel (taxol, a chemotherapeutic drug) were conjugated to the remaining nonacetylated primary amino groups. The appropriate control dendrimer conjugates have been synthesized as well. Characterization of the G5 PAMAM dendrimer and its nanosize conjugates, including the molecular weight and number of primary amine groups, has been determined by multiple analytical methods such as gel permeation chromatography (GPC), nuclear magnetic resonance spectroscopy (NMR), potentiometric titration, high-performance liquid chromatography (HPLC), and UV spectroscopy. These multifunctional dendrimer conjugates have been tested in vitro for targeted delivery of chemotherapeutic and imaging agents to specific cancer cells. We present here the synthesis, characterization, and functionality of these dendrimer conjugates.

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Induction of Apoptosis by Hybrid Liposomes for Human Breast Tumor Cells along with Activation of Caspases.
Nagami H, Matsumoto Y, Ueoka R.
Biol Pharm Bull. 2006 Feb;29(2):380-1.
[ expand abstract ]

Highly inhibitory effects of hybrid liposomes on the growth of human breast tumor cells in vitro were obtained. It is worthy to note that induction of apoptosis through activation of caspases by hybrid liposomes was clearly observed.

Therapeutic possibilities of plasmonically heated gold nanoparticles.
Pissuwan D, Valenzuela SM, Cortie MB.
Trends Biotechnol. 2006 Feb;24(2):62-7.
[ expand abstract ]

Nanoparticles of gold, which are in the size range 10-100nm, undergo a plasmon resonance with light. This is a process whereby the electrons of the gold resonate in response to incoming radiation causing them to both absorb and scatter light. This effect can be harnessed to either destroy tissue by local heating or release payload molecules of therapeutic importance. Gold nanoparticles can also be conjugated to biologically active moieties, providing possibilities for targeting to particular tissues. Here, we review the progress made in the exploitation of the plasmon resonance of gold nanoparticles in photo-thermal therapeutic medicine.

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Characterization and cytotoxicity of mixed polyethyleneglycol modified liposomes containing doxorubicin.
Sadzuka Y, Sugiyama I, Tsuruda T, Sonobe T.
Int J Pharm. 2006 Jan 31; [Epub ahead of print] .
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Liposomes are recognized as one of the useful drug carriers, but have many problems to overcome before their clinical application. Liposomes, bonding peculiarly with serum protein (opsonization), are taken up by reticuloendothelial system (RES) cells in the liver and spleen. It is known that polyethyleneglycol (PEG) modification of the liposome surface induces the formation of a fixed aqueous layer around the liposomes due to the interaction between the PEG-polymer and water molecule, and thus prevents the attraction of opsonins. Namely, PEG-modified liposomes are able to escape trapping by the RES cells, and have a prolonged circulation time. In this study, the effects of different anchors with the same PEG molecular weight on the cell uptake and cytotoxicity of mixed PEG-modified liposomal doxorubicin (DOX) were examined. The fixed aqueous layer thickness (FALT) of liposomes covered with mixtures of PEG-molecules which differ in their chain length were increased, compared to that of the single PEG2000-modified liposome. Mixed PEG-modification of liposomes with different anchors (PEG2000-(1-monomethoxypolyethyleneglycol-2,3-distearoylglycerol (DSG): cholesterol (CHO)=1:1)-modified liposome) led to an increase in the FALT, compared to that of each single PEG-modification. The uptake of DOX into Ehrlich ascites carcinoma cells by the liposomes covered with PEG-CHO was higher than the other liposomes. Thus, liposomes covered with PEG-DSG and PEG-CHO have an enhanced cytotoxicity. In conclusion, it was confirmed that mix-modified liposomes using PEG-lipid with different anchors were superior.

Nano-oncology: drug delivery, imaging, and sensing.
Portney NG, Ozkan M.
Anal Bioanal Chem. 2006 Jan 27;:1-11 .
[ expand abstract ]

Innovation in the last decade has endowed nanotechnology with an assortment of tools for delivery, imaging, and sensing in cancer research-stealthy nanoparticle vectors circulating in vivo, assembled with exquisite molecular control, capable of selective tumor targeting and potent delivery of therapeutics; intense and photostable quantum dot-based tumor imaging, enabling multicolor detection of cell receptors with a single optical excitation source; arrays of semiconducting nanowire and carbon nanotube sensor elements for selective multiplexed sensing of cancer markers without the need for probe labeling. These rapidly emerging tools are indicative of a burgeoning field ready to expand into medical applications. This review attempts to outline most of the current nanoparticle toolset for therapeutic release by liposomes, dendrimers, smart polymers, and virus-based systems. Advantages of nanoparticle-based imaging and targeting by use of nanoshells and quantum dots are also explored. Finally, emerging nanoelectronics-based sensing and a global discussion on the utility of each nanoparticle system addresses their fundamental advantages and shortcomings in cancer research.

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Engineered nanoparticles as precise drug delivery systems.
Yih TC, Al-Fandi M.
J Cell Biochem. 2006 Jan 26; [Epub ahead of print] .
[ expand abstract ]

With the remarkable development of nanotechnology in recent years, new drug delivery approaches based on the state-of-the-art nanotechnology have been receiving significant attention. Nanoparticles, an evolvement of nanotechnology, are increasingly considered as a potential candidate to carry therapeutic agents safely into a targeted compartment in an organ, particular tissue or cell. These particles are colloidal structures with a diameter smaller than 1,000 nm, and therefore can penetrate through diminutive capillaries into the cell's internal machinery. This innovative delivery technique might be a promising technology to meet the current challenges in drug delivery. When loaded with a gene or drug agent, nanoparticles can become nanopills, which can effectively treat problematical diseases such as cancer. This article summarizes different types of nanoparticles drug delivery systems under investigation and their prospective therapeutic applications. Also, this article presents a closer look at the advances, current challenges, and future direction of nanoparticles drug delivery systems. J. Cell. Biochem. (c) 2006 Wiley-Liss, Inc.

Delivery of different length poly(l-lysine)-conjugated ODN to HepG2 cells using N-stearyllactobionamide-modified liposomes and their enhanced cellular biological effects.
Wang S, Cheng L, Yu F, Pan W, Zhang J.
Int J Pharm. 2006 Jan 18; [Epub ahead of print] .
[ expand abstract ]

Short (14-20-mer range) synthetic oligodeoxynucleotides (ODNs) allow specific modulation of cellular gene expression at various stages, thus providing a versatile tool for fundamental studies and a rational approach to anticancer chemotherapy. However, several problems, such as metabolic stability, efficient cell internalization of ODNs and their efficient entrapment into liposomes continue to markedly limit this approach. To improve the target specificity and biological activity of ODN, three different length of poly(l-lysine) (PLL) were conjugated to ODN and these conjugates were encapsulated in N-stearyllactobionamide (N-SLBA)-modified liposomes, N-SLBA is a ligand for the asialoglycoprotein receptor. Then, we investigated their effects on cell cycle and survivin protein levels of HepG2 cells. The results showed that the encapsulation efficiency was improved because the polycationic charges of PLL neutralized the polyanionic charges of ODN. Among them, PLL (M(W) 2000 and 10,000)-conjugated ODN encapsulated in N-SLBA liposomes induced apoptosis of HepG2 cells and highly inhibited survivin gene expression.

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Carbon nanotubes as intracellular transporters for proteins and DNA: an investigation of the uptake mechanism and pathway.
Kam NW, Liu Z, Dai H.
Angew Chem Int Ed Engl. 2006 Jan 16;45(4):577-81.
[ expand abstract ]

No abstract available

Application of TPGS in polymeric nanoparticulate drug delivery system.
Mu L, Seow PH.
Colloids Surf B Biointerfaces. 2006 Jan 15;47(1):90-7.
[ expand abstract ]

d-alpha-Tocopheryl polyethylene glycol 1000 succinate (TPGS) has great potential in pharmacology and nanotechnology. The present work investigated the molecular behaviour of TPGS at the air-water interface, its effect on a model bio-membrane composed of dipalmitoylphosphatidylcholine (DPPC) lipid monolayer, and the interaction between the TPGS coated nanoparticles with the lipid model membrane. Paclitaxel loaded polymeric nanoparticles with TPGS as surfactant stabiliser were fabricated and characterised in terms of their drug incorporation capability and release kinetics. The result showed that TPGS exhibited notable effect on the surface properties of air-water interface as well as the lipid monolayer. The inter-particle force and the interaction between nanoparticles and lipid monolayer varied with the surface substance. The penetration of various nanoparticles into the model membrane indicated that an optimal balance between hydrophilicity and hydrophobicity on nanoparticle surface is needed to achieve an effective cellular uptake of nanoparticles. The results also demonstrate that the drug incorporation capability and the release characteristics of drug-loaded nanoparticles can be influenced by surfactant stabiliser.

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Formulation optimization for the nanoparticles-in-microsphere hybrid oral delivery system using factorial design.
Bhavsar MD, Tiwari SB, Amiji MM.
J Control Release. 2006 Jan 10;110(2):422-30.
[ expand abstract ]

The tremendous progress witnessed in the field of biotechnology with respect to discovery of therapeutic and antigenic proteins has propelled the need for development of suitable oral delivery devices for these and other macromolecules. In this study, we report the encapsulation of fluorescein isothiocyanate (FITC)-labeled gelatin nanoparticles into poly(epsilon-caprolactone) (PCL) microsphere (nanoparticle-in-microsphere oral delivery system, NiMOS) by double emulsion like technique and the influence of variables such as polymer concentration in organic phase, amount of nanoparticles added as internal phase, and the speed of homogenization on particle size of NiMOS using a 3(3) randomized full factorial design. A statistical model with interaction terms was derived to predict the particle size of the hybrid system. The results from multiple linear regression analysis and Student's t-test revealed that for obtaining large particles of NiMOS, a high polymer concentration and low speed of homogenization was necessary. In contrast, to obtain particles of smaller size, high speed of homogenization was found to be very important. The mathematical model obtained was validated for prediction of particle size. The encapsulation of gelatin nanoparticles in PCL microsphere was confirmed by fluorescent microscopy. Based on the statistical model we were also successful in producing NiMOS of less than 10 mum in size, which could be used as oral delivery system for therapeutic and antigenic macromolecules.

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Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles.
Owens DE 3rd, Peppas NA.
Int J Pharm. 2006 Jan 3;307(1):93-102.
[ expand abstract ]

The process of opsonization is one of the most important biological barriers to controlled drug delivery. Injectable polymeric nanoparticle carriers have the ability to revolutionize disease treatment via spatially and temporally controlled drug delivery. However, opsonin proteins present in the blood serum quickly bind to conventional non-stealth nanoparticles, allowing macrophages of the mononuclear phagocytic system (MPS) to easily recognize and remove these drug delivery devices before they can perform their designed therapeutic function. To address these limitations, several methods have been developed to mask or camouflage nanoparticles from the MPS. Of these methods, the most preferred is the adsorption or grafting of poly(ethylene glycol) (PEG) to the surface of nanoparticles. Addition of PEG and PEG-containing copolymers to the surface of nanoparticles results in an increase in the blood circulation half-life of the particles by several orders of magnitude. This method creates a hydrophilic protective layer around the nanoparticles that is able to repel the absorption of opsonin proteins via steric repulsion forces, thereby blocking and delaying the first step in the opsonization process.

Phase II study of temozolomide plus pegylated liposomal doxorubicin in the treatment of brain metastases from solid tumours.
Caraglia M, Addeo R, Costanzo R, Montella L, Faiola V, Marra M, Abbruzzese A, Palmieri G, Budillon A, Grillone F, Venuta S, Tagliaferri P, Del Prete S.
Cancer Chemother Pharmacol. 2006 Jan;57(1):34-9.
[ expand abstract ]

OBJECTIVE: A combination regimen of temozolomide (TMZ) and pegylated liposomal doxorubicin has been evaluated in the treatment of brain metastases from solid tumours. STUDY DESIGN: Nineteen consecutive patients (pts) have been enrolled in a prospective phase II trial and treated with TMZ 200 mg/m2 (days 1-5) and pegylated liposomal doxorubicin 35 mg/m2 (day 1) every 28 days. The study was prospectively projected according to the Simon's two-stage optimal design. RESULTS: Major toxicities have been grade III neutropenia and thrombocytopenia in one patient (pt) and grade III erythrodisesthesia in two pts. Three pts achieved a complete response (CR) and four a partial response (PR), for an overall response rate of 36.8% (95% CI: 19.1-59.2), which exceeded the target activity in the study design. A significant improvement in quality of life was demonstrated by FACT-G analysis. The median Progression Free Survival (PFS) was 5.5 (95% CI: 2.7-8.2) months while the median Overall Survival (OS) was 10.0 months (95% CI: 6.3-13.7). CONCLUSIONS: The TMZ/pegylated liposomal doxorubicin regimen was well tolerated with an encouraging activity in brain metastases from solid tumours.

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Doxorubicin-encapsulated thermosensitive liposomes modified with poly(N-isopropylacrylamide-co-acrylamide): drug release behavior and stability in the presence of serum.
Han HD, Shin BC, Choi HS.
Eur J Pharm Biopharm. 2006 Jan;62(1):110-6.
[ expand abstract ]

In the field of the temperature sensitive drug delivery systems, we studied on the surface modification of liposomes by using poly(N-isopropylacrylamide-co-acrylamide) (PNIPAM-AAM) and polyethyleneglycol (PEG) to increase the release of doxorubicin (DOX) from liposomes and prolong the stability of liposomes in the presence of serum. The release of DOX from the PNIPAM-AAM/PEG modified liposomes is enhanced around the transition temperature of the polymer. In addition, the stability of the PNIPAM-AAM/PEG modified liposomes in serum shows a high level comparing with polymer unmodified liposomes. These results suggest that the modification on the surface of liposomes with both PNIPAM-AAM and PEG enhances the drug release from liposomes and reduces the protein adsorption in serum.

Cytotoxic and antitumor activity of liposome-incorporated sclareol against cancer cell lines and human colon cancer xenografts.
Hatziantoniou S, Dimas K, Georgopoulos A, Sotiriadou N, Demetzos C.
Pharmacol Res. 2006 Jan;53(1):80-7.
[ expand abstract ]

The aim of this study was to design and prepare liposome-incorporated sclareol--a highly lipophilic natural product-to overcome its water insolubility and develop suitable formulations for in vivo administration. The bioactive labdane-type diterpene sclareol was incorporated into liposomes composed of egg phosphatidylcholine and dipalmitoylphosphatidylglycerol prepared by the thin-film hydration method followed by sonication. A formulation of egg phosphatidylcholine/dipalmitoylphosphatidylglycerol/sclareol (9:0.1:5 molar ratio) was developed and characterized. The lipid recovery and the sclareol to lipid molar ratio were measured using high-performance thin-layer chromatography/flame ionization detection. In vitro drug release was measured in supplemented RPMI-1640 at 37 degrees C. The liposomal and the free sclareol were initially tested in vitro for their activity against human cancer cell lines using the sulphorhodamine B assay. Liposomes incorporating sclareol at a drug to lipid molar ratio of 0:43, suggesting an incorporation efficiency of almost 80%, showed reduced growth rate of human colon cancer tumors (HCT116) developed in SCID mice, without any significant side effects.

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Self-assembled nanoparticles based on glycol chitosan bearing hydrophobic moieties as carriers for doxorubicin: in vivo biodistribution and anti-tumor activity.
Hyung Park J, Kwon S, Lee M, Chung H, Kim JH, Kim YS, Park RW, Kim IS, Bong Seo S, Kwon IC, Young Jeong S.
Biomaterials. 2006 Jan;27(1):119-26.
[ expand abstract ]

Self-assembled nanoparticles, formed by polymeric amphiphiles, have been demonstrated to accumulate in solid tumors by the enhanced permeability and retention effect, following intravenous administration. In this study, hydrophobically modified glycol chitosans capable of forming nano-sized self-aggregates were prepared by chemical conjugation of fluorescein isothiocyanate or doxorubicin to the backbone of glycol chitosan. Biodistribution of self-aggregates (300 nm in diameter) was evaluated using tissues obtained from tumor-bearing mice, to which self-aggregates were systemically administered via the tail vein. Irrespective of the dose, a negligible quantity of self-aggregates was found in heart and lung, whereas a small amount (3.6-3.8% of dose) was detected in liver for 3 days after intravenous injection of self-aggregates. The distributed amount of self-aggregates gradually increased in tumor as blood circulation time increased. The concentration of self-aggregates in blood was as high as 14% of dose at 1 day after intravenous injection and was still higher than 8% even at 3 days. When self-aggregates loaded with doxorubicin were administered into the tumor-bearing mice via the tail vein, they exhibited lower toxicity than but comparable anti-tumor activity to free doxorubicin. These results revealed the promising potential of self-aggregates on the basis of glycol chitosan as a carrier for hydrophobic anti-tumor agents.

Apoptosis Induction in Human Lymphoma and Leukemia Cell Lines by Transfection via Dendrosomes Carrying Wild-Type p53 cDNA.
Massumi M, Ziaee AA, Sarbolouki MN.
Biotechnol Lett. 2006 Jan;28(1):61-6.
[ expand abstract ]

The efficiency of dendrosomes (novel dendritic spheroidal nanoparticle gene porters) were assessed in transferring wild-type p53 cDNA into two human acute lymphoblastic leukemia cell lines (MOLT-4 and CCRF-CEM) derived from T cells and erythroleukemic cell line K562. Flow cytometric studies showed a 65% and 45% enhancement in apoptosis and necrosis of K562 and CCRF-CEM cells transfected with complex of dendrosomes and wild-type p53 cDNA in comparison to controls. The cytotoxicity studies on T lymphoma cells revealed that dendrosomes have a low cytotoxicity in comparison to lipofectin.

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A liposomal formulation of doxorubicin, composed of hexadecylphosphocholine (HePC): physicochemical characterization and cytotoxic activity against human cancer cell lines.
Papagiannaros A, Hatziantoniou S, Dimas K, Papaioannou GT, Demetzos C.
Biomed Pharmacother. 2006 Jan;60(1):36-42.
[ expand abstract ]

The overall goal of this study was to prepare a novel liposomal formulation of doxorubicin, composed of hexadecylphosphocholine (HePC), as a combined formulation and to study its activity against cancer cells and peripheral blood mononuclear cells (PBMCs), in terms of efficacy and toxicity. Liposomes composed of HePC/egg phosphatidylcholine/stearylamine (HePC/EPC/SA) 10:10:0.1 (molar ratio) (1) and EPC/SA 10:0.1 (molar ratio) (2) were prepared and doxorubicin was encapsulated using the pH gradient method. Determination of lipids and doxorubicin has been achieved by high-performance thin-layer chromatography coupled with a flame-ionization detector. Prepared liposomes were characterized for their size distribution and their zeta-potential at each step of the preparation procedure. In vitro release studies have been evaluated in buffer and culture medium at 25 and 37 degrees C for 24 hours period. Liposomal formulations, free doxorubicin and HePC were tested against cancer cell lines and PBMCs, using sulforhodamine B (SRB) assay. Doxorubicin was encapsulated into the liposomes 1 and 2 at a drug to lipid molar ratio of 1.08 and 0.77, respectively, with an entrapping efficiency almost 100% in both cases. Doxorubicin was retained into liposome 1 up to 70% at 25 degrees C in TES, while up to 80% was released from 1 when liposomes were incubated at 37 degrees C either in culture medium or in the TES buffer at 24 hours. The activity of doxorubicin was retained or slightly improved when entrapped into liposomes 1 and 2, while liposomal formulation 1 encapsulating doxorubicin was found to be less toxic against normal cells (PBMCs). The combination of HePC and doxorubicin in one combined formulations justified as an improvement of the therapeutic index (TI) of doxorubicin in terms of efficacy and toxicity.

Pharmacokinetics of intravenously administered stealth liposomal doxorubicin modulated with verapamil in rats.
Wang JC, Liu XY, Lu WL, Chang A, Zhang Q, Goh BC, Lee HS.
Eur J Pharm Biopharm. 2006 Jan;62(1):44-51.
[ expand abstract ]

Treatment of cancer through co-administration of anticancer drugs and multidrug resistance (MDR) modulators as a strategy to overcome drug resistance has been extensively explored. However, success has been limited by pharmacokinetic interactions because of non-specific blockade of P-glycoprotein (P-gp) in normal tissues or inability to reach relevant concentrations clinically. We hypothesized that stealth liposomal co-encapsulation of doxorubicin (DOX) with a P-glycoprotein inhibitor, verapamil (DARSLs), may overcome these limitations. Using intravenous (i.v.) administrations, the effects of verapamil (VER) either free (FV) or liposome co-encapsulated with DOX (DARSLs) on the pharmacokinetics and tissue distribution characteristics of DOX either as free (FD) or liposome-encapsulated (LD) were evaluated in normal rats. FV increased (P<0.05) the plasma AUC of free DOX (FD). Preparations containing LD had significant prolonged systemic exposure and slow tissue distribution of DOX. LDFV (liposomal DOX with free verapamil) and DARSLs shared similar DOX pharmacokinetics but the latter showed slower DOX distribution in most tissues studied and slower (P<0.05) DOX biliary transport. The addition of VER into LD in these two preparations significantly increased the AUC (P<0.01) and reduced the clearance (P<0.01) of DOX when compared to LD. Specifically, DARSLs reduced initial DOX distribution to the heart (P<0.05) corresponding to initial alleviation (P<0.05) of bradycardia when compared to other DOX with VER preparations. In conclusion, liposomal co-encapsulation of DOX with VER has promise of significant therapeutic advantages, and should be explored further in therapeutic studies with animal tumor xenograft models.

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Application of boron-entrapped stealth liposomes to inhibition of growth of tumour cells in the in vivo boron neutron-capture therapy model.
Yanagie H, Maruyama K, Takizawa T, Ishida O, Ogura K, Matsumoto T, Sakurai Y, Kobayashi T, Shinohara A, Rant J, Skvarc J, Ilic R, Kuhne G, Chiba M, Furuya Y, Sugiyama H, Hisa T, Ono K, Kobayashi H, Eriguchi M.
Biomed Pharmacother. 2006 Jan;60(1):43-50.
[ expand abstract ]

Tumour cell destruction in boron neutron-capture therapy (BNCT) is due to the nuclear reaction between (10)B and thermal neutrons. It is necessary for effective BNCT therapy to accumulate (10)B atoms in the tumour cells. The delivery system consisted of polyethylene-glycol (PEG) binding liposomes (DPPC/cholesterol/DSPC-PEG2000) with an entrapped (10)B-compound and we evaluated the cytotoxic effects of intravenously injected (10)B-PEG-liposomes on human pancreatic carcinoma xenografts in nude mice with thermal neutron irradiation. After thermal neutron irradiation of mice injected with (10)B-PEG-liposomes, growth of AsPC-1 tumours was suppressed relative to controls. Injection of (10)B-PEG-liposomes caused the greatest tumour suppression with thermal neutron irradiation in vivo. These results suggest that intravenous injection of (10)B-PEG-liposomes can increase the retention of (10)B atoms by tumour cells, causing suppression of tumour growth in vivo, after thermal neutron irradiation.

Vaccine Efficacy of Fusogenic Liposomes Containing Tumor Cell-Lysate against Murine B16BL6 Melanoma.
Yoshikawa T, Okada N, Tsujino M, Gao JQ, Hayashi A, Tsutsumi Y, Mayumi T, Yamamoto A, Nakagawa S.
Biol Pharm Bull. 2006 Jan;29(1):100-4.
[ expand abstract ]

Recent advances in tumor immunology have facilitated the development of cancer immunotherapy targeting tumor-associated antigens (TAAs). However, because TAAs were identified in only a few types of human cancer, novel vaccine strategies that utilize tumor cell-lysate (TCL), including unidentified TAAs as an antigen source, are needed. Herein, we describe the utility of fusogenic liposomes (FLs) as TCL-delivery carriers for both ex vivo dendritic cell-based vaccination and in vivo direct immunization in the murine B16BL6 melanoma model. As a result, both in vivo direct immunization and ex vivo immunization induced anti-B16 melanoma prophylactic effects. Ex vivo dendritic cell (DC)-mediated vaccination strategy exert more potent anti-tumor effect than direct immunization. Our results suggest that this flexible system is a promising approach for the development of versatile cancer immunotherapy regimes.

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Nanoparticles of poly(lactide)/vitamin E TPGS copolymer for cancer chemotherapy: synthesis, formulation, characterization and in vitro drug release.
Zhang Z, Feng SS.
Biomaterials. 2006 Jan;27(2):262-70.
[ expand abstract ]

Paclitaxel is one of the best anticancer drugs, which has excellent therapeutic effects against a wide spectrum of cancers. The formulation of paclitaxel used in its currently clinical administration includes Cremophor EL, which has been found to cause serious side effects. Nanoparticle formulation of paclitaxel may provide an ideal solution for this problem and achieve a sustained chemotherapy. A novel copolymer, poly(lactide)-vitamin E TPGS (PLA-TPGS), was synthesized from lactide and d-alpha-tocopheryl polyethylene glycol 1000 succinate by bulk polymerization for nanoparticle formulation of anticancer drugs. 1H NMR, FTIR and GPC were used to detect molecular structure of the copolymer. Paclitaxel-loaded PLA-TPGS nanoparticles were fabricated by a modified solvent extraction/evaporation technique with or without emulsifier involved, which were characterized by laser light scattering for size and size distribution; field emission scanning electron microscopy for surface morphology; zeta potential for surface charge; X-ray photoelectron spectroscopy for surface chemistry. The drug encapsulation efficiency and the in vitro drug release kinetics were measured by high-performance liquid chromatography. Formulation optimization was pursued. The particles were found of around 300 nm in size and narrow size distribution. Of all, 89% drug encapsulation efficiency has been achieved for nanoparticles of 5% drug loading. The drug release from PLA-TPGS nanoparticles was found to be biphasic with an initial burst of 17% in the first day, followed by a sustained pattern with 51% release after 31 days.

2005

Polymeric nanoparticulate delivery system for Indocyanine green: Biodistribution in healthy mice.
Saxena V, Sadoqi M, Shao J.
Int J Pharm. 2005 Dec 28; [Epub ahead of print].
[ expand abstract ]

The objective of this study is to investigate the biodistribution of Indocyanine green (ICG) in healthy mice, when delivered through polymeric nanoparticles. The poly(dl-lactic-co-glycolic acid) (PLGA) nanoparticles entrapping ICG were engineered and characterized. The extraction method for ICG recovery from biological samples was developed. The biodistribution of ICG was determined in healthy C57BL/6 mice (female, 10-week old) when delivered through PLGA nanoparticles in comparison to free ICG solution, using a fluorometric assay method. The extraction method for ICG shows efficiency above 80% for various organs and plasma. When nanoparticles were used to deliver ICG, 2-8 times higher concentrations of ICG was deposited in various organs, with 5-10 times higher plasma levels till 4h, after an i.v. dose as compared to free ICG solution. In conclusion, the nanoparticle formulation significantly increased the ICG concentration and circulation time in plasma as well as the ICG uptake, accumulation and retention in various organs. Overall, this study represents the first step in exploring and establishing the potential of nanoparticles as an ICG-delivery system for use in tumor-diagnosis and photodynamic therapy.

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Synthesis, characterization and stability of dendrimer prodrugs.
Najlah M, Freeman S, Attwood D, D'Emanuele A.
Int J Pharm. 2005 Dec 26; [Epub ahead of print] .
[ expand abstract ]

The design, synthesis and characterization of a series of zero generation (G0) PAMAM dendrimer-based prodrugs for the potential enhancement of drug solubility and bioavailability are described. Naproxen, a poorly water-soluble drug, was conjugated to dendrimers either directly by an amide bond or by ester bonds using either l-lactic acid or diethylene glycol as a linker. All of the prodrugs were more hydrophilic than the parent drug, as evaluated by drug partitioning between 1-octanol and phosphate buffer (pH 7.4). Hydrolysis of the conjugates was measured at 37 degrees C in hydrochloric acid buffer (pH 1.2), phosphate buffer (pH 7.4), borate buffer (pH 8.5) and in 80% human plasma. The amide conjugate and both ester conjugates were chemically stable at all pHs over 48h of incubation. Naproxen was enzymatically released from both ester conjugates in plasma; the lactic ester conjugate hydrolyzed slowly with only 25% of naproxen released after 24h, the diethylene glycol ester conjugate cleaved rapidly following pseudo first order kinetics (t(1/2)=51min). G0 PAMAM dendrimer prodrugs with an appropriate linker (diethylene glycol) show good potential as carriers for oral delivery.

Therapeutic possibilities of plasmonically heated gold nanoparticles.
Pissuwan D, Valenzuela SM, Cortie MB.
Trends Biotechnol. 2005 Dec 24; [Epub ahead of print] .
[ expand abstract ]

Nanoparticles of gold, which are in the size range 10-100nm, undergo a plasmon resonance with light. This is a process whereby the electrons of the gold resonate in response to incoming radiation causing them to both absorb and scatter light. This effect can be harnessed to either destroy tissue by local heating or release payload molecules of therapeutic importance. Gold nanoparticles can also be conjugated to biologically active moieties, providing possibilities for targeting to particular tissues. Here, we review the progress made in the exploitation of the plasmon resonance of gold nanoparticles in photo-thermal therapeutic medicine

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Systemic trafficking of plant virus nanoparticles in mice via the oral route.
Rae CS, Wei Khor I, Wang Q, Destito G, Gonzalez MJ, Singh P, Thomas DM, Estrada MN, Powell E, Finn MG, Manchester M.
Virology. 2005 Dec 20;343(2):224-35.
[ expand abstract ]

The plant virus, cowpea mosaic virus (CPMV), is increasingly being used as a nanoparticle platform for multivalent display of peptides. A growing variety of applications have employed the CPMV display technology including vaccines, antiviral therapeutics, nanoblock chemistry, and materials science. CPMV chimeras can be inexpensively produced from experimentally infected cowpea plants and are completely stable at 37 degrees C and low pH, suggesting that they could be used as edible or mucosally-delivered vaccines or therapeutics. However, the fate of CPMV particles in vivo, or following delivery via the oral route, is unknown. To address this question, we examined CPMV in vitro and in vivo. CPMV was shown to be stable under simulated gastric conditions in vitro. The pattern of localization of CPMV particles to mouse tissues following oral or intravenous dosing was then determined. For several days following oral or intravenous inoculation, CPMV was found in a wide variety of tissues throughout the body, including the spleen, kidney, liver, lung, stomach, small intestine, lymph nodes, brain, and bone marrow. CPMV particles were detected after cardiac perfusion, suggesting that the particles entered the tissues. This pattern was confirmed using methods to specifically detect the viral capsid proteins and the internal viral RNA. The stability of CPMV virions in the gastrointestinal tract followed by their systemic dissemination supports their use as orally bioavailable nanoparticles.

Epidermal Growth Factor Receptor-Targeted Immunoliposomes Significantly Enhance the Efficacy of Multiple Anticancer Drugs In vivo.
Mamot C, Drummond DC, Noble CO, Kallab V, Guo Z, Hong K, Kirpotin DB, Park JW.
Cancer Res. 2005 Dec 15;65(24):11631-8.
[ expand abstract ]

We previously reported the development of epidermal growth factor receptor (EGFR)-targeted immunoliposomes that bind and internalize in tumor cells which overexpress EGFR and/or mutant EGFR variant III (EGFRvIII), enabling intracellular delivery of potent anticancer agents in vitro. We now describe in vivo proof-of-concept for this approach for the delivery of multiple anticancer drugs in EGFR-overexpressing tumor models. Anti-EGFR immunoliposomes were constructed modularly with Fab' fragments of cetuximab (IMC-C225), covalently linked to liposomes containing probes and/or anticancer drugs. Pharmacokinetic and biodistribution studies confirmed long circulation times (t(1/2) = 21 hours) and efficient accumulation in tumors (up to 15% ID/g) irrespective of the presence of the targeting ligand. Although total accumulations of anti-EGFR immunoliposomes and nontargeted liposomes in EGFR-overexpressing tumors were comparable, only immunoliposomes internalized extensively within tumor cells (92% of analyzed cells versus <5% for nontargeted liposomes), indicating different mechanisms of delivery at the cellular level. In vivo therapy studies in a series of xenograft models featuring overexpression of EGFR and/or EGFRvIII showed the superiority of immunoliposomal delivery of encapsulated drugs, which included doxorubicin, epirubicin, and vinorelbine. For each of these drugs, anti-EGFR immunoliposome delivery showed significant antitumor effects and was significantly superior to all other treatments, including the corresponding free or liposomal drug (P < 0.001-0.003). We conclude that anti-EGFR immunoliposomes provide efficient and targeted drug delivery of anticancer compounds and may represent a useful new treatment approach for tumors that overexpress the EGFR. (Cancer Res 2005; 65(24): 11631-8).

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Non-linear pharmacodynamics in a non-viral gene delivery system: Positive non-linear relationship between dose and transfection efficiency.
Moriguchi R, Kogure K, Iwasa A, Akita H, Harashima H.
J Control Release. 2005 Dec 14; [Epub ahead of print].
[ expand abstract ]

A remarkable non-linearity was found between dose and transfection activities of non-viral gene delivery systems, such as a Lipofectamine/DNA complex and an octaarginine-modified multifunctional envelope-type nano device (R8-MEND). We measured the nuclear delivery of pDNA to distinguish the non-linearity in intracellular pharmacokinetics or pharmacodynamics after transfection with R8-MEND at different doses. A remarkable positive non-linearity was found in the pharmacodynamics when the dose was increased. Even dummy pDNA enhanced the efficiency of transcription and/or translation per pDNA in the nucleus, but empty liposomes did not. These results suggest the importance of controlled pharmacodynamics as well as the importance of intracellular pharmacokinetics for the rational design of non-viral gene delivery systems.

Targeted drug delivery with dendrimers: Comparison of the release kinetics of covalently conjugated drug and non-covalent drug inclusion complex.
Patri AK, Kukowska-Latallo JF, Baker JR Jr.
Adv Drug Deliv Rev. 2005 Dec 14;57(15):2203-14.
[ expand abstract ]

Dendrimers have unique characteristics including monodispersity and modifiable surface functionality, along with highly defined size and structure. This makes these polymers attractive candidates as carriers in drug delivery applications. Drug delivery can be achieved by coupling a drug to polymer through one of two approaches. Hydrophobic drugs can be complexed within the hydrophobic dendrimer interior to make them water-soluble or drugs can be covalently coupled onto the surface of the dendrimer. Using both methods we compared the efficacy of generation 5 PAMAM dendrimers in the targeted drug delivery of methotrexate coupled to the polymer. The amine-terminated dendrimers bind to negatively charged membranes of cells in a non-specific manner and can cause toxicity in vitro and in vivo. To reduce toxicity and to increase aqueous solubility, modifications were made to the surface hydroxyl groups of the dendrimers. For targeted drug delivery, the dendrimer was modified to have a neutral terminal functionality for use with surface-conjugated folic acid as the targeting agent. The complexation of methotrexate within a dendrimer changes the water insoluble drug into a stable and readily water-soluble compound. When this dendrimer complexed drug, however, was placed in a solution of phosphate buffered saline, the methotrexate was immediately released and displayed diffusion characteristics identical to free methotrexate. Covalently coupled methotrexate dendrimer conjugates were stable under identical conditions in water and buffered saline. Cytotoxicity tests showed that methotrexate as the dendrimer inclusion complex had an activity identical to the free drug in vitro. In contrast, folic acid targeted dendrimer with covalently conjugated methotrexate specifically killed receptor-expressing cells by intracellular delivery of the drug through receptor-mediated endocytosis. This study demonstrates that while drug as a dendrimer inclusion complex is readily released and active in vitro, covalently conjugated drug to dendrimer is better suited for specifically targeted drug delivery.

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Vacancy Engineered Ceria Nanostructures for Protection from Radiation-Induced Cellular Damage.
Tarnuzzer RW, Colon J, Patil S, Seal S.
Nano Lett. 2005 Dec 14;5(12):2573-2577.
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The ability of engineered cerium oxide nanoparticles to confer radioprotection was examined. Human normal and tumor cells were treated with nanoceria and irradiated, and cell survival was measured. Treatment of normal cells conferred almost 99% protection from radiation-induced cell death, whereas the same concentration showed almost no protection of tumor cells. For the first time, nanoceria is shown to confer radioprotection to a normal human breast line but not to a human breast tumor line, MCF-7.

Genospheres: self-assembling nucleic acid-lipid nanoparticles suitable for targeted gene delivery.
Hayes ME, Drummond DC, Kirpotin DB, Zheng WW, Noble CO, Park JW, Marks JD, Benz CC, Hong K.
Gene Ther. 2005 Dec 8; [Epub ahead of print].
[ expand abstract ]

We describe the assembly of a cationic lipid-nucleic acid nanoparticle from a liquid monophase containing water and a water miscible organic solvent where both lipid and DNA components are separately soluble prior to their combination. Upon removal of the organic solvent, stable and homogenously sized (70-100 nm) lipid-nucleic acid nanoparticles (Genospherestrade mark) were formed. The low accessibility (<15%) of the nanoparticle-encapsulated DNA to a DNA intercalating dye indicated well-protected nucleic acids and high DNA incorporation efficiencies. It was demonstrated that Genospheres could be stably stored under a variety of conditions including a lyophilized state where no appreciable increase in particle size or DNA accessibility was observed following reconstitution. Finally, Genospheres were made target-specific by insertion of an antibody-lipopolymer (anti-HER2 scFv (F5)-PEG-DSPE) conjugate into the particle. The target specificity (>100-fold) in HER2 overexpressing SK-BR-3 breast cancer cells was dependent on the degree of PEGylation, where the incorporation of high amounts of PEG-lipid on the particle surface (up to 5 mol%) had only a minor effect on the transfection activity of the targeted Genospheres. In summary, this work describes a novel, readily scalable method for preparing highly stable immunotargeted nucleic acid delivery vehicles capable of achieving a high degree of specific transfection activity.

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Preferential extravasation and accumulation of liposomal vincristine in tumor comparing to normal tissue enhances antitumor activity.
Shan S, Flowers C, Peltz CD, Sweet H, Maurer N, Kwon EJ, Krol A, Yuan F, Dewhirst MW.
Cancer Chemother Pharmacol. 2005 Dec 8;1-11.
[ expand abstract ]

To quantitatively evaluate the extravasation, accumulation and selectivity to tumor tissues of liposomal vincristine (LV), dorsal skin-fold window chambers on athymic mice with or without LX-1, a human small cell lung cancer, xenograft implants and fluorescent intravital microscopy imaging were used. In vitro studies show that minimal loss of fluorescence marker DiI from liposomes occurs after 4 days of inoculation in murine plasma, and the release profiles of DiI-LV and LV were essentially the same with approximately 40% of the encapsulated vincristine sulfate (VCR) released after 26 h. Significantly faster extravasation of DiI-LV from tumor vessels was shown compared to non-tumor tissue after single dose i.v. administration. The relative interstitial amounts at 60 min (RIA(60)) for tumor and non-tumor tissues were 0.837+/-0.314 and 0.012+/-0.091, respectively (P=0.01). DiI-LV accumulation was significantly higher in tumor than in normal tissue, which continued beyond 48 h. Both DiI-LV and LV showed significant antitumor effects in window chambers and in flank tumors, compared with controls and VLS alone. The preferential extravasation of DiI-LV from tumor vasculature as well as its differential retention in tumor tissue provides the basis for the enhancement in antitumor activity of LV over VCR.

Formulation optimization for the nanoparticles-in-microsphere hybrid oral delivery system using factorial design.
Bhavsar MD, Tiwari SB, Amiji MM.
J Control Release. 2005 Dec 7; [Epub ahead of print].
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The tremendous progress witnessed in the field of biotechnology with respect to discovery of therapeutic and antigenic proteins has propelled the need for development of suitable oral delivery devices for these and other macromolecules. In this study, we report the encapsulation of fluorescein isothiocyanate (FITC)-labeled gelatin nanoparticles into poly(epsilon-caprolactone) (PCL) microsphere (nanoparticle-in-microsphere oral delivery system, NiMOS) by double emulsion like technique and the influence of variables such as polymer concentration in organic phase, amount of nanoparticles added as internal phase, and the speed of homogenization on particle size of NiMOS using a 3(3) randomized full factorial design. A statistical model with interaction terms was derived to predict the particle size of the hybrid system. The results from multiple linear regression analysis and Student's t-test revealed that for obtaining large particles of NiMOS, a high polymer concentration and low speed of homogenization was necessary. In contrast, to obtain particles of smaller size, high speed of homogenization was found to be very important. The mathematical model obtained was validated for prediction of particle size. The encapsulation of gelatin nanoparticles in PCL microsphere was confirmed by fluorescent microscopy. Based on the statistical model we were also successful in producing NiMOS of less than 10 mum in size, which could be used as oral delivery system for therapeutic and antigenic macromolecules.

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Liposome formulation of a novel hydrophobic aryl-imidazole compound for anti-cancer therapy.
Liu J, Lee H, Huesca M, Young A, Allen C.
Cancer Chemother Pharmacol. 2005 Dec 7;1-13.
[ expand abstract ]

Purpose: A cholesterol-free liposome formulation formed from mixtures of egg phosphatidylcholine (ePC) and poly (ethylene glycol) conjugated distearoylphosphatidylethanolamine (DSPE-PEG 2000) was optimized and evaluated for delivery of a novel anti-cancer agent ML220 (2-(5-bromo-1H-indol-3-yl)-1H-phenanthro [9,10-d] imidazole). Results and Discussion: ML220 is highly lipophilic with a water solubility of 0.14 mug/ml and calculated log P of 5.69. The ML220-loaded liposomes had a unimodal size-distribution and a mean diameter of 89 nm. The drug to lipid ratio in the formulation was 1:3.5 (mol:mol) and the drug loading efficiency was 83% providing a more than 50,000-fold increase in the water solubility of ML220. The formulation was demonstrated to be stable in vitro at 37 degrees C for over 2 weeks with a delayed drug release profile. Evaluation of the subacute toxicity of the liposome formulated drug in C3H mice revealed no overt signs of toxicity. Also, a biexponential drug plasma concentration pattern was found upon evaluation of the pharmacokinetics in Balb/C mice. The in vivo evaluation of the anti-cancer activity in a human colon HT29 carcinoma model revealed a significant delay in tumor growth. Conclusion: Overall, the ePC/DSPE-PEG liposomes were demonstrated to be a suitable delivery system for ML220. These studies also highlight the potential of cholesterol-free liposomes as a formulation strategy for highly lipophilic drugs.

Tumor-Targeting Nanoimmunoliposome Complex for Short Interfering RNA Delivery.
Pirollo KF, Zon G, Rait A, Zhou Q, Yu W, Hogrefe R, Chang EH.
Hum Gene Ther. 2005 Dec 7; [Epub ahead of print].
[ expand abstract ]

The potential of short interfering RNA (siRNA) to be developed for therapeutic use against cancer depends on the availability of an efficient tumor-specific delivery vehicle. We have previously shown that a nanoscale nonviral liposome-based complex that includes an anti-transferrin receptor single-chain antibody fragment as the targeting moiety can, when systemically administered, specifically and efficiently target primary and metastatic tumors and deliver molecules useful in gene medicine, including plasmid DNA and antisense oligonucleotides. Here we explore the ability of this complex to deliver a fluorescein-labeled siRNA to tumor cells in vivo and examine the intracellular localization in vitro by confocal microscopy. We show that the immunoliposome-siRNA complex maintains its nanoscale size and, using three separate tumor models, can efficiently and specifically deliver siRNA to both primary and metastatic disease after systemic delivery, thus increasing the possibility for translating the potent effects of siRNA observed in vitro into clinically useful therapeutics.

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Oligoarginine vectors for intracellular delivery: Design and cellular-uptake mechanisms.
Futaki S.
Biopolymers. 2005 Dec 6; [Epub ahead of print].
[ expand abstract ]

Intracellular delivery using membrane-permeable peptide vectors is a recently developed methodology that has successfully been employed to transport various bioactive molecules into cells to modify cell functions. The efficient delivery of proteins, peptides, nucleic acids, liposomes and so on has been accomplished using this methodology by conjugation of a peptide vector with the cargo molecules. The potentials of this approach for medical and pharmaceutical applications has also attracted our attention. Arginine-rich peptides, including a basic peptide segment derived from the human immunodeficiency virus type 1 (HIV-1) Tat protein, are categorized into one of the most frequently used peptide vectors, and the efforts of designing novel vectors have been ongoing. Internalization of these peptides has previously been regarded as not employing endocytosis. However, recent reevaluations have demonstrated the significant involvement of endocytosis in the cellular uptake of these peptides. These arginine-rich peptide vectors share many common features in internalization. However, there seems to be certain simultaneous dissimilarities observed in the modes of internalization among these peptides. In this review, the structural features of these arginine-rich peptide vectors have been focused on and the current understandings of their internalization mechanisms are summarized. (c) 2005 Wiley Periodicals, Inc. Biopolymers, 2005.

Rerouting lipoprotein nanoparticles to selected alternate receptors for the targeted delivery of cancer diagnostic and therapeutic agents.
Zheng G, Chen J, Li H, Glickson JD.
Proc Natl Acad Sci U S A. 2005 Dec 6;102(49):17757-62.
[ expand abstract ]

We report that a lipoprotein-based nanoplatform generated by conjugating tumor-homing molecules to the protein components of naturally occurring lipoproteins reroutes them from their normal lipoprotein receptors to other selected cancer-associated receptors. Multiple copies of these targeting moieties may be attached to the same nanoparticle, or a variety of different targeting moieties can be attached. Such a diverse set of tumor-homing molecules could be used to create a variety of conjugated lipoproteins as multifunctional, biocompatible nanoplatforms with a broad application to both cancer imaging and treatment. The same principle can be applied to imaging and treatment of other diseases and for monitoring specific tissues. To validate this concept, we prepared a low-density lipoprotein (LDL)-based folate receptor (FR)-targeted agent by conjugating folic acid to the Lys residues of the apolipoprotein B (apoB)-100 protein. To demonstrate the ability of the lipoprotein-based nanoplatform to deliver surfaceloaded and core-loaded payloads, the particles were labeled either with the optical reporter 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine that was intercalated in the phospholipid monolayer or with the lipophilic photodynamic therapy agent, tetra-t-butyl-silicon phthalocyanine bisoleate, that was reconstituted into the lipid core. Cellular localization of the labeled LDL was monitored by confocal microscopy and flow cytometry in FR-overexpressing KB cells, in FR-nonexpressing CHO and HT-1080 cells, and in LDL receptor-overexpressing HepG(2) cells. These studies demonstrate that the folic acid conjugation to the Lys side-chain amino groups blocks binding to the normal LDL receptor and reroutes the resulting conjugate to cancer cells through their FRs.

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Nano-carriers for DNA delivery to the lung based upon a TAT-derived peptide covalently coupled to PEG-PEI.
Kleemann E, Neu M, Jekel N, Fink L, Schmehl T, Gessler T, Seeger W, Kissel T.
J Control Release. 2005 Dec 5;109(1-3):299-316.
[ expand abstract ]

Gene therapy aimed at the respiratory epithelium holds therapeutic potential for diseases such as cystic fibrosis and lung cancer. Polyethylenimine (PEI) has been utilized for gene delivery to the airways. In this study, we describe a new modification of PEI, in which an oligopeptide related to the protein transduction domain of HIV-1 TAT was covalently coupled to 25 kDa PEI (PEI) through a heterobifunctional polyethylenglycol (PEG) spacer resulting in a TAT-PEG-PEI conjugate. Improved DNA reporter gene complexation and protection was observed for small ( approximately 90 nm) polyplexes as well as significantly improved stability against polyanions, Alveofact(R), bronchial alveolar lining fluid and DNase. To determine polyplex toxicity in vitro, MTT assays were performed and, for in vivo testing, the mice bronchial alveolar lavage was investigated for total cell counts, quantity of neutrophils, total protein and TNF-alpha concentration. All parameters suggest significantly lower toxicity for TAT-PEG-PEI. Transfection efficiencies of both PEI and TAT-PEG-PEI polyplexes with DNA were studied under in vitro conditions (A549) and in mice after intratracheal instillation. While luciferase expression in A549 cells was much lower for TAT-PEG-PEI (0.2 ng/mg protein) than for PEI (2 ng/mg), significantly higher transfection efficiencies for TAT-PEG-PEI were detected in mice. Reporter gene expression was distributed through bronchial and alveolar tissue. Thus, TAT-PEG-PEI represents a new approach to non-viral gene carriers for lung therapy, comprising protection for plasmid DNA, low toxicity and significantly enhanced transfection efficiency under in vivo conditions.

Phase II study of sphingosomal vincristine in patients with recurrent or refractory adult acute lymphocytic leukemia.
Thomas DA, Sarris AH, Cortes J, Faderl S, O'brien S, Giles FJ, Garcia-Manero G, Rodriguez MA, Cabanillas F, Kantarjian H.
Cancer. 2005 Dec 5; [Epub ahead of print].
[ expand abstract ]

BACKGROUND: Outcomes with salvage therapy for patients with recurrent or refractory acute lymphocytic leukemia (ALL) are poor, with complete response (CR) rates reported to be 20-30% and a median survival ranging from 2-6 months. New agents are needed to reduce the recurrence rate after frontline chemotherapy. Vincristine is an important component of ALL therapy. In animal models, the encapsulation of vincristine into sphingomyelin liposomes or "sphingosomes" for injection (SV) has improved efficacy compared with conventional vincristine. METHODS: A Phase II clinical trial of single-agent SV given at a dose of 2.0 mg/m(2) every 2 weeks was conducted in patients with recurrent or refractory ALL. Approximately half of the 16 patients who received SV had a first CR duration of less than 1 year, 19% had failed standard induction chemotherapy, and 50% had Philadelphia chromosome-positive disease. SV was the first salvage attempt in 69% of the patients. RESULTS: The overall response rate in the 14 evaluable patients was 14% (1 CR and 1 partial response). Five patients (36%) had transient reductions in bone marrow leukemia infiltrate with subsequent regrowth of the leukemia between SV infusions. Toxicity with limited treatment (median number of doses was two; range, one to five doses) was minimal with expected peripheral neuropathy. CONCLUSIONS: Further study of SV in patients with ALL is warranted. A Phase I-II clinical trial of weekly SV with pulse dexamethasone currently is ongoing.

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Liposomal, nanoparticle, and conjugated formulations of anticancer agents.
Zamboni WC.
Clin Cancer Res. 2005 Dec 1;11(23):8230-4.
[ expand abstract ]

No abstract available

Applications of carbon nanotubes in drug delivery.
Bianco A, Kostarelos K, Prato M.
Curr Opin Chem Biol. 2005 Dec;9(6):674-9.
[ expand abstract ]

The development of new and efficient drug delivery systems is of fundamental importance to improve the pharmacological profiles of many classes of therapeutic molecules. Many different types of drug delivery systems are currently available. Within the family of nanomaterials, carbon nanotubes (CNT) have emerged as a new alternative and efficient tool for transporting and translocating therapeutic molecules. CNT can be functionalised with bioactive peptides, proteins, nucleic acids and drugs, and used to deliver their cargos to cells and organs. Because functionalised CNT display low toxicity and are not immunogenic, such systems hold great potential in the field of nanobiotechnology and nanomedicine.

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Nanoscale polymer carriers to deliver chemotherapeutic agents to tumours.
Cegnar M, Kristl J, Kos J.
Expert Opin Biol Ther. 2005 Dec;5(12):1557-69.
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Nanoscale polymer carriers have the potential to enhance the therapeutic efficacy of antitumour drugs as they can regulate their release, improve their stability and prolong circulation time by protecting the drug from elimination by phagocytic cells or premature degradation. Moreover, nanoscale polymeric carriers are capable of accumulating in tumour cells and tissues due to enhanced permeability and retention effect or by active targeting bearing ligands designed to recognise overexpressed tumour-associated antigens. The diversity in the polymer structures being studied as drug carriers in cancer therapy allows an optimal solution for a particular drug to be provided regarding its delivery and efficacy, and thus the patient's quality of life. This review is focused on the different types of nanoscale polymer carriers used for the delivery of chemotherapeutic agents and on the factors that affect their cellular uptake and trafficking.

Therapeutic potential of nanoparticulate systems for macrophage targeting.
Chellat F, Merhi Y, Moreau A, Yahia L.
Biomaterials. 2005 Dec;26(35):7260-75.
[ expand abstract ]

The use of non-viral nanoparticulate systems for the delivery of therapeutic agents is receiving considerable attention for medical and pharmaceutical applications. This increasing interest results from the fact that these systems can be designed to meet specific physicochemical requirements, and they display low toxic and immunogenic effects. Among potential cellular targets by drug-loaded nanoparticles, macrophages are considered because they play a central role in inflammation and they act as reservoirs for microorganisms that are involved with deadly infectious diseases. The most common and potent drugs used in macrophage-mediated diseases treatment often induce unwanted side effects, when applied as a free form, due to the necessity of high doses to induce a satisfactory effect. This could result in their systemic spreading, a lack of bioavailability at the desired sites, and a short half-life. Therefore, the use of drug-loaded nanoparticles represents a good alternative to avoid, or at least decrease, side effects and increase efficacy. In this manuscript, we present an overview of the usefulness of nanoparticles for macrophage-mediated therapies in particular. We discuss, though not exhaustively, the potential of therapeutic agent-loaded nanoparticles for some macrophage-mediated diseases. We also underline the most important parameters that affect the interaction mechanisms of the macrophages and the physicochemical aspects of the particulate systems that may influence their performance in macrophage-targeted therapies.

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Sonic activation of molecularly-targeted nanoparticles accelerates transmembrane lipid delivery to cancer cells through contact-mediated mechanisms: Implications for enhanced local drug delivery.
Crowder KC, Hughes MS, Marsh JN, Barbieri AM, Fuhrhop RW, Lanza GM, Wickline SA.
Ultrasound Med Biol. 2005 Dec;31(12):1693-700.
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Liquid perfluorocarbon nanoparticles serve as sensitive and specific targeted contrast and drug delivery vehicles by binding to specific cell surface markers. We hypothesized that application of acoustic energy at diagnostic power levels could promote nanoparticle-associated drug delivery by stimulating increased interaction between the nanoparticle's lipid layer and the targeted cell's plasma membrane. Ultrasound (mechanical index = 1.9) applied with a conventional ultrasound imaging system to nanoparticles targeted to alpha(v)beta(3)-integrins on C32 melanoma cancer cells in vitro produced no untoward effects. Within 5 min, lipid delivery from nanoparticles into cell cytoplasm was dramatically augmented. We also demonstrate the operation of a potential physical mechanism for this effect, the acoustic radiation force on the nanoparticles, which may contribute to the enhanced lipid delivery. Accordingly, we propose that local delivery of lipophilic substances (e.g., drugs) from targeted nanoparticles directly into cell cytoplasm can be augmented rapidly and safely with conventional ultrasound imaging devices through nondestructive mechanisms. (E-mail: ).

Nanotechnology for drug and gene therapy: the importance of understanding molecular mechanisms of delivery.
Labhasetwar V.
Curr Opin Biotechnol. 2005 Dec;16(6):674-80.
[ expand abstract ]

Nanotechnology, although not a new concept, has gained significant momentum in recent years. This stems partly from the realization that nanosystems have significantly different biological properties from large-sized systems (e.g. implants or microparticles) that could be used effectively to overcome problems in drug and gene therapy. In drug therapy, we face the problems of inefficacy or nonspecific effects; hence, nanosystems are being developed for targeted drug therapy. In gene therapy using non-viral systems, the main issues are relatively transient gene expression and lower efficiency than viral vectors. Research efforts have focused on understanding the barriers in gene delivery so that non-viral systems can be developed that are as effective as viral systems in gene transfection. Understanding the molecular mechanisms that underlie the interactions of nanosystems with the cell, their uptake properties and retention will be crucial for the successful development of these systems.

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Polymeric Nanoparticle Preparation that Eradicates Tumors.
McCarthy JR, Perez JM, Bruckner C, Weissleder R.
Nano Lett. 2005 Dec;5(12):2552-6.
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We report the production of poly(lactic-co-glycolic acid) nanoparticles that encapsulate the photosensitizer meso-tetraphenylporpholactol. These nanoparticles are stable and nonphototoxic upon systemic administration. Upon cellular internalization, the photosensitizer is released from the nanoparticle and becomes highly phototoxic. Irradiation with visible light results in cell-specific killing of several cancer cell lines. Importantly, in vivo experiments show complete eradication of cancers in mouse models. The concept of photosensitizers with selective phototoxicity should have widespread applications in cancer therapy.

The influence of oral tumor cell proliferation activity and membrane potential on the transfection efficiency of a cationic liposome.
Nakase M, Okumura K, Kamei T, Nakamura S, Inui M, Tagawa T.
Oncol Rep. 2005 Dec;14(6):1487-91.
[ expand abstract ]

The transfection efficiency of cationic liposomes varies according to cell type, but the specific cellular characteristics that affect transfection efficiency have not yet been defined. We investigated whether the transfection efficiency of cationic liposomes correlates with cell proliferation activity or cell membrane potential in oral malignant melanoma (HMG) and oral osteosarcoma cell lines (HOSM-1 and HOSM-2). The cell membrane potential was assessed by uptake of a cationic probe. Three oral tumor cell lines were exposed to a cationic liposome complexed with a beta-galactosidase expression plasmid, and beta-galactosidase expression was compared. Cell proliferation was about 2-fold higher in HOSM-1 cells than in HMG cells. The cell membrane potential in HMG and HOSM-1 cells was comparable, while the membrane potential in HOSM-2 cells was 1.6-fold higher. beta-galactosidase expression was measured by X-Gal staining in 7.0% of HMG, 17.0% of HOSM-1 and 11.5% of HOSM-2 cells. The present study demonstrates that gene therapy with cationic liposomes may be a promising new strategy for treatment of oral malignant melanoma and osteosarcoma. In addition, the transfection efficiency of cationic liposomes appears to be influenced by cell proliferation activity, but not cell membrane potential.

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Liquid filled nanoparticles as a drug delivery tool for protein therapeutics.
Venkatesan N, Yoshimitsu J, Ito Y, Shibata N, Takada K.
Biomaterials. 2005 Dec;26(34):7154-63.
[ expand abstract ]

In the present study, an attempt was made to study the feasibility of nanoparticulate adsorbents in the presence of an absorption enhancer, as a drug delivery tool for the administration of erythropoietin (EPO) to the small intestine. Liquid filled nano- and micro-particles (LFNPS/LFMPS) were prepared using solid adsorbents such as porous silicon dioxide (Sylysia 550), carbon nanotubes (CNTs), carbon nanohorns, fullerene, charcoal and bamboo charcoal. Surfactants such as a saturated polyglycolysed C8-C18 glyceride (Gelucire 44/14), PEG-8 capryl/caprylic acid glycerides (Labrasol) and polyoxyethylene hydrogenated castor oil derivative (HCO-60) were used as an absorption enhancer at 50mg/kg along with casein/lactoferrin as enzyme inhibitors. The absorption of EPO was studied by measuring serum EPO levels by an ELISA method after small intestinal administration of EPO-LFNPS preparation to rats at the EPO dose level of 100 IU/kg. Among the adsorbents studied, CNTs showed the highest serum EPO level of 62.7 +/- 11.6 mIU/ml. In addition, with the use of casein, EPO absorption was improved, C(max) 143.1 +/- 15.2 mIU/ml. Labrasol showed the highest absorption enhancing effect after intra-jejunum administration than Gelucire 44/14 and HCO-60, 25.6 +/- 3.2 and 22.2 +/- 3.6 mIU/ml, respectively. Jejunum was found to be the best absorption site for the absorption of EPO from LFNPS. The use of CNTs as LFNPS, improved the bioavailability of EPO to 11.5% following intra-small intestinal administration.

Pegylated liposomal doxorubicin (Lipo-Dox(R)) for platinum-resistant or refractory epithelial ovarian carcinoma: A Taiwanese gynecologic oncology group study with long-term follow-up.
Chou HH, Wang KL, Chen CA, Wei LH, Lai CH, Hsieh CY, Yang YC, Twu NF, Chang TC, Yen MS.
Gynecol Oncol. 2005 Nov 30; [Epub ahead of print].
[ expand abstract ]

OBJECTIVES.: To evaluate the efficacy and safety of a distearoylphosphatidylcholine pegylated liposomal doxorubicin, Lipo-Dox(R), in platinum-resistant or refractory epithelial ovarian cancer. METHODS.: A multicenter phase II trial enrolled women with platinum-resistant or refractory epithelial ovarian carcinoma and naive to anthracycline. Eligible patients had either measurable tumor(s) or elevated serum CA 125 titer. Lipodox was initiated with a dose of 45 mg/m(2) at a 4-week interval with subsequent escalation or reduction. A total of six cycles were scheduled. RESULTS.: 29 patients, 20 with platinum-resistant and 9 with platinum refractory tumors, were enrolled. Lipo-Dox was given for an average of 4.6 cycles per patient with a total of 134 cycles. Among the 26 evaluable patients, one achieved CR, 5 PR and 9 SD. The overall response rate was 23.1% (95% CI, 6.8%-39.3%) with a median response duration of 11.6 weeks. 5 of the 6 responses were in patients with resistant disease. The median progression-free duration in the SD patients was 25.7 weeks. With a median follow-up of 13.8 months, the median progression-free and median overall survivals in the 26 patients were 5.4 months and 13.8 months, respectively. Hand-foot skin reaction occurred in 4.5% and skin pigmentation in 11.2% of all treatment cycles, all were Grade 1/2. Nausea and vomiting occurred in 14.2%, while anemia, leukopenia and thrombocytopenia occurred in 20.9%, 32.8% and 9% of cycle, respectively, and were mostly Grade 1 or 2. CONCLUSION.: Lipo-Dox, the third liposome encapsulated doxorubicin, at 45 mg/m(2) every 4 weeks, is effective against recurrent, platinum-resistant epithelial ovarian cancers.

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Paclitaxel-loaded PLGA nanoparticles: potentiation of anticancer activity by surface conjugation with wheat germ agglutinin.
Mo Y, Lim LY.
J Control Release. 2005 Nov 28;108(2-3):244-62.
[ expand abstract ]

PURPOSE: To potentiate the anticancer activity of paclitaxel-loaded PLGA nanoparticles through surface conjugation with wheat germ agglutinin (WGA). METHODS: PLGA nanoparticles loaded with paclitaxel and isopropyl myristate (IPM) as release modifier were prepared by a solvent evaporation method. WGA was conjugated to the nanoparticle surface to give novel WIT-NP of 330+/-3 nm. In vitro cytotoxicity of WIT-NP against malignant (A549 and H1299) and normal (CCL-186) pulmonary cell lines was evaluated alongside control formulations. IC50 doses were determined by the MTT assay, while cellular apoptosis was detected by cell nuclei staining and DeadEndtrade mark Fluorometric TUNEL assay. Cell cycle arrest was confirmed by flow cytometry. Cellular uptake of 3[H]-paclitaxel from the test and control formulations was also quantified. In vivo anticancer efficacy was evaluated in the SCID mice model engrafted with the A549 tumor nodule. RESULTS: WIT-NP had superior anti-proliferation activity against the A549 and H1299 cell lines compared with conventional paclitaxel formulations as measured by IC50 doses. This was attributed to a more efficient intracellular accumulation of paclitaxel via WGA-receptor-mediated endocytosis and IPM-facilitated intracellular paclitaxel release. WIT-NP activity was associated with paclitaxel-induced apoptosis and cell arrest in the G2/M phase. A single intratumoral injection of WIT-NP at paclitaxel dose of 10 mg/kg inhibited the growth of A549 tumor nodules without inducing significant weight loss in the SCID mice over a period of 25 days. Tumor doubling time was greater than 25 days, compared with 11 days for nodules treated with conventional paclitaxel formulation. CONCLUSION: The formulation of WIT-NP, in which WGA is conjugated to the surface of paclitaxel and IPM-loaded PLGA nanoparticles, significantly potentiates the anticancer activity of paclitaxel.

Barriers to carrier mediated drug and gene delivery to brain tumors.
Huynh GH, Deen DF, Szoka FC Jr.
J Control Release. 2005 Nov 27; [Epub ahead of print].
[ expand abstract ]

Brain tumor patients face a poor prognosis despite significant advances in tumor imaging, neurosurgery and radiation therapy. Potent chemotherapeutic drugs fail when used to treat brain tumors because biochemical and physiological barriers limit drug delivery into the brain. In the past decade a number of strategies have been introduced to increase drug delivery into the brain parenchyma. In particular, direct drug administration into the brain tumor has shown promising results in both animal models and clinical trials. This technique is well suited for the delivery of liposome and polymer drug carriers, which have the potential to provide a sustained level of drug and to reach cellular targets with improved specificity. We will discuss the current approaches that have been used to increase drug delivery into the brain parenchyma in the context of fluid and solute transport into, through and from the brain, with a focus on liposome and polymer drug carriers.
The phototoxicity of photofrin was enhanced by PEGylated liposome in vitro.
Sadzuka Y, Tokutomi K, Iwasaki F, Sugiyama I, Hirano T, Konno H, Oku N, Sonobe T.
Cancer Lett. 2005 Nov 18; [Epub ahead of print]

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Synthesis and biological activity of anticancer ether lipids that are specifically released by phospholipase A2 in tumor tissue.
Andresen TL, Jensen SS, Madsen R, Jorgensen K.
J Med Chem. 2005 Nov 17;48(23):7305-14.
[ expand abstract ]

In recent years, photodynamic therapy (PDT) with a photosensitizer and laser has been given attention, especially for the treatment of superficial cancers, such as lung, gastric, bladder and cervical cancer. In this study, in order to enhance the efficacy of PDT, photofrin liposome (PF-Lip) was prepared with dimyristoylphosphatidylcholine, dimyristoylphosphatidylglycerol and cholesterol. Polyethyleneglycol modified photofrin liposome (PF-PEG-Lip) was prepared by modification of PF-Lip with monomethoxypolyethyleneglycol-2. 3-dimyristoylglycerol.

Synthesis and biological activity of anticancer ether lipids that are specifically released by phospholipase A2 in tumor tissue.
Andresen TL, Jensen SS, Madsen R, Jorgensen K.
J Med Chem. 2005 Nov 17;48(23):7305-14.
[ expand abstract ]

The clinical use of anticancer lipids is severely limited by their ability to cause lysis of red blood cells prohibiting intravenous injection. Novel delivery systems are therefore required in order to develop anticancer ether lipids (AELs) into clinically useful anticancer drugs. In a recent article (J. Med. Chem. 2004, 47, 1694) we showed that it is possible to construct liposome systems composed of masked AELs that are activated by secretory phospholipase A2 in cancerous tissue. We present here the synthesis of six AELs and evaluate the biological activity of these bioactive lipids. The synthesized AEL 1-6 were tested against three different cancer cell lines. It was found that the stereochemistry of the glycerol headgroup in AEL-2 and 3 has a dramatic effect on the cytotoxicity of the lipids. AEL 1-4 were furthermore evaluated for their ability to prevent phosphorylation of the apoptosis regulating kinase Akt, and a correlation was found between their cytotoxic activity and their ability to inhibit Akt phosphorylation.

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Therapeutic Nanoreactors: Combining Chemistry and Biology in a Novel Triblock Copolymer Drug Delivery System.
Ranquin A, Versees W, Meier W, Steyaert J, Van Gelder P.
Nano Lett. 2005 Nov 9;5(11):2220-2224.
[ expand abstract ]

Triblock copolymeric nanoreactors are introduced as an alternative for liposomes as encapsulating carrier for prodrug activating enzymes. Inosine-adenosine-guanosine preferring nucleoside hydrolase of Trypanosoma vivax, a potential prodrug activating enzyme, was encapsulated in nanometer-sized vesicles constructed of poly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-(2-methyloxazoline) triblock copolymers. The nanoreactor is functionalized by incorporation of bacterial porins, OmpF or Tsx, in the reactor wall. Efficient cleavage of three natural substrates and one prodrug, 2-fluoroadenosine, by the nanoreactors was demonstrated.

The NK-lysin derived peptide NK-2 preferentially kills cancer cells with increased surface levels of negatively charged phosphatidylserine.
Schroder-Borm H, Bakalova R, Andra J.
FEBS Lett. 2005 Nov 7;579(27):6128-34.
[ expand abstract ]

The NK-lysin derived peptide NK-2 is a potent antibacterial, but non-toxic to a human keratinocyte cell line and of low hemolytic activity. Its target selectivity is based upon a strong binding preference to membranes containing anionic phospholipids, which are normally not found on the surface of human cells. Here, we analyzed the interaction of NK-2 with normal human lymphocytes and seven different human cancer cell lines and demonstrate that some of these cells expose negatively charged surface phosphatidylserine (PS), which presumably facilitates killing of the cells by NK-2. This is underlined by the specific intercalation of the peptide into PS-containing liposomes analyzed by fluorescence-resonance energy transfer spectroscopy.

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Poly(Ethylene Oxide)-Modified Poly(beta-Amino Ester) Nanoparticles as a pH-Sensitive System for Tumor-Targeted Delivery of Hydrophobic Drugs: Part 2. In Vivo Distribution and Tumor Localization Studies.
Shenoy D, Little S, Langer R, Amiji M.
Pharm Res. 2005 Nov 3; [Epub ahead of print].
[ expand abstract ]

PURPOSE: This study was carried out to determine the biodistribution profiles and tumor localization potential of poly(ethylene oxide) (PEO)-modified poly(beta-amino ester) (PbAE) as a novel, pH-sensitive biodegradable polymeric nanoparticulate system for tumor-targeted drug delivery. METHODS: The biodistribution studies of PEO-modified PbAE and PEO-modified poly(varepsilon-caprolactone) (PCL), a non-pH-sensitive polymer, nanoparticle systems were carried out in normal mice using (111)indium-oxine [(111)In] as a lipophilic radiolabel encapsulated within the polymeric matrix, and the distribution of the nanoparticles was studied in plasma and all the vital organs following intravenous administration. Solid tumors were developed on nude mice using human ovarian carcinoma xenograft (SKOV-3) and the change in concentrations of tritium [(3)H]-labeled paclitaxel encapsulated in polymeric nanoparticles was examined in blood, tumor mass, and liver. RESULTS: Study in normal mice with a gamma-emitting isotope [(111)In] provided a thorough biodistribution analysis of the PEO-modified nanoparticulate carrier systems, whereas (3)H-paclitaxel was useful to understand the change in concentration and tumor localization of anticancer compound directly in major sites of distribution. Both PEO-PbAE and PEO-PCL nanoparticles showed long systemic circulating properties by virtue of surface modification with PEO-containing triblock block copolymer (Pluronic(R)) stabilizer. Although the PCL nanoparticles showed higher uptake by the reticuloendothelial system, the PbAE nanoparticles effectively delivered the encapsulated payload into the tumor mass. CONCLUSIONS: PEO-modified PbAE nanoparticles showed considerable passive tumor targeting potential in early stages of biodistribution via the enhanced permeation and retention (EPR) mechanism. This prompts a detailed biodistribution profiling of the nanocarrier for prolonged periods to provide conclusive evidence for superiority of the delivery system.

Novel mucoadhesion tests for polymers and polymer-coated particles to design optimal mucoadhesive drug delivery systems.
Takeuchi H, Thongborisute J, Matsui Y, Sugihara H, Yamamoto H, Kawashima Y.
Adv Drug Deliv Rev. 2005 Nov 3;57(11):1583-94.
[ expand abstract ]

To design an effective particulate drug delivery system having mucoadhesive function, several mucoadhesion tests for polymers and the resultant particulate systems were developed. Mucin particle method is a simple mucoadhesion test for polymers, in which the commercial mucin particles are used. By measuring the change in particle size or zeta potential of the mucin particle in a certain concentration of polymer solution, we could estimate the extent of their mucoadhesive property. BIACORE method is also a novel mucoadhesion test for polymers. On passing through the mucin suspension on the polymer-immobilized chip of BIACORE instrument, the interaction was quantitatively evaluated with the change in its response diagram. By using these mucoadhesion tests, we detected a strong mucoadhesive property of several types of chitosan and Carbopol. Evaluation of mucoadhesive property of polymer-coated particulate systems was demonstrated with the particle counting method developed by us. To detect the mucoadhesive phenomena in the intestinal tract, we observed the rat intestine with the confocal laser scanning microscope (CLSM) after oral administration of the particulate systems. The resultant photographs clearly showed a longer retention of submicron-sized chitosan-coated liposomes (ssCS-Lip) in the intestinal tract than other liposomal particles tested such as non-coated liposomes and chitosan-coated multilamellar one. These observations explained well the superiority of the ssCS-Lip as drug carrier in oral administration of calcitonin in rats than other liposomal particles.

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Phase III trial of nanoparticle albumin-bound paclitaxel compared with polyethylated castor oil-based paclitaxel in women with breast cancer.
Gradishar WJ, Tjulandin S, Davidson N, Shaw H, Desai N, Bhar P, Hawkins M, O'Shaughnessy J.
J Clin Oncol. 2005 Nov 1;23(31):7794-803.
[ expand abstract ]

PURPOSE: ABI-007, the first biologically interactive albumin-bound paclitaxel in a nanameter particle, free of solvents, was compared with polyethylated castor oil-based standard paclitaxel in patients with metastatic breast cancer (MBC). This phase III study was performed to confirm preclinical studies demonstrating superior efficacy and reduced toxicity of ABI-007 compared with standard paclitaxel. PATIENTS AND METHODS: Patients were randomly assigned to 3-week cycles of either ABI-007 260 mg/m(2) intravenously without premedication (n = 229) or standard paclitaxel 175 mg/m(2) intravenously with premedication (n = 225). RESULTS: ABI-007 demonstrated significantly higher response rates compared with standard paclitaxel (33% v 19%, respectively; P = .001) and significantly longer time to tumor progression (23.0 v 16.9 weeks, respectively; hazard ratio = 0.75; P = .006). The incidence of grade 4 neutropenia was significantly lower for ABI-007 compared with standard paclitaxel (9% v 22%, respectively; P < .001) despite a 49% higher paclitaxel dose. Febrile neutropenia was uncommon (< 2%), and the incidence did not differ between the two study arms. Grade 3 sensory neuropathy was more common in the ABI-007 arm than in the standard paclitaxel arm (10% v 2%, respectively; P < .001) but was easily managed and improved rapidly (median, 22 days). No hypersensitivity reactions occurred with ABI-007 despite the absence of premedication and shorter administration time. CONCLUSION: ABI-007 demonstrated greater efficacy and a favorable safety profile compared with standard paclitaxel in this patient population. The improved therapeutic index and elimination of corticosteroid premedication required for solvent-based taxanes make the novel albumin-bound paclitaxel ABI-007 an important advance in the treatment of MBC.

Nanoparticle albumin-bound paclitaxel for metastatic breast cancer.
Harries M, Ellis P, Harper P.
J Clin Oncol. 2005 Nov 1;23(31):7768-71.
[ expand abstract ]

No abstract available

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The role of nanobiotechnology in drug discovery.
Jain KK.
Drug Discov Today. 2005 Nov 1;10(21):1435-42.
[ expand abstract ]

The application of nanotechnology in life sciences, nanobiotechnology, is already having an impact on diagnostics and drug delivery. Now, researchers are starting to use nanotechnology in the field of drug discovery. This review explains how several technologies, including nanoparticles and nanodevices such as nanobiosensors and nanobiochips, are used to improve drug discovery and development. Nanoscale assays can contribute significantly to cost-saving in screening campaigns. In addition, some nanosubstances (such as fullerenes) could be potential drugs for the future. Although there might be some safety concerns with respect to the in vivo use of nanoparticles, studies are in place to determine the nature and extent of adverse events. Future prospects for the application of nanotechnology in healthcare and for the development of personalized medicine appear to be excellent.

Phase I and pharmacokinetics trial of ABI-007, a novel nanoparticle formulation of paclitaxel in patients with advanced nonhematologic malignancies.
Nyman DW, Campbell KJ, Hersh E, Long K, Richardson K, Trieu V, Desai N, Hawkins MJ, Von Hoff DD.
J Clin Oncol. 2005 Nov 1;23(31):7785-93.
[ expand abstract ]

PURPOSE: ABI-007 is a novel solvent-free, albumin-bound, 130-nm particle formulation of paclitaxel designed to avoid solvent-related toxicities and to deliver paclitaxel to tumors via molecular pathways involving an endothelial cell-surface albumin receptor (gp60) and an albumin-binding protein expressed by tumor cells and secreted into the tumor interstitium (secreted protein acid rich in cysteine). This study determined the maximum-tolerated dose (MTD) of ABI-007 monotherapy administered weekly (three weekly doses, repeated every 4 weeks) and assessed the pharmacokinetics of paclitaxel administered as ABI-007. PATIENTS AND METHODS: Patients with advanced nonhematologic malignancies received ABI-007 without premedication at dose levels from 80 to 200 mg/m(2) as a 30-minute intravenous infusion once a week for 3 weeks, followed by 1 week of rest (one cycle). RESULTS: Thirty-nine patients were treated with an average of five cycles of ABI-007; 33% of patients received > or = six cycles of treatment. MTDs for heavily and lightly pretreated patients were 100 and 150 mg/m(2), respectively; and the dose-limiting toxicities were grade 4 neutropenia and grade 3 peripheral neuropathy, respectively. Maximum paclitaxel concentration and area under the curve increased linearly with dose. Dose-dependent changes in plasma clearance did not occur. Partial responses were observed in five patients with breast, lung, and ovarian cancers, all of whom had previously been treated with paclitaxel containing polyoxyethylated castor oil in the formulation. CONCLUSION: This study demonstrated that weekly ABI-007 can be administered at doses exceeding those typically used for paclitaxel containing polyoxyethylated castor oil. Pharmacokinetics were linear over the dose range studied. Antitumor responses occurred in patients previously treated with paclitaxel containing polyoxyethylated castor oil.

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Paclitaxel repackaged in an albumin-stabilized nanoparticle: handy or just a dandy?
Sparreboom A, Baker SD, Verweij J.
J Clin Oncol. 2005 Nov 1;23(31):7765-7.
[ expand abstract ]

No abstract available

BioNanoMedicine: a nanotechnology platform for the 21st century.
Appasani K.
Expert Rev Mol Diagn. 2005 Nov;5(6):839-40.
[ expand abstract ]

No abstract available

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Transfer of lipophilic drugs between liposomal membranes and biological interfaces: consequences for drug delivery.
Fahr A, van Hoogevest P, May S, Bergstrand N, S Leigh ML.
Eur J Pharm Sci. 2005 Nov;26(3-4):251-65.
[ expand abstract ]

This review paper describes the present knowledge on the interaction of lipophilic, poorly water soluble, drugs with liposomal membranes and the reversibility of this interaction. This interaction is discussed in the context of equilibrium and spontaneous transfer kinetics of the drug, when the liposomes are brought in co-dispersion with other artificial or natural phospholipid membranes in an aqueous medium. The focus is on drugs, which have the potential to partition (dissolve) in a lipid membrane but do not perturb membranes. The degree of interaction is described as solubility of a drug in phospholipid membranes and the kinetics of transfer of a lipophilic drug between membranes. Finally, the consequences of these two factors on the design of lipid based carriers for oral, as well as parenteral use, for lipophilic drugs and lead selection of oral lipophilic drugs is described. Since liposomes serve as model-membranes for natural membranes, the assessment of lipid solubility and transfer kinetics of lipophilic drug using liposome formulations may additionally have predictive value for bioavailability and biodistribution and the pharmacokinetics of lipophilic drugs after parenteral as well as oral administration.

Targeted adriamycin delivery to MXT-B2 metastatic mammary carcinoma cells by transferrin liposomes: effect of adriamycin ADR-to-lipid ratio.
Lopez-Barcons LA, Polo D, Llorens A, Reig F, Fabra A.
Oncol Rep. 2005 Nov;14(5):1337-43.
[ expand abstract ]

In the protein-targeted therapy for cancer, transferrin (Tf) is used to reach a selective and specific target in cancer cells. Tf is used conjugated to chemotherapeutic drugs, insulin, toxins, antibodies, polymers, nanoparticles, lipoplexes and liposomes. Using this latter approach, hydrophobically derivatized Tf was incorporated to liposomal bilayers. The biological activity of Tf-liposome was tested using MXT-B2 cells, a metastatic mammary carcinoma cell line. In Tf binding assays, the Scatchard analysis indicated 4.5x10(5) Tf receptors/cell. In cell growth assays, Tf-liposomes stimulated cell growth in a dose-dependent manner, up to a maximum of 32% of the total free Tf stimulation. Following this, we prepared Tf-liposomes encapsulating adriamycin (ADR) at two different ADR-to-lipid ratios. In vitro cytotoxicity assays against MXT-B2 cells gave IC(50) values 2.1-times lower for Tf-liposomal ADR in comparison to control liposomal ADR. However, similar IC(50) values were found for low ADR-to-lipid ratio Tf-liposomal ADR, as well as for control liposomal ADR. The free Tf added in excess increased the IC(50) value of Tf-liposomal ADR by 51%, while the IC(50) value of control liposomal ADR was unaffected, supporting a receptor-mediated mechanism of targeting by Tf. In addition, the lower IC(50) value is correlated with a higher total of ADR accumulation in the cells.

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Dendrimer delivery of an anti-VEGF oligonucleotide into the eye: a long-term study into inhibition of laser-induced CNV, distribution, uptake and toxicity.
Marano RJ, Toth I, Wimmer N, Brankov M, Rakoczy PE.
Gene Ther. 2005 Nov;12(21):1544-50.
[ expand abstract ]

We have performed a long-term study into the use of a lipophilic amino-acid dendrimer to deliver an anti-vascular endothelial growth factor (VEGF) oligonucleotide (ODN-1) into the eyes of rats and inhibit laser-induced choroidal neovascularization (CNV). In addition, the uptake, distribution and retinal tolerance of the dendrimer plus oligonucleotide conjugates were examined. Analysis of fluorescein angiograms of laser photocoagulated eyes revealed that dendrimer plus ODN-1 significantly inhibited (P<0.05) the development of CNV for 4-6 months by up to 95% in the initial stages. Eyes similarly injected with ODN-1 alone showed no significant difference (P>0.05) in mean severity score at 2 months (2.86+/-0.09), 4 months (2.15+/-0.17) or 6 months (2.7+/-0.12) compared to the vehicle-injected controls. Furthermore, we showed that intravitreally injected ODN-1 tagged with 6-fam was absorbed by a wide area of the retina and penetrated all of the retinal cell layers to the retinal pigment epithelium. Ophthalmological examinations indicated that the dendrimers plus ODN-1 conjugates were well tolerated in vivo, which was later confirmed using immunohistochemistry, which showed no observable increase in antigens associated with inflammation. We conclude that the use of such dendrimers may provide a viable mechanism for the delivery of therapeutic oligonucleotides for the treatment of angiogenic eye diseases.

Immunoliposome targeting to mesangial cells: a promising strategy for specific drug delivery to the kidney.
Tuffin G, Waelti E, Huwyler J, Hammer C, Marti HP.
J Am Soc Nephrol. 2005 Nov;16(11):3295-305.
[ expand abstract ]

Mesangial cell-mediated nephropathies are a frequent cause of ESRD. Specific drug delivery to mesangial cells might be more effective and better tolerated than existing systemic treatments. Rat mesangial cells are characterized by Thy1.1 antigen expression. Therefore, OX7-coupled immunoliposomes (OX7-IL) were prepared by coupling liposomes with F(ab') fragments of OX7 mAb directed against Thy1.1 antigen. As the glomerular endothelium is fenestrated and no basement membrane separates glomerular capillaries from the mesangium, mesangial cells represent a particularly suitable target for drug delivery by OX7-IL. Therefore, the targeting efficacy of OX7-IL to mesangial cells was investigated. Specific targeting in vitro was obtained, and intravenous injection of OX-7-IL to rats showed a specific targeting of all mesangial cells in both kidneys. OX7-IL showed marked accumulation in the cytoplasm of rat mesangial cells, both in vitro and in vivo. This renal targeting was blocked when free OX7 F((ab')2) fragments were co-administered with OX7-IL. Rats that were given a single intravenous injection of low-dose doxorubicin encapsulated in OX7-IL showed extensive glomerular damage, whereas other parts of the kidney and other organs were spared. Free doxorubicin and the liposomal formulation of this agent had no effect. Thus, immunoliposomes are a very promising delivery system for the treatment of kidney diseases.

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Coformulated N-Octanoyl-glucosylceramide Improves Cellular Delivery and Cytotoxicity of Liposomal Doxorubicin.
Veldman RJ, Koning GA, van Hell A, Zerp S, Vink SR, Storm G, Verheij M, van Blitterswijk WJ.
J Pharmacol Exp Ther. 2005 Nov;315(2):704-10.
[ expand abstract ]

The anticancer agent doxorubicin is in certain cases administered as a long-circulating liposomal formulation. Due to angiogenesis-related structural abnormalities in the endothelial lining of many neoplasms, these complexes tend to extravasate and accumulate in the tumor stroma. However, delivery of doxorubicin is still not optimal since liposomes are not taken up directly by tumor cells. Instead, doxorubicin is gradually released into the interstitial space, and the subsequent uptake by surrounding cells is a limiting step in the delivery process. We recently demonstrated that plasma membrane-inserted short-chain sphingomyelin facilitates the cellular uptake of free doxorubicin. Here, we report that N-octanoyl-glucosylceramide acts equally potent but is itself less toxic. When coformulated with liposomal doxorubicin, this short-chain glycosphingolipid administered to cultured A431 epidermoid carcinoma cells led to superior (up to 4-fold) cellular doxorubicin accumulation and cytotoxicity, compared with control doxorubicin liposomes. These results were fully reproducible when N-octanoyl-glucosylceramide was postinserted into Caelyx, a commercial liposomal doxorubicin preparation. The doxorubicin-potentiating effect of N-octanoyl-glucosylceramide-enriched liposomes proved relatively insensitive to high serum concentrations, indicating that in vivo application is a feasible option. N-Octanoyl-glucosylceramide enrichment might thus represent a major improvement of conventional liposomal doxorubicin formulations.

Immunoliposomes directed toward vcam-1 interact specifically with activated endothelial cells-a potential tool for specific drug delivery.
Voinea M, Manduteanu I, Dragomir E, Capraru M, Simionescu M.
Pharm Res. 2005 Nov;22(11):1906-17.
[ expand abstract ]

PURPOSE: Immunoliposomes can be potentially used as carriers for drug delivery to specific cells. The aim of this paper was to exploit the overexpression of vascular cell adhesion molecule-1 (VCAM-1) on activated human endothelial cells (HEC) for targeting of anti-VCAM-1 coupled liposomes with the intent for further use as drug carriers. METHODS: TNF-alpha-activated HEC were exposed to liposomes, either plain or coupled with antibodies to VCAM-1 (L-VCAM-1) or to irrelevant IgG (L-IgG); nonactivated HEC subjected to the same conditions were used as control. For binding studies, the cells were incubated with fluorescently labeled liposomes at 4 degrees C, and after 2 h, fluorescence intensity was assessed by flow cytometry; specificity of binding was determined by performing the experiments in the presence of excess anti-VCAM-1. Cellular internalization of liposomes was studied employing radioactively or fluorescently labelled liposomes; to detect the mechanisms of uptake, experiments were performed in the presence of agents that interfere in the endocytotic pathway. Transmigration of liposomes was monitored in a two-chamber culture model. The effect of L-VCAM-1 binding to HEC on intracellular calcium ([Ca(2+)](i)) and distribution of actin was determined by fluorimetry and fluorescence microscopy. RESULTS: (1) L-VCAM-1 binds selectively and specifically to TNF-alpha activated HEC. (2) Approximately 50% of L-VCAM-1 is taken up by receptor-mediated endocytosis via clathrin-coated vesicles. (3) Binding of L-VCAM-1 to HEC surface induces a rise in [Ca(2+)](i) and reorganization of actin filaments. (4) A small percentage of liposomes migrates across HEC. CONCLUSION: The data indicate that VCAM-1 may be an appropriate target for specific drug delivery to activated HEC using immunoliposomes.

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Nanotechnology for drug and gene therapy: the importance of understanding molecular mechanisms of delivery.
Labhasetwar V.
Curr Opin Biotechnol. 2005 Oct 28; [Epub ahead of print].
[ expand abstract ]

Nanotechnology, although not a new concept, has gained significant momentum in recent years. This stems partly from the realization that nanosystems have significantly different biological properties from large-sized systems (e.g. implants or microparticles) that could be used effectively to overcome problems in drug and gene therapy. In drug therapy, we face the problems of inefficacy or nonspecific effects; hence, nanosystems are being developed for targeted drug therapy. In gene therapy using non-viral systems, the main issues are relatively transient gene expression and lower efficiency than viral vectors. Research efforts have focused on understanding the barriers in gene delivery so that non-viral systems can be developed that are as effective as viral systems in gene transfection. Understanding the molecular mechanisms that underlie the interactions of nanosystems with the cell, their uptake properties and retention will be crucial for the successful development of these systems.

A liposomal formulation of doxorubicin, composed of hexadecylphosphocholine (HePC): physicochemical characterization and cytotoxic activity against human cancer cell lines.
Papagiannaros A, Hatziantoniou S, Dimas K, Papaioannou GT, Demetzos C.
Biomed Pharmacother. 2005 Oct 25; [Epub ahead of print].
[ expand abstract ]

The overall goal of this study was to prepare a novel liposomal formulation of doxorubicin, composed of hexadecylphosphocholine (HePC), as a combined formulation and to study its activity against cancer cells and peripheral blood mononuclear cells (PBMCs), in terms of efficacy and toxicity. Liposomes composed of HePC/egg phosphatidylcholine/stearylamine (HePC/EPC/SA) 10:10:0.1 (molar ratio) (1) and EPC/SA 10:0.1 (molar ratio) (2) were prepared and doxorubicin was encapsulated using the pH gradient method. Determination of lipids and doxorubicin has been achieved by high-performance thin-layer chromatography coupled with a flame-ionization detector. Prepared liposomes were characterized for their size distribution and their zeta-potential at each step of the preparation procedure. In vitro release studies have been evaluated in buffer and culture medium at 25 and 37 degrees C for 24 hours period. Liposomal formulations, free doxorubicin and HePC were tested against cancer cell lines and PBMCs, using sulforhodamine B (SRB) assay. Doxorubicin was encapsulated into the liposomes 1 and 2 at a drug to lipid molar ratio of 1.08 and 0.77, respectively, with an entrapping efficiency almost 100% in both cases. Doxorubicin was retained into liposome 1 up to 70% at 25 degrees C in TES, while up to 80% was released from 1 when liposomes were incubated at 37 degrees C either in culture medium or in the TES buffer at 24 hours. The activity of doxorubicin was retained or slightly improved when entrapped into liposomes 1 and 2, while liposomal formulation 1 encapsulating doxorubicin was found to be less toxic against normal cells (PBMCs). The combination of HePC and doxorubicin in one combined formulations justified as an improvement of the therapeutic index (TI) of doxorubicin in terms of efficacy and toxicity.

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Cytotoxic and antitumor activity of liposome-incorporated sclareol against cancer cell lines and human colon cancer xenografts.
Hatziantoniou S, Dimas K, Georgopoulos A, Sotiriadou N, Demetzos C.
Pharmacol Res. 2005 Oct 24; [Epub ahead of print].
[ expand abstract ]

The aim of this study was to design and prepare liposome-incorporated sclareol-a highly lipophilic natural product-to overcome its water insolubility and develop suitable formulations for in vivo administration. The bioactive labdane-type diterpene sclareol was incorporated into liposomes composed of egg phosphatidylcholine and dipalmitoylphosphatidylglycerol prepared by the thin-film hydration method followed by sonication. A formulation of egg phosphatidylcholine/dipalmitoylphosphatidylglycerol/sclareol (9:0.1:5 molar ratio) was developed and characterized. The lipid recovery and the sclareol to lipid molar ratio were measured using high-performance thin-layer chromatography/flame ionization detection. In vitro drug release was measured in supplemented RPMI-1640 at 37 degrees C. The liposomal and the free sclareol were initially tested in vitro for their activity against human cancer cell lines using the sulphorhodamine B assay. Liposomes incorporating sclareol at a drug to lipid molar ratio of 0:43, suggesting an incorporation efficiency of almost 80%, showed reduced growth rate of human colon cancer tumors (HCT116) developed in SCID mice, without any significant side effects.

Microfabricated drug delivery devices.
Hilt JZ, Peppas NA.
Int J Pharm. 2005 Oct 24; [Epub ahead of print].
[ expand abstract ]

We review newest developments in the design and fabrication of drug delivery devices based on micropatterned structures. Electronic devices have now reached a stage of dimensions comparable to those of biological macromolecules. This raises exciting possibilities for combining microelectronics and biotechnology to develop new technologies with unprecedented power and versatility. While molecular electronics use the unique self-assembly, switching and dynamic capabilities of molecules to miniaturize electronic devices, nanoscale biosystems use the power of microelectronics to design ultrafast/ultrasmall biocompatible devices, including implants, that can revolutionize the field of bioengineering. Thus, in recent years we have seen an explosion in the field of novel microfabricated and nanofabricated devices for drug delivery. Such devices seek to develop a platform of well controlled functions in the micro- or nano-level. They include nanoparticulate systems, recognitive molecular systems, biosensing devices, and microfabricated and microelectronic devices.

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Development of Stealth Liposome Formulation of 2'-Deoxyinosine as 5-Fluorouracil Modulator: In Vitro and In Vivo Study.
Fanciullino R, Giacometti S, Aubert C, Fina F, Martin PM, Piccerelle P, Ciccolini J.
Pharm Res. 2005 Oct 21; [Epub ahead of print].
[ expand abstract ]

PURPOSE: The aims of this study were to develop a stealth, pegylated liposomal formulation of 2'-deoxyinosine (d-Ino), a 5-fluorouracil (5-FU) modulator, to evaluate its efficacy in vitro and in tumor-bearing mice, and to study its pharmacokinetics in rats. METHOD: After designing a pegylated liposome encapsulating d-Ino (L-d-Ino), we evaluated its efficacy as 5-FU modulator in vitro. Antiproliferative assays, thymidylate synthase (TS) inhibition, and apoptosis studies were carried out to check whether an optimization of 5-FU action was achieved on the 5-FU-resistant SW620 cell line. Animal pharmacokinetic and ex vivo studies were next performed to confirm that L-d-Ino displayed a slower plasma elimination pattern than free d-Ino. Finally, effects on tumor growth of L-d-Ino + 5-FU combination was evaluated in xenografted mice. RESULTS: We developed a stable, sterile, and homogenous 100-nm population of pegylated liposomes encapsulating 30% of d-Ino. Liposomal d-Ino exhibited a strong potential as 5-FU modulator in vitro by enhancing TS inhibition and subsequent apoptosis induction, while displaying a better pharmacokinetic profile in animals, with a near seven times clearance reduction as compared with the free form. When used in tumor-bearing mice in combination with 5-FU, our results showed next that the association led to 70% of tumor reduction with a doubling median survival time as compared with untreated animals, whereas 5-FU alone was ineffective. CONCLUSION: Our data show that liposomal d-Ino, through an optimized pharmacokinetic profile, displays apotenteffect as fluoropyrimidines modulator, both in vitro and in xenografted mice. Besides, we showed here that itispossible to reverse a resistant phenotype to 5-FU, a major drug extensively described in clinical oncology.

Micellar electrokinetic capillary chromatography reveals differences in intracellular metabolism between liposomal and free doxorubicin treatment of human leukemia cells.
Eder AR, Arriaga EA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2005 Oct 20; [Epub ahead of print].
[ expand abstract ]

Doxil((R)) is a pegylated liposome formulation of the anthracycline doxorubicin. To better explain observed differences in the toxicity of Doxil((R)) and free doxorubicin in solution, the intracellular metabolism of the formulations after treatment in CCRF-CEM and CEM/C2 human leukemia cell lines was investigated. Using micellar electrokinetic capillary chromatography with laser-induced fluorescence detection, with a 63 zepto (10(-21)) mole doxorubicin limit of detection, five common metabolites and doxorubicin were detected upon treatment with both of these drug delivery systems. Two unique metabolites appeared with the Doxil((R)) and two unique metabolites appeared with the free doxorubicin delivery systems. For common metabolites, the relative amount of metabolite generated from Doxil((R)) was approximately 10 times higher than for free doxorubicin.

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Novel biomimetic polymersomes as polymer therapeutics for drug delivery.
Xu JP, Ji J, Chen WD, Shen JC.
J Control Release. 2005 Oct 20;107(3):502-12.
[ expand abstract ]

Novel amphiphilic diblock copolymers, cholesterol-end-capped poly(2-methacryloyloxyethyl phosphorylcholine) (CMPC), which have poly(2-methacryloyloxyethyl phosphorylcholine) (poly(MPC)) as hydrophilic segment and cholesterol as hydrophobic segment, was specially designed as drug delivery systems. Fluorescence probe technique and transmission electron microscope (TEM) characterizations indicated that this novel amphiphilic copolymer formed micelles structure in water and the critical micelle concentration (CMC) was determined to be 1.57 x 10(-7) mol/l. A commercial obtained polymeric amphiphiles, Cholesterol end capped PEO (CPEO), which had a similar structure with CMPC, was used as a control in the cytotoxicity test. While CPEO showed obvious cytotoxicity, cytotoxicity of this novel amphiphiles was not observed as indicated by cell culture. Anti-cancer drug adriamycin (ADR) was incorporated into the micelles by oil-in-water method. The size of the drug-containing micelles was less than 200 nm, and the size distribution of the drug-containing micelles showed a narrow and monodisperse unimodal pattern. The release rate of ADR from the nanosphere was slow and the release continued over 7 days and the release rate decreased with the increase of molecular weights of the copolymer and the amount of the drug entrapped. These experimental results suggested that the nanoparticles prepared from CMPC block copolymers could be a good candidate for injectable drug delivery carrier.

Applications of carbon nanotubes in drug delivery.
Bianco A, Kostarelos K, Prato M.
Curr Opin Chem Biol. 2005 Oct 15; [Epub ahead of print].
[ expand abstract ]

The development of new and efficient drug delivery systems is of fundamental importance to improve the pharmacological profiles of many classes of therapeutic molecules. Many different types of drug delivery systems are currently available. Within the family of nanomaterials, carbon nanotubes (CNT) have emerged as a new alternative and efficient tool for transporting and translocating therapeutic molecules. CNT can be functionalised with bioactive peptides, proteins, nucleic acids and drugs, and used to deliver their cargos to cells and organs. Because functionalised CNT display low toxicity and are not immunogenic, such systems hold great potential in the field of nanobiotechnology and nanomedicine.

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The novel chelator lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA(3)-DTDA) promotes stable binding of His-tagged proteins to liposomal membranes: Potent anti-tumor responses induced by simultaneously targeting antigen, cytokine and costimulatory signals to T cells.
van Broekhoven CL, Altin JG.
Biochim Biophys Acta. 2005 Oct 15;1716(2):104-16.
[ expand abstract ]

Recent studies indicate that the chelator lipid nitrilotriacetic acid ditetradecylamine (NTA-DTDA) can be used to engraft T cell costimulatory molecules onto tumor cell membranes, potentially circumventing the need for genetic manipulation of the cells for development of cell- or membrane-based tumor vaccines. Here, we show that a related lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA(3)-DTDA, which has three NTA moieties in its headgroup instead of one) is several-fold more effective than NTA-DTDA at promoting stable His-tagged protein engraftment. IAsys biosensor studies show that binding of His-tagged B7.1 (B7.1-6H) to NTA(3)-DTDA-containing membranes, exhibit a faster on-rate and a slower off-rate, compared to membranes containing NTA-DTDA. Also, NTA(3)-DTDA-containing liposomes and plasma membrane vesicles (PMV) engrafted with B7.1-6H and CD40-6H exhibit greater binding to T cells, in vitro and in vivo. Engrafted NTA(3)-DTDA-containing PMV encapsulated cytokines such as IL-2, IL-12, GM-CSF and IFN-gamma, allowing targeted delivery of both antigen and cytokine to T cells, and stimulation of antigen-specific T cell proliferation and cytotoxicity. Importantly, use of B7.1-CD40-engrafted PMV containing IL-2 and IL-12 as a vaccine in DBA/2J mice induced protection against challenge with syngeneic tumor cells (P815 mammary mastocytoma), and regression of established tumors. The results show that stable protein engraftment onto liposomal membranes using NTA(3)-DTDA can be used to simultaneously target associated antigen, costimulatory molecules and cytokines to T cells in vivo, inducing strong anti-tumor responses and immunotherapeutic effect.

Mitochondrial delivery of mastoparan with transferrin liposomes equipped with a pH-sensitive fusogenic peptide for selective cancer therapy.
Yamada Y, Shinohara Y, Kakudo T, Chaki S, Futaki S, Kamiya H, Harashima H.
Int J Pharm. 2005 Oct 13;303(1-2):1-7.
[ expand abstract ]

Mastoparan (MP), a potent facilitator of mitochondrial permeability transition (PT), could be used as an antitumor agent, if it were encapsulated in a tumor selective delivery system. We recently developed transferrin-modified liposomes (Tf-L) with a pH-sensitive fusogenic peptide (GALA), which delivers an encapsulated fluorescent marker into cytosol efficiently. In this study, we encapsulated MP into Tf-L with GALA for the selective delivery to mitochondria of tumor cells. The MP showed potent PT activity at concentrations above 25muM in a homogenate of K 562 cells as well as in isolated mitochondria in the presence of phosphate. Tf-L equipped with cholesteryl GALA can release encapsulated sulforhodamine B, while Tf-L failed, as evidenced by confocal laser scanning microscopy. The MP, which was delivered with Tf-L with GALA, released cytochrome c (cyt c) from mitochondria to the cytosol, while free MP released cyt c not only to the cytosol but also extracellulary. These results demonstrate the utility of MP in Tf-L with GALA for cancer therapy.

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Multihydroxylated [Gd@C(82)(OH)(22)](n)() Nanoparticles: Antineoplastic Activity of High Efficiency and Low Toxicity.
Chen C, Xing G, Wang J, Zhao Y, Li B, Tang J, Jia G, Wang T, Sun J, Xing L, Yuan H, Gao Y, Meng H, Chen Z, Zhao F, Chai Z, Fang X.
Nano Lett. 2005 Oct 12;5(10):2050-2057.
[ expand abstract ]

[Gd@C(82)(OH)(22)](n)() particles (22 nm in a saline solution) of a dose level as low as 10(-7) mol/kg exhibit a very high antineoplastic efficiency ( approximately 60%) in mice. A dose increment of 1 x 10(-7) mol/kg increases the tumor inhibition rate 26%. [Gd@C(82)(OH)(22)](n)() particles have a strong capacity to improve immunity and interfere with tumor invasion in normal muscle cells, nearly without toxicity in vivo and in vitro. Unlike conventional antineoplastic chemicals, the high antitumor efficiency of nanoparticles is not due to toxic effects to cells because they do not kill the tumor cells directly and only about 0.05% of the used dose is found in the tumor tissues. Results suggest that fullerene derivatives with proper surface modifications and sizes may help realize the dream of tumor chemotherapeutics of high-efficacy and low-toxicity.

Paclitaxel-loaded PLGA nanoparticles: Potentiation of anticancer activity by surface conjugation with wheat germ agglutinin.
Mo Y, Lim LY.
J Control Release. 2005 Oct 4; [Epub ahead of print].
[ expand abstract ]

PURPOSE: To potentiate the anticancer activity of paclitaxel-loaded PLGA nanoparticles through surface conjugation with wheat germ agglutinin (WGA). METHODS: PLGA nanoparticles loaded with paclitaxel and isopropyl myristate (IPM) as release modifier were prepared by a solvent evaporation method. WGA was conjugated to the nanoparticle surface to give novel WIT-NP of 330+/-3 nm. In vitro cytotoxicity of WIT-NP against malignant (A549 and H1299) and normal (CCL-186) pulmonary cell lines was evaluated alongside control formulations. IC50 doses were determined by the MTT assay, while cellular apoptosis was detected by cell nuclei staining and DeadEndtrade mark Fluorometric TUNEL assay. Cell cycle arrest was confirmed by flow cytometry. Cellular uptake of (3)[H]-paclitaxel from the test and control formulations was also quantified. In vivo anticancer efficacy was evaluated in the SCID mice model engrafted with the A549 tumor nodule. RESULTS: WIT-NP had superior anti-proliferation activity against the A549 and H1299 cell lines compared with conventional paclitaxel formulations as measured by IC50 doses. This was attributed to a more efficient intracellular accumulation of paclitaxel via WGA-receptor-mediated endocytosis and IPM-facilitated intracellular paclitaxel release. WIT-NP activity was associated with paclitaxel-induced apoptosis and cell arrest in the G2/M phase. A single intratumoral injection of WIT-NP at paclitaxel dose of 10 mg/kg inhibited the growth of A549 tumor nodules without inducing significant weight loss in the SCID mice over a period of 25 days. Tumor doubling time was greater than 25 days, compared with 11 days for nodules treated with conventional paclitaxel formulation. CONCLUSION: The formulation of WIT-NP, in which WGA is conjugated to the surface of paclitaxel and IPM-loaded PLGA nanoparticles, significantly potentiates the anticancer activity of paclitaxel.

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Enhanced intracellular delivery and improved antitumor efficacy of doxorubicin by sterically stabilized liposomes modified with a synthetic RGD mimetic.
Xiong XB, Huang Y, Lu WL, Zhang X, Zhang H, Nagai T, Zhang Q.
J Control Release. 2005 Oct 3;107(2):262-75.
[ expand abstract ]

While sterically stabilized liposomes (SSL) can passively accumulate into tumor tissue due to the effect of enhanced permeability and retention (EPR), the intracellular uptake of the entrapped anticancer drugs by the tumor cells should be a determinant step for their antitumor activities. Therefore, strategies that can enhance the intracellular uptake of SSL into tumor cells could lead to an improved therapeutic efficacy for the drugs. To check this possibility, RGD-mimetic-modified SSL (RGDm-SSL) were constructed aimed to achieve tumor accumulation as well as enhanced intracellular delivery, and were loaded with doxorubicin (DOX), an anticancer drug. Flow cytometry and confocal microscopy reveal that RGDm-SSL facilitated the DOX uptake into the melanoma cells via integrin-mediated endocytosis. DOX-loaded RGDm-SSL (RGDm-SSL-DOX) displayed higher cytotoxicity on melanoma cells than DOX-loaded SSL (SSL-DOX). Tissue distribution and therapeutic experiments were examined in C57BL/6 mice carrying melanoma B16 tumors. RGDm-SSL-DOX displayed similar DOX accumulation in tumor tissue to that of SSL-DOX but showed significantly lower DOX level in blood and remarkably higher DOX level in spleen than SSL-DOX. Administration of RGDm-SSL-DOX at a dose of 5 mg DOX/kg resulted in effective retardation of tumor growth and prolonged survival times compared with SSL-DOX. These results suggest that RGDm-modified SSL may be a promising intracellular targeting carrier for efficient delivery of chemotherapeutic agents into tumor cells.

Development of tumor targeting bioprobes ((111)In-chimeric L6 monoclonal antibody nanoparticles) for alternating magnetic field cancer therapy.
DeNardo SJ, DeNardo GL, Miers LA, Natarajan A, Foreman AR, Gruettner C, Adamson GN, Ivkov R.
Clin Cancer Res. 2005 Oct 1;11(19 Pt 2):7087s-7092s.
[ expand abstract ]

OBJECTIVES: (111)In-chimeric L6 (ChL6) monoclonal antibody (mAb)-linked iron oxide nanoparticle (bioprobes) pharmacokinetics, tumor uptake, and the therapeutic effect of inductively heating these bioprobes by externally applied alternating magnetic field (AMF) were studied in athymic mice bearing human breast cancer HBT 3477 xenografts. Tumor cell radioimmunotargeting of the bioprobes and therapeutic and toxic responses were determined. METHODS: Using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCl, (111)In-7,10-tetra-azacyclododecane-N, N',N'',N'''-tetraacetic acid-ChL6 was conjugated to the carboxylated polyethylene glycol on dextran-coated iron oxide 20 nm particles, one to two mAbs per nanoparticle. After magnetic purification and sterile filtration, pharmacokinetics, histopathology, and AMF/bioprobe therapy were done using (111)In-ChL6 bioprobe doses (20 ng/2.2 mg ChL6/ bioprobe), i.v. with 50 microg ChL6 in athymic mice bearing HBT 3477; a 153 kHz AMF was given 72 hours postinjection for therapy with amplitudes of 1,300, 1,000, or 700 Oe. Weights, blood counts, and tumor size were monitored and compared with control mice receiving nothing, or AMF or bioprobes alone. RESULTS: (111)In-ChL6 bioprobe binding in vitro to HBT 3477 cells was 50% to 70% of that of (111)In-ChL6. At 48 hours, tumor, lung, kidney, and marrow uptakes of the (111)In-ChL6 bioprobes were not different from that observed in prior studies of (111)In-ChL6. Significant therapeutic responses from AMF/bioprobe therapy were shown with up to eight times longer mean time to quintuple tumor volume with therapy compared with no treatment (P = 0.0013). Toxicity was only seen in the 1,300 Oe AMF cohort, with 4 of 12 immediate deaths and skin erythema. Electron micrographs showed bioprobes on the surfaces of the HBT 3477 cells of excised tumors and tumor necrosis 24 hours after AMF/bioprobe therapy. CONCLUSION: This study shows that mAb-conjugated nanoparticles (bioprobes), when given i.v., escape into the extravascular space and bind to cancer cell membrane antigen, so that bioprobes can be used in concert with externally applied AMF to deliver thermoablative cancer therapy.

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Combined radiofrequency ablation and adjuvant liposomal chemotherapy: effect of chemotherapeutic agent, nanoparticle size, and circulation time.
Ahmed M, Lukyanov AN, Torchilin V, Tournier H, Schneider AN, Goldberg SN.
J Vasc Interv Radiol. 2005 Oct;16(10):1365-71.
[ expand abstract ]

PURPOSE: To evaluate the effects of liposomal chemotherapeutic agent, nanoparticle size, and liposome circulation time on tissue coagulation and intratumoral drug uptake when radiofrequency (RF) ablation is combined with adjuvant intravenous liposomal chemotherapy in an animal breast tumor model. MATERIALS AND METHODS: Ninety-one R3230 mammary adenocarcinoma nodules were implanted in 48 Fischer rats. First, standardized RF ablation was combined with intravenous liposomal doxorubicin, cisplatin, or 5-fluorouracil (35 tumors each). Second, three different-sized doxorubicin-containing nanoparticle preparations were combined with standardized RF ablation. Last, two doxorubicin-containing liposome preparations with different blood elimination half-lives were combined with RF ablation. Coagulation diameter and interstitial doxorubicin concentration were measured 48 hours after treatment and compared with use of statistical analysis. RESULTS: All combinations of RF with liposomal chemotherapy caused significantly greater tumor necrosis than RF alone (P < .05). Significantly increased necrosis was observed with intravenous liposomal RF/doxorubicin and RF/cisplatin compared with intravenous liposomal RF/5-fluorouracil (P < .01). Greater coagulation was observed with RF combined with 100-nm nanoparticles compared with 20-nm or 250-nm nanoparticles (P = .01 and P = .04, respectively). Additionally, greater intratumoral doxorubicin uptake was observed in the group treated with 20-nm nanoparticles compared with those treated with other sizes of nanoparticles (P < .05). RF plus liposomal doxorubicin produced greater coagulation and intratumoral doxorubicin uptake than RF plus 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (P < .05). CONCLUSION: When combined with RF ablation, modification of adjuvant intravenous liposomal chemotherapy, including nanoparticle size, circulation time, and chemotherapeutic agent, can influence intratumoral drug accumulation and tissue coagulation.

Targeting ECM-Integrin Interaction with Liposome-Encapsulated Small Interfering RNAs Inhibits the Growth of Human Prostate Cancer in a Bone Xenograft Imaging Model.
Bisanz K, Yu J, Edlund M, Spohn B, Hung MC, Chung LW, Hsieh CL.
Mol Ther. 2005 Oct;12(4):634-43.
[ expand abstract ]

The intricate intracellular communication between stromal and epithelial cells, which involves cell-cell-, cell-insoluble extracellular matrix- (ECM), and cell-soluble factor-mediated signaling processes, is an attractive target for therapeutic intervention in hormone-refractory and bone-metastatic prostate cancer. In the present study we demonstrated that androgen-independent PC3 prostate cancer cells adhered to and migrated on vitronectin (VN), a major noncollagenous ECM in mature bone, through the expression of alphav-containing integrin receptors alphavbeta1 and alphavbeta5 on the cell surface, as determined by antibody function blocking assay and flow cytometry analysis. Small interfering RNAs (siRNAs) targeting human integrin alphav markedly reduced their respective mRNA and protein expression in cells, resulting in nearly complete reduction in VN-mediated cancer progression in vitro. In vivo quantitative bioluminescence analysis of human prostate cancer bone xenografts demonstrated for the first time that intratumoral administration of liposome-encapsulated human alphav-siRNAs significantly inhibits the growth of luciferase-tagged PC3 tumors in skeleton, which was associated with decreased integrin alphav expression and increased apoptosis in tumor cells. This integrin-based gene therapy is particularly suitable for the treatment of prostate cancer bone metastasis.

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Selective targeting of antibody-conjugated nanoparticles to leukemic cells and primary T-lymphocytes.
Dinauer N, Balthasar S, Weber C, Kreuter J, Langer K, von Briesen H.
Biomaterials. 2005 Oct;26(29):5898-906.
[ expand abstract ]

In the present study, surface-modified nanoparticles based on biodegradable material were used for antibody coupling in order to get a selective drug carrier systems. Gelatin nanoparticles were prepared by a desolvation process. Sulfhydryl groups were introduced which enabled the linkage of NeutrAvidin (NAv). Antibodies specific for the CD3 antigen on lymphocytic cells were conjugated to the nanoparticles surface. The binding of biotinylated anti-CD3 antibody was achieved by NAv-biotin-complex formation. Cellular binding and uptake were determined by flow cytometry and confocal laser scanning microscopy (CLSM). Cell-type-specific targeting of anti-CD3-conjugated nanoparticles into CD3-positive human T-cell leukemia cells and primary T-lymphocytes could be shown. Celluar uptake and effective internalization of antibody-conjugated nanoparticles into CD3 expressing cells were demonstrated. Uptake rates of about 84% into T-cell leukemia cells were observed. To confirm selectivity of T-cell targeting, competition experiments were carried out adding excessive free anti-CD3 prior to nanoparticle incubation leading to significantly reduced cellular uptake of antibody-conjugated nanoparticles. Further analysis on the mechanism of uptake confirmed a receptor-mediated endocytotic process. Protein-based nanoparticles conjugated with an antibody against a specific cellular antigen hold promise as selective drug delivery systems for specific cell types.

Poly(d,l-lactide-co-glycolide)/montmorillonite nanoparticles for oral delivery of anticancer drugs.
Dong Y, Feng SS.
Biomaterials. 2005 Oct;26(30):6068-76.
[ expand abstract ]

This research developed a novel bioadhesive drug delivery system, poly(d,l-lactide-co-glycolide)/montmorillonite (PLGA/MMT) nanoparticles, for oral delivery of paclitaxel. Paclitaxel-loaded PLGA/MMT nanoparticles were prepared by the emulsion/solvent evaporation method. MMT was incorporated in the formulation as a matrix material component, which also plays the role of a co-emulsifier in the nanoparticle preparation process. Paclitaxel-loaded PLGA/MMT nanoparticles were found to be of spherical shape with a mean size of around 310 nm and polydispersity of less than 0.150. Adding MMT component to the matrix material appears to have little influence on the particles size and the drug encapsulation efficiency. The drug release pattern was found biphasic with an initial burst followed by a slow, sustained release, which was not remarkably affected by the MMT component. Cellular uptake of the fluorescent coumarin 6-loaded PLGA/MMT nanoparticles showed that MMT enhanced the cellular uptake efficiency of the pure PLGA nanoparticles by 57-177% for Caco-2 cells and 11-55% for HT-29 cells, which was dependent on the amount of MMT and the particle concentration in incubation. Such a novel formulation is expected to possess extended residence time in the gastrointestinal (GI) tract, which promotes oral delivery of paclitaxel.

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Folate-linked nanoparticle-mediated suicide gene therapy in human prostate cancer and nasopharyngeal cancer with herpes simplex virus thymidine kinase.
Hattori Y, Maitani Y.
Cancer Gene Ther. 2005 Oct;12(10):796-809.
[ expand abstract ]

For targeted gene delivery to human prostate cancer LNCaP and PC-3 cells and nasopharyngeal cancer KB cells, we developed a folate-linked nanoparticle (NP-F), and evaluated the potential of NP-F-mediated suicide gene therapy in the cells and xenografts with herpes simplex virus thymidine kinase (HSV-tk) and connexin 43 (Cx43). An NP-F-plasmid DNA complex (NP-F nanoplex) showed high DNA transfection efficiency in KB, LNCaP and PC-3 cells. Cell growth inhibition in the presence of ganciclovir (GCV) was enhanced with HSV-tk and Cx43 genes in LNCaP cells. In suicide gene therapy, the tumor growths of KB and LNCaP xenografts were significantly inhibited when an NP-F nanoplex of the HSV-tk gene, and HSV-tk and Cx43 genes, respectively, was injected intratumorally and GCV was administered intraperitoneally. These findings suggested that the NP-F is a potential target vector in prostate and nasopharyngeal cancer for suicide gene therapy.

Folate receptor-mediated drug targeting: From therapeutics to diagnostics.
Hilgenbrink AR, Low PS.
J Pharm Sci. 2005 Oct;94(10):2135-46.
[ expand abstract ]

Folate targeted drug delivery has emerged as an alternative therapy for the treatment and imaging of many cancers and inflammatory diseases. Due to its small molecular size and high binding affinity for cell surface folate receptors (FR), folate conjugates have the ability to deliver a variety of molecular complexes to pathologic cells without causing harm to normal tissues. Complexes that have been successfully delivered to FR expressing cells, to date, include protein toxins, immune stimulants, chemotherapeutic agents, liposomes, nanoparticles, and imaging agents. This review will summarize the applications of folic acid as a targeting ligand and highlight the various methods being developed for delivery of therapeutic and imaging agents to FR-expressing cells.

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Increased efficiency of cisplatin-resistant cell lines to DNA-mediated gene transfer with cationic liposome.
Sato T, Serikawa T, Sekine M, Aoki Y, Tanaka K.
J Obstet Gynaecol Res. 2005 Oct;31(5):368-74.
[ expand abstract ]

Aim: Because of its effectiveness against many gynecologic malignancies, chemotherapy including cisplatin is mainly used as the first-line chemotherapy for epithelium ovarian cancer. However, one of the major problems that is well recognized is that tumor cells can easily acquire resistance to cisplatin. Various trials were carried out in order to establish treatment against cisplatin-resistant tumor cells. Method: Using both in vivo and in vitro studies, we examined whether or not the newly developed liposome could be used to demonstrate sufficient transfection activity as the anticancer reagent for cisplatin-resistant tumor cells. Result: With our newly developed liposome, GTE 319 and GTE 321, the lac-Z gene was more efficiently transfected in cisplatin-resistant variant cells, mEIIL-R, KF-ra and KF-rb, than in parental cells, mEIIL and KF, using X-gal staining. In cytotoxic assay, transfection of herpes simplex thymidine kinase (HSV-tk) gene conjugated with GTE319 or GTE 321, and cultivation with aciclovir for 5 days revealed accelerated tumor-inhibition activity in all of the cisplatin-resistant tumor cells compared with that in the naive parental cells. In addition, the high anti-tumor effect was obtained from intratumoral local injection of the tk gene conjugated with GTE-321 liposome following aciclovir administration against KF-rb-transplanted tumor formed in nude mouse hypodermic. Conclusion: These results suggest that gene therapy using a newly developed liposome-conjugated suicide gene can be an attractive approach for treatment against cisplatin-resistant ovarian cancer cells.

Method of laser activated nano-thermolysis for elimination of tumor cells.
Lapotko D, Lukianova E, Potapnev M, Aleinikova O, Oraevsky A.
Cancer Lett. 2005 Sep 30; [Epub ahead of print].
[ expand abstract ]

We describe novel ex vivo method for elimination of tumor cells from cell suspension, Laser Activated Nanothermolysis and propose this method for purging of bone marrow and blood transplants. K562 and human lympholeukemia cells were eliminated in experiments by laser-induced micro-bubbles that emerge inside individual target cells around selectively formed clusters of light-absorbing gold nanoparticles. Pretreatment of tumor cells with specific monoclonal antibodies and Ig-conjugated 30-nm gold particles allowed the formation of clusters of 10-20 on the surface of cell membrane. Electron microscopy found the nanoparticulate clusters inside the cells. Total (100%) elimination of K562 cells targeted with specific antibodies was achieved with single laser pulses with optical fluence of 5J/cm(2) at the wavelength of 532 nm without damage to the same cells targeted without specific antibodies. Total elimination of human lymphoblasts from suspension of normal stem cells was achieved by a single laser pulse with the optical fluence of 1.7J/cm(2), while the damage level of normal cells was 16%.

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Doxorubicin-PAMAM dendrimer complex attached to liposomes: Cytotoxic studies against human cancer cell lines.
Papagiannaros A, Dimas K, Papaioannou GT, Demetzos C.
Int J Pharm. 2005 Sep 30;302(1-2):29-38.
[ expand abstract ]

Liposomes composed of HePC:EPC:SA 10:10:0.1 (molar ratio) (1) and EPC:SA 10:0.1 (molar ratio) (2) were prepared and were used for incorporating the doxorubicin-PAMAM complex (3:1 molar ratio) (3). The doxorubicin-PAMAM complex was attached to liposomes and the incorporation efficiency was 91 and 95% for 1 and 2, respectively. The incorporation efficiency for doxorubicin into PAMAM was almost 97% while doxorubicin to PAMAM molar ratio was 3.56+/-0.04. The release rate of doxorubicin as doxorubicin-PAMAM complex from liposomes 1 and 2 and from the complex 3, was studied using buffers and 50% RPMI cell culture medium at 37 and 25 degrees C. The low release rate of doxorubicin as well as the high incorporation efficiency of doxorubicin-PAMAM complex into liposomes are considered as beneficial factors concerning the activity of doxorubicin. The cytotoxic activity of the liposomal formulation 1 incorporating doxorubicin-PAMAM complex, based on doxorubicin activity, was compared to that of 2 incorporating doxorubicin-PAMAM complex and to that of 3. The results showed that complex 1 was the most active formulation against all cancer cell lines compared to that of 2 and 3. Liposomal formulations composed of lipids and of a drug-dendrimer complex could be characterized as modulatory liposomal controlled release system (MLCRS), and could provide benefits to the delivery of drugs and modulate their release.

Thermosensitive Nanospheres with a Gold Layer Revealed as Low-Cytotoxic Drug Vehicles.
Qin J, Jo YS, Ihm JE, Kim DK, Muhammed M.
Langmuir. 2005 Sep 27;21(20):9346-9351.
[ expand abstract ]

In this paper, the positive effect of a gold layer on cell viability is demonstrated by examining the results given by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfop henyl)-2H-tetrazolium (MTS) assay and two-color cell fluorescence viability (TCCV) assay. These cytotoxicity tests were performed with human cervical adenocarcinoma cells (HeLa cell line) and transformed African green monkey kidney fibroblast cells (Cos-7 cell line). To fabricate the nanostructures as drug vehicles, first, poly(l,l-lactide-co-ethylene glycol) (PLLA-PEG) and poly(N-isopropylacrylamide-co-d,d-lactide) (PNIPAAm-PDLA) were synthesized, and then two kinds of thermosensitive nanospheres comprising "shell-in-shell" structures without a gold layer (PLLA-PEG@PNIPAAm-PDLA) and with a gold layer (Au@PLLA-PEG@PNIPAAm-PDLA) were constructed by a modified double-emulsion method (MDEM). Both of them displayed a unique thermosensitive character exhibiting the lower critical solubility temperature (LCST) at 36.7 degrees C which was confirmed by UV-vis spectroscopy and differential scanning calorimetry (DSC). The release profiles of entrapped bovine serum albumin (BSA) were monitored at 22 and 37 degrees C, respectively, to reveal the thermal dependence on the release rate. In cell viability tests, both PLLA-PEG@PNIPAAm-PDLA and Au@PLLA-PEG@PNIPAAm-PDLA showed excellent cell viability, and furthermore, Au@PLLA-PEG@PNIPAAm-PDLA, particularly at high doses, exhibited more enhanced cell viability than PLLA-PEG@PNIPAAm-PDLA. This effect is mainly attributed to the gold layer which binds the protein molecules first and consequently facilitates transmembrane uptake of essential nutrients in the cell media, resulting in favorable cell proliferation.

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Poly(amidoamine) dendrimer-based multifunctional engineered nanodevice for cancer therapy.
Majoros IJ, Thomas TP, Mehta CB, Baker JR Jr.
J Med Chem. 2005 Sep 22;48(19):5892-9.
[ expand abstract ]

Multifunctional cancer therapeutic nanodevices have been designed and synthesized using the poly(amidoamine) (PAMAM) dendrimer as a carrier. Partial acetylation of the generation 5 (G5) PAMAM dendrimer was utilized to neutralize a fraction of the primary amino groups, provide enhanced solubility of the dendrimer during the conjugation reaction of fluorescein isothiocyanate (FITC) (in dimethyl sulfoxide (DMSO)), and prevent nonspecific targeting interactions (in vitro and in vivo) during delivery. The remaining nonacetylated primary amino groups were utilized for conjugation of the functional molecules fluorescein isothiocyanate (FITC, an imaging agent), folic acid (FA, targets overexpressed folate receptors on specific cancer cells), and methotrexate (MTX, chemotherapeutic drug). The appropriate control nanodevices have been synthesized as well. The G5 PAMAM dendrimer molecular weight and number of primary amino groups were determined by gel permeation chromatography (GPC) and potentiometric titration for stoichiometric design of ensuing conjugation reactions. Additionally, dendrimer conjugates were characterized by multiple analytical methods including GPC, nuclear magnetic resonance spectroscopy (NMR), high performance liquid chromatography (HPLC), and UV spectroscopy. The fully characterized nanodevices can be used for the targeted delivery of chemotherapeutic and imaging agents to specific cancer cells. Here, we present a more extensive investigation of our previously reported synthesis of this material with improvements directed toward scale-up synthesis and clinical trials.

Systemic trafficking of plant virus nanoparticles in mice via the oral route.
Rae CS, Wei Khor I, Wang Q, Destito G, Gonzalez MJ, Singh P, Thomas DM, Estrada MN, Powell E, Finn MG, Manchester M.
Virology. 2005 Sep 22; [Epub ahead of print].
[ expand abstract ]

The plant virus, cowpea mosaic virus (CPMV), is increasingly being used as a nanoparticle platform for multivalent display of peptides. A growing variety of applications have employed the CPMV display technology including vaccines, antiviral therapeutics, nanoblock chemistry, and materials science. CPMV chimeras can be inexpensively produced from experimentally infected cowpea plants and are completely stable at 37 degrees C and low pH, suggesting that they could be used as edible or mucosally-delivered vaccines or therapeutics. However, the fate of CPMV particles in vivo, or following delivery via the oral route, is unknown. To address this question, we examined CPMV in vitro and in vivo. CPMV was shown to be stable under simulated gastric conditions in vitro. The pattern of localization of CPMV particles to mouse tissues following oral or intravenous dosing was then determined. For several days following oral or intravenous inoculation, CPMV was found in a wide variety of tissues throughout the body, including the spleen, kidney, liver, lung, stomach, small intestine, lymph nodes, brain, and bone marrow. CPMV particles were detected after cardiac perfusion, suggesting that the particles entered the tissues. This pattern was confirmed using methods to specifically detect the viral capsid proteins and the internal viral RNA. The stability of CPMV virions in the gastrointestinal tract followed by their systemic dissemination supports their use as orally bioavailable nanoparticles.

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Doxorubicin-encapsulated thermosensitive liposomes modified with poly(N-isopropylacrylamide-co-acrylamide): Drug release behavior and stability in the presence of serum.
Han HD, Shin BC, Choi HS.
Eur J Pharm Biopharm. 2005 Sep 21; [Epub ahead of print].
[ expand abstract ]

In the field of the temperature sensitive drug delivery systems, we studied on the surface modification of liposomes by using poly(N-isopropylacrylamide-co-acrylamide) (PNIPAM-AAM) and polyethyleneglycol (PEG) to increase the release of doxorubicin (DOX) from liposomes and prolong the stability of liposomes in the presence of serum. The release of DOX from the PNIPAM-AAM/PEG modified liposomes is enhanced around the transition temperature of the polymer. In addition, the stability of the PNIPAM-AAM/PEG modified liposomes in serum shows a high level comparing with polymer unmodified liposomes. These results suggest that the modification on the surface of liposomes with both PNIPAM-AAM and PEG enhances the drug release from liposomes and reduces the protein adsorption in serum.

On the study of BSA-loaded calcium-deficient hydroxyapatite nano-carriers for controlled drug delivery.
Liu TY, Chen SY, Liu DM, Liou SC.
J Control Release. 2005 Sep 20;107(1):112-21.
[ expand abstract ]

Calcium-deficient hydroxyapatite (CDHA) nano-crystals incorporated with bovine serum albumin (BSA) to form BSA-loaded nano-carriers were synthesized via both in-situ and ex-situ processes. Amount of BSA uptake by the CDHA nano-crystals and subsequent release behaviors of the BSA-loaded nano-carriers were investigated. The amount of BSA uptake by CDHA decreases with increasing pH but a larger amount was observed in the ex-situ compared to in-situ process above pH=8.0. The release profile showed a bursting behavior for the nano-carrier prepared via the ex-situ process, which is probably due to the desorption of BSA molecules. In contrast, for the sample synthesized via the in-situ process at a higher pH level, a slower release profile without bursting behavior due to the dissolution of the BSA-incorporated CDHA crystal is seen from high solution TEM that indicates different extent of interaction between BSA and CDHA. On the other hand, for the nano-carriers prepared via the same process at lower pH level, a two-stage release profile was detected. An initial bursting release is due to the desorption of BSA from the CDHA surface, followed by a slow release as a result of the dissolution of the BSA-incorporated nano-crystals along its c-axis direction.

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Liposomalization of SN-38 as active metabolite of CPT-11.
Sadzuka Y, Takabe H, Sonobe T.
J Control Release. 2005 Sep 20; [Epub ahead of print].
[ expand abstract ]

Although many drugs have been developed for the treatment of disease, some drugs have complications such as adverse effects, and antitumor agents should target tumors or cells more selectively. It is therefore necessary to develop drug delivery systems, and liposomes are reportedly useful as an effective drug carrier. An antitumor agent, CPT-11, inhibits DNA synthesis by the inhibition of topoisomerase1 and has a strong antitumor activity. SN-38 is converted from CPT-11 as an active metabolite by carboxylesterase in the liver. As SN-38 is insoluble, it has not been applied at the clinical stage as an injection. It is expected that SN-38 liposomalization may increase its usefulness in cancer chemotherapy. Our purpose is to have a clinical application of SN-38 by a novel method of liposomalization to expand the application for the other insolubility drugs. As SN-38 is hydrophobic, SN-38-trapped liposome preparation was attempted using the Bangham method, which is effective for general preparation. However, a high ratio of SN-38 trapped in liposome was not achieved, and this was not improved by the freezing-thawing method or the freeze-drying method. On the other hand, the ratio of SN-38 trapped in liposome by the modified remote loading method was about 4 times that by the Bangham method, and the ratio by the film loading method, novel method of liposomal preparation, reached 2 times and 8 times that by the modified remote loading method and Bangham method, respectively, showing a remarkable increase. In conclusion, it was suggested that the preparation of SN-38 liposome using the film loading method effectively entraps SN-38. Thus, it is expected that SN-38 liposome can be applied as an injection. This preparation method is useful if application is possible in the other insolubility drugs.

Synthetic anticancer gene medicine exploits intrinsic antitumor activity of cationic vector to cure established tumors.
Dufes C, Keith WN, Bilsland A, Proutski I, Uchegbu IF, Schatzlein AG.
Cancer Res. 2005 Sep 15;65(18):8079-84.
[ expand abstract ]

The systemic delivery of genetic therapies required for the treatment of inaccessible tumors and metastases remains a challenge despite the development of various viral and synthetic vector systems. Here we show that a synthetic vector system based on polypropylenimine dendrimers has the desired properties of a systemic delivery vehicle and mediates efficient transgene expression in tumors after i.v. administration. The systemic tumor necrosis factor alpha (TNFalpha) gene therapy was efficacious in the experimental treatment of established A431 epidermoid carcinoma, C33a cervix carcinoma, and LS174T colorectal adenocarcinoma. Specifically, the systemic injection of dendrimer nanoparticles containing a TNFalpha expression plasmid regulated by telomerase gene promoters (hTR and hTERT) leads to transgene expression, regression of remote xenograft murine tumors, and long-term survival of up to 100% of the animals. Interestingly, these dendrimers and, to a lesser extent, other common polymeric transfection agents also exhibit plasmid-independent antitumor activity, ranging from pronounced growth retardation to complete tumor regression. The genetic therapy as well as treatment with dendrimer alone was well tolerated with no apparent signs of toxicity in the animals. The combination of intrinsic dendrimer activity and transcriptionally targeted TNFalpha when complexed was significantly more potent than either treatment alone or when both were administered in sequence. The combination of pharmacologically active synthetic transfection agent and transcriptionally targeted antitumor gene creates an efficacious gene medicine for the systemic treatment of experimental solid tumors.

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A Fullerene-Paclitaxel Chemotherapeutic: Synthesis, Characterization, and Study of Biological Activity in Tissue Culture.
Zakharian TY, Seryshev A, Sitharaman B, Gilbert BE, Knight V, Wilson LJ.
J Am Chem Soc. 2005 Sep 14;127(36):12508-12509.
[ expand abstract ]

A fullerene-paclitaxel conjugate has been synthesized as a slow-release drug for aerosol liposome delivery of paclitaxel for lung cancer therapy. The conjugate was designed to release paclitaxel via enzymatic hydrolysis and subsequently has shown a half-life of release of 80 min in bovine plasma. A liposome formulation of the conjugate has been prepared using dilauroylphosphatidylcholine (DLPC), and its IC(50) is virtually identical to the IC(50) for a paclitaxel-DLPC formulation in human epithelial lung carcinoma A549 cells. With both clinically relevant kinetics of hydrolysis and significant cytotoxicity in tissue culture, the conjugate holds promise for enhanced therapeutic efficacy of paclitaxel in vivo.

Functionalization of Carbon Nanotubes via Cleavable Disulfide Bonds for Efficient Intracellular Delivery of siRNA and Potent Gene Silencing.
Kam NW, Liu Z, Dai H.
J Am Chem Soc. 2005 Sep 14;127(36):12492-3.
[ expand abstract ]

We present a novel functionalization scheme for single-walled carbon nanotubes (SWNTs) to afford nanotube-biomolecule conjugates with the incorporation of cleavable bonds to enable controlled molecular releasing from nanotube surfaces, thus creating "smart" nanomaterials with high potential for chemical and biological applications. With this versatile functionalization, we demonstrate transporting, enzymatic cleaving and releasing of DNA from SWNT transporters, and subsequent nuclear translocation of DNA oligonucleotides in mammalian cells. We further show highly efficient delivery of siRNA by SWNTs and achieving more potent RNAi functionality than a widely used conventional transfection agent. Thus, the novel functionalization of SWNTs with cleavable bonds is highly promising for a wide range of applications including gene and protein therapy.

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A multifunctional envelope-type nano device for novel gene delivery of siRNA plasmids.
Moriguchi R, Kogure K, Akita H, Futaki S, Miyagishi M, Taira K, Harashima H.
Int J Pharm. 2005 Sep 14;301(1-2):277-85.
[ expand abstract ]

A multifunctional envelope-type nano device (MEND) for use in the delivery of siRNA expression plasmids is described. The plasmid DNA encoding anti-luciferase short interfering RNA (siRNA) was condensed by poly-l-lysine (PLL) and packaged into the MEND. The silencing effect of the MEND(PLL) showed a 96% inhibition of luciferase activities in a co-transfection study. The silencing effect was maintained at more than 60%, even under the 100-fold diluted conditions. In the luciferase transformed cells, however, the MEND(PLL) showed no significant silencing effect (10%), indicating heterogeneity in transfection by the MEND(PLL). To solve this problem, the DNA condensing agents were optimized by comparing PLL, stearyl octaarginine (STR-R8) and protamine (Prot). No difference in silencing effect (95-97%) was found among these MENDs in a co-transfection study. However, the MEND(Prot) showed a 70% silencing effect in the transformed cells. These results suggest that the MEND(Prot) has less heterogeneity in transfection, while the MEND(PLL) and the MEND(STR-R8) have large heterogeneities. These results demonstrate that MEND(Prot) is a promising gene delivery system for siRNA expression plasmids with less heterogeneity associated with the transfection.

A Fullerene-Paclitaxel Chemotherapeutic: Synthesis, Characterization, and Study of Biological Activity in Tissue Culture.
Zakharian TY, Seryshev A, Sitharaman B, Gilbert BE, Knight V, Wilson LJ.
J Am Chem Soc. 2005 Sep 14;127(36):12508-12509.
[ expand abstract ]

A fullerene-paclitaxel conjugate has been synthesized as a slow-release drug for aerosol liposome delivery of paclitaxel for lung cancer therapy. The conjugate was designed to release paclitaxel via enzymatic hydrolysis and subsequently has shown a half-life of release of 80 min in bovine plasma. A liposome formulation of the conjugate has been prepared using dilauroylphosphatidylcholine (DLPC), and its IC(50) is virtually identical to the IC(50) for a paclitaxel-DLPC formulation in human epithelial lung carcinoma A549 cells. With both clinically relevant kinetics of hydrolysis and significant cytotoxicity in tissue culture, the conjugate holds promise for enhanced therapeutic efficacy of paclitaxel in vivo.

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Synthesis and Characterization of mPEG-PLA Prodrug Micelles.
Hans M, Shimoni K, Danino D, Siegel SJ, Lowman A.
Biomacromolecules. 2005 Sep 12;6(5):2708-2717.
[ expand abstract ]

Polymeric prodrugs of mPEG-PLA-haloperidol (methoxypoly(ethylene glycol)-b-poly(lactic acid)) can self-assemble into nanoscale micelle-like structures in aqueous solutions. mPEG-PLA-haloperidol was prepared and characterized using (1)H and (13)C NMR. The conjugation efficiency was found to be 64.8 +/- 21%. Micelles that form spontaneously upon solubilization of the mPEG-PLA and the polymeric prodrugs in water were characterized using a variety of techniques. The mPEG-PLA and prodrug micelles were found to have diameters of 28.73 +/- 1.45 and 49.67 +/- 4.29 nm, respectively, using dynamic light scattering (DLS). The micelle size and polydispersity were also evaluated with cryogenic transmission electron microscopy (cryo-TEM) and were consistent with the DLS results. Cryo-TEM and proton NMR confirmed that the micelles were spherical in shape. DLS was also used to determine the aggregation numbers of the micelles. The aggregation numbers ranged from 351 to 603. The change in aggregation number was dependent on the total drug incorporation into the micelle core. Critical micelle concentrations were determined for the various micelle/drug formulations and found to range from 3 to 14 mug/mL. Finally, drug was incorporated into the micelle core using the conjugate, free drug with a saturated aqueous phase during production, or a combination of both techniques. Drug incorporation could be increased from 3% to 20% (w/w) using the different formulations.

Intratumoral injection of immature dendritic cells enhances antitumor effect of hyperthermia using magnetic nanoparticles.
Tanaka K, Ito A, Kobayashi T, Kawamura T, Shimada S, Matsumoto K, Saida T, Honda H.
Int J Cancer. 2005 Sep 10;116(4):624-33.
[ expand abstract ]

Dendritic cells (DCs) are potent antigen-presenting cells that play a pivotal role in regulating immune responses in cancer and have recently been shown to be activated by heat shock proteins (HSPs). We previously reported that HSP70 expression after hyperthermia induces antitumor immunity. Our hyperthermia system using magnetite cationic liposomes (MCLs) induced necrotic cell death that was correlated with HSP70 release. In the present study, we investigated the therapeutic effects of DC therapy combined with MCL-induced hyperthermia on mouse melanoma. In an in vitro study, when immature DCs were pulsed with mouse B16 melanoma cells heated at 43 degrees C, major histocompatibility complex (MHC) class I/II, costimulatory molecules CD80/CD86 and CCR7 in the DCs were upregulated, thus resulting in DC maturation. C57BL/6 mice bearing a melanoma nodule were subjected to combination therapy using hyperthermia and DC immunotherapy in vivo by means of tumor-specific hyperthermia using MCLs and directly injected immature DCs. Mice were divided into 4 groups: group I (control), group II (hyperthermia), group III (DC therapy) and group IV (hyperthermia + DC therapy). Complete regression of tumors was observed in 60% of mice in group IV, while no tumor regression was seen among mice in the other groups. Increased cytotoxic T lymphocyte (CTL) and natural killer (NK) activity was observed on in vitro cytotoxicity assay using splenocytes in the cured mice treated with combination therapy, and the cured mice rejected a second challenge of B16 melanoma cells. This study has important implications for the application of MCL-induced hyperthermia plus DC therapy in patients with advanced malignancies as a novel cancer therapy.

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Novel biomimetic polymersomes as polymer therapeutics for drug delivery.
Xu JP, Ji J, Chen WD, Shen JC.
J Control Release. 2005 Sep 8; [Epub ahead of print].
[ expand abstract ]

Novel amphiphilic diblock copolymers, cholesterol-end-capped poly(2-methacryloyloxyethyl phosphorylcholine) (CMPC), which have poly(2-methacryloyloxyethyl phosphorylcholine) (poly(MPC)) as hydrophilic segment and cholesterol as hydrophobic segment, was specially designed as drug delivery systems. Fluorescence probe technique and transmission electron microscope (TEM) characterizations indicated that this novel amphiphilic copolymer formed micelles structure in water and the critical micelle concentration (CMC) was determined to be 1.57x10(-7) mol/l. A commercial obtained polymeric amphiphiles, Cholesterol end capped PEO (CPEO), which had a similar structure with CMPC, was used as a control in the cytotoxicity test. While CPEO showed obvious cytotoxicity, cytotoxicity of this novel amphiphiles was not observed as indicated by cell culture. Anti-cancer drug adriamycin (ADR) was incorporated into the micelles by oil-in-water method. The size of the drug-containing micelles was less than 200 nm, and the size distribution of the drug-containing micelles showed a narrow and monodisperse unimodal pattern. The release rate of ADR from the nanosphere was slow and the release continued over 7 days and the release rate decreased with the increase of molecular weights of the copolymer and the amount of the drug entrapped. These experimental results suggested that the nanoparticles prepared from CMPC block copolymers could be a good candidate for injectable drug delivery carrier.

In vitro effects of chitosan nanoparticles on proliferation of human gastric carcinoma cell line MGC803 cells.
Qi LF, Xu ZR, Li Y, Jiang X, Han XY.
World J Gastroenterol. 2005 Sep 7;11(33):5136-41.
[ expand abstract ]

AIM: To investigate the effects of chitosan nanoparticles on proliferation of human gastric carcinoma cell line MGC803 in vitro and the possible mechanisms involved. METHODS: Chitosan nanoparticles were characterized by particle size, zeta potential, and morphology. After treatment with various concentrations of chitosan nanoparticles (25, 50, 75, 100 mug/mL) at various time intervals, cell proliferation, ultrastructural changes, DNA fragmentation, mitochondrial membrane potential (MMP), cell cycle phase distribution and apoptotic peaks of MGC803 cells were analyzed by MTT assay, electron microscopy, DNA agarose gel electrophoresis, and flow cytometry. RESULTS: Chitosan nanoparticles exhibited a small particle size as 65 nm and a high surface charge as 52 mV. Chitosan nanoparticles markedly inhibited cell proliferation of MGC803 cells with an IC(50) value of 5.3 mug/mL 48 h after treatment. After treatment with chitosan nanoparticles, the typical necrotic cell morphology was observed by electron microscopy, a typical DNA degradation associated with necrosis was determined by DNA agarose electrophoresis. Flow cytometry showed the loss of MMP and occurrence of apoptosis in chitosan nanoparticles-treated cells. CONCLUSION: Chitosan nanoparticles effectively inhibit the proliferation of human gastric carcinoma cell line MGC803 in vitro through multiple mechanisms, and may be a beneficial agent against human carcinoma.

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Highly Active and Stable DNAzyme-Carbon Nanotube Hybrids.
Yim TJ, Liu J, Lu Y, Kane RS, Dordick JS.
J Am Chem Soc. 2005 Sep 7;127(35):12200-1.
[ expand abstract ]

We report herein the generation and characterization of DNAzyme-carbon nanotube conjugates. Biotinylated DNAzyme was attached to streptavidin-functionalized MWNTs. The resultant DNAzyme-MWNT conjugates exhibited Michaelis-Menten kinetics and multiple catalytic turnovers. The specific activity of the nanoscale conjugates was approximately (1)/(3) of that for freely soluble DNAzyme. Such high activity may allow myriad applications ranging from the directed assembly of nanomaterials to nanoscale cellular therapeutics that rely on the RNA cleaving ability of DNAzymes.

Interleukin-2/liposomes potentiate immune responses to a soluble protein cancer vaccine in mice.
Johnston D, Reynolds SR, Bystryn JC.
Cancer Immunol Immunother. 2005 Sep 6;:1-8 [Epub ahead of print].
[ expand abstract ]

A critical element in improving the potency of cancer vaccines, especially pure protein or peptide antigens, is to develop procedures that can strongly but safely increase their ability to induce immune responses. Here, we describe that encapsulation of a pure protein antigen and interleukin-2 (IL-2) together into liposomes significantly improves immune responses and tumor protection. Groups of C57Bl/6 mice were immunized weekly x4 with -0.1 mg of ovalbumin (OVA) injected subcutaneously in PBS or encapsulated in liposomes with or without human recombinant IL-2. Control groups included mice immunized to irradiated E.G7-OVA cells (that express ovalbumin), or to PBS. Sera were collected and pooled by immunization group at baseline and at weeks 2 and 4 to measure antibody responses to OVA by ELISA. Splenocytes obtained at week 4 were tested for anti-OVA cellular responses by ELISPOT. Mice were then challenged to a lethal dose of E.G7-OVA cells to measure tumor-protective immunity. IL-2 liposomes caused no detectable toxicity. Antibody, CD8(+) T cell, and tumor-protective immune responses were markedly enhanced in mice immunized to OVA + IL-2 in liposomes compared to mice immunized to OVA, either alone or encapsulated into liposomes without IL-2. These results indicate that IL-2 liposomes enhance antibody, cellular, and tumor-protective immune responses to immunization with a soluble protein. This may provide a simple, safe, and effective way to enhance the immunogenicity of vaccines that consist of pure protein antigens.

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Current strategies for the development of peptide-based anti-cancer therapeutics.
Borghouts C, Kunz C, Groner B.
J Pept Sci. 2005 Sep 2; [Epub ahead of print].
[ expand abstract ]

The completion of the human genome sequence and the development of new techniques, which allow the visualisation of comprehensive gene expression patterns, has led to the identification of a large number of gene products differentially expressed in tumours and corresponding normal tissues. The task at hand is the sorting of these genes into correlative and causative ones. Correlative genes are merely changed as a consequence of transformation and have no decisive effects upon transformation. In contrast, causative genes play a direct role in the process of cellular transformation and the maintenance of the transformed state, which can be exploited for therapeutic purposes. Oncogenes and tumour suppressor genes are prime targets for the development of new inhibitors and gene therapeutic strategies. However, many target oncogene products do not exhibit enzymatic activity that can be inhibited by conventional small molecular weight compounds. They exert their functions through regulated protein-protein or protein-DNA interactions and might require other compounds for efficient interference with such functions. Peptides are emerging as a novel class of drugs for cancer therapy, which could fulfil these tasks. Peptide therapy aims at the specific inhibition of inappropriately activated oncogenes. This review will focus on the selection procedures, which can be employed to identify useful peptides for the treatment of cancer. Before peptide-based therapeutics can become useful, it will be necessary to increase their stability by modifications or the use of scaffolds. Additionally, various delivery methods including liposomes and particularly the use of protein transduction domains (PTDs) have to be explored. These strategies will yield highly specific and more effective peptides and improve the potential of peptide-based anti-cancer therapeutics. Copyright (c) 2005 European Peptide Society and John Wiley & Sons, Ltd.

Simultaneous monitoring of temperature and T(1): Methods and preliminary results of application to drug delivery using thermosensitive liposomes.
Bos C, Lepetit-Coiffe M, Quesson B, Moonen CT.
Magn Reson Med. 2005 Sep 2; [Epub ahead of print].
[ expand abstract ]

A method is presented to obtain temperature and longitudinal relaxivity measurements simultaneously and in near real-time. Quantitative relaxivity values are obtained from the signal magnitude from fast Look-Locker EPI data, whereas phase information from all signal samples on the recovery curve is combined to provide temperature values using the proton resonance frequency method. The utility of this technique is illustrated in an in vitro experiment with thermosensitive liposomes, which are studied as potential micro vehicles for local drug delivery. The method allowed measuring the evolution of relaxivity during RF-heating of liposomes containing a paramagnetic contrast agent, demonstrating increase of liposome permeability near the phase transition temperature. Potential applications are monitoring of local drug delivery using thermosensitive liposomes, and confirmation of reaching the liposomes' threshold temperature during thermal therapy. Magn Reson Med, 2005. (c) 2005 Wiley-Liss, Inc.

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Liposome-DNA complexes infused intravenously inhibit tumor angiogenesis and elicit antitumor activity in dogs with soft tissue sarcoma.
Kamstock D, Guth A, Elmslie R, Kurzman I, Liggitt D, Coro L, Fairman J, Dow S.
Cancer Gene Ther. 2005 Sep 2; [Epub ahead of print].
[ expand abstract ]

Intravenous gene delivery using liposome-DNA complexes (LDC) has previously been shown to elicit antitumor activity, but only in rodent tumor models. Therefore, we conducted a study to determine in a large animal spontaneous tumor model whether intravenous infusions of LDC could target gene expression to cutaneous tumor tissues and whether repeated treatments had an effect on tumor growth or angiogenesis. A total of 13 dogs with cutaneous soft tissue sarcomas were enrolled in the study and were randomized to receive a series of 6 weekly infusions of LDC containing either canine endostatin DNA or DNA encoding an irrelevant gene (luciferase). Serial tumor biopsies were obtained to assess transgene expression, tumor microvessel density (MVD), and intratumoral leukocyte inflammatory responses. We found that intravenous infusion of LDC did not result in detectable gene expression in cutaneous tumor tissues. However, two of 13 treated dogs had objective tumor responses and eight dogs had stable disease during the treatment period. In addition, a significant decrease in tumor MVD was noted in six of 12 treated dogs at the completion of six treatments. These results suggest that intravenous infusions of LDC may elicit nonspecific antitumor activity and inhibit tumor angiogenesis.

Multicenter phase II trial of ABI-007, an albumin-bound paclitaxel, in women with metastatic breast cancer.
Ibrahim NK, Samuels B, Page R, Doval D, Patel KM, Rao SC, Nair MK, Bhar P, Desai N, Hortobagyi GN.
J Clin Oncol. 2005 Sep 1;23(25):6019-26.
[ expand abstract ]

PURPOSE: ABI-007 is a novel nanoparticle, albumin-bound paclitaxel that is free of solvents. This multicenter phase II study was designed to evaluate the efficacy and safety of ABI-007 for the treatment of metastatic breast cancer (MBC). PATIENTS AND METHODS: Sixty-three women with histologically confirmed and measurable MBC received 300 mg/m2 ABI-007 by intravenous infusion over 30 minutes every 3 weeks without premedication. Forty-eight patients received prior chemotherapy; 39 patients received no prior treatment for metastatic disease. RESULTS: Overall response rates (complete or partial responses) were 48% (95% CI, 35.3% to 60.0%) for all patients. For patients who received ABI-007 as first-line and greater than first-line therapy for their metastatic disease, the respective response rates were 64% (95% CI, 49.0% to 79.2%) and 21% (95% CI, 7.1% to 42.1%). Median time to disease progression was 26.6 weeks, and median survival was 63.6 weeks. No severe hypersensitivity reactions were reported despite the lack of premedication. Toxicities observed were typical of paclitaxel and included grade 4 neutropenia (24%), grade 3 sensory neuropathy (11%), and grade 4 febrile neutropenia (5%). Patients received a median of six treatment cycles; 16 patients had 25% dose reductions because of toxicities, and two of these patients had subsequent dose reductions. CONCLUSION: ABI-007, the first biologically interactive albumin-bound form of paclitaxel in the nanoparticle state, uses the natural carrier albumin rather than synthetic solvents to deliver paclitaxel and allows for safe administration of high paclitaxel doses without premedication, resulting in significant antitumor activity in patients with MBC, including those receiving the drug as first-line therapy.

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Specific Delivery of Therapeutic RNAs to Cancer Cells via the Dimerization Mechanism of phi29 Motor pRNA.
Guo S, Tschammer N, Mohammed S, Guo P.
Hum Gene Ther. 2005 Sep;16(9):1097-109.
[ expand abstract ]

The application of small RNA in therapy has been hindered by the lack of an efficient and safe delivery system to target specific cells. Packaging RNA (pRNA), part of the DNA-packaging motor of bacteriophage phi29(Phi29), was manipulated by RNA nanotechnology to make chimeric RNAs that form dimers via interlocking right- and left-hand loops. Fusing pRNA with receptor-binding RNA aptamer, folate, small interfering RNA (siRNA), ribozyme, or another chemical group did not disturb dimer formation or interfere with the function of the inserted moieties. Incubation of cancer cells with the pRNA dimer, one subunit of which harbored the receptor-binding moiety and the other harboring the gene-silencing molecule, resulted in their binding and entry into the cells, and subsequent silencing of anti/proapoptotic genes. The chimeric pRNA complex was found to be processed into functional double-stranded siRNA by Dicer (RNA-specific endonuclease). Animal trials confirmed the suppression of tumorigenicity of cancer cells by ex vivo delivery. It has been reported [Shu, D., Moll, W.-D., Deng, Z., Mao, C., and Guo, P. (2004). Nano Lett. 4:1717-1724] that RNA can be used as a building block for bottom-up assembly in nanotechnology. The assembly of proteinfree 25-nm RNA nanoparticles reported here will allow for repeated long-term administration and avoid the problems of short retention time of small molecules and the difficulties in the delivery of particles larger than 100 nm.

Hydrophobic drug delivery by self-assembling triblock copolymer-derived nanospheres.
Sheihet L, Dubin RA, Devore D, Kohn J.
Biomacromolecules. 2005 Sep-Oct;6(5):2726-31.
[ expand abstract ]

We describe the synthesis and characterization of a family of biocompatible ABA-triblock copolymers that comprised of hydrophilic A-blocks of poly(ethylene glycol) and hydrophobic B-blocks of oligomers of suberic acid and desaminotyrosyl-tyrosine esters. The triblock copolymers spontaneously self-assemble in aqueous solution into nanospheres, with hydrodynamic diameters between 40 and 70 nm, that do not dissociate under chromatographic and ultracentrifugation conditions. These nanospheres form strong complexes with hydrophobic molecules, including the fluorescent dye 5-dodecanoylaminofluorescein (DAF) and the antitumor drug, paclitaxel, but not with hydrophilic molecules such as fluorescein and Oregon Green. The nanosphere-paclitaxel complexes retain in vitro the high antiproliferative activity of paclitaxel, demonstrating that these nanospheres may be useful for delivery of the hydrophobic drugs.

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Mitochondrial delivery of mastoparan with transferrin liposomes equipped with a pH-sensitive fusogenic peptide for selective cancer therapy.
Yamada Y, Shinohara Y, Kakudo T, Chaki S, Futaki S, Kamiya H, Harashima H.
Int J Pharm. 2005 Aug 31; [Epub ahead of print].
[ expand abstract ]

Mastoparan (MP), a potent facilitator of mitochondrial permeability transition (PT), could be used as an antitumor agent, if it were encapsulated in a tumor selective delivery system. We recently developed transferrin-modified liposomes (Tf-L) with a pH-sensitive fusogenic peptide (GALA), which delivers an encapsulated fluorescent marker into cytosol efficiently. In this study, we encapsulated MP into Tf-L with GALA for the selective delivery to mitochondria of tumor cells. The MP showed potent PT activity at concentrations above 25muM in a homogenate of K 562 cells as well as in isolated mitochondria in the presence of phosphate. Tf-L equipped with cholesteryl GALA can release encapsulated sulforhodamine B, while Tf-L failed, as evidenced by confocal laser scanning microscopy. The MP, which was delivered with Tf-L with GALA, released cytochrome c (cyt c) from mitochondria to the cytosol, while free MP released cyt c not only to the cytosol but also extracellulary. These results demonstrate the utility of MP in Tf-L with GALA for cancer therapy.

Pharmacokinetic and cytotoxic studies of pegylated liposomal daunorubicin.
Song H, Zhang J, Han Z, Zhang X, Li Z, Zhang L, Fu M, Lin C, Ma J.
Cancer Chemother Pharmacol. 2005 Aug 30;:1-8 [Epub ahead of print].
[ expand abstract ]

Pegylated liposomes have been studied for nearly two decades. However, fewer pharmacological studies about its application in daunorubicin (DNR) than those in doxorubicin have been reported. In order to conduct a complete pharmacokinetic study, radiolabeled DNR was encapsulated in pegylated liposomes. Its in vitro drug release kinetics was determined to be in a slow manner, which was reflected in its cytotoxic effect on four cell lines. The lethal dose, plasma pharmacokinetics as well as tissue distribution of the formulation were evaluated in comparison with free DNR. The results revealed that liposomal daunorubicin significantly reduced the toxicity of the drug, with a half lethal dose of 29.35 mg/kg, compared with 5.45 mg/kg for free drug. Pharmacokinetic study of liposomal DNR demonstrated a slower clearance rate, an elevated area under the concentration-time curve, as well as increased half-lives compared to free drug. In addition, an altered tissue distribution of liposomal DNR was observed, with lower cardiac accumulation. Taken together, pegylated liposome-loaded DNR may be a promising anticancer drug and worth further therapeutic study.

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Ultra-deformable liposomes containing bleomycin: in vitro stability and toxicity on human cutaneous keratinocyte cell lines.
Lau KG, Hattori Y, Chopra S, O'Toole EA, Storey A, Nagai T, Maitani Y.
Int J Pharm. 2005 Aug 26;300(1-2):4-12.
[ expand abstract ]

Formulations of ultra-deformable liposomes containing bleomycin (Bleosome) have previously been described and proposed for topical treatment of skin cancer [Lau, K.G., Chopra, S., Maitani, Y., 2003. Entrapment of bleomycin in ultra-deformable liposomes. S. T. P. Pharm. Sci. 13, 237-239]. In this study, the stability of various Bleosome formulations was characterised and a purification process was established to isolate Bleosome for testing on cultures of either human cutaneous keratinocytes (NEB-1) immortalised by human papilloma virus (HPV)-type 16, or a spontaneously immortalised human squamous cell carcinoma (SCC) from a primary tumour. Bleosome facilitated entrapment of high concentrations of active bleomycin and samples purified by gel-filtration chromatography remained stable during 7 days of storage at 4 degrees C or at room temperature. Serially-diluted samples of this purified, high-strength product, 'high dose' were applied onto keratinocyte cell cultures to elucidate Bleosome LD50 profiles. In vitro data revealed that the LD50 of bleomycin encapsulated in Bleosome was approximately three-fold higher than free bleomycin solution for SCC cells, and nearly 30 times higher for NEB-1 cells. However, Bleosome containing 30 microg/ml of active bleomycin killed more than twice as many SCC cells than NEB-1 cells. At that concentration, the potency of liposomal bleomycin on causing cell death of SCC cells was found to be similar to that of free bleomycin solution.This effect was not seen on NEB-1 cells. It seems that SCC cells were particularly susceptible to Bleosome containing high levels of bleomycin. Results from these experiments promote the development of a novel product for the topical treatment of skin cancer.

Pharmacokinetics of intravenously administered stealth liposomal doxorubicin modulated with verapamil in rats.
Wang JC, Liu XY, Lu WL, Chang A, Zhang Q, Goh BC, Lee HS.
Eur J Pharm Biopharm. 2005 Aug 26; [Epub ahead of print].
[ expand abstract ]

Treatment of cancer through co-administration of anticancer drugs and multidrug resistance (MDR) modulators as a strategy to overcome drug resistance has been extensively explored. However, success has been limited by pharmacokinetic interactions because of non-specific blockade of P-glycoprotein (P-gp) in normal tissues or inability to reach relevant concentrations clinically. We hypothesized that stealth liposomal co-encapsulation of doxorubicin (DOX) with a P-glycoprotein inhibitor, verapamil (DARSLs), may overcome these limitations. Using intravenous (i.v.) administrations, the effects of verapamil (VER) either free (FV) or liposome co-encapsulated with DOX (DARSLs) on the pharmacokinetics and tissue distribution characteristics of DOX either as free (FD) or liposome-encapsulated (LD) were evaluated in normal rats. FV increased (P<0.05) the plasma AUC of free DOX (FD). Preparations containing LD had significant prolonged systemic exposure and slow tissue distribution of DOX. LDFV (liposomal DOX with free verapamil) and DARSLs shared similar DOX pharmacokinetics but the latter showed slower DOX distribution in most tissues studied and slower (P<0.05) DOX biliary transport. The addition of VER into LD in these two preparations significantly increased the AUC (P<0.01) and reduced the clearance (P<0.01) of DOX when compared to LD. Specifically, DARSLs reduced initial DOX distribution to the heart (P<0.05) corresponding to initial alleviation (P<0.05) of bradycardia when compared to other DOX with VER preparations. In conclusion, liposomal co-encapsulation of DOX with VER has promise of significant therapeutic advantages, and should be explored further in therapeutic studies with animal tumor xenograft models.

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Enhanced intracellular delivery and improved antitumor efficacy of doxorubicin by sterically stabilized liposomes modified with a synthetic RGD mimetic.
Xiong XB, Huang Y, Lu WL, Zhang X, Zhang H, Nagai T, Zhang Q.
J Control Release. 2005 Aug 24; [Epub ahead of print].
[ expand abstract ]

While sterically stabilized liposomes (SSL) can passively accumulate into tumor tissue due to the effect of enhanced permeability and retention (EPR), the intracellular uptake of the entrapped anticancer drugs by the tumor cells should be a determinant step for their antitumor activities. Therefore, strategies that can enhance the intracellular uptake of SSL into tumor cells could lead to an improved therapeutic efficacy for the drugs. To check this possibility, RGD-mimetic-modified SSL (RGDm-SSL) were constructed aimed to achieve tumor accumulation as well as enhanced intracellular delivery, and were loaded with doxorubicin (DOX), an anticancer drug. Flow cytometry and confocal microscopy reveal that RGDm-SSL facilitated the DOX uptake into the melanoma cells via integrin-mediated endocytosis. DOX-loaded RGDm-SSL (RGDm-SSL-DOX) displayed higher cytotoxicity on melanoma cells than DOX-loaded SSL (SSL-DOX). Tissue distribution and therapeutic experiments were examined in C57BL/6 mice carrying melanoma B16 tumors. RGDm-SSL-DOX displayed similar DOX accumulation in tumor tissue to that of SSL-DOX but showed significantly lower DOX level in blood and remarkably higher DOX level in spleen than SSL-DOX. Administration of RGDm-SSL-DOX at a dose of 5 mg DOX/kg resulted in effective retardation of tumor growth and prolonged survival times compared with SSL-DOX. These results suggest that RGDm-modified SSL may be a promising intracellular targeting carrier for efficient delivery of chemotherapeutic agents into tumor cells.

Effectiveness of submicron-sized, chitosan-coated liposomes in oral administration of peptide drugs.
Takeuchi H, Matsui Y, Sugihara H, Yamamoto H, Kawashima Y.
Int J Pharm. 2005 Aug 23; [Epub ahead of print].
[ expand abstract ]

The mucoadhesive behavior of chitosan-coated liposomes in the intestinal tract of the rat was examined to elucidate their particle size effects on the absorption of an entrapped drug, calcitonin. The intestine was removed from rats after oral administration of liposomes containing a fluorescent dye, and its various parts were observed with confocal laser scanning microscopy. Penetration of submicron-sized liposomes (ssLip) or chitosan-coated ssLip (ssCS-Lip) into the mucosa was observed, while such behavior was not observed for the multilamellar liposomes, even when coated with chitosan (CS-Lip). The retentive property of ssCS-Lip was confirmed by measuring the amount of dye in each part of the intestine. The pharmacologic effects of calcitonin-loaded liposomes of different particle size were measured after oral administration in rats. The pharmacologic effect of oral administration of ssLip coated with chitosan was detected up to 120h after administration. The extensive pharmacologic effect of ssCS-Lip was attributed to their prolonged retention in the intestinal mucosa, partly owing to their penetrative property into the intestinal mucosa. The chitosan-coated ssLip, with their higher retentive property in the intestinal tract, are much more effective than ssLip and CS-Lip in improving the enteral absorption of peptide drugs.

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Drastic effect of nanoapatite particles on liposome-mediated mRNA delivery to mammalian cells.
Zohra FT, Chowdhury EH, Nagaoka M, Akaike T.
Anal Biochem. 2005 Aug 23; [Epub ahead of print].
[ expand abstract ]

No abstract available.

Vaccination with mRNAs encoding tumor-associated antigens and granulocyte-macrophage colony-stimulating factor efficiently primes CTL responses, but is insufficient to overcome tolerance to a model tumor/self antigen.
Hess PR, Boczkowski D, Nair SK, Snyder D, Gilboa E.
Cancer Immunol Immunother. 2005 Aug 20;:1-12 [Epub ahead of print].
[ expand abstract ]

Immunization of mice with dendritic cells transfected ex vivo with tumor-associated antigen (TAA)-encoding mRNA primes cytotoxic T lymphocytes (CTL) that mediate tumor rejection. Here we investigated whether direct injection of TAA mRNA, encapsulated in cationic liposomes, could function similarly as cancer immunotherapy. Intradermal and intravenous injection of ovalbumin (OVA) mRNA generated specific CTL activity and inhibited the growth of OVA-expressing tumors. Vaccination studies with DNA have demonstrated that co-administration of antigen (Ag)- and cytokine-encoding plasmids potentiate the T cell response; in analogous fashion, the inclusion of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA enhanced OVA-specific cytotoxicity. The ability of this GM-CSF-augmented mRNA vaccine to treat an established spontaneous tumor was evaluated in the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mouse, using the SV40 large T Ag (TAg) as a model tumor/self Ag. Repeated vaccination elicited vigorous TAg-specific CTL activity in nontransgenic mice, but tumor-bearing TRAMP mice remained tolerant. Adoptive transfer of naive splenocytes into TRAMP mice prior to the first vaccination restored TAg reactivity, and slowed tumor progression. The data from this study suggests that vaccination with TAA mRNA is a simple and effective means of priming antitumor CTL, and that immunogenicity of the vaccine can be augmented by co-delivery of GM-CSF mRNA. Nonetheless, limitations of such vaccines in overcoming tolerance to tumor/self Ag may mandate prior or simultaneous reconstitution of the autoreactive T cell repertoire for this form of immunization to be effective.

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Carbon nanotubes as multifunctional biological transporters and near-infrared agents for selective cancer cell destruction.
Shi Kam NW, O'connell M, Wisdom JA, Dai H.
Proc Natl Acad Sci U S A. 2005 Aug 16;102(33):11600-5.
[ expand abstract ]

Biological systems are known to be highly transparent to 700- to 1,100-nm near-infrared (NIR) light. It is shown here that the strong optical absorbance of single-walled carbon nanotubes (SWNTs) in this special spectral window, an intrinsic property of SWNTs, can be used for optical stimulation of nanotubes inside living cells to afford multifunctional nanotube biological transporters. For oligonucleotides transported inside living cells by nanotubes, the oligos can translocate into cell nucleus upon endosomal rupture triggered by NIR laser pulses. Continuous NIR radiation can cause cell death because of excessive local heating of SWNT in vitro. Selective cancer cell destruction can be achieved by functionalization of SWNT with a folate moiety, selective internalization of SWNTs inside cells labeled with folate receptor tumor markers, and NIR-triggered cell death, without harming receptor-free normal cells. Thus, the transporting capabilities of carbon nanotubes combined with suitable functionalization chemistry and their intrinsic optical properties can lead to new classes of novel nanomaterials for drug delivery and cancer therapy

Immunoliposomes Directed Toward VCAM-1 Interact Specifically with Activated Endothelial Cells-A Potential Tool for Specific Drug Delivery.
Voinea M, Manduteanu I, Dragomir E, Capraru M, Simionescu M.
Pharm Res. 2005 Aug 13; [Epub ahead of print].
[ expand abstract ]

PURPOSE: Immunoliposomes can be potentially used as carriers for drug delivery to specific cells. The aim of this paper was to exploit the overexpression of vascular cell adhesion molecule-1 (VCAM-1) on activated human endothelial cells (HEC) for targeting of anti-VCAM-1 coupled liposomes with the intent for further use as drug carriers. METHODS: TNF-alpha-activated HEC were exposed to liposomes, either plain or coupled with antibodies to VCAM-1 (L-VCAM-1) or to irrelevant IgG (L-IgG); nonactivated HEC subjected to the same conditions were used as control. For binding studies, the cells were incubated with fluorescently labeled liposomes at 4 degrees C, and after 2 h, fluorescence intensity was assessed by flow cytometry; specificity of binding was determined by performing the experiments in the presence of excess anti-VCAM-1. Cellular internalization of liposomes was studied employing radioactively or fluorescently labelled liposomes; to detect the mechanisms of uptake, experiments were performed in the presence of agents that interfere in the endocytotic pathway. Transmigration of liposomes was monitored in a two-chamber culture model. The effect of L-VCAM-1 binding to HEC on intracellular calcium ([Ca(2+)](i)) and distribution of actin was determined by fluorimetry and fluorescence microscopy. RESULTS: (1) L-VCAM-1 binds selectively and specifically to TNF-alpha activated HEC. (2) Approximately 50% of L-VCAM-1 is taken up by receptor-mediated endocytosis via clathrin-coated vesicles. (3) Binding of L-VCAM-1 to HEC surface induces a rise in [Ca(2+)](i) and reorganization of actin filaments. (4) A small percentage of liposomes migrates across HEC. CONCLUSION: The data indicate that VCAM-1 may be an appropriate target for specific drug delivery to activated HEC using immunoliposomes.

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Carbon nanotubes as multifunctional biological transporters and near-infrared agents for selective cancer cell destruction.
Kam NW, O'connell M, Wisdom JA, Dai H.
Proc Natl Acad Sci U S A. 2005 Aug 8; [Epub ahead of print].
[ expand abstract ]

Biological systems are known to be highly transparent to 700- to 1,100-nm near-infrared (NIR) light. It is shown here that the strong optical absorbance of single-walled carbon nanotubes (SWNTs) in this special spectral window, an intrinsic property of SWNTs, can be used for optical stimulation of nanotubes inside living cells to afford multifunctional nanotube biological transporters. For oligonucleotides transported inside living cells by nanotubes, the oligos can translocate into cell nucleus upon endosomal rupture triggered by NIR laser pulses. Continuous NIR radiation can cause cell death because of excessive local heating of SWNT in vitro. Selective cancer cell destruction can be achieved by functionalization of SWNT with a folate moiety, selective internalization of SWNTs inside cells labeled with folate receptor tumor markers, and NIR-triggered cell death, without harming receptor-free normal cells. Thus, the transporting capabilities of carbon nanotubes combined with suitable functionalization chemistry and their intrinsic optical properties can lead to new classes of novel nanomaterials for drug delivery and cancer therapy.

Interaction of functional dendrimers with multilamellar liposomes: design of a model system for studying drug delivery.
Pantos A, Tsiourvas D, Nounesis G, Paleos CM.
Langmuir. 2005 Aug 2;21(16):7483-90.
[ expand abstract ]

Multilamellar liposomes consisting of phosphatidylcholine-cholesterol-dihexadecyl phosphate (19:9.5:1 molar ratio) and dispersed in aqueous or phosphate buffer solutions were interacted with poly(propylene imine) dendrimers which were partially functionalized with guanidinium groups. The remaining toxic external primary amino groups of the dendrimers were reacted with propylene oxide, affording the corresponding hydroxylated derivatives. Microscopic, zeta-potential, and dynamic light scattering techniques have shown that liposomal-dendrimeric molecular recognition occurs due to the interaction between the complementary phosphate and guanidinium groups. Calcein liposomal entrapment experiments demonstrate a limited leakage, i.e., less than 13%, following liposomes interaction with the modified dendrimers. Calorimetric studies indicate that the enthalpy of the interaction is dependent on the number of guanidinium groups present at the dendrimeric surface and the medium. The process is reversible, and redispersion of the aggregates occurs by adding concentrated phosphate buffer. Two corticosteroid drugs, i.e., betamethasone dipropionate and betamethasone valerate, were encapsulated into the functionalized dendrimers. Drug transport from guanidinylated dendrimers to multilamellar liposomes ranges from 40% to 85%, and it is also dependent on the medium and the degree of dendrimer guanidinylation.

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Phase I Study of Liposome-DNA Complexes Encoding the Interleukin-2 Gene in Dogs with Osteosarcoma Lung Metastases.
Dow S, Elmslie R, Kurzman I, Macewen G, Pericle F, Liggitt D.
Hum Gene Ther. 2005 Aug;16(8):937-46.
[ expand abstract ]

Systemic gene delivery using cationic liposome-DNA complexes (LDCs) has been shown to elicit potent antitumor activity in mice with tumor metastases to the lungs. However, intravenous gene delivery for treatment of established cancer has not been evaluated previously in a spontaneous, large animal model. We therefore evaluated the safety, toxicity, and efficacy of intravenous gene delivery, using LDCs in dogs with established tumor metastases. Twenty dogs with chemotherapy-resistant osteosarcoma metastases to the lungs received a series of intravenous infusions of cationic liposomes and plasmid DNA encoding the canine interleukin-2 (IL-2) cDNA. Effects of intravenous gene delivery on immune activation, clinical and hematologic parameters, tumor responses, and survival times were assessed. We found that slow intravenous administration of IL-2 LDCs resulted in detectable IL-2 transgene expression in lung tissues of dogs. Repeated intravenous infusions of LDCs were well tolerated by dogs with lung tumor metastases and elicited systemic immune activation, as reflected by fever, leukogram changes, monocyte activation, and increased natural killer cell activity. Three of 20 dogs experienced partial or complete regression of lung metastases after infusion of IL-2 LDCs. Overall survival times were significantly increased in treated dogs compared with historical control animals with the same stage of disease. We conclude that repeated intravenous infusion of LDCs in cancerbearing dogs is safe and well tolerated at low doses and may be capable of eliciting antitumor activity in some animals with advanced tumor metastases.

Targeting delivery of oligonucleotide and plasmid DNA to hepatocyte via galactosylated chitosan vector.
Gao S, Chen J, Dong L, Ding Z, Yang YH, Zhang J.
Eur J Pharm Biopharm. 2005 Aug;60(3):327-34.
[ expand abstract ]

Delivery of oligonucleotide to specific cells and maintenance of its biological function are important for nucleic acid therapy. The objective of this paper is to demonstrate that galactosylated low molecular weight chitosan (gal-LMWC) is a safe and effective vector of antisense oligonucleotide (ASO) and plasmid DNA for the hepatocyte targeting delivery. Gal-LMWC has been successfully prepared and MTT cytotoxic assay shows that cytotoxicity of gal-LMWC is lower than that of high molecular weight chitosan (HMWC) and low molecular weight chitosan (LMWC) in HepG2 cells. Using a complex coacervation process, gal-LMWC can form stable nano-complexes with plasmid DNA or with ASO by the electrostatic interaction. The morphometrics, particle size, and the zeta potential of gal-LMWC/ASO complexes and gal-LMWC/plasmid DNA complexes are very similar. The transfection efficiency by using gal-LMWC vector is significantly higher than that of naked DNA or naked ASO in HepG2 cells. Transfection efficiency of gal-LMWC/ASO complexes and gal-LMWC/plasmid DNA complexes depends on the molar ratio of the positive chitosan amino group and the negative DNA phosphate group (N/P ratio) strongly. Inhibition experiments confirm that the enhanced transfection efficiency is due to the ASGR mediated endocytosis of the gal-LMWC/ASO complexes or gal-LMWC/DNA complexes. These results suggest that gal-LMWC can be used in gene therapy to improve the transfection efficiency in vitro and in vivo.

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Lipid carrier systems for targeted drug and gene delivery.
Hashida M, Kawakami S, Yamashita F.
Chem Pharm Bull (Tokyo). 2005 Aug;53(8):871-80.
[ expand abstract ]

For effective chemotherapy, it is necessary to deliver therapeutic agents selectively to their target sites, since most drugs are associated with both beneficial effects and side effects. The use of lipid dispersion carrier systems, such as lipid emulsions and liposomes, as carriers of lipophilic drugs has attracted particular interest. A drug delivery system can be defined as a methodology for manipulating drug distribution in the body. Since drug distribution depends on the carrier, administration route, particle size of the carrier, lipid composition of the carrier, electric charge of the carrier and ligand density of the targeting carrier, these factors must be optimized. Recently, the lipid carrier system has also been applied to gene delivery systems for gene therapy. However, in both drug and gene medicine cases, a lack of cell-selectivity limits the wide application of this kind of drug and/or gene therapy. Therefore, lipid carrier systems for targeted drug and gene delivery must be developed for the rational therapy. In this review, we shall focus on the progress of research into lipid carrier systems for drug and gene delivery following systemic or local injection.

Liposomal encapsulated anti-cancer drugs.
Hofheinz RD, Gnad-Vogt SU, Beyer U, Hochhaus A.
Anticancer Drugs. 2005 Aug;16(7):691-707.
[ expand abstract ]

Among several drug delivery systems, liposomal encapsulated anti-cancer agents represent an advanced and versatile technology. Several formulations of liposomal anthracyclines are approved, e.g. for the treatment of metastatic breast cancer (pegylated and non-pegylated liposomal doxorubicin) or AIDS-related Kaposi's sarcoma (pegylated liposomal doxorubicin and liposomal daunorubicin). Meanwhile, virtually all anti-cancer drugs have been encapsulated in liposomes using different technologies. This review will summarize preclinical and clinical data of approved and exemplary emerging liposomal anti-cancer agents.

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Nanotechnology-based Drug Delivery for Cancer.
Jain KK.
Technol Cancer Res Treat. 2005 Aug;4(4):407-16.
[ expand abstract ]

Nanobiotechnologies have been applied to improve drug delivery and to overcome some of the problems of drug delivery in cancer. These can be classified into many categories that include use of various nanoparticles, nanoencapsulation, targeted delivery to tumors of various organs, and combination with other methods of treatment of cancer such as radiotherapy. Nanoparticles are also used for gene therapy for cancer. Some of the technologies enable combination of diagnostics with therapeutics which will be important for the personalized management of cancer. Some of the limitations of these technologies and prospects for future development are discussed.

Editorial: targeted drug delivery for cancer.
Jain KK.
Technol Cancer Res Treat. 2005 Aug;4(4):311-4.
[ expand abstract ]

Cancer drug delivery is no longer simply wrapping the drug in new formulations for different routes of delivery. Knowledge and experience from other technologies such as nanotechnology, advanced polymer chemistry, and electronic engineering, are being brought together in developing novel methods of drug delivery. Advances in our knowledge of molecular biology of cancer and pathways involved in malignant transformation of cells are revolutionizing the approach to cancer treatment with a focus is on targeted cancer therapy. The newer approaches to cancer treatment not only supplement the conventional chemotherapy and radiotherapy but also aim to prevent damage to the normal tissues and overcome drug resistance. Innovative methods of cancer treatment require new concepts of drug delivery in cancer.

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Selective tumor targeting by enhanced permeability and retention effect. Synthesis and antitumor activity of polyphosphazene-platinum (II) conjugates.
Jun YJ, Kim JI, Jun MJ, Sohn YS.
J Inorg Biochem. 2005 Aug;99(8):1593-601
[ expand abstract ]

Nanosized polyphosphazene-platinum (II) conjugates with a wide range of molecular weight from 24,000 to 115,000 were synthesized to study their tumor selectivity by enhanced permeability and retention (EPR) effect and their antitumor activity. It has been found from biodistribution study that the present polyphosphazene-Pt(II) conjugates exhibit high tumor selectivity by EPR effect with the tumor to tissue ratio (TTR) from 3.6 to 13 depending on the molecular size. These polymer conjugates have shown excellent in vivo antitumor activity against both murine and human cancer cell lines. In particular, xenograft trials of the conjugates have shown outstanding tumor inhibition effect on the stomach cancer cell line, YCC-3, which is one of the least responsive to the anticancer agents currently in clinical use, although the reason is not clearly explainable yet. The high in vivo activity seems to be attributed to the controlled-release of the antitumor active platinum (II) moiety, [GlyGluPt(dach] (dach=trans-(+/-)-1,2-diaminocyclohexane) from the phosphazene backbone by degradation in aqueous solution.

Antitumor activity of cationic liposome-mediated Bax gene transfer in osteosarcoma cells: induction of apoptosis and caspase-independent cell death.
Okumura K, Nakase M, Nakamura S, Inui M, Hiramoto K, Tagawa T.
Int J Oncol. 2005 Aug;27(2):433-8.
[ expand abstract ]

The purpose of this study was to evaluate the anti-tumor effects of osteosarcoma (HOSM-1) cells via transfer of the Bax gene using a cationic liposome. We evaluated the levels of Bax, Bcl-xL, Bcl-2 and cytochrome c expression by Western blot analysis, and caspase-9 and -3 activities were determined in a colorimetric assay. Apoptosis was detected using a TUNEL assay, and cell growth inhibition was determined in an MTT assay. Following Bax gene transfer, release of cytochrome c to the cytosol was detected, the activities of caspase-9 and -3 increased, and TUNEL-positive cells (37.5%) were detected. Cell survival rate was 50.8% under these conditions. Induction of apoptosis was inhibited by a caspase inhibitor (zVAD-fmk), but only a slight increase in cell survival rate occurred. Hence, since not only apoptosis but also caspase-independent cell death is induced in HOSM-1 cells, we anticipate that Bax gene therapy with cationic liposomes will be useful for osteosarcoma.

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Intracellular delivery of doxorubicin with RGD-modified sterically stabilized liposomes for an improved antitumor efficacy: In vitro and in vivo.
Xiong XB, Huang Y, Lu WL, Zhang X, Zhang H, Nagai T, Zhang Q.
J Pharm Sci. 2005 Aug;94(8):1782-93.
[ expand abstract ]

Passive targeting by sterically stabilized liposomes (SSL), once combined with efficient intracellular delivery, may be a very useful strategy to improve the antitumor efficacy for the anticancer agents. The arginine-glycine-aspartic acid tripeptide (RGD) is known to serve as a recognition motif for several different integrins located on cell surface. In this study, the RGD tripeptide was coupled to the distal end of the poly (ethylene glycol)-coated liposomes (RGD-SSL) aimed to achieve increased tumor accumulation and enhanced intracellular uptake. DOX-loaded RGD-SSL (RGD-SSL-DOX), DOX-loaded SSL (SSL-DOX), and free DOX were compared with respect to their in vitro uptake and cytotoxicity and their in vivo biodistribution and therapeutic efficacy in tumor-bearing mice. Flow cytometry and confocal microscopy studies revealed that RGD-SSL could facilitate the DOX uptake into melanoma cells by integrin-mediated endocytosis. RGD-SSL-DOX displayed higher cytotoxicity on melanoma cells than SSL-DOX. While RGD-SSL-DOX demonstrated prolonged circulation time and increased tumor accumulation as SSL-DOX did, it showed remarkably higher splenic uptake than SSL-DOX. Mice receiving RGD-SSL-DOX (5 mg DOX/kg) showed effective retardation in tumor growth compared with those receiving same dose of SSL-DOX, free DOX solution, or saline. These results suggest that RGD-modified SSL may be a feasible intracellular targeting carrier for efficient delivery of chemotherapeutic agents into tumor cells.

Cancer: one step at a time.
Mooney D.
Nature. 2005 Jul 28;436(7050):468-9.
[ expand abstract ]

No abstract

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Temporal targeting of tumour cells and neovasculature with a nanoscale delivery system.
Sengupta S, Eavarone D, Capila I, Zhao G, Watson N, Kiziltepe T, Sasisekharan R.
Nature. 2005 Jul 28;436(7050):568-72.
[ expand abstract ]

In the continuing search for effective treatments for cancer, the emerging model is the combination of traditional chemotherapy with anti-angiogenesis agents that inhibit blood vessel growth. However, the implementation of this strategy has faced two major obstacles. First, the long-term shutdown of tumour blood vessels by the anti-angiogenesis agent can prevent the tumour from receiving a therapeutic concentration of the chemotherapy agent. Second, inhibiting blood supply drives the intra-tumoural accumulation of hypoxia-inducible factor-1alpha (HIF1-alpha); overexpression of HIF1-alpha is correlated with increased tumour invasiveness and resistance to chemotherapy. Here we report the disease-driven engineering of a drug delivery system, a 'nanocell', which overcomes these barriers unique to solid tumours. The nanocell comprises a nuclear nanoparticle within an extranuclear pegylated-lipid envelope, and is preferentially taken up by the tumour. The nanocell enables a temporal release of two drugs: the outer envelope first releases an anti-angiogenesis agent, causing a vascular shutdown; the inner nanoparticle, which is trapped inside the tumour, then releases a chemotherapy agent. This focal release within a tumour results in improved therapeutic index with reduced toxicity. The technology can be extended to additional agents, so as to target multiple signalling pathways or distinct tumour compartments, enabling the model of an 'integrative' approach in cancer therapy.

Liposome transfected to plasmid-encoding endostatin gene combined with radiotherapy inhibits liver cancer growth in nude mice.
Zheng AQ, Song XR, Yu JM, Wei L, Wang XW.
World J Gastroenterol. 2005 Jul 28;11(28):4439-42.
[ expand abstract ]

AIM: To evaluate whether intratumoral injection of liposome-endostatin complexes could enhance the antitumor efficacy of radiation therapy in human liver carcinoma (BEL7402) model. METHODS: Recombinant plasmid pcDNA3.End was transfected into human liver carcinoma cell line (BEL7402) with lipofectamine to produce conditioned medium. Then BEL7402 cells and human umbilical vein endothelial cells (HUVECs) were treated with the conditioned medium. Cell cycle and apoptosis were analyzed by flow cytometer and endothelial cell proliferation rates were determined by MTT assay. The antitumor efficacy of endostatin gene combined with ionizing radiation in mouse xenograft liver tumor was observed. RESULTS: Endostatin significantly suppressed the S phase fraction and increased the apoptotic index in HUVECs. In contrast, endostatin treatment had no effect on BEL7402 cell apoptosis (2.1+/-0.3% vs 8.9+/-1.3%, t = 8.83, P = 0.009<0.01) or cell cycle distribution (17.2+/-2.3% vs 9.8+/-1.2%, t = 4.94, P = 0.016<0.05). The MTT assay showed that endostatin significantly inhibited the proliferation of HUVECs by 46.4%. The combination of local endostatin gene therapy with radiation therapy significantly inhibited the growth of human liver carcinoma BEL7402 xenografts, the inhibition rate of tumor size was 69.8% on d 28 compared to the untreated group. The tumor volume in the pcDNA3.End combined with radiation therapy group (249+/-83 mm(3)) was significantly different from that in the untreated group (823+/-148 mm(3), t = 5.86, P = 0.009<0.01) or in the pcDNA3 group (717+/-94 mm(3), t = 6.46, P = 0.003<0.01). Endostatin or the radiation alone also inhibited the growth of liver tumor in vivo, but their inhibition effects were weaker than those of endostatin combined with radiation, the inhibition rates on d 28 were 44.7% and 40.1%, respectively. CONCLUSION: Endostatin not only significantly suppresses tumor growth but also enhances the antitumor efficacy of radiation therapy in human carcinoma xenograft.

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Preparation, cellular transport, and activity of polyamidoamine-based dendritic nanodevices with a high drug payload.
Kolhe P, Khandare J, Pillai O, Kannan S, Lieh-Lai M, Kannan RM.
Biomaterials. 2005 Jul 26; [Epub ahead of print].
[ expand abstract ]

Dendrimers are emerging as a relatively new class of polymeric biomaterials with applications in drug delivery, and imaging. Achieving a high drug payload in dendrimers, and understanding the therapeutic effect of the dendrimer-drug conjugates are receiving increasing attention. A high drug payload nanodevice was obtained by covalent conjugation of ibuprofen to a polyamidoamine (PAMAM-G4-OH) dendrimer. Using DCC as a coupling agent, 58 molecules of ibuprofen were covalently conjugated to one molecule of generation 4 PAMAM-OH dendrimer. Cellular entry of the fluoroisothiocynate (FITC)-labeled dendrimer-drug conjugate was evaluated in vitro by using human lung epithelial carcinoma A549 cells by flow cytometry, confocal microscopy and UV/Visible spectroscopy. The pharmacological activity of the dendrimer-ibuprofen conjugate was compared to pure ibuprofen at various time points by measuring the suppression of prostaglandin E2. Significant amounts of the conjugate entered the cells rapidly within 15min. Suppression of prostaglandin was noted within 30min for the dendrimer-drug conjugates versus 1h for the free ibuprofen. The results suggest that dendrimers with high drug payload improve the drug's efficacy by enhanced cellular delivery, and may produce a rapid pharmacological response. These dendrimer-drug conjugates can potentially be further modified by attaching antibodies and ligands for targeted drug delivery.

Preparation and in vitro evaluation of chitosan nanoparticles containing a caspase inhibitor.
Aktas Y, Andrieux K, Alonso MJ, Calvo P, Gursoy RN, Couvreur P, Capan Y.
Int J Pharm. 2005 Jul 25;298(2):378-83.
[ expand abstract ]

The aim of this work was to develop a formulation for Z-DEVD-FMK, a peptide which is a caspase inhibitor and has been used in experimental animal studies for a decade. Peptide loaded chitosan nanoparticles were obtained by ionotropic gelation process and Z-DEVD-FMK was quantified by an HPLC method. The influence of the initial peptide concentration on the nanoparticle characteristics and release behavior was evaluated. The CS nanoparticles have a particle diameter (Z-average) ranging from approximately 313-412 nm and a positive zeta potential (20-28 mV). The formulation with the initial peptide concentration of 400 ng/ml provided the highest loading capacity (0.46%) and the highest extent of release (65% at 24 h) suggesting the possibility to achieve a therapeutic dose. According to the data obtained, this chitosan-based nanotechnology opens new and interesting perspectives for anticaspase activity.

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A methodology to study intracellular distribution of nanoparticles in brain endothelial cells.
Garcia-Garcia E, Andrieux K, Gil S, Kim HR, Le Doan T, Desmaele D, d'Angelo J, Taran F, Georgin D, Couvreur P.
Int J Pharm. 2005 Jul 25;298(2):310-4.
[ expand abstract ]

Cell internalisation and intracellular distribution of PEG-coated polyhexadecylcyanoacrylate (PEG-PHDCA) nanoparticles in rat brain endothelial cells (RBEC) have been investigated. A cell fractionation method has been developed based on the selective permeabilisation of RBEC plasma membrane by digitonin. By interacting with membrane cholesterol, digitonin creates pores allowing the release of soluble and diffusible species outside the cell. The selectivity of plasma membrane permeabilisation was controlled by using compartment markers such as lactate dehydrogenase (LDH) for cytoplasm and cathepsin B for lysosomes. An optimal digitonin concentration of 0.003% (w/v) has been identified to induce a pattern of membrane permeabilisation corresponding to the extraction of 72% LDH and less than 15% of Cathepsin B. Membrane permeabilisation at this digitonin concentration allows one to distinguish between the cell cytoplasm and its endo/lysosomal fraction. This methodology was applied to investigate the intracellular distribution of the nanoparticles after their incubation with the RBEC. The results showed that PEG-PHDCA nanoparticles were able to be internalised to a higher extent than PHDCA nanoparticles (after 20 min incubation). Additionally, these nanoparticles displayed different patterns of intracellular capture, depending on their specific surface composition: PEG-PHDCA nanoparticles were 48% in the plasma membrane, 24% in the cytoplasm, 20% in vesicular compartments and 8% associated with the fraction of the nucleus, the cytoskeleton and caveolae suggesting that PEG-PHDCA nanoparticle uptake by RBEC is specific and presumably due to endocytosis. Confocal microscopy studies confirmed the cellular uptake of PEG-PHDCA nanoparticles.

Polyethylenimine-Grafted Multiwalled Carbon Nanotubes for Secure Noncovalent Immobilization and Efficient Delivery of DNA.
Liu Y, Wu DC, Zhang WD, Jiang X, He CB, Chung TS, Goh SH, Leong KW.
Angew Chem Int Ed Engl. 2005 Jul 25;44(30):4782-4785.
[ expand abstract ]

No abstract

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Preparation and in vitro anticancer activity of wheat germ agglutinin (WGA)-conjugated PLGA nanoparticles loaded with paclitaxel and isopropyl myristate.
Mo Y, Lim LY.
J Control Release. 2005 Jul 25; [Epub ahead of print].
[ expand abstract ]

The purpose of this study was to develop a novel lectin-conjugated isopropyl myristate (IPM)-incorporated PLGA nanoparticle system (NP) for the local delivery of paclitaxel to the lungs. Wheat germ agglutinin (WGA) was conjugated onto preformed IPM- and paclitaxel-loaded PLGA NPs by a two-step carbodiimide method following comparative uptake studies of Concanavalin A, Ricinus communis-120 and WGA on A549, H1299 and CCL-186 cells. WIT-NP with mean diameter of 331 nm and zeta potential of -4.3 mV were prepared with yield of 66% and paclitaxel encapsulation efficiency of 61%. Particle size was expanded by surface conjugation with WGA, while zeta potential was reduced by the addition of IPM and WGA. In vitro paclitaxel release profile was not affected by WGA but initial drug release was enhanced by adding IPM into the formulation. The WIT-NP showed a burst-release of about 32% of the paclitaxel load within the first 5 h followed by a slow zero-order release of another 7% of the drug load in the next 115 h. Compared with the clinical paclitaxel formulation, paclitaxel-loaded nanoparticles without IPM or WGA, or paclitaxel-loaded nanoparticles with only IPM or WGA, the WIT-NP had superior in vitro cytotoxicity against A549 and H1299 cells. IC50 for WIT-NP after 5 and 24 h incubation with A549 cells were not significantly different (15.5 and 15 muM, respectively) whereas the clinical formulation was not cytotoxic after 5 h but had IC50 of 14 muM after 24 h incubation. WIT-NP exhibited stronger cell-killing effect because of more efficient cellular uptake via WGA-receptor-mediated endocytosis and IPM-facilitated release of paclitaxel from the NPs.

Fusogenic liposome delivers encapsulated nanoparticles for cytosolic controlled gene release.
Kunisawa J, Masuda T, Katayama K, Yoshikawa T, Tsutsumi Y, Akashi M, Mayumi T, Nakagawa S.
J Control Release. 2005 Jul 20;105(3):344-53.
[ expand abstract ]

Therapeutic agents based on DNA or RNA oligonucleotides (e.g., antisense DNA oligonucleotide, small interfering RNA) require a regulation of their kinetics in cytoplasm to maintain an optimal concentration during the treatment period. In this respect, delivery of functional nanoparticles containing these drugs into cytoplasm has been thought to have a potential for the cytosolic controlled gene release. In this study, we establish a protocol for the encapsulation of nanoparticles into liposome, which is further fused with ultra violet-inactivated Sendai virus to compose fusogenic liposomes. When nanoparticles were encapsulated in conventional liposomes, endocytosis-mediated uptake of nanoparticles was observed. In contrast, numerous amounts of nanoparticles were delivered into the cytoplasm without any cytotoxicity when the particles were encapsulated in fusogenic liposomes. Additionally, fusogenic liposome showed a high ability to deliver nanoparticles containing DNA oligonucleotides into cytoplasm. These results indicate that this combinatorial nanotechnology using fusogenic liposome and nanoparticle is a valuable system for regulating the intracellular pharmacokinetics of gene-based drugs.

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Targeting ECM-Integrin Interaction with Liposome-Encapsulated Small Interfering RNAs Inhibits the Growth of Human Prostate Cancer in a Bone Xenograft Imaging Model.
Bisanz K, Yu J, Edlund M, Spohn B, Hung MC, Chung LW, Hsieh CL.
Mol Ther. 2005 Jul 19; [Epub ahead of print].
[ expand abstract ]

The intricate intracellular communication between stromal and epithelial cells, which involves cell-cell-, cell-insoluble extracellular matrix- (ECM), and cell-soluble factor-mediated signaling processes, is an attractive target for therapeutic intervention in hormone-refractory and bone-metastatic prostate cancer. In the present study we demonstrated that androgen-independent PC3 prostate cancer cells adhered to and migrated on vitronectin (VN), a major noncollagenous ECM in mature bone, through the expression of alphav-containing integrin receptors alphavbeta1 and alphavbeta5 on the cell surface, as determined by antibody function blocking assay and flow cytometry analysis. Small interfering RNAs (siRNAs) targeting human integrin alphav markedly reduced their respective mRNA and protein expression in cells, resulting in nearly complete reduction in VN-mediated cancer progression in vitro. In vivo quantitative bioluminescence analysis of human prostate cancer bone xenografts demonstrated for the first time that intratumoral administration of liposome-encapsulated human alphav-siRNAs significantly inhibits the growth of luciferase-tagged PC3 tumors in skeleton, which was associated with decreased integrin alphav expression and increased apoptosis in tumor cells. This integrin-based gene therapy is particularly suitable for the treatment of prostate cancer bone metastasis.

Nanoparticles of poly(lactide)/vitamin E TPGS copolymer for cancer chemotherapy: Synthesis, formulation, characterization and in vitro drug release.
Zhang Z, Feng SS.
Biomaterials. 2005 Jul 15; [Epub ahead of print].
[ expand abstract ]

Paclitaxel is one of the best anticancer drugs, which has excellent therapeutic effects against a wide spectrum of cancers. The formulation of paclitaxel used in its currently clinical administration includes Cremophor EL, which has been found to cause serious side effects. Nanoparticle formulation of paclitaxel may provide an ideal solution for this problem and achieve a sustained chemotherapy. A novel copolymer, poly(lactide)-vitamin E TPGS (PLA-TPGS), was synthesized from lactide and d-alpha-tocopheryl polyethylene glycol 1000 succinate by bulk polymerization for nanoparticle formulation of anticancer drugs. (1)H NMR, FTIR and GPC were used to detect molecular structure of the copolymer. Paclitaxel-loaded PLA-TPGS nanoparticles were fabricated by a modified solvent extraction/evaporation technique with or without emulsifier involved, which were characterized by laser light scattering for size and size distribution; field emission scanning electron microscopy for surface morphology; zeta potential for surface charge; X-ray photoelectron spectroscopy for surface chemistry. The drug encapsulation efficiency and the in vitro drug release kinetics were measured by high-performance liquid chromatography. Formulation optimization was pursued. The particles were found of around 300nm in size and narrow size distribution. Of all, 89% drug encapsulation efficiency has been achieved for nanoparticles of 5% drug loading. The drug release from PLA-TPGS nanoparticles was found to be biphasic with an initial burst of 17% in the first day, followed by a sustained pattern with 51% release after 31 days.

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Targeting of the antivascular drug combretastatin to irradiated tumors results in tumor growth delay.
Pattillo CB, Sari-Sarraf F, Nallamothu R, Moore BM, Wood GC, Kiani MF.
Pharm Res. 2005 Jul;22(7):1117-20.
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PURPOSE: The aim of the study is to evaluate the effects of targeting the antivascular drug combretastatin to irradiated mouse melanomas. METHODS: Combretastatin was incorporated into liposomes with surfaces modified by the addition of cyclo(Arg-Gly-Asp-D: -Phe-Cys) (RGD) to create an immunoliposome (IL). This addition of RGD allows the liposome to be preferentially targeted to alpha(v)beta(3), an integrin up-regulated in the vasculature of irradiated tumors. C57BL mice bearing a transplanted B16-F10 melanoma were randomly assigned to one of the following treatment groups: untreated, a single dose of 5-Gy radiation (IR), IL (14.5 mg/kg of combretastatin), 5-Gy radiation plus IL, and a systemic administration of free drug (81.0 mg/kg of combretastatin). RESULTS: In this transplanted tumor model, there was no significant increase in the volume of the IL + IR (5 Gy) treated tumors during the initial 6 days posttreatment; all other treatment groups exhibited exponential growth curves after day 3. The IL + IR (5 Gy) treatment resulted in a 5.1-day tumor growth delay compared to untreated controls. CONCLUSIONS: These findings indicate that preferential targeting of antivascular drugs to irradiated tumors results in significant tumor growth delay

Encapsulation efficiency of water-soluble and insoluble drugs in liposomes prepared by the microencapsulation vesicle method.
Nii T, Ishii F.
Int J Pharm. 2005 Jul 14;298(1):198-205.
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The microencapsulation vesicle (MCV) method is a liposome preparation technique that reproducibly produces liposomes with homogeneous particle sizes with a high encapsulation efficiency. Liposomes encapsulating water-soluble drugs, lipophilic drugs and an amphiphilic drug were prepared by the MCV method and the encapsulation efficiency of the drugs was examined. Three kinds of egg yolk lecithin with different iodine values, i.e., purified egg yolk lecithin (PEL), partially hydrogenated purified egg yolk lecithin (R-20) and completely hydrogenated purified egg yolk lecithin (R-5), were used for membrane materials in order to explore the possible effects of membrane rigidity or surface area on the encapsulation efficiency of the drug. Water-soluble 5-fluorouracil showed 12-15% encapsulation efficiency, which was higher than those reported in the literature (less than 10%). With the MCV method, theoretically the initial drug-containing water phase was always separated from the dispersion medium by the lecithin-containing oil phase, which was advantageous to maintaining a higher encapsulation efficiency of the water-soluble drug. The encapsulation efficiency of lipophilic ibuprofen and flurbiprofen was around 90%. As for ketoprofen and liposomes were not formed when using hydrogenated egg yolk lecithin R-5, while the encapsulation efficiency using PEL or R-20 was around 80%. Amphiphilic amitriptyline hydrochloride resulted in a slightly higher encapsulation efficiency when dissolved in the water than the chloroform. Among the three kinds of lecithin, the most unsaturated PEL tended to show a higher encapsulation efficiency, probably due to differences in the packing geometry of the hydrophobic carbon chains in the membrane bilayer. The encapsulation efficiency of these drugs strongly correlated to the logP(octanol/water) and also tended to correlate to the logP(chloroform/water) for the order of the logP(chloroform/water) was almost the same as the order of the logP(octanol/water) in the drugs examined. As far as the results of this study, the logP(octanol/water) was considered to be a better indicator of the encapsulation efficiency of a drug in the MCV method.

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Radiosensitivity of human colon cancer cell enhanced by immunoliposomal docetaxel.
Wang QW, Lu HL, Song CC, Liu H, Xu CG.
World J Gastroenterol. 2005 Jul 14;11(26):4003-7.
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AIM: To enhance the radiosensitivity of human colon cancer cells by docetaxel. METHODS: Immunoliposomal docetaxel was prepared by coupling monoclonal antibody against carcinoembryonic antigen to cyanuric chloride at the PEG terminus of liposome. LoVo adenocarcinoma cell line was treated with immunoliposomal docetaxel or/and irradiation. MTT colorimetric assay was used to estimate cytotoxicity of immunoliposomal docetaxel and radiotoxicity. Cell cycle redistribution and apoptosis were determined with flow cytometry. Survivin expression in LoVo cells was verified by immunohistochemistry. D801 morphologic analysis system was used to semi-quantify immunohistochemical staining of survivin. RESULTS: Cytotoxicity was induced by immunoliposomal docetaxel alone in a dose-dependent manner. Immunoli-posomal docetaxel yielded a cytotoxicity effect at a low dose of 2 nmol/L. With a single dose irradiation, the relative surviving fraction of LoVo cells showed a dose-dependent response, but there were no significant changes as radiation delivered from 4 to 8 Gy. Compared with liposomal docetaxel or single dose irradiation, strongly radiopotentiating effects of immunoliposomal docetaxel on LoVo cells were observed. A low dose of immunoliposomal docetaxel could yield sufficient radiosensitivity. Immunoliposomal docetaxel were achieved both specificity of the conjugated antibody and drug radiosensitization. Combined with radiation, immunoliposomal docetaxel significantly increased the percentage of G(2)/M cells and induced apoptosis, but significantly decreased the percentage of cells in G(2)/G(1) and S phase by comparison with liposomal docetaxel. Immunohistochemical analysis showed that the brown stained survivin was mainly in cytoplasm of LoVo cells. Semi-quantitative analysis of the survivin immunostaining showed that the expression of survivin in LoVo cells under irradiation with immunoliposomal docetaxel was significantly decreased. CONCLUSION: Immunoliposomal docetaxel is strongly effective for target radiosensitation in LoVo colon carcinoma cells, and may offer the potential to improve local radiotherapy.

Novel Functionalized Biodegradable Polymers for Nanoparticle Drug Delivery Systems.
Kallinteri P, Higgins S, Hutcheon GA, St Pourcain CB, Garnett MC.
Biomacromolecules. 2005 Jul 11;6(4):1885-1894.
[ expand abstract ]

We have prepared and screened a library of novel functionalized polymers for development of nanoparticle drug delivery systems. The polymer backbone consisting of two ester-linked, nontoxic, biological monomers, glycerol and adipic acid, was prepared using a hydrolytic enzyme. The specificity of the chosen enzyme yields a linear polymer with one free pendant hydroxyl group per repeat unit, which can be further functionalized. This protocol gives control over the backbone polymer molecular weight, together with the ability to incorporate various amounts of different fatty acyl substituents. These functionalized polymers are able to self-assemble into well-defined small particles of high homogeneity with a very low toxicity. They are able to incorporate a water soluble drug, dexamethasone phosphate, with a high efficiency and drug loading which varies with the polymer specification. The above characteristics strongly suggest that these polymers could be developed into useful nanoparticulate drug delivery systems.

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Phase I Study of Liposome-DNA Complexes Encoding the Interleukin-2 Gene in Dogs with Osteosarcoma Lung Metastases.
Dow S, Elmslie R, Kurzman I, Macewen G, Pericle F, Liggitt D.
Hum Gene Ther. 2005 Jul 7; [Epub ahead of print].
[ expand abstract ]

Systemic gene delivery using cationic liposome-DNA complexes (LDCs) has been shown to elicit potent antitumor activity in mice with tumor metastases to the lungs. However, intravenous gene delivery for treatment of established cancer has not been evaluated previously in a spontaneous, large animal model. We therefore evaluated the safety, toxicity, and efficacy of intravenous gene delivery, using LDCs in dogs with established tumor metastases. Twenty dogs with chemotherapy-resistant osteosarcoma metastases to the lungs received a series of intravenous infusions of cationic liposomes and plasmid DNA encoding the canine interleukin-2 (IL-2) cDNA. Effects of intravenous gene delivery on immune activation, clinical and hematologic parameters, tumor responses, and survival times were assessed. We found that slow intravenous administration of IL-2 LDCs resulted in detectable IL-2 transgene expression in lung tissues of dogs. Repeated intravenous infusions of LDCs were well tolerated by dogs with lung tumor metastases and elicited systemic immune activation, as reflected by fever, leukogram changes, monocyte activation, and increased natural killer cell activity. Three of 20 dogs experienced partial or complete regression of lung metastases after infusion of IL-2 LDCs. Overall survival times were significantly increased in treated dogs compared with historical control animals with the same stage of disease. We conclude that repeated intravenous infusion of LDCs in cancerbearing dogs is safe and well tolerated at low doses and may be capable of eliciting antitumor activity in some animals with advanced tumor metastases.

Encapsulation and Controlled Release of a Hydrophobic Drug Using a Novel Nanoparticle-Forming Hyperbranched Polyester.
Zou J, Shi W, Wang J, Bo J.
Macromol Biosci. 2005 Jul 7;5(7):662-668.
[ expand abstract ]

An amphiphilic, hyperbranched polymer suitable for use in controlled drug delivery is reported. This polymer was obtained by modification of the hyperbranched aliphatic polyester Boltorntrade mark H20 (H20) with succinic anhydride and then glycidyl methacrylate, and formed nanoparticles in aqueous solution. The critical association concentration was 7.4 x 10(-3) g . L(-1), as determined by fluorescence spectroscopy using pyrene as a molecular probe. A static/dynamic laser light scattering (LLS) study revealed that the average particle size was 39.4 nm with a low particle size distribution (PDI = 0.04), and that each particle was composed of about 350 amphiphilic molecules. Daidzein, a hydrophobic traditional Chinese medicine, was encapsulated during particle formation and the release properties were determined. The optimal feeding concentration of daidzein to hyperbranched polyester was 4.9 x 10(-5) g . mL(-1) to 5.0 x 10(-3) g . mL(-1) with a loading efficiency of 76.1%. In the presence of the enzyme Lipase PS, the drug loaded nanoparticles degraded in a random one-by-one manner and released the drug over a few days. This system is therefore a novel controlled drug release system based on nanoparticles formed of hyperbranched polyester.Encapsulation of daidzein by hyperbranched polyester particles.

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Polyethylenimine-Grafted Multiwalled Carbon Nanotubes for Secure Noncovalent Immobilization and Efficient Delivery of DNA.
Liu Y, Wu DC, Zhang WD, Jiang X, He CB, Chung TS, Goh SH, Leong KW.
Angew Chem Int Ed Engl. 2005 Jul 1; [Epub ahead of print].
[ expand abstract ]

No abstract available.

Microfluidic Gradient-Generating Device for Pharmacological Profiling.
Pihl J, Sinclair J, Sahlin E, Karlsson M, Petterson F, Olofsson J, Orwar O.
Anal Chem. 2005 Jul 1;77(13):3897-3903.
[ expand abstract ]

We describe an on-chip microfluidic gradient-generating device that generates concentration gradients spanning nearly 5 orders of magnitude starting from a single concentration. The exiting stream of drugs held at different concentrations remains laminar in a recording chamber and can be presented as 24 discrete solutions to a cell-based sensor. The high-performance characteristics of the device are demonstrated by pharmacological screening of voltage-gated K(+) channels (hERG) and ligand-gated GABA(A) receptors using scanning-probe patch-clamp measurements. Multiple data point dose-response curves and IC(50) and EC(50) values were rapidly obtained, typically in less than 30 min, through its combined functionality of gradient generation and open-volume laminar flow. The device facilitates rapid pharmacological profiling of ion channel and GPCR effectors and enables the acquisition of large numbers of data points with minute sample consumption and handling.

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Applied nanotechnology for the management of breakthrough cancer pain.
Sprintz M, Benedetti C, Ferrari M.
Minerva Anestesiol. 2005 Jul-Aug;71(7-8):419-23.
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Pain, often considered the 5th vital sign, plays a significant role in the pathophysiology of cancer. Often addressed as an afterthought, untreated or under-treated cancer-related pain can have deleterious effects on a patient's physical and psychological well-being. Additionally, patients with breakthrough cancer pain tend to have more intense and more frequent background pain than patients without breakthrough pain. Currently, only oral transmucosal fentanyl citrate (OTFC) has reached the market for the treatment of cancer-related breakthrough pain. OTFC is an excellent first step in addressing the unmet need for symptomatic relief of breakthrough cancer pain; however, there is much room for improvement. Nanoscale science and engineering advancements have the long-term potential to bring revolutionary changes in society and across virtually all physical, biological and engineering disciplines, particularly medicine. Nanotechnology offers the potential to address multiple, major unmet problems in the diagnosis, treatment and symptom management of a large variety of diseases and conditions, including cancer. Nanotechnology can engender transformational progress in crucial aspects of the fight against cancer, spanning the continuum that ranges from prevention, to early detection, screening and monitoring, to innovative diagnostics and therapeutic modalities in the era of patient-centered, molecular medicine. Specifically, the authors will discuss their current research in the field of biomedical nanotechnology and its application to the management of breakthrough cancer pain.

Cationic lipid-protamine-DNA (LPD) complexes for delivery of antisense c-myc oligonucleotides.
Junghans M, Loitsch SM, Steiniger SC, Kreuter J, Zimmer A.
Eur J Pharm Biopharm. 2005 Jul;60(2):287-94.
[ expand abstract ]

In the present study, cationic lipid-peptide-DNA-complexes (LPDs) consisting of AH-Chol-liposomes and protamine-phosphodiester-oligonucleotide-particles (proticles) were introduced as carriers for antisense therapy. The LPDs were physically characterized, and a possible mechanism for adsorption of oligonucleotides (ODNs) was suggested. An increase in stability of ODNs against DNase I and serum nuclease digestion by these carriers was demonstrated. The hydrodynamic diameter increased after incubation with FCS which could be attributed to a protein coating of the particle surface. However, in cell culture medium lower particle sizes of the complexes occurred. In an antisense c-myc in vitro model, the effect of LPDs was tested using U937 cells. The C-MYC level was reduced after treatment of these antisense ODN carrier complexes. Furthermore, no changes in target mRNA concentration of the treated cells was found by reverse transcription and competitive multiplex-PCR.

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Phase I trial of liposomal encapsulated doxorubicin (MyocetTM; D-99) and weekly docetaxel in advanced breast cancer patients.
Mrozek E, Rhoades CA, Allen J, Hade EM, Shapiro CL.
Ann Oncol. 2005 Jul;16(7):1087-93.
[ expand abstract ]

BACKGROUND: : We conducted a phase I trial to determine the safety and maximum tolerated dose (MTD) of non-pegylated liposome-encapsulated doxorubicin (Myocettrade mark; D-99) administered with weekly docetaxel in metastatic breast cancer (MBC) patients. PATIENTS AND METHODS: : Twenty-one patients with no prior chemotherapy for MBC received D-99 (60 or 50 mg/m(2)) intravenously (i.v.) on day 1 and escalating doses of docetaxel (25, 30 and 35 mg/m(2)) i.v. on days 1 and 8 in cohorts of three to six patients. Treatment cycles were repeated every 21 days for a maximum of six cycles. RESULTS: : The maximum tolerated dose (MTD) was 50 mg/m(2) of D-99 in combination with 25 mg/m(2) of weekly docetaxel. The most common grade 4 toxicity was neutropenia that occurred in 42 (41%) of treatment cycles, with 10 hospitalizations for febrile neutropenia. Serious protocol-defined cardiac events occurred in three (14%) patients, with two (10%; 95% confidence interval [CI] 1% to 30%) developing congestive heart failure (CHF) after a total cumulative anthracycline dose (adjuvant doxorubicin + D-99) of 540 mg/m(2). CONCLUSIONS: : D-99 in combination with weekly docetaxel, at the doses and schedule as administered in this trial, is not recommended for phase II testing. Additional trials, using different doses and schedules, are required to evaluate the potential side-effects and efficacy of D-99 and docetaxel.

Substantial increases in idarubicin plasma concentration by liposome encapsulation mediates improved antitumor activity.
Dos Santos N, Waterhouse D, Masin D, Tardi PG, Karlsson G, Edwards K, Bally MB.
J Control Release. 2005 Jun 20;105(1-2):89-105.
[ expand abstract ]

Idarubicin has been successfully encapsulated in cholesterol-free liposomes, however, little is known about how the rate of drug release from circulating liposomes influences therapeutic activity. The studies described herein assess the attributes of a liposome formulation required to significantly increase the plasma levels of idarubicin and further establish whether increases in the circulation longevity of the drug mediate improved antitumor activity. Pharmacokinetic assessments of 6 different (3)[H]-labelled liposome formulations were compared to free idarubicin. The highest idarubicin plasma concentrations were observed with DSPC/DSPE-PEG(2000) liposomes formulated with 2 mol% DSPE-PEG(2000) and 150 mM (iso-osmotic) internal citrate concentration. It was shown that increased levels of PEG-lipid incorporation augmented IDA release and the optimal liposomal formulation needed to be prepared under iso-osmotic conditions. For efficacy studies in a murine leukemia model, groups of 12-14 mice were treated i.v. with saline or equivalent doses (1, 2, 3 mg/kg) of free or liposomal IDA. Liposomal treatment groups exhibited a higher % increase in life span (ILS) as compared to equivalent doses of free drug. Efficacy studies completed in two drug resistant models, P388/ADR and MDA435LCC6/MDR1, demonstrated that neither the free nor liposomal formulation of idarubicin was therapeutically active. Encapsulation of IDA in liposomes increased antitumor activity in an IDA sensitive model, however, the significant increase in plasma drug levels was not sufficient to overcome multidrug resistance.

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Novel mechanism of hybrid liposomes-induced apoptosis in human tumor cells.
Matsumoto Y, Iwamoto Y, Matsushita T, Ueoka R.
Int J Cancer. 2005 Jun 20;115(3):377-82.
[ expand abstract ]

Hybrid liposomes can be prepared by simply ultrasonicating a mixture of vesicular and micellar molecules in a buffer solution. The physical properties of these liposomes, such as size, membrane fluidity, phase transition temperature and hydrophobicity can be controlled by changing the composition. Hybrid liposomes composed of dimyristoylphosphatidylcholine and polyoxyethylene (10) dodecyl ether were found to inhibit the growth of human promyelocytic leukemia (HL-60) cells without using any drugs. Induction of apoptosis by hybrid liposomes in HL-60 cells was verified on the basis of fluorescence microscopy and flow cytometry analysis, after fusion and accumulation of hybrid liposomes, which was revealed on the basis of microphysiometer. We elucidated the pathways of apoptosis induced by the hybrid liposomes. That is, hybrid liposomes fused and accumulated in tumor cell membranes, and the apoptosis signal first passed through mitochondria, caspase-9 and caspase-3, second through Fas, caspase-8, caspase-3 and then reached the nucleus. Hybrid liposomes themselves can induce apoptosis in human tumor cells along with high inhibitory effects on the growth of tumor cells. (c) 2005 Wiley-Liss, Inc.

Liquid filled nanoparticles as a drug delivery tool for protein therapeutics.
Venkatesan N, Yoshimitsu J, Ito Y, Shibata N, Takada K.
Biomaterials. 2005 Jun 18; [Epub ahead of print].
[ expand abstract ]

In the present study, an attempt was made to study the feasibility of nanoparticulate adsorbents in the presence of an absorption enhancer, as a drug delivery tool for the administration of erythropoietin (EPO) to the small intestine. Liquid filled nano- and micro-particles (LFNPS/LFMPS) were prepared using solid adsorbents such as porous silicon dioxide (Sylysia 550), carbon nanotubes (CNTs), carbon nanohorns, fullerene, charcoal and bamboo charcoal. Surfactants such as a saturated polyglycolysed C8-C18 glyceride (Gelucire 44/14), PEG-8 capryl/caprylic acid glycerides (Labrasol) and polyoxyethylene hydrogenated castor oil derivative (HCO-60) were used as an absorption enhancer at 50mg/kg along with casein/lactoferrin as enzyme inhibitors. The absorption of EPO was studied by measuring serum EPO levels by an ELISA method after small intestinal administration of EPO-LFNPS preparation to rats at the EPO dose level of 100IU/kg. Among the adsorbents studied, CNTs showed the highest serum EPO level of 62.7+/-11.6mIU/ml. In addition, with the use of casein, EPO absorption was improved, C(max) 143.1+/-15.2mIU/ml. Labrasol showed the highest absorption enhancing effect after intra-jejunum administration than Gelucire 44/14 and HCO-60, 25.6+/-3.2 and 22.2+/-3.6mIU/ml, respectively. Jejunum was found to be the best absorption site for the absorption of EPO from LFNPS. The use of CNTs as LFNPS, improved the bioavailability of EPO to 11.5% following intra-small intestinal administration.

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Enhancement of the tumour inhibitory activity, in vivo, of diospyrin, a plant-derived quinonoid, through liposomal encapsulation.
Hazra B, Kumar B, Biswas S, Pandey BN, Mishra KP.
Toxicol Lett. 2005 Jun 17;157(2):109-17.
[ expand abstract ]

Diospyrin, a bisnaphthoquinonoid plant product, shows inhibitory activity against murine tumour in vivo and human cancer cell lines in vitro. Efforts have further been made to obtain synthetic derivatives of diospyrin with the objective of improved therapeutic effects. With the goal to reduce the toxicity towards normal cells and enhance the efficacy to tumour cells, diospyrin was encapsulated in liposomal vesicle and its antitumour potential was observed on the growth of Ehrlich ascites tumour in Swiss mice. It was found that the longevity of the tumour-bearing mice was significantly enhanced by treatment with liposomal diospyrin as compared with the free drug. Biochemical assay of liver function enzymes, viz. LDH, AP, GOT and GPT in blood serum of the tumour-bearing mice showed substantial alterations in the activity of these enzymes. These parameters were, however, restored to near normal level when the drug treatment was given encapsulated in a liposome. Histopathological studies on the liver tissues indicated a near normal pathological status in the treated animals despite being challenged by tumour cells. This study on diospyrin has shown, for the first time, an enhancement of its antitumour effect in vivo through liposomal encapsulation.

New magnetic nano-absorbent for the determination of n-octanol/water partition coefficients.
Gao X, Yu CH, Tam KY, Tsang SC.
J Pharm Biomed Anal. 2005 Jun 15;38(2):197-203.
[ expand abstract ]

A novel and generic miniaturization methodology for the determination of partition coefficient values of organic compounds in n-octanol/water by using magnetic nanoparticles is, for the first time, described. We have successfully designed, synthesised and characterised new colloidal stable porous silica-encapsulated magnetic nanoparticles of controlled dimensions. These nanoparticles absorbing a tiny amount of n-octanol in their porous silica over-layer are homogeneously dispersed into a bulk aqueous phase (pH 7.40) containing an organic compound prior to magnetic separation. The small size of the particles and the efficient mixing allow a rapid establishment of the partition equilibrium of the organic compound between the solid supported n-octanol nano-droplets and the bulk aqueous phase. UV-vis spectrophotometry is then applied as a quantitative method to determine the concentration of the organic compound in the aqueous phase both before and after partitioning (after magnetic separation). logD values of organic compounds of pharmaceutical interest (0.65-3.50), determined by this novel methodology, were found to be in excellent agreement with the values measured by the shake-flask method in two independent laboratories, which are also consistent with the literature data. It was also found that this new technique gives a number of advantages such as providing an accurate measurement of logD value, a much shorter experimental time and a smaller sample size required. With this approach, the formation of a problematic emulsion, commonly encountered in shake-flask experiments, is eliminated. It is envisaged that this method could be applicable to the high throughput logD screening of drug candidates.

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Nanoparticle targeting of anticancer drug improves therapeutic response in animal model of human epithelial cancer.
Kukowska-Latallo JF, Candido KA, Cao Z, Nigavekar SS, Majoros IJ, Thomas TP, Balogh LP, Khan MK, Baker JR Jr.
Cancer Res. 2005 Jun 15;65(12):5317-24.
[ expand abstract ]

Prior studies suggested that nanoparticle drug delivery might improve the therapeutic response to anticancer drugs and allow the simultaneous monitoring of drug uptake by tumors. We employed modified PAMAM dendritic polymers <5 nm in diameter as carriers. Acetylated dendrimers were conjugated to folic acid as a targeting agent and then coupled to either methotrexate or tritium and either fluorescein or 6-carboxytetramethylrhodamine. These conjugates were injected i.v. into immunodeficient mice bearing human KB tumors that overexpress the folic acid receptor. In contrast to nontargeted polymer, folate-conjugated nanoparticles concentrated in the tumor and liver tissue over 4 days after administration. The tumor tissue localization of the folate-targeted polymer could be attenuated by prior i.v. injection of free folic acid. Confocal microscopy confirmed the internalization of the drug conjugates into the tumor cells. Targeting methotrexate increased its antitumor activity and markedly decreased its toxicity, allowing therapeutic responses not possible with a free drug.

Suppression of pancreatic tumor progression by systemic delivery of a pancreatic-cancer-specific promoter driven Bik mutant.
Li Z, Ding Q, Li Y, Miller SA, Abbruzzese JL, Hung MC.
Cancer Lett. 2005 Jun 10; [Epub ahead of print].
[ expand abstract ]

Pancreatic cancer is highly aggressive with extremely poor prognosis. Developing a pancreatic cancer specific promoter (PCSP) is one approach for pancreatic cancer gene therapy. We have modified the promoter of cholecystokinin type A receptor (CCKAR), named CCK/Mpd, which possesses a relatively high activity in pancreatic cancer cells as compared with normal cells. The CCK/Mpd promoter-driven luciferase exhibits a better tumor specific tissue distribution than the CMV promoter-driven luciferase when systemically administered in vivo. Notably, we demonstrate a treatment efficacy by using CCK/Mpd-Bik-DD/liposome in a nude mice xenograft model, suggesting the feasibility of PCSP-based gene therapy in pancreatic cancer treatment.

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Synthesis, liposomal formulation and thermal effects on phospholipid bilayers of leuprolide.
Saroglou V, Hatziantoniou S, Smyrniotakis M, Kyrikou I, Mavromoustakos T, Zompra A, Magafa V, Cordopatis P, Demetzos C.
J Pept Sci. 2005 Jun 7; [Epub ahead of print].
[ expand abstract ]

A novel liposomal formulation was developed for the encapsulation of the oligopeptide leuprolide (GlpHisTrpSerTyr-D-LeuLeuArgProNHEt), a potent analogue of gonadotropin releasing hormone used in the treatment of advanced prostate cancer, endometriosis and precocious puberty. Leuprolide was synthesized using solid phase methodology on a {3-[(ethyl-Fmoc-amino)-methyl]-1-indol-1-yl}-acetyl AM resin and Fmoc/tBu chemistry. The new liposomal formulation, called 'liposomes in liposomes' is composed of egg phosphatidylcholine:dipalmitoylphosphatidylglycerol in a molar ratio of 98.91:1.09 (internal liposomes) and egg phosphatidylcholine:dipalmitoylphosphatidylglycerol:cholesterol in a molar ratio of 68.71:0.76:30.53 (external liposomes). It offers high encapsulation efficiency (73.8% for leuprolide); it can provide new delivery characteristics and it may have possible advantages in future applications regarding the encapsulation and delivery of bioactive peptides to target tissues. Furthermore, the physicochemical characteristics (size distribution and zeta-potential) of the liposomal formulations and the thermal effects on leuprolide in model lipidic bilayers composed of dipalmitoylphosphatidylcholine were studied using differential scanning calorimetry. Finally, the dynamic effects of leuprolide in an egg phosphatidylcholine/cholesterol system were examined using solid state (13)C MAS NMR spectroscopy.

Iron Oxide Nanoparticles for Sustained Delivery of Anticancer Agents.
Jain TK, Morales MA, Sahoo SK, Leslie-Pelecky DL, Labhasetwar V.
Mol Pharm. 2005 Jun 6;2(3):194-205.
[ expand abstract ]

We have developed a novel water-dispersible oleic acid (OA)-Pluronic-coated iron oxide magnetic nanoparticle formulation that can be loaded easily with high doses of water-insoluble anticancer agents. Drug partitions into the OA shell surrounding iron oxide nanoparticles, and the Pluronic that anchors at the OA-water interface confers aqueous dispersity to the formulation. Neither the formulation components nor the drug loading affected the magnetic properties of the core iron oxide nanoparticles. Sustained release of the incorporated drug is observed over 2 weeks under in vitro conditions. The nanoparticles further demonstrated sustained intracellular drug retention relative to drug in solution and a dose-dependent antiproliferative effect in breast and prostate cancer cell lines. This nanoparticle formulation can be used as a universal drug carrier system for systemic administration of water-insoluble drugs while simultaneously allowing magnetic targeting and/or imaging.

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Strategies for cytosolic delivery of liposomal macromolecules.
Fretz MM, Mastrobattista E, Koning GA, Jiskoot W, Storm G.
Int J Pharm. 2005 Jun 4; [Epub ahead of print].
[ expand abstract ]

Potential approaches to achieve cytosolic delivery of liposomal macromolecules are presented. These approaches include: (1) the co-encapsulation of fusogenic peptides into targeted drug-containing liposomes (2) coupling of the HIV-1-derived cell-penetrating peptide TAT to the surface of liposomes and (3) photochemical internalization, based on photochemically inducible permeabilization of endocytic vesicles.

Preparation and characterisation of liposomes encapsulating ketoprofen-cyclodextrin complexes for transdermal drug delivery.
Maestrelli F, Gonzalez-Rodriguez ML, Rabasco AM, Mura P.
Int J Pharm. 2005 Jun 4; [Epub ahead of print].
[ expand abstract ]

Multilamellar vesicle (MLV) liposomes containing ketoprofen-cyclodextrin complexes intended for drug topical delivery were prepared, with the aim of simultaneously exploiting the favourable properties of both carriers. Drug complexes with beta-cyclodextrin (betaCyd) and hydroxypropyl-betaCyd (HPbetaCyd), prepared by coevaporation and sealed-heating methods, were characterised by differential scanning calorimetry, hot stage microscopy, scanning electron microscopy and tested for dissolution properties. The coevaporated system with HPbetaCyd was the most effective, enabling an about 11-fold increase in drug dissolution. Drug and drug-Cyd systems were incorporated in MLV liposomes prepared by the thin layer evaporation technique. All liposomal formulations were characterised for encapsulation efficiency, particle size and morphology, using dialysis, light scattering and transmission electron microscopy techniques, respectively. MLV formation was negatively influenced by the presence of Cyd; nevertheless, it was possible to prepare stable MLVs containing ketoprofen-Cyd complexes. The presence of the Cyd complex affected MLV dimensions but not their lamellar structure. The complex with HPbetaCyd, in virtue of its greater stability than the betaCyd one, allowed higher percentages of encapsulation and gave rise to more stable MLV systems. Permeability studies of drug and drug-Cyd complexes, as such or incorporated in liposomes, performed both across artificial membranes and rat skin, highlighted a favourable effect of Cyd on drug permeation rate, due to its solubilizing action; by contrast, unexpectedly, no skin-permeation enhancer property of liposomes has been evidenced. Confocal laser scanning microscopy studies carried out with the rhodamine-Cyd complex as fluorescent marker, confirmed such results, showing that the label permeated deeper across rat skin layers when it was in solution than when entrapped in liposomes.

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Synthesis and Application of Integrin Targeting Lipopeptides in Targeted Gene Delivery.
Waterhouse JE, Harbottle RP, Keller M, Kostarelos K, Coutelle C, Jorgensen MR, Miller AD.
Chembiochem. 2005 Jun 3; [Epub ahead of print].
[ expand abstract ]

One of the main problems facing gene therapy is the ability to target the delivery of DNA to specific cells of choice. Recently, we developed a synthetic nonviral vector platform system known as LMD (liposome:mu:DNA) that was designed for further modular upgrading with tool-kits of chemical components. First-generation LMD systems were prepared from DC-Chol/DOPE cationic liposomes (DC-Chol=3beta-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol, DOPE=dioleoyl-L-alpha-phosphatidylethanolamine), mu peptide from the adenovirus core and plasmid DNA (pDNA). Here we report attempts to realise peptide-targeted gene delivery that build upon the LMD platform. Our strategy was to prepare novel lipopeptides with a lipid moiety designed to insert into the outer lipid bilayer of LMD particles whilst simultaneously presenting a peptide moiety for cell-surface receptor binding. One main functional peptide sequence was selected (PLAEIDGIELA; tenascin peptide sequence) known to target alpha(9)beta(1)-integrin proteins predominant on upper-airway epithelial cells. This sequence was investigated along with a corresponding control sequence. The syntheses of two classes (A and B) of lipopeptides are reported; the syntheses of class A lipopeptides requires a modification of Mitsunobu chemistry that could be of general utility to facilitate Mitsunobu reactions in other diverse systems. "Targeted" LMD and LD transfections with class A or B lipopeptides exhibit nonspecific peptide enhancements (up to one order of magnitude) over nonlipopeptide control transfections but few specific effects. Specific targeting effects can be seen if the overall LMD or LD particle cationic charge is lowered, but nonspecific effects are never eliminated. Whilst promising, these data now highlight the need for in vivo data and even a new modular, aqueous chemistry for the controlled adaptation of LMD particles in buffer in order for successful peptide-targeted, synthetic, nonviral gene delivery to be realised.

Fusogenic liposome delivers encapsulated nanoparticles for cytosolic controlled gene release.
Kunisawa J, Masuda T, Katayama K, Yoshikawa T, Tsutsumi Y, Akashi M, Mayumi T, Nakagawa S.
J Control Release. 2005 Jun 2; [Epub ahead of print].
[ expand abstract ]

Therapeutic agents based on DNA or RNA oligonucleotides (e.g., antisense DNA oligonucleotide, small interfering RNA) require a regulation of their kinetics in cytoplasm to maintain an optimal concentration during the treatment period. In this respect, delivery of functional nanoparticles containing these drugs into cytoplasm has been thought to have a potential for the cytosolic controlled gene release. In this study, we establish a protocol for the encapsulation of nanoparticles into liposome, which is further fused with ultra violet-inactivated Sendai virus to compose fusogenic liposomes. When nanoparticles were encapsulated in conventional liposomes, endocytosis-mediated uptake of nanoparticles was observed. In contrast, numerous amounts of nanoparticles were delivered into the cytoplasm without any cytotoxicity when the particles were encapsulated in fusogenic liposomes. Additionally, fusogenic liposome showed a high ability to deliver nanoparticles containing DNA oligonucleotides into cytoplasm. These results indicate that this combinatorial nanotechnology using fusogenic liposome and nanoparticle is a valuable system for regulating the intracellular pharmacokinetics of gene-based drugs.

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Targeting and inhibition of cell growth by an engineered dendritic nanodevice.
Thomas TP, Majoros IJ, Kotlyar A, Kukowska-Latallo JF, Bielinska A, Myc A, Baker JR Jr.
J Med Chem. 2005 Jun 2;48(11):3729-35.
[ expand abstract ]

The cellular uptake and cytotoxicity of an engineered multifunctional dendritic nanodevice containing folic acid (FA) as the targeting molecule, methotrexate (MTX) as the chemotherapeutic drug, and fluorescein (FI) as the detecting agent were studied in vitro. FI and FA were conjugated to the generation 5 poly(amidoamine) (G5) dendrimer carrier through a thiourea and amide linkage and MTX was conjugated through an ester linkage to the carrier to generate the trifunctional dendritic device, G5-FI-FA-MTX. This trifunctional dendrimer-drug conjugate bound to FA receptor-expressing KB cells in a dose-dependent and saturable manner. Confocal microscopic analysis demonstrated cellular internalization of the conjugate. G5-FI-FA-MTX induced a time- and dose-dependent inhibition of cell growth in KB cells. The targeted dendrimer conjugates G5-FI-FA-MTX and G5-FA-MTX inhibited cell growth in KB cells, whereas the nontargeted G5-MTX failed to induce growth inhibition. These studies show the potential of G5-FI-FA-MTX or G5-FA-MTX for targeting and growth suppression of tumor cells that overexpress FA-receptors.

Neuroblastoma targeting by c-myb-selective antisense oligonucleotides entrapped in anti-GD(2) immunoliposome: immune cell-mediated anti-tumor activities.
Brignole C, Marimpietri D, Pagnan G, Paolo DD, Zancolli M, Pistoia V, Ponzoni M, Pastorino F.
Cancer Lett. 2005 Jun 1; [Epub ahead of print].
[ expand abstract ]

Liposome encapsulation of anticancer agents results in reduced side effects of the entrapped drug and improved therapeutic efficacy. The external surface of the lipidic envelope can be coupled with antibodies directed against tumor-associated antigens. The resulting immunoliposomes allow to increase the therapeutic index of cytotoxic drugs while minimizing their systemic toxicity. In this regard, the disialoganglioside GD(2) is a very promising tumor-associated antigen since it is expressed at high intensity on human neuroblastoma cells, but is detected only in normal cerebellum and peripheral nerves. Immunoliposomes can be used as vectors to deliver antisense oligonucleotides to cancer cells with the aim to modulate oncogene expression. Furthermore, antisense oligonucleotides have attracted much interest because of their ability to stimulate immune responses. Here, we will describe a novel experimental therapeutic approach for neuroblastoma based on anti-GD(2) liposomal c-myb-selective antisense oligonucleotides.

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Vascular endothelial growth factor gene delivery by magnetic DNA nanospheres ameliorates limb ischemia in rabbits(1).
Jiang H, Zhang T, Sun X.
J Surg Res. 2005 Jun 1;126(1):48-54.
[ expand abstract ]

BACKGROUND: Critical limb ischemia often leads to disability and limb loss. Vascular endothelial growth factor (VEGF), delivered either as recombinant protein or as gene therapy, has been shown to promote arteriogenesis and angiogenesis in animal models of limb ischemia. However, most of the studies used a nonspecific targeting system. MATERIALS AND METHODS: Magnetic DNA nanospheres containing expression plasmids encoding VEGF were synthesized, and their morphology, magnetropism, and stability were analyzed. The magnetic DNA nanospheres were administrated via an artery into a rabbit limb ischemia model. The expression of VEGF and vascularization were examined by immunohistochemistry. The angiography was taken to evaluate arteriogenesis. RESULTS: Magnetic DNA nanospheres were very stable and showed a high magnetropism. Gene delivery of such nanospheres via artery under a magnetic field led to the overexpression of VEGF in situ. The capillary density and capillary to muscle fiber ratio were doubled compared with those of the control animals. The arteriogenesis also was promoted in VEGF gene therapy group compared with controls but at later interval than capillary angiogenesis. CONCLUSIONS: Our results suggest that intra-arterial VEGF gene delivery by magnetic DNA nanosphere promotes angiogenesis and arteriogenesis and presents a potent therapeutic strategy for critical limb ischemia.

Liposomes targeted via two different antibodies: Assay, B-cell binding and cytotoxicity.
Laginha K, Mumbengegwi D, Allen T.
Biochim Biophys Acta. 2005 Jun 1;1711(1):25-32.
[ expand abstract ]

The selective toxicity of anticancer drugs can be improved with the use of antibody-targeted liposomes. We hypothesize that liposomes targeted via antibodies against two or more receptor populations will increase the apparent receptor density on the target cells, resulting in improved therapeutic affects. A fluorescent assay was developed, using the fluorophores Alexa Fluor(R) 350 and 532 to label monoclonal antibodies (mAb), and used to quantitate two different mAb populations coupled to the same liposome surface to within +/-10% of the values obtained with radiolabeled antibody ((125)I) tracers. The binding and uptake of targeted liposomes by B lymphoma (Namalwa) cells were examined for either individual populations of alphaCD19-targeted or alphaCD20-targeted liposomes, mixed populations (1:1) of alphaCD19-targeted liposomes plus alphaCD20-targeted liposomes, and dual-targeted liposomes, i.e., equal amount of both alphaCD19 and alphaCD20 on the same liposomes. At similar antibody densities, the binding and uptake of the dual-targeted liposomes were greater than that of either individually targeted liposomes alone, and showed additivity. At the same total lipid and antibody densities, 1:1 mixtures of individually targeted liposomes gave similar results to dual-targeted liposomes. Cytotoxicity was also improved, with DXR-loaded dual-targeted liposomes appearing to have higher cytotoxicity than 1:1 mixtures of individually targeted liposomes.

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Comparative preclinical and clinical pharmacokinetics of a cremophor-free, nanoparticle albumin-bound paclitaxel (ABI-007) and paclitaxel formulated in Cremophor (Taxol).
Sparreboom A, Scripture CD, Trieu V, Williams PJ, De T, Yang A, Beals B, Figg WD, Hawkins M, Desai N.
Clin Cancer Res. 2005 Jun 1;11(11):4136-43.
[ expand abstract ]

PURPOSE: To compare the preclinical and clinical pharmacokinetic properties of paclitaxel formulated as a Cremophor-free, albumin-bound nanoparticle (ABI-007) and formulated in Cremophor-ethanol (Taxol). EXPERIMENTAL DESIGN: ABI-007 and Taxol were given i.v. to Harlan Sprague-Dawley male rats to determine pharmacokinetic and drug disposition. Paclitaxel pharmacokinetic properties also were assessed in 27 patients with advanced solid tumors who were randomly assigned to treatment with ABI-007 (260 mg/m(2), 30 minutes; n = 14) or Taxol (175 mg/m(2), 3 hours; n = 13), with cycles repeated every 3 weeks. RESULTS: The volume of distribution at steady state and clearance for paclitaxel formulated as Cremophor-free nanoparticle ABI-007 were significantly greater than those for paclitaxel formulated with Cremophor (Taxol) in rats. Fecal excretion was the main elimination pathway with both formulations. Consistent with the preclinical data, paclitaxel clearance and volume of distribution were significantly higher for ABI-007 than for Taxol in humans [21.13 versus 14.76 L/h/m(2) (P = 0.048) and 663.8 versus 433.4 L/m(2) (P = 0.040), respectively]. CONCLUSIONS: Paclitaxel formulated as ABI-007 differs from paclitaxel formulated as Taxol, with a higher plasma clearance and a larger volume of distribution. This finding is consistent with the absence of paclitaxel-sequestering Cremophor micelles after administration of ABI-007. This unique property of ABI-007 could be important for its therapeutic effectiveness.

Nanotechnology blooms, at last (Review).
Gordon EM, Hall FL.
Oncol Rep. 2005 Jun;13(6):1003-7.
[ expand abstract ]

Clinical trials for deadly pancreatic cancer have recently opened on two continents to evaluate the safety and efficacy of engineered nanoparticles guided by a targeted delivery system (TDS) to overcome the daunting barriers of turbulence, dilution, filtration, and inactivation encountered in the human circulatory system to deliver a killing designer gene to metastatic tumors that are refractory to conventional chemotherapy. The first patients receiving multiple intravenous infusions of the TDS-encapsulated genetic bullets have all responded favorably, prompting the FDA to grant orphan drug status for the nanobiotic medicine, Rexin-G, to assist in the development of this new cancer treatment. This review/commentary is an effort to translate the arcane terminology of physiology, biochemistry, and molecular genetics into the more generally accessible language of nanotechnology and medical delivery. While the text is somewhat erudite and laden at times with inconspicuous literary allusions, descriptions of the elegant bioengineering of nano-scale gene delivery vehicles are clear and the numerous references to classical mechanics of the Industrial Age are helpful.

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Tumor-targeted gene delivery using poly(ethylene glycol)-modified gelatin nanoparticles: in vitro and in vivo studies.
Kaul G, Amiji M.
Pharm Res. 2005 Jun;22(6):951-61.
[ expand abstract ]

PURPOSE: To develop safe and effective systemically administered nonviral gene therapy vectors for solid tumors, DNA-containing poly(ethylene glycol)-modified (PEGylated) gelatin nanoparticles were fabricated and evaluated in vitro and in vivo. METHODS: Reporter plasmid DNA encoding for beta-galactosidase (pCMV-beta) was encapsulated in gelatin and PEGylated gelatin nanoparticles using a water-ethanol solvent displacement method under controlled pH and temperature. Lewis lung carcinoma (LLC) cells in culture were transfected with the pCMV-beta in the control and nanoparticle formulations. Periodically, the expression of beta-galactosidase in the cells was measured quantitatively using an enzymatic assay for the conversion of o-nitrophenyl-beta-D: -galactopyranoside (ONPG) to o-nitrophenol (ONP). Qualitative expression of beta-galactosidase in LLC cells was observed by staining with 5-bromo-4-chloro-3-indolyl-beta-D: -galactopyranoside (X-gal). Additionally, the plasmid DNA-encapsulated gelatin and PEGylated gelatin nanoparticles were administered intravenously (i.v.) and intratumorally (i.t.) to LLC-bearing female C57BL/6J mice. At various time points postadministration, the animals were sacrificed and transgene expression in the tumor and liver was determined quantitatively by the ONPG to ONP enzymatic conversion assay and qualitatively by X-gal staining. RESULTS: Almost 100% of the pCMV-beta was encapsulated in gelatin and PEGylated gelatin nanoparticles (mean diameter 200 nm) at 0.5% (w/w) concentration. PEGylated gelatin nanoparticles efficiently transfected the LLC cells and the beta-galactosidase expression, as measured by the ONPG to ONP enzymatic conversion assay at 420 nm absorbance, increased starting from 12 h until 96 h post-transfection. The efficient expression of LLC cells was also evident by the X-gal staining method that shows blue color formation. The in vivo studies showed significant expression of beta-galactosidase in the tumor following administration of DNA-containing PEGylated gelatin nanoparticles to LLC-bearing mice by both i.v. and i.t. routes. Following i.v. administration of pCMV-beta in PEGylated gelatin nanoparticles, for instance, the absorbance at 420 nm per gram of tumor increased from 0.60 after 12 h to 0.85 after 96 h of transfection. After i.t. administration, the absorbance values increased from 0.90 after 12 h to almost 1.4 after 96 h. CONCLUSIONS: The in vitro and in vivo results of this study clearly show that a long-circulating, biocompatible and biodegradable, DNA-encapsulating nanoparticulate system would be highly desirable for systemic delivery of genetic constructs to solid tumors.

Targeting liposomes with protein drugs to the blood-brain barrier in vitro.
Visser CC, Stevanovic S, Voorwinden LH, Bloois L, Gaillard PJ, Danhof M, Crommelin DJ, Boer AG.
Eur J Pharm Sci. 2005 Jun;25(2-3):299-305.
[ expand abstract ]

In this study, we aim to target pegylated liposomes loaded with horseradish peroxidase (HRP) and tagged with transferrin (Tf) to the BBB in vitro. Liposomes were prepared with the post-insertion technique: micelles of polyethylene glycol (PEG) and PEG-Tf were inserted into pre-formed liposomes containing HRP. Tf was measured indirectly by measuring iron via atomic absorption spectroscopy. All liposomes were around 100nm in diameter, contained 5-13mug HRP per mumol phospholipid and 63-74 Tf molecules per liposome (lipo Tf) or no Tf (lipo C). Brain capillary endothelial cells (BCEC) were incubated with liposomes at 4 degrees C (to determine binding) or at 37 degrees C (to determine association, i.e. binding+endocytosis) and the HRP activity, rather than the HRP amount was determined in cell lysates. Association of lipo Tf was two- to three-fold higher than association of lipo C. Surprisingly, the binding of lipo Tf at 4 degrees C was four-fold higher than the association of at 37 degrees C. Most likely this high binding and low endocytosis is explained by intracellular degradation of endocytosed HRP. In conclusion, we have shown targeting of liposomes loaded with protein or peptide drugs to the BCEC and more specifically to the lysosomes. This is an advantage for the treatment of lysosomal storage disease. However, drug targeting to other intracellular targets also results in intracellular degradation of the drug. Our experiments suggest that liposomes release some of their content within the BBB, making targeting of liposomes to the TfR on BCEC an attractive approach for brain drug delivery.

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Enhanced Intracellular Uptake of Sterically Stabilized Liposomal Doxorubicin in Vitro Resulting in Improved Antitumor Activity in Vivo.
Xiong XB, Huang Y, Lu WL, Zhang H, Zhang X, Zhang Q.
Pharm Res. 2005 Jun;22(6):933-939.
[ expand abstract ]

PURPOSE: To investigate the correlation between the in vitro intracellular uptake and the in vivo antitumor activity of anticancer drugs delivered by sterically stabilized liposomes (SSL). METHODS: Arginine-glycine-aspartic acid (RGD) peptide or RGD mimetic (RGDm) was coupled onto the surface of SSL to obtain the cell-binding carrier to facilitate the intracellular delivery of the encapsulated drugs. DOX-loaded SSL (SSL-DOX), DOX-loaded RGD-modified SSL (RGD-SSL-DOX) and DOX-loaded RGDm-modified SSL (RGDm-SSL-DOX) were prepared by lipid film dispersion followed by remote loading of DOX. The intracellular uptake of DOX from the various liposomal formulations was evaluated in vitro with melanoma B16 cells, and the pharmacokinetics, biodistribution, and antitumor activity were compared in C57BL/6 mice carrying melanoma B16 tumors. RESULTS: In vitro intracellular uptake of DOX by B16 cells and in vivo antitumor activity in terms of tumor growth inhibition and mice survival time prolongation for various liposomal DOX were in the following order: RGD-SSL-DOX > RGDm-SSL-DOX > SSL-DOX. The mean survival time of the mice treated with RGD-SSL-DOX, RGDm-SSL-DOX, and SSL-DOX was 55, 49, and 44 days, respectively. The three liposomal DOX formulations produced very close DOX accumulation in tumor, which is significantly higher than that of free DOX. RGD- or RGDm-SSL-DOX demonstrated prolonged circulation time similar to that of SSL-DOX, whereas they showed significantly lower DOX level in blood and remarkably higher uptake by spleen than SSL-DOX. CONCLUSIONS: Enhanced intracellular uptake of DOX encapsulated in SSL could produce an improved therapeutic effect for the melanoma B16 tumors. Enhancing intracellular delivery of the anticancer drugs encapsulated in SSL may be a promising strategy to improve their therapeutic efficacy for solid tumors.

Formulating Paclitaxel in nanoparticles alters its disposition.
Yeh TK, Lu Z, Wientjes MG, Au JL.
Pharm Res. 2005 Jun;22(6):867-74.
[ expand abstract ]

PURPOSE: Paclitaxel is active and widely used to treat multiple types of solid tumors. The commercially available paclitaxel formulation uses Cremophor/ethanol (C/E) as the solubilizers. Other formulations including nanoparticles have been introduced. This study evaluated the effects of nanoparticle formulation of paclitaxel on its tissue distribution. METHODS: We compared the plasma and tissue pharmacokinetics of paclitaxel-loaded gelatin nanoparticles and the C/E formulation. Mice were given paclitaxel-equivalent doses of 10 mg/kg by intravenous injection. RESULTS: The nanoparticle and C/E formulations showed significant differences in paclitaxel disposition; the nanoparticles yielded 40% smaller area under the blood concentration-time curve and faster blood clearance of total paclitaxel concentrations (sum of free, protein-bound, and nanoparticle-entrapped drug). The two formulations also showed different tissue specificity. The rank order of tissue-to-blood concentration ratios was liver > small intestine > kidney >> large intestine > spleen = stomach > lung > heart for the nanoparticles, and liver > small intestine > large intestine > stomach > lung >/= kidney > spleen > heart for the C/E formulation. The nanoparticles also showed longer retention and higher accumulation in organs and tissues (average of 3.2 +/- 2.3-fold), especially in the liver, small intestine, and kidney. The most striking difference was an 8-fold greater drug accumulation and sustained retention in the kidney. CONCLUSIONS: These data indicate that formulation of paclitaxel affects its clearance and distribution into tissues, with preferential accumulation of nanoparticles in the liver, spleen, small intestine, and kidney.

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Applicability of anti-neovascular therapy to drug-resistant tumor: suppression of drug-resistant P388 tumor growth with neovessel-targeted liposomal adriamycin.
Shimizu K, Asai T, Fuse C, Sadzuka Y, Sonobe T, Ogino K, Taki T, Tanaka T, Oku N.
Int J Pharm. 2005 May 30;296(1-2):133-41.
[ expand abstract ]

Anti-neovascular therapy, one of the effective anti-angiogenic chemotherapy, damages new blood vessels by cytotoxic agents delivered to angiogenic endothelial cells and results in indirect eradication of tumor cells. We previously reported that liposomes-modified with a pentapeptide, Ala-Pro-Arg-Pro-Gly (APRPG-Lip) homing to angiogenic site, highly accumulated in tumor tissue, and APRPG-Lip encapsulating adriamycin (APRPG-LipADM) effectively suppressed tumor growth in tumor-bearing mice. In the present study, we examined the topological distribution of fluorescence-labeled APRPG-LipADM as well as TUNEL-stained cells in an actual tumor specimen obtained from Colon 26 NL-17 carcinoma-bearing mice. The fluorescence-labeled APRPG-Lip dominantly localized to vessel-like structure: a part of which was also stained with anti-CD31 antibody. Furthermore, TUNEL-stained cells were co-localized to the same structure. These data indicated that APRPG-LipADM bound to angiogenic endothelial cells and induced apoptosis of them. We also investigated the applicability of anti-neovascular therapy using APRPG-LipADM to ADM-resistant P388 solid tumor. As a result, APRPG-LipADM significantly suppressed tumor growth in mice bearing the ADM-resistant tumor. These data suggest that APRPG-LipADM is applicable to various kinds of tumor including drug-resistant tumor since it targets angiogenic endothelial cells instead of tumor cells, and eradicates tumor cells through damaging the neovessels.

Targeting Cancer Cells with DNA-Assembled Dendrimers: A Mix and Match Strategy for Cancer.
Choi Y, Baker JR Jr.
Cell Cycle. 2005 May 24;4(5) [Epub ahead of print].
[ expand abstract ]

The unique biology of cancer requires the development of a multifunctional drug delivery system which can be efficiently manufactured to target subtle molecular alterations that distinguish a cancer cells from the many types of healthy cells found in the body. We sought to produce dendrimers conjugated to different bio-functional moieties [fluorescein (FITC) and folic acid (FA)], then link them together using complementary DNA oligonucleotides to produce clustered molecules that target cancer cells that over-express the high affinity folate receptor. This study demonstrates a unique molecular platform based on the DNA-directed assembly of dendritic polymers for the delivery of different agents to cancer cells. While only nanometers in diameter (the size of proteins), this DNA-linked dendrimer nanocluster platform is considered to allows for the delivery of drugs, genetic materials, and imaging agents to cancer cells, offering the potential for developing combinatorial therapeutics.

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Accelerated blood clearance of PEGylated liposomes following preceding liposome injection: Effects of lipid dose and PEG surface-density and chain length of the first-dose liposomes.
Ishida T, Harada M, Wang XY, Ichihara M, Irimura K, Kiwada H.
J Control Release. 2005 May 20; [Epub ahead of print].
[ expand abstract ]

We recently reported that a second dose of polyethylene glycol (PEG) (M.W. 2000)-modified liposomes (mPEG(2000)-liposomes) is rapidly cleared from the blood and accumulates in the liver when injected twice in the same rat or mouse at several-day intervals (referred to as the "accelerated blood clearance (ABC) phenomenon"). In the present study we observed that a high dose (5 mumol/kg) of conventional liposomes (CL: without a PEG-coating) can induce the same phenomenon, while a low lipid dose (0.001 mumol/kg) did not. The induction of the phenomenon by mPEG(2000)-liposomes decreased with increasing first dose (0.001-5 mumol/kg). We observed a strong inverse relationship between the dose of initially injected PEG(2000)-liposomes and the extent to which the ABC phenomenon was induced: the higher the dose the smaller the phenomenon. Increasing the PEG density at the liposome surface beyond 5 mol% attenuated rather than induced the induction of the phenomenon, but elongation of the PEG chain length up to M.W. 5000, had no effect. In a series of hematological, serum-biochemical and histopathological safety evaluations we observed neither acute toxicity nor any signs of hepatic damage during the induction of the ABC phenomenon. Morphological examination of the liver by transmission electron microscopy (TEM) showed extensive accumulation of the second dose of mPEG(2000)-liposomes in the Kupffer cells, even already after 15 min, suggesting that the PEG liposomes had somehow lost the protective effect of the surface-grafted PEG against rapid clearance. The observations reported in this paper may have a considerable impact on the design and engineering of PEGylated liposomal formulations for use in multiple drug therapy.

Phase II study of a liposome-entrapped cisplatin analog (L-NDDP) administered intrapleurally and pathologic response rates in patients with malignant pleural mesothelioma.
Lu C, Perez-Soler R, Piperdi B, Walsh GL, Swisher SG, Smythe WR, Shin HJ, Ro JY, Feng L, Truong M, Yalamanchili A, Lopez-Berestein G, Hong WK, Khokhar AR, Shin DM.
J Clin Oncol. 2005 May 20;23(15):3495-501.
[ expand abstract ]

PURPOSE: To determine pathologic response rates to liposome-entrapped cis-bisneodecanoato-trans-R,R-1,2-diaminocyclohexane platinum(II) (L-NDDP) administered intrapleurally in patients with malignant pleural mesothelioma. PATIENTS AND METHODS: Thirty-three patients with malignant pleural mesothelioma and free-flowing pleural effusions received intrapleural L-NDDP once every 3 weeks at a dose of 450 mg/m2. Thoracoscopic evaluation with pleural biopsies was performed before therapy and then after every two cycles. The primary end point was pathologic response as determined by thoracoscopic biopsy. RESULTS: After at least two cycles, post-treatment pleural biopsy analysis was negative in 14 patients for a pathologic response rate of 42% (95% CI, 25% to 61%). Median survival was 11.2 months. There were three treatment-related deaths secondary to peritonitis, cellulitis at the thoracoscopy site, and empyema. Grade 3 nonhematologic toxicities included infection, fever, dyspnea, and anorexia, which occurred in five (15%), one (3%), one (3%), and one (3%) patients, respectively. There were no grade 4 nonhematologic toxicities. Grade 3 or 4 neutropenia, thrombocytopenia, and anemia occurred in five (15%), three (9%), and two (6%) patients, respectively. Two patients with pathologic responses subsequently underwent pleural decortication. Both surgical specimens revealed residual tumor in regions that were not in direct communication with the pleural space. CONCLUSION: Intrapleural L-NDDP therapy in this patient population is feasible with significant but manageable toxicity. Although pathologic responses are highly encouraging, areas of mesothelioma that are not in direct communication with the pleural space will evade drug exposure and limit efficacy in some patients. The optimal role of intrapleural L-NDDP therapy currently remains to be determined.

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Encapsulation of doxorubicin into thermosensitive liposomes via complexation with the transition metal manganese.
Chiu GN, Abraham SA, Ickenstein LM, Ng R, Karlsson G, Edwards K, Wasan EK, Bally MB.
J Control Release. 2005 May 18;104(2):271-288.
[ expand abstract ]

In the present study, doxorubicin was encapsulated into two thermosensitive liposome formulations which were composed of DPPC/MSPC/DSPE-PEG(2000) (90/10/4 mole ratio) or DPPC/DSPE-PEG(2000) (95/5 mole ratio). Doxorubicin loading was achieved through the use of a pH gradient or a novel procedure that involved doxorubicin complexation with manganese. Regardless of the initial drug-to-lipid ratios (D:L), the final D:L reached a maximum of 0.05 (w/w) when doxorubicin was encapsulated via a pH gradient for both thermosensitive liposome formulations. In contrast, the final maximum D:L achieved through manganese complexation was 0.2 (w/w), and this loading method did not affect temperature-induced drug release, with 85% of drug released from MSPC-containing liposomes within 10 min at 42 degrees C but <5% released over 60 min at 37 degrees C. When the thermosensitive liposomes prepared via the two different loading methods were injected into mice, similar plasma elimination profiles were observed. Cryo-transmission electron microscopy analysis indicated the presence of doxorubicin fiber bundles in liposomes loaded via pH gradient, compared to a stippled and diffuse morphology in those loaded via manganese complexation. To investigate the effect of intraliposomal pH on drug precipitate morphology, the A23187 ionophore (mediates Mn(2+)/H(+) exchange) was added to liposomes loaded with doxorubicin-manganese complex, and the stippled and diffuse appearance could be converted to one exhibiting fiber bundles after acidification of the liposome core. This suggests that the formation of doxorubicin-manganese complex is favored when the intraliposomal pH is >6.5. During the conversion to the fiber bundle morphology, no doxorubicin release was observed when A23187 was added to liposomes exhibiting a 0.05 (w/w), whereas a significant release was noted when the initial D:L was 0.2 (w/w). Following acidification of the liposomal interior and establishment of an apparent new D:L equilibrium, the measured D:L ratio was 0.05 (w/w). In conclusion, the manganese complexation loading method increased the encapsulation efficiency of doxorubicin in thermosensitive liposomes with no major impact on temperature-triggered drug release or pharmacokinetics.

Novel biocompatible phosphorylcholine-based self-assembled nanoparticles for drug delivery.
Salvage JP, Rose SF, Phillips GJ, Hanlon GW, Lloyd AW, Ma IY, Armes SP, Billingham NC, Lewis AL.
J Control Release. 2005 May 18;104(2):259-70.
[ expand abstract ]

Major challenges associated with nano-sized drug delivery systems include removal from systemic circulation by phagocytic cells and controlling appropriate drug release at target sites. 2-methacryloyloxyethyl phosphorylcholine (MPC) has been copolymerised in turn with two pH responsive comonomers (2-(diethylamino)ethyl methacrylate (DEA) and 2-(diisopropylamino)ethyl methacrylate (DPA), to develop novel biocompatible drug delivery vehicles. Micelles were prepared from a series of copolymers with varying block compositions and their colloidal stability and dimensions were assessed over a range of solution pH using photon correlation spectroscopy. The drug loading capacities of these micelles were evaluated using Orange OT dye as a model compound. The cytotoxicity of the micelles was assessed using an in vitro assay. The MPC-DEA diblock copolymers formed micelles at around pH 8 and longer DEA block lengths allowed higher drug loadings. However, these micelles were not stable at physiological pH. In contrast, MPC-DPA diblock copolymers formed micelles of circa 30 nm diameter at physiological pH. In vitro assays indicated that these MPC-DPA diblock copolymers had negligible cytotoxicities. Thus novel non-toxic biocompatible micelles of appropriate size and good colloidal stability with pH-modulated drug uptake and release can be readily produced using MPC-DPA diblock copolymers.

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Folate-linked nanoparticle-mediated suicide gene therapy in human prostate cancer and nasopharyngeal cancer with herpes simplex virus thymidine kinase.
Hattori Y, Maitani Y.
Cancer Gene Ther. 2005 May 13; [Epub ahead of print].
[ expand abstract ]

For targeted gene delivery to human prostate cancer LNCaP and PC-3 cells and nasopharyngeal cancer KB cells, we developed a folate-linked nanoparticle (NP-F), and evaluated the potential of NP-F-mediated suicide gene therapy in the cells and xenografts with herpes simplex virus thymidine kinase (HSV-tk) and connexin 43 (Cx43). An NP-F-plasmid DNA complex (NP-F nanoplex) showed high DNA transfection efficiency in KB, LNCaP and PC-3 cells. Cell growth inhibition in the presence of ganciclovir (GCV) was enhanced with HSV-tk and Cx43 genes in LNCaP cells. In suicide gene therapy, the tumor growths of KB and LNCaP xenografts were significantly inhibited when an NP-F nanoplex of the HSV-tk gene, and HSV-tk and Cx43 genes, respectively, was injected intratumorally and GCV was administered intraperitoneally. These findings suggested that the NP-F is a potential target vector in prostate and nasopharyngeal cancer for suicide gene therapy.Cancer Gene Therapy advance online publication, 13 May 2005; doi:10.1038/sj.cgt.7700844.

Microgel-Based Engineered Nanostructures and Their Applicability with Template-Directed Layer-by-Layer Polyelectrolyte Assembly in Protein Encapsulation.
Shenoy DB, Sukhorukov GB.
Macromol Biosci. 2005 May 12;5(5):451-458.
[ expand abstract ]

A novel strategy for the fabrication of microcapsules is elaborated by employing biomacromolecules and a dissolvable template. Calcium carbonate (CaCO(3)) microparticles were used as sacrificial templates for the two-step deposition of polyelectrolyte coatings by surface controlled precipitation (SCP) followed by the layer-by-layer (LbL) adsorption technique to form capsule shells. When sodium alginate was used for inner shell assembly, template decomposition with an acid resulted in simultaneous formation of microgel-like structures due to calcium ion-induced gelation. An extraction of the calcium after further LbL treatment resulted in microcapsules filled with the biopolymer. The hollow as well as the polymer-filled polyelectrolyte capsules were characterized using confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), and scanning force microscopy (SFM). The results demonstrated multiple functionalities of the CaCO(3) core - as supporting template, porous core for increased polymer accommodation/immobilization, and as a source of shell-hardening material. The LbL treatment of the core-inner shell assembly resulted in further surface stabilization of the capsule wall and supplementation of a nanostructured diffusion barrier for encapsulated material. The polymer forming the inner shell governs the chemistry of the capsule interior and could be engineered to obtain a matrix for protein/drug encapsulation or immobilization. The outer shell could be used to precisely tune the properties of the capsule wall and exterior.Confocal laser scanning microscopy (CLSM) image of microcapsules (insert is after treating with rhodamine 6G to stain the capsule wall).

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Entrapment of some compounds into biocompatible nano-sized particles and their releasing properties.
Watanabe J, Iwamoto S, Ichikawa S.
Colloids Surf B Biointerfaces. 2005 May 10;42(2):141-6.
[ expand abstract ]

Two types of biocompatible nanoparticles with an average diameter of around 200 nm were formed only by mixing hydrolysates of chitosan and carboxymethyl cellulose (CMC). Nanoparticle A was produced from chitosanase hydrolysate of chitosan and cellulase hydrolysate of carboxymethyl cellulose, and nanoparticle B was produced from lysozyme hydrolysate of chitosan and the carboxymethyl cellulose hydrolysate. Negatively charged or amphoteric compounds were first mixed with chitosan hydrolysate and then added to carboxymethyl cellulose hydrolysate to effectively entrap them in the particles. Positively charged compounds could also be effectively entrapped by mixing the hydrolysates and the compound in the reverse order. Negatively charged compounds with high molecular weights were maintained in the particles even at the higher pH levels than the pK(a) of the amino groups of chitosan. Entrapped compounds were gradually released from nanoparticle A by lysozyme treatment. In contrast, there was no release from nanoparticle B. These results indicate that nanoparticle A can be applied to controlled-release drug delivery systems, and that nanoparticle B is stably retained in the body without releasing the entrapped compounds.

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Successful treatment of photo-damaged skin of nano-scale atRA particles using a novel transdermal delivery.
Yamaguchi Y, Nagasawa T, Nakamura N, Takenaga M, Mizoguchi M, Kawai S, Mizushima Y, Igarashi R.
J Control Release. 2005 May 5;104(1):29-40.
[ expand abstract ]

We show a novel drug delivery system (DDS) for improved all-trans retinoic acid (atRA) therapy for external treatments of photo-damaged skin. We prepared inorganic-coated atRA nanoparticles, in turn an egg-like structure in nano-scale (Nano-atRA), using boundary-organized reaction droplets. The interfacial properties of organic architectures, in atRA micelles, were used to template the nucleation of inorganic minerals. As a result, irritation and inflammation associated with atRA therapy were substantially reduced due to the complete encapsulation of the carboxylic function. Both irritative symptoms and physicochemical instability of the atRA micelle were improved. Since Nano-atRA which is prepared following to this new DDS system developmentally improved the permeability to the stratum corneum, the remarkable pharmacological effects were resulted in comparison with atRA as such as follows: (1) thicker epidermis than classical atRA treatment and (2) the overexpression of mRNA for heparin-binding epidermal growth factor (HB-EGF) as the provocation epidermal hyperplasia. Furthermore, we found a surprising boost in production of hyaluronan (HA) among the intercellular spaces of the basal and spinous cell layers in epidermis. Nano-atRA technology for atRA therapy could not only efficiently regulate keratinocyte cell proliferation and differentiation, but also markedly produce the additional benefit. Severely injured human skin by chronic ultraviolet irradiation will completely repair due to the accelerated turnover of skin tissue, which is induced by Nano-atRA.

Preparation and characterisation of antibody modified gelatin nanoparticles as drug carrier system for uptake in lymphocytes.
Balthasar S, Michaelis K, Dinauer N, von Briesen H, Kreuter J, Langer K.
Biomaterials. 2005 May;26(15):2723-32.
[ expand abstract ]

Established methods of protein chemistry can be used for the effective attachment of drug targeting ligands to the surface of protein-based nanoparticles. In the present work gelatin nanoparticles were used for the attachment of biotinylated anti-CD3 antibodies by avidin-biotin-complex formation. These antibody modified nanoparticles represent a promising carrier system for the specific drug targeting to T-lymphocytes. The objective of this work was the comprehensive quantification of every chemical reaction step during the preparation procedure of these cell specific nanoparticles. Gelatin nanoparticles were formed by a two-step desolvation process. After the first desolvation step the remaining sediment and the supernatant were analysed for molecular weight distribution by size exclusion chromatography (SEC). Nanoparticles then were formed using the high molecular gelatin fraction and subsequently were stabilised by glutaraldehyde crosslinking. A part of the detectable amino groups on the particle surface was reacted with 2-iminothiolane in order to introduce reactive sulfhydryl groups. The thiolated nanoparticles were coupled to NeutrAvidin (NAv) which previously was activated with the heterobifunctional crosslinker sulfo-MBS. All these reaction steps were quantified by photometry or gravimetry. The functionality of NAv after covalent conjugation was confirmed by a biotin-4-fluorescein assay. The NAv-modified nanoparticles then were used for the binding of biotinylated anti-CD3 antibodies by avidin-biotin-complex formation. A highly effective attachment of the ligand was ascertained by different, indirect methods: immunoblotting and fluorimetry. Therefore, a well-defined nanoparticle system with drug targeting ligand modification was established that holds promise for further effective preclinical testing.

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DNA encapsulated magnesium and manganous phosphate nanoparticles: potential non-viral vectors for gene delivery.
Bhakta G, Mitra S, Maitra A.
Biomaterials. 2005 May;26(14):2157-63.
[ expand abstract ]

Nanoparticles of Mg and Mn (II) phosphates encapsulating pDNA were prepared. The sizes of these DNA loaded particles in aqueous dispersion were about 100-130 nm diameter, and they aggregated with the progression of time. Although magnesium phosphate nanoparticles were crystalline, the manganous phosphate nanoparticles were found to be amorphous in nature. Nanoparticle dissolution and pDNA release were studied using atomic absorption spectroscopy and gel electrophoresis experiments. These inorganic phosphate nanoparticles dissolved in mild acidic pH ( approximately 5) releasing pDNA indicating that DNA release in the endosomal compartment is possible. In vitro transfection in HeLa cells demonstrated that while magnesium phosphate nanoparticles showed 100% efficiency, manganous phosphate nanoparticles exhibited about 85% transfection efficiency compared to that of 'polyfect', as control.

In Vitro Cytotoxicity of Stealth Liposomes Co-encapsulating Doxorubicin and Verapamil on Doxorubicin-Resistant Tumor Cells.
Wang J, Goh B, Lu W, Zhang Q, Chang A, Liu XY, Tan TM, Lee H.
Biol Pharm Bull. 2005 May;28(5):822-8.
[ expand abstract ]

Multidrug resistance (MDR) is a major obstacle to successful clinical cancer chemotherapy. A novel doxorubicin anti-resistant Stealth liposomes (DARSLs), prepared by co-encapsulating doxorubicin (DOX) and verapamil (VER) into stealth liposomes, has been developed. The average particle size of DARSLs was 118.1+/-22.3 nm. Encapsulation efficiencies of DOX and VER in DARSLs were greater than 95% and 70%, respectively. The IC(50) of DARSLs as measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) assay in multidrug resistant rat prostate cancer Mat-LyLu-B2 (MLLB2) cells was 0.079+/-0.017 muM, 13 fold less than that for liposomal DOX with free VER (LDFV 0.96+/-0.46 muM) but only about 2 times less than FDFV. The IC(50) cytotoxicity on MLLB2 cells of the various formulations was as follows: DARSLs approximately LDLV<FDFV<FDLV<LDFV<LD<FD, (LD: liposomal DOX; LV: liposomal VER; FD: free DOX; FV: free VER). Similar cytotoxicities were shown between DARSLs and FDFV in DOX-resistant human uterus sarcoma MES-SA/DX5 cells, reversing DOX-resistance to that shown by FD on DOX-sensitive MES-SA cells. For MLLB2 cells, DARSLs was the most cytotoxic, but its intracellular concentration of DOX, measured as mean cellular fluorescence with flow cytometry was lower (p<0.01) than that observed with the FDFV formulation. In conclusion, DARSLs was an effective DOX formulation which could overcome drug resistance in DOX-resistant tumor cells, but its mechanisms of action may be complex.

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Inhibition of Mitosis by Glycopeptide Dendrimer Conjugates of Colchicine.
Lagnoux D, Darbre T, Schmitz ML, Reymond JL.
Chemistry. 2005 Apr 29; [Epub ahead of print].
[ expand abstract ]

Glycopeptide dendrimers have been prepared bearing four or eight identical glycoside moieties at their surface (beta-glucose, alpha-galactose, alpha-N-acetyl-galactose, or lactose), natural amino acids within the branches (Ser, Thr, His, Asp, Glu, Leu, Val, Phe), 2,3-diaminopropionic acid as the branching unit, and a cysteine residue at the core. These dendrimers have been used as drug-delivery devices for colchicine. Colchicine was attached to the dendrimers at the cysteine thiol group through a disulfide or thioether linkage. The biological activities of the glycopeptide dendrimer conjugates were evaluated in HeLa tumor cells and non-transformed mouse embryonic fibroblasts (MEFs). The concentrations of glycopeptide dendrimer drug conjugates required to achieve inhibition of cell proliferation by interference with the tubulin system were found to be higher (IC(50) > 1 muM) compared to the required colchicine concentration. On the other hand, the glycopeptide dendrimer conjugates inhibited the proliferation of HeLa cells 20-100 times more effectively than the proliferation of MEFs. In comparison, non-glycosylated dendrimers and colchicine itself showed a selectivity of 10-fold or less for HeLa cells.

Carbon nanotubes as intracellular protein transporters: generality and biological functionality.
Kam NW, Dai H.
J Am Chem Soc. 2005 Apr 27;127(16):6021-6.
[ expand abstract ]

Various proteins adsorb spontaneously on the sidewalls of acid-oxidized single-walled carbon nanotubes. This simple nonspecific binding scheme can be used to afford noncovalent protein-nanotube conjugates. The proteins are found to be readily transported inside various mammalian cells with nanotubes acting as the transporter via the endocytosis pathway. Once released from the endosomes, the internalized protein-nanotube conjugates can enter into the cytoplasm of cells and perform biological functions, evidenced by apoptosis induction by transported cytochrome c. Carbon nanotubes represent a new class of molecular transporters potentially useful for future in vitro and in vivo protein delivery applications.

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Induction of effective and antigen-specific antitumour immunity by a liposomal ErbB2/HER2 peptide-based vaccination construct.
Roth A, Rohrbach F, Weth R, Frisch B, Schuber F, Wels WS.
Br J Cancer. 2005 Apr 25;92(8):1421-9.
[ expand abstract ]

Efficient delivery of tumour-associated antigens to appropriate cellular compartments of antigen-presenting cells is of prime importance for the induction of potent, cell-mediated antitumour immune responses. We have designed novel multivalent liposomal constructs that co-deliver the p63-71 cytotoxic T Lymphocyte epitope derived from human ErbB2 (HER2), and HA307-319, a T-helper (Th) epitope derived from influenza haemagglutinin. Both peptides were conjugated to the surface of liposomes via a Pam(3)CSS anchor, a synthetic lipopeptide with potent adjuvant activity. In a murine model system, vaccination with these constructs completely protected BALB/c mice from subsequent s.c. challenge with ErbB2-expressing, but not ErbB2-negative, murine renal carcinoma (Renca) cells, indicating the induction of potent, antigen-specific immune responses. I.v. re-challenge of tumour-free animals 2 months after the first tumour cell inoculation did not result in the formation of lung tumour nodules, suggesting that long-lasting, systemic immunity had been induced. While still protecting the majority of vaccinated mice, a liposomal construct lacking the Th epitope was less effective than the diepitope construct, also correlating with a lower number of CD8(+) IFN-gamma(+) T-cells identified upon ex vivo peptide restimulation of splenocytes from vaccinated animals. Importantly, in a therapeutic setting treatment with the liposomal vaccines resulted in cures in the majority of tumour-bearing mice and delayed tumour growth in the remaining ones. Our results demonstrate that liposomal constructs which combine Tc and Th peptide antigens and lipopeptide adjuvants can induce efficient, antigen-specific antitumour immunity, and represent promising synthetic delivery systems for the design of specific antitumour vaccines.British Journal of Cancer (2005) 92, 1421-1429. doi:10.1038/sj.bjc.6602526 www.bjcancer.com Published online 5 April 2005.

Liposome-polycation-DNA (LPD) particle as a carrier and adjuvant for protein-based vaccines: Therapeutic effect against cervical cancer.
Cui Z, Huang L.
Cancer Immunol Immunother. 2005 Apr 22; [Epub ahead of print].
[ expand abstract ]

With the successful identification of many tumor-specific antigens, tumor-associated antigens, and the potential of using unfractioned tumor cell derivatives as tumor antigens, a system and/or adjuvant that can deliver these antigens and help them to induce strong and effective anti-tumor immune responses is greatly needed. Previously, we reported that a MHC class I-restricted peptide epitope derived from human papillomavirus (HPV) 16 E7 protein, when incorporated into a clinically proven safe LPD (liposome-polycation-DNA) particle, was able to effectively eradicate tumors established in mice. Cervical cancer is the second most common cancer among women worldwide. HPV infection is clearly linked to this cancer. Vaccines based on the early (E) gene products of HPV could be effective in controlling it. However, besides the fact that epitope vaccines have many limitations particularly, concerning the diverse HLAs in humans, the use of the epitope as an antigen prevented us from fully characterizing the immune responses induced by the LPD as a vaccine carrier and/or adjuvant in previous studies. In the present study, by using the HPV 16 E7 protein as an antigen, we first showed that LPD, as a vaccine carrier and adjuvant induced strong and robust immune responses, both cellular and antibody. We then showed that immunization with LPD particles incorporated with either the wild type HPV 16 E7 protein or a potentially safer mutant induced strong immune responses that caused complete regressions of a model cervical cancer tumor established in murines. LPD could be a potent vaccine carrier and/or adjuvant for many antigens.

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Phase I trial of liposomal encapsulated doxorubicin (MyocetTM; D-99) and weekly docetaxel in advanced breast cancer patients.
Mrozek E, Rhoades CA, Allen J, Hade EM, Shapiro CL.
Ann Oncol. 2005 Apr 22; [Epub ahead of print].
[ expand abstract ]

Background: We conducted a phase I trial to determine the safety and maximum tolerated dose (MTD) of non-pegylated liposome-encapsulated doxorubicin (Myocet(TM); D-99) administered with weekly docetaxel in metastatic breast cancer (MBC) patients. Patients and methods: Twenty-one patients with no prior chemotherapy for MBC received D-99 (60 or 50 mg/m(2)) intravenously (i.v.) on day 1 and escalating doses of docetaxel (25, 30 and 35 mg/m(2)) i.v. on days 1 and 8 in cohorts of three to six patients. Treatment cycles were repeated every 21 days for a maximum of six cycles. Results: The maximum tolerated dose (MTD) was 50 mg/m(2) of D-99 in combination with 25 mg/m(2) of weekly docetaxel. The most common grade 4 toxicity was neutropenia that occurred in 42 (41%) of treatment cycles, with 10 hospitalizations for febrile neutropenia. Serious protocol-defined cardiac events occurred in three (14%) patients, with two (10%; 95% confidence interval [CI] 1% to 30%) developing congestive heart failure (CHF) after a total cumulative anthracycline dose (adjuvant doxorubicin + D-99) of 540 mg/m(2). Conclusions: D-99 in combination with weekly docetaxel, at the doses and schedule as administered in this trial, is not recommended for phase II testing. Additional trials, using different doses and schedules, are required to evaluate the potential side-effects and efficacy of D-99 and docetaxel.

Haloperidol-associated stealth liposomes: a potent carrier for delivering genes to human breast cancer cells.
Mukherjee A, Prasad TK, Rao NM, Banerjee R.
J Biol Chem. 2005 Apr 22;280(16):15619-27.
[ expand abstract ]

Sigma receptors are membrane-bound proteins that are overexpressed in certain human malignancies including breast cancer. These receptors show very high affinity for various sigma ligands including neuroleptics like haloperidol. We hypothesized that in associating haloperidol-linked lipid into the cationic lipid-DNA complex, we can specifically target and deliver genes to breast cancer cells that overexpress sigma receptors. In the present study, haloperidol was chemically modified to conjugate at the distal end of the polyethylene glycollinked phospholipid, which was then incorporated into the cationic liposome known to condense and deliver genes inside cells. The resulting haloperidol-conjugated targeted lipoplex showed at least 10-fold higher (p < 0.001) reporter gene expression in MCF-7 cells than control lipoplex. The reporter gene expression of the targeted lipoplex was significantly blocked by haloperidol (p < 0.001) and by another sigma ligand, 1,3-ditolylguanidine (p < 0.001) in the majority of cationic lipid to DNA charge ratios (+/-). Spironolactone-mediated sigma receptor down-regulation enabled MCF-7 to show 10-fold lower transgene expression with targeted lipoplex compared with that obtained in spironolactone-untreated cells. The targeted lipoplex generated nonspecific gene expression in sigma receptor-nonexpressing human cancer cells such as Hela, KB, HepG2, and Chinese hamster ovary cells. Moreover, the transgene expression remained unabated in physiologically relevant serum concentrations. This is the first study to demonstrate that haloperidol-targeted gene delivery systems can mediate efficient targeting of genes to sigma receptor-overexpressing breast cancer cells, thereby becoming a novel class of therapeutics for the treatment of human cancers.

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Phase 1 trial of the proteasome inhibitor bortezomib and pegylated liposomal doxorubicin in patients with advanced hematologic malignancies.
Orlowski RZ, Voorhees PM, Garcia RA, Hall MD, Kudrik FJ, Allred T, Johri AR, Jones PE, Ivanova A, Van Deventer HW, Gabriel DA, Shea TC, Mitchell BS, Adams J, Esseltine DL, Trehu EG, Green M, Lehman MJ, Natoli S, Collins JM, Lindley CM, Dees EC.
Blood. 2005 Apr 15;105(8):3058-65.
[ expand abstract ]

Proteasome inhibitors, a novel class of chemotherapeutic agents, enhance the antitumor efficacy of anthracyclines in vitro and in vivo. We therefore sought to determine the maximum tolerated dose (MTD) and dose-limiting toxicities of bortezomib and pegylated liposomal doxorubicin (PegLD). Bortezomib was given on days 1, 4, 8, and 11 from 0.90 to 1.50 mg/m2 and PegLD on day 4 at 30 mg/m2 to 42 patients with advanced hematologic malignancies. Grade 3 or 4 toxicities in at least 10% of patients included thrombocytopenia, lymphopenia, neutropenia, fatigue, pneumonia, peripheral neuropathy, febrile neutropenia, and diarrhea. The MTD based on cycle 1 was 1.50 and 30 mg/m2 of bortezomib and PegLD, respectively. However, due to frequent dose reductions and delays at this level, 1.30 and 30 mg/m2 are recommended for further study. Pharmacokinetic and pharmacodynamic studies did not find significant drug interactions between these agents. Antitumor activity was seen against multiple myeloma, with 8 of 22 evaluable patients having a complete response (CR) or near-CR, including several with anthracycline-refractory disease, and another 8 having partial responses (PRs). One patient with relapsed/refractory T-cell non-Hodgkin lymphoma (NHL) achieved a CR, whereas 2 patients each with acute myeloid leukemia and B-cell NHL had PRs. Bortezomib/PegLD was safely administered in this study with promising antitumor activity, supporting further testing of this regimen.

Fluorescent dendrimers with a peptide cathepsin B cleavage site for drug delivery applications.
Fuchs S, Otto H, Jehle S, Henklein P, Schluter AD.
Chem Commun (Camb). 2005 Apr 14;(14):1830-2.
[ expand abstract ]

The synthesis of a multifunctionally equipped first generation (G1) dendrimer carrying a pentapeptide with a cathepsin[space]B cleavage site, chelating ligands for Pt(2+)-complexation, and a dansyl fluorescence marker is described and an investigation of its cellular uptake as well as intracellular localization by confocal fluorescence microscopy reported.

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Pharmacokinetics and biodistribution of RGD-targeted doxorubicin-loaded nanoparticles in tumor-bearing mice.
Bibby DC, Talmadge JE, Dalal MK, Kurz SG, Chytil KM, Barry SE, Shand DG, Steiert M.
Int J Pharm. 2005 Apr 11;293(1-2):281-90.
[ expand abstract ]

We report the biodistribution and pharmacokinetics (PK) of a cyclic RGD-doxorubicin-nanoparticle (NP) formulation in tumor-bearing mice. The NP core was composed of inulin multi-methacrylate with a targeting peptide, cyclic RGD, covalently attached to the NPs via PEG-400. Seventy-two percent of the doxorubicin was attached to the NP matrix via an amide bond; 28% of doxorubicin was entrapped as unconjugated drug. The PK of total, unconjugated and metabolized doxorubicin was examined for 5 days following intravenous (i.v.) administration of the NP formulation (250mug doxorubicin equiv.), revealing a bi-exponential fix with a terminal half-life of 5.99h. In addition, the biodistribution studies revealed decreasing drug concentrations over time in the heart, lung, kidney and plasma and accumulating drug concentrations in the liver, spleen and tumor. The drug concentration in these latter tissues peaked between 24 and 48h with the liver, spleen and tumor containing 56, 3.5 and 1.8% of the administered dose at t=48h, respectively. In contrast to all of the organs studied, the tumors contained high levels of a doxorubicin metabolite.

NK105, a paclitaxel-incorporating micellar nanoparticle formulation, can extend in vivo antitumour activity and reduce the neurotoxicity of paclitaxel.
Hamaguchi T, Matsumura Y, Suzuki M, Shimizu K, Goda R, Nakamura I, Nakatomi I, Yokoyama M, Kataoka K, Kakizoe T.
Br J Cancer. 2005 Apr 11;92(7):1240-6.
[ expand abstract ]

Paclitaxel (PTX) is one of the most effective anticancer agents. In clinical practice, however, high incidences of adverse reactions of the drug, for example, neurotoxicity, myelosuppression, and allergic reactions, have been reported. NK105, a micellar nanoparticle formulation, was developed to overcome these problems and to enhance the antitumour activity of PTX. Via the self-association process, PTX was incorporated into the inner core of the micelle system by physical entrapment through hydrophobic interactions between the drug and the well-designed block copolymers for PTX. NK105 was compared with free PTX with respect to their in vitro cytotoxicity, in vivo antitumour activity, pharmacokinetics, pharmacodynamics, and neurotoxicity. Consequently, the plasma area under the curve (AUC) values were approximately 90-fold higher for NK105 than for free PTX because the leakage of PTX from normal blood vessels was minimal and its capture by the reticuloendothelial system minimised. Thus, the tumour AUC value was 25-fold higher for NK105 than for free PTX. NK105 showed significantly potent antitumour activity on a human colorectal cancer cell line HT-29 xenograft as compared with PTX (P<0.001) because the enhanced accumulation of the drug in the tumour has occurred, probably followed by its effective and sustained release from micellar nanoparticles. Neurotoxicity was significantly weaker with NK105 than with free PTX. The neurotoxicity of PTX was attenuated by NK105, which was demonstrated by both histopathological (P<0.001) and physiological (P<0.05) methods for the first time. The present study suggests that NK105 warrants a clinical trial for patients with metastatic solid tumours.

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Poly(ethylene oxide)-modified poly(varepsilon-caprolactone) nanoparticles for targeted delivery of tamoxifen in breast cancer.
Shenoy DB, Amiji MM.
Int J Pharm. 2005 Apr 11;293(1-2):261-70.
[ expand abstract ]

This study was carried out to evaluate and compare the biodistribution profile of tamoxifen when administered intravenously (i.v.) as a simple solution or when encapsulated in polymeric nanoparticulate formulations, with or without surface-stabilizing agents. Tamoxifen-loaded, poly(ethylene oxide)-modified poly(varepsilon-caprolactone) (PEO-PCL) nanoparticles were prepared by solvent displacement process that allowed in situ surface modification via physical adsorption of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock polymeric stabilizer (Pluronic((R))). The nanoparticles were characterized for particle size and surface charge. Presence of PEO chains on nanoparticle surface was ascertained by electron spectroscopy for chemical analysis (ESCA). In vivo biodistribution studies were carried out in Nu/Nu athymic mice bearing a human breast carcinoma xenograft, MDA-MB-231 using tritiated [(3)H]-tamoxifen as radio-marker for quantification. PEO-PCL nanoparticles with an average diameter of 150-250nm, having a smooth spherical shape, and a positive surface charge were obtained with the formulation procedure. About 90% drug encapsulation efficiency was achieved when tamoxifen was loaded at 10% by weight of the polymer. Aqueous wettability, suspendability, and ESCA results showed surface hydrophilization of the PCL nanoparticles by the Pluronics((R)). The primary site of accumulation for the drug-loaded nanoparticles after i.v. administration was the liver, though up to 26% of the total activity could be recovered in tumor at 6h post-injection for PEO-modified nanoparticles. PEO-PCL nanoparticles exhibited significantly increased level of accumulation of the drug within tumor with time as well as extended their presence in the systemic circulation than the controls (unmodified nanoparticles or the solution form). Pluronic((R)) surfactants (F-68 and F-108) presented simple means for efficient surface modification and stabilization of PCL nanoparticles to achieve preferential tumor-targeting and a circulating drug reservoir for tamoxifen.

Intratumoral injection of immature dendritic cells enhances antitumor effect of hyperthermia using magnetic nanoparticles.
Tanaka K, Ito A, Kobayashi T, Kawamura T, Shimada S, Matsumoto K, Saida T, Honda H.
Int J Cancer. 2005 Apr 11; [Epub ahead of print].
[ expand abstract ]

Dendritic cells (DCs) are potent antigen-presenting cells that play a pivotal role in regulating immune responses in cancer and have recently been shown to be activated by heat shock proteins (HSPs). We previously reported that HSP70 expression after hyperthermia induces antitumor immunity. Our hyperthermia system using magnetite cationic liposomes (MCLs) induced necrotic cell death that was correlated with HSP70 release. In the present study, we investigated the therapeutic effects of DC therapy combined with MCL-induced hyperthermia on mouse melanoma. In an in vitro study, when immature DCs were pulsed with mouse B16 melanoma cells heated at 43 degrees C, major histocompatibility complex (MHC) class I/II, costimulatory molecules CD80/CD86 and CCR7 in the DCs were upregulated, thus resulting in DC maturation. C57BL/6 mice bearing a melanoma nodule were subjected to combination therapy using hyperthermia and DC immunotherapy in vivo by means of tumor-specific hyperthermia using MCLs and directly injected immature DCs. Mice were divided into 4 groups: group I (control), group II (hyperthermia), group III (DC therapy) and group IV (hyperthermia + DC therapy). Complete regression of tumors was observed in 60% of mice in group IV, while no tumor regression was seen among mice in the other groups. Increased cytotoxic T lymphocyte (CTL) and natural killer (NK) activity was observed on in vitro cytotoxicity assay using splenocytes in the cured mice treated with combination therapy, and the cured mice rejected a second challenge of B16 melanoma cells. This study has important implications for the application of MCL-induced hyperthermia plus DC therapy in patients with advanced malignancies as a novel cancer therapy.

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Properties of various phospholipid mixtures as emulsifiers or dispersing agents in nanoparticle drug carrier preparations.
Ishii F, Nii T.
Colloids Surf B Biointerfaces. 2005 Apr 10;41(4):257-62.
[ expand abstract ]

The characteristics of mixed phospholipids were examined when used as dispersing agents and emulsifiers. Synthesized phospholipids were mixed to investigate the potential effects of different hydrophilic or lipophilic groups on emulsification and dispersion. To examine the effects of the hydrophilic polar head group on the dispersing or emulsifying potency of phospholipids, l-alpha-phosphatidylcholine dimyristoyl (DMPC) and l-alpha-phosphatidylethanolamine dimyristoyl (DMPE) were mixed in various ratios. Moreover, all combinations of two kinds of phosphatidylcholines (PCs) out of l-alpha-phosphatidylcholine dilauroyl (DLPC), DMPC, l-alpha-phosphatidylcholine dipalmitoyl (DPPC) and l-alpha-phosphatidylcholine distearoyl (DSPC) were tested (50:50, w/w) to examine the effects of the hydrophobic carbon chains on the dispersing or emulsifying potency of phospholipids. Mean diameters of vesicles and O/W emulsions prepared by sonication were measured. Vesicles prepared with DMPC-DMPE mixtures gave larger particle sizes than those of DMPC alone. Particle sizes of vesicles prepared with a mixture of two kinds of PCs increased when adding a PC with a longer carbon chain, while particle sizes in a mixture with a PC having a shorter carbon chain was comparable to those in pure PC. In vesicles that were generated by hydration of phospholipids and had a bilayer form, the physical form of the phospholipids consisting of bilayers was thought to be an important factor influencing particle sizes. Among the emulsions, DMPC-DMPE mixtures gave a similar droplet size to DMPC alone. Droplet size in emulsions prepared with a mixture of two kinds of PCs had a strong positive correlation with the total number of carbons, which corresponds to hydrophilic-lipophilic balance (HLB). In O/W emulsions, in which phospholipids were absorbed at water-oil interfaces and which have a single layer form, HLB was thought to be a major factor in the determination of particle size; likewise with non-ionic emulsifiers.

A bio-recognition device developed onto nano-crystals of carbonate apatite for cell-targeted gene delivery.
Chowdhury EH, Akaike T.
Biotechnol Bioeng. 2005 Apr 6; [Epub ahead of print].
[ expand abstract ]

The DNA delivery to mammalian cells is an essential tool for analyzing gene structure, regulation, and function. The approach holds great promise for the further development of gene therapy techniques and DNA vaccination strategies to treat and control diseases. Here, we report on the establishment of a cell-specific gene delivery and expression system by physical adsorption of a cell-recognition molecule on the nano-crystal surface of carbonate apatite. As a model, DNA/nano-particles were successfully coated with asialofetuin to facilitate uptake by hepatocyte-derived cell lines through the asialoglycoprotein receptor (ASGPr) and albumin to prevent non-specific interactions of the particles with cell-surface. The resulting composite particles with dual surface properties could accelerate DNA uptake and enhance expression to a notable extent. Nano-particles coated with transferrin in the same manner dramatically enhanced transgene expression in the corresponding receptor-bearing cells and thus our newly developed strategy represents a universal phenomenon for anchoring a bio-recognition macromolecule on the apatite crystal surface for targeted gene delivery, having immediate applications in basic research laboratories and great promise for gene therapy.

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Induction of effective and antigen-specific antitumour immunity by a liposomal ErbB2/HER2 peptide-based vaccination construct.
Roth A, Rohrbach F, Weth R, Frisch B, Schuber F, Wels WS.
Br J Cancer. 2005 Apr 5; [Epub ahead of print].
[ expand abstract ]

Efficient delivery of tumour-associated antigens to appropriate cellular compartments of antigen-presenting cells is of prime importance for the induction of potent, cell-mediated antitumour immune responses. We have designed novel multivalent liposomal constructs that co-deliver the p63-71 cytotoxic T Lymphocyte epitope derived from human ErbB2 (HER2), and HA307-319, a T-helper (Th) epitope derived from influenza haemagglutinin. Both peptides were conjugated to the surface of liposomes via a Pam(3)CSS anchor, a synthetic lipopeptide with potent adjuvant activity. In a murine model system, vaccination with these constructs completely protected BALB/c mice from subsequent s.c. challenge with ErbB2-expressing, but not ErbB2-negative, murine renal carcinoma (Renca) cells, indicating the induction of potent, antigen-specific immune responses. I.v. re-challenge of tumour-free animals 2 months after the first tumour cell inoculation did not result in the formation of lung tumour nodules, suggesting that long-lasting, systemic immunity had been induced. While still protecting the majority of vaccinated mice, a liposomal construct lacking the Th epitope was less effective than the diepitope construct, also correlating with a lower number of CD8(+) IFN-gamma(+) T-cells identified upon ex vivo peptide restimulation of splenocytes from vaccinated animals. Importantly, in a therapeutic setting treatment with the liposomal vaccines resulted in cures in the majority of tumour-bearing mice and delayed tumour growth in the remaining ones. Our results demonstrate that liposomal constructs which combine Tc and Th peptide antigens and lipopeptide adjuvants can induce efficient, antigen-specific antitumour immunity, and represent promising synthetic delivery systems for the design of specific antitumour vaccines.

In vitro photodynamic effects of phthalocyaninatosilicon covalently linked to 2,2,6,6-tetramethyl-1-piperidinyloxy radicals on cancer cells.
Ishii K, Takayanagi A, Shimizu S, Abe H, Sogawa K, Kobayashi N.
Free Radic Biol Med. 2005 Apr 1;38(7):920-7.
[ expand abstract ]

In this paper, we have investigated the ability to sensitize the phototoxicity toward HeLa cells in vitro, of tetra-tert-butylphthalocyaninatosilicon (SiPc) covalently linked to one or two 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) radicals (R1c or R2c), which are shown as photosensitizers efficiently producing singlet oxygen ((1)Delta(g)). Addition of R1c or R2c encapsulated in liposomes to cultures, followed by irradiation with a 680-nm dye laser, resulted in a highly significant phototoxicity toward HeLa cells, in contrast to negligible phototoxicity observed with (dihydroxy)SiPc (R0). EPR measurements indicate that R1c and R2c exist in some degree as nitroxide radicals even in HeLa cells. Electronic absorption spectra indicate that the degree of aggregation increases in the order R2c < R1c < R0. Thus, the high phototoxicity of R1c and R2c toward HeLa cells is reasonably interpreted by both the large singlet oxygen yield and the inhibition of aggregation due to the bulky TEMPO radicals. This increase in photodynamic effect on HeLa cells is an unusual and important example for increasing the photobiological reaction yields using paramagnetic radicals.

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Dendrimer modified magnetite nanoparticles for protein immobilization.
Pan BF, Gao F, Gu HC.
J Colloid Interface Sci. 2005 Apr 1;284(1):1-6.
[ expand abstract ]

A cascading polyamidoamine (PAMAM) dendrimer was synthesized on the surface of magnetite nanoparticles to allow enhanced immobilization of bovine serum albumin (BSA). Characterization of the synthesis revealed exponential doubling of the surface amine from generations one through four starting with an amino silane initiator. Furthermore, transmission electron microscopy (TEM) revealed clear dispersion of the dendrimer-modified magnetite nanoparticles in methanol solution. The dendrimer-modified magnetite nanoparticles were used to carry out magnetic immobilization of BSA. BSA immobilizing efficiency increased with increasing generation from one to five and BSA binding amount of magnetite nanoparticles modified with G5 dendrimer was 7.7 times as much as that of magnetite nanoparticles modified with only aminosilane. There are two major factors that improve the BSA binding capacity of dendrimer-modified magnetite nanoparticles: one is that the increased surface amine can be conjugated to BSA by a chemical bond through glutaraldehyde; the other is that the available area has increased due to the repulsion of surface positive charge.

Strategic approaches for overcoming peptide and protein instability within biodegradable nano- and microparticles.
Bilati U, Allemann E, Doelker E.
Eur J Pharm Biopharm. 2005 Apr;59(3):375-88.
[ expand abstract ]

This paper reviews the major factors that are closely involved in peptide and protein degradation during the preparation of biodegradable nano- and microparticles. The various means usually employed for overcoming these obstacles are described, in order to bring to the fore the strategies for protein stabilization. Both processing and formulation parameters can be modified and are distinctly considered from a strategic point of view. We describe how partial or full protein stability retention within the carriers and during drug release might be achieved by individual or combined optimized strategies. Additionally, problems commonly encountered during protein quantification, stability determination and release are briefly reviewed. Artefacts that might occur during sampling and analytical procedures and which might hinder critical interpretation of results are discussed.

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Enhancement of drug delivery in tumors by using interaction of nanoparticles with ultrasound radiation.
Larina IV, Evers BM, Ashitkov TV, Bartels C, Larin KV, Esenaliev RO.
Technol Cancer Res Treat. 2005 Apr;4(2):217-26.
[ expand abstract ]

Efficacy and safety of cancer chemo- and biotherapy are limited by poor penetration of anti-cancer drugs from blood into tumor cells. Tumor blood vessel wall, slow diffusion in the interstitium, and cancer cell membrane create physiological barriers for anti-cancer drugs, in particular promising macromolecular agents. Recently, we proposed to use selective accumulation of exogenous nano- and microparticles in tumors followed by ultrasound-induced cavitation for safe and efficient drug and gene delivery. In this paper, we first investigated the influence of polystyrene nanoparticles (100 and 280 nm in diameter and concentration up to 0.2% w/w) on cavitation threshold in water at the frequency of 20 kHz. Then, using optimal irradiation parameters found in the first part of this work, we studied efficacy of cancer chemotherapy with this technique. The experiments were performed in athymic nude mice bearing human colon KM20 tumors, which are highly resistant to chemotherapy. Ultrasound with the frequency of 20 kHz in combination with i.v. injected polystyrene nanoparticles was applied to enhance delivery of chemotherapeutic agent 5-fluorouracil. Our studies demonstrated that ultrasound irradiation in combination with the nanoparticle and drug injections significantly decreased tumor volume and resulted in complete tumor regression at optimal irradiation conditions, while the volume of control (non-irradiated) tumors increased despite drug injections. These data suggest that ultrasound-induced drug delivery may improve efficacy of current cancer treatment regimens.

Increased Gene Expression by Cationic Liposomes (TFL-3) in Lung Metastases Following Intravenous Injection.
Li W, Ishida T, Okada Y, Oku N, Kiwada H.
Biol Pharm Bull. 2005 Apr;28(4):701-6.
[ expand abstract ]

We recently showed that size, not surface charge, is a major determinant of the in vitro lipofection efficiency of pDNA/TFL-3 complex (lipoplex), even in the presence of serum. In this study, the effect of lipoplex size as a result of interaction with serum proteins on in vitro lipofection and the relationship of this with in vivo lipofection was examined in a murine lung metastasis model. As previously described, the pDNA to lipid ratio (P/L ratio) affected both the size and zeta potential of the lipoplex. In vitro studies also indicated that transgene expression in B16BL6 cells was largely dependent on the size of the lipoplex, both in the absence or presence (50% (v/v)) of serum. An in vivo lipofection experiment showed that predominant gene expression in lungs occurred only in tumor-bearing mice, not in normal mice. Based on the in vitro study, this tumor-related gene expression was not related to lipoplex size in the presence of serum (50% (v/v)), suggesting that the size alteration, as the result of interactions with serum proteins in the blood stream may not play an important role in the case of systemic injections. In addition, the efficient gene expression in tumor-bearing lung was not related to the progression of lung metastases. The area-specific gene expression in tumor-bearing lungs, which was largely dependent on the P/L ratio of the lipoplexes, was observed by fluorescent microscopy. Although the underlying mechanism for the area-specific transgene expression is not clear, it may be related to the interaction of lipoplexes with tumor cells, vascular endothelial cells under angiogenesis and normal cells in the lungs. The possibility that TFL-3 is a useful utility to the targeted delivery of pDNA to lungs and tumor-related lipofection is demonstrated. This result suggests that area-specific gene expression in lung metastases may be achieved by controlling the physicochemical properties of the lipoplex, i.e. the P/L ratio.

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Dendrimers of citric acid and poly (ethylene glycol) as the new drug-delivery agents.
Namazi H, Adeli M.
Biomaterials. 2005 Apr;26(10):1175-83.
[ expand abstract ]

Citric acid-polyethylene glycol-citric acid (CPEGC) triblock dendrimers as biocompatible compounds containing G(1), G(2) and G(3) were applied as the drug-delivery systems. Some of the small size molecules and drugs are trapped with the above-synthesized dendrimers. The guest molecules, which are hydrophobic when trapped into the suitable sites of dendrimers, are becoming soluble in aqueous solution. The quantity of trapped molecules and drugs such as 5-amino salicylic acid (5-ASA), pyridine, mefenamic acid, and diclofenac was measured. The drug/dendrimer complexes remained in room temperature for about 10 months and after this long time they were stable and the drugs were not released. Also, the controlled release of the above-mentioned molecules and drugs in vitro conditions was investigated. The structure definition and controlled release of the molecules and drugs were carried out using different spectroscopy methods.

Systemic delivery of RafsiRNA using cationic cardiolipin liposomes silences Raf-1 expression and inhibits tumor growth in xenograft model of human prostate cancer.
Pal A, Ahmad A, Khan S, Sakabe I, Zhang C, Kasid UN, Ahmad I.
Int J Oncol. 2005 Apr;26(4):1087-91.
[ expand abstract ]

Raf-1, a protein serine-threonine kinase, plays a critical role in mitogen-activated protein kinase kinase (MKK/MEK)- mitogen-activated protein kinase (extracellular signal-regulated kinase) (MAPK/ERK) pathways. We show here that systemically delivered novel cationic cardiolipin liposomes (NeoPhectin-AT) containing a small interfering RNA (siRNA) against Raf-1 silence the expression of Raf-1 in tumor tissues and inhibit tumor growth in xenograft model of human prostate cancer. The knockdown of Raf-1 expression by siRNA is also associated with down-regulation of cyclin D1 expression in vivo.

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Pharmacokinetics and adverse reactions of a new liposomal cisplatin (Lipoplatin): phase I study.
Stathopoulos GP, Boulikas T, Vougiouka M, Deliconstantinos G, Rigatos S, Darli E, Viliotou V, Stathopoulos JG.
Oncol Rep. 2005 Apr;13(4):589-95.
[ expand abstract ]

Lipoplatin, a new liposomal cisplatin formulation, is formed from cisplatin and liposomes composed of dipalmitoyl phosphatidyl glycerol (DPPG), soy phosphatidyl choline (SPC-3), cholesterol and methoxy-polyethylene glycol-distearoyl phosphatidylethanolamine (mPEG2000-DSPE). Following intravenous infusion, the nanoparticles (110 nm) are distributed into tissues and concentrate preferentially at tumor sites supposedly via extravasation through the leaky tumor vasculature. This study was designed to investigate the pharmacokinetics and the toxicity of this new liposomal cisplatin in patients with pretreated advanced malignant tumors. The drug was infused for 8 h every 14 days at escalating doses. Twenty-seven patients were included and 3-5 patients were selected for each dosage level; levels started at 25 mg/m2 and were increased by 25 to 125 mg/m2. Three patients were also treated at higher dose levels, one each at 200, 250 and 300 mg/m2. Blood was taken at certain time intervals in order to estimate total platinum plasma levels. At level 5 (125 mg/m2), grades 1 and 2 GI tract and hematological toxicities were detected. No nephrotoxicity was observed. Seven additional patients were added at the 4th level (100 mg/m2) for further pharmacokinetic evaluation. Measurement of platinum levels in the plasma of patients as a function of time showed that a maximum platinum level is attained at 6-8 h. The half-life of Lipoplatin was 60-117 h depending on the dose. Urine excretion reached about 40% of the infused dose in 3 days. The data demonstrate that Lipoplatin up to a dose of 125 mg/m2 every 14 days has no nephrotoxicity and it lacks the serious side effects of cisplatin.

Development and characterization of PLGA nanospheres and nanocapsules containing xanthone and 3-methoxyxanthone.
Teixeira M, Alonso MJ, Pinto MM, Barbosa CM.
Eur J Pharm Biopharm. 2005 Apr;59(3):491-500.
[ expand abstract ]

The aim of the present work was to develop and characterize two different nanosystems, nanospheres and nanocapsules, containing either xanthone (XAN) or 3-methoxyxanthone (3-MeOXAN), with the final goal of improving the delivery of these poorly water-soluble compounds. The xanthones-loaded nanospheres (nanomatrix systems) and nanocapsules (nanoreservoir systems), made of poly(DL-lactide-co-glycolide) (PLGA), were prepared by the solvent displacement technique. The following characteristics of nanoparticle formulations were determined: particle size and morphology, zeta potential, incorporation efficiency, thermal behaviour, in vitro release profiles and physical stability at 4 degrees C. The nanospheres had a mean diameter <170 nm, a narrow size distribution (polydispersity index <0.1), and a negative surface charge (zeta potential <-36 mV). Their incorporation efficiencies were 33% for XAN and 42% for 3-MeOXAN. The presence of the xanthones did not affect the nanospheres size and zeta potential. DSC studies indicated that XAN and 3-MeOXAN were dispersed at a molecular level within the polymeric nanomatrix. Nanocapsules were also nanometric (mean size <300 nm) and exhibited a negative charge (zeta potential <-36 mV). Their incorporation efficiency values (>77%) were higher than those corresponding to nanospheres for both xanthones. The release of 3-MeOXAN from nanocapsules was similar to that observed for the correspondent nanoemulsion, indicating that drug release is mainly governed by its partition between the oil core and the external aqueous medium. In contrast, the release of XAN from nanocapsules was significantly slower than from the nanoemulsion, a behaviour that suggests an interaction of the drug with the polymer. Nanocapsule formulations exhibited good physical stability at 4 degrees C during a 4-month period for XAN and during a 3-month period for 3-MeOXAN.

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Pegylated nanoparticles based on poly(methyl vinyl ether-co-maleic anhydride): preparation and evaluation of their bioadhesive properties.
Yoncheva K, Lizarraga E, Irache JM.
Eur J Pharm Sci. 2005 Apr;24(5):411-9.
[ expand abstract ]

Pegylated nanoparticles based on poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA) were prepared by simple solvent displacement method, in the absence of catalysts or specific chemical conditions. Pegylation efficiency increased with the increasing of molecular weight and bulk concentration of poly(ethylene glycols) (PEGs) investigated. In fact, the use of PEG with molecular weight less than 1000Da did not lead to its attachment. (1)H NMR spectroscopy was performed in order to estimate the conformation state of PEG-chains and to predict the nanoparticle structure. Pegylation with PEG 2000 gave surface modified nanoparticles ("brush" conformation), while the chains of PEG 1000 were distributed either in the core or physically adsorbed on the nanoparticle surface. The capacity of nanoparticles to adsorb mucin at pH 7.4 was significantly higher for PEG 1000-NP than for PEG 2000-NP. The "brush" layer seemed to decrease the interaction between PEG 2000-NP and mucin, which facilitated their penetration through the mucus gel. As a consequence, PEG 2000-NP displayed higher capacity to develop adhesive interactions with rat intestinal mucosa in vivo. Independent on the weaker bioadhesive potential of PEG 1000-NP, both types of pegylated nanoparticles demonstrated very high affinity to the intestinal mucosa rather than to the stomach wall, which could be established for drug targeting to the small intestine.

Binding and condensation of plasmid DNA onto functionalized carbon nanotubes: toward the construction of nanotube-based gene delivery vectors.
Singh R, Pantarotto D, McCarthy D, Chaloin O, Hoebeke J, Partidos CD, Briand JP, Prato M, Bianco A, Kostarelos K.
J Am Chem Soc. 2005 Mar 30;127(12):4388-96.
[ expand abstract ]

Carbon nanotubes (CNTs) constitute a class of nanomaterials that possess characteristics suitable for a variety of possible applications. Their compatibility with aqueous environments has been made possible by the chemical functionalization of their surface, allowing for exploration of their interactions with biological components including mammalian cells. Functionalized CNTs (f-CNTs) are being intensively explored in advanced biotechnological applications ranging from molecular biosensors to cellular growth substrates. We have been exploring the potential of f-CNTs as delivery vehicles of biologically active molecules in view of possible biomedical applications, including vaccination and gene delivery. Recently we reported the capability of ammonium-functionalized single-walled CNTs to penetrate human and murine cells and facilitate the delivery of plasmid DNA leading to expression of marker genes. To optimize f-CNTs as gene delivery vehicles, it is essential to characterize their interactions with DNA. In the present report, we study the interactions of three types of f-CNTs, ammonium-functionalized single-walled and multiwalled carbon nanotubes (SWNT-NH3+; MWNT-NH3+), and lysine-functionalized single-walled carbon nanotubes (SWNT-Lys-NH3+), with plasmid DNA. Nanotube-DNA complexes were analyzed by scanning electron microscopy, surface plasmon resonance, PicoGreen dye exclusion, and agarose gel shift assay. The results indicate that all three types of cationic carbon nanotubes are able to condense DNA to varying degrees, indicating that both nanotube surface area and charge density are critical parameters that determine the interaction and electrostatic complex formation between f-CNTs with DNA. All three different f-CNT types in this study exhibited upregulation of marker gene expression over naked DNA using a mammalian (human) cell line. Differences in the levels of gene expression were correlated with the structural and biophysical data obtained for the f-CNT:DNA complexes to suggest that large surface area leading to very efficient DNA condensation is not necessary for effective gene transfer. However, it will require further investigation to determine whether the degree of binding and tight association between DNA and nanotubes is a desirable trait to increase gene expression efficiency in vitro or in vivo. This study constitutes the first thorough investigation into the physicochemical interactions between cationic functionalized carbon nanotubes and DNA toward construction of carbon nanotube-based gene transfer vector systems.

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Drug interaction and location in liposomes: correlation with polar surface areas.
El Maghraby GM, Williams AC, Barry BW.
Int J Pharm. 2005 Mar 23;292(1-2):179-85.
[ expand abstract ]

An important step in liposome characterization is to determine the location of a drug within the liposome. This work thus investigated the interaction of dipalmitoylphosphatidylcholine liposomes with drugs of varied water solubility, polar surface area (PSA) and partition coefficient using high sensitivity differential scanning calorimetry. Lipophilic estradiol (ES) interacted strongest with the acyl chains of the lipid membrane, followed by the somewhat polar 5-fluorouracil (5-FU). Strongly hydrophilic mannitol (MAN) showed no evidence of interaction but water soluble polymers inulin (IN) and an antisense oligonucleotide (OLG), which have very high PSAs, interacted with the lipid head groups. Accordingly, the drugs could be classified as: hydrophilic ones situated in the aqueous core and which may interact with the head groups; those located at the water-bilayer interface with some degree of penetration into the lipid bilayer; those lipophilic drugs constrained within the bilayer.

Induction of apoptosis of human lung carcinoma cells by hybrid liposomes containing polyoxyethylenedodecyl ether.
Iwamoto Y, Matsumoto Y, Ueoka R.
Int J Pharm. 2005 Mar 23;292(1-2):231-9.
[ expand abstract ]

Hybrid liposomes can be prepared by simply ultrasonicating a mixture of vesicular and micellar molecules in aqueous solution. A clear solution of hybrid liposomes composed of 90 mol% dimyristoylphosphatidylcholine (DMPC) and 10 mol% polyoxyethylene(23)dodecyl ether (C12(EO)23) having a hydrodynamic diameter of 100-120 nm was obtained. Highly inhibitory effects of hybrid liposomes of 90 mol% DMPC/10 mol% C12(EO)23 on the growth of human lung carcinoma (RERF-LC-OK and A549) cells without any drugs were obtained. Induction of apoptosis by hybrid liposomes in RERF-LC-OK and A549 cells was verified on the basis of fluorescence microscopy, agarose gel electrophoresis of DNA and flow cytometry. We elucidated the pathway of apoptosis induced by hybrid liposomes as follows: (a) accumulation of hybrid liposomes in tumor cell membrane was revealed using microphysiometer. (b) Reduction of mitochodrial membrane potential and activation of caspase-9, -3 and -8 were obtained, indicating that apoptotic signal by hybrid liposomes should pass through mitochondria and these caspases. It is worthy to note that such a novel mechanism of apoptosis induced by hybrid liposomes without any drugs was performed for the first time in human lung carcinoma cells.

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Towards a better understanding of the dissociation behavior of liposome-oligonucleotide complexes in the cytosol of cells.
Lucas B, Remaut K, Sanders NN, Braeckmans K, De Smedt SC, Demeester J.
J Control Release. 2005 Mar 21;103(2):435-50.
[ expand abstract ]

To obtain real breakthroughs in antisense therapy, it is necessary to understand the cellular behavior of antisense delivery systems. Fluorescence fluctuation spectroscopy (FFS), which measures in time fluorescence fluctuations in the excitation volume of a microscope and which can thus be applied on a cellular scale, shows potential for this purpose. In this study dual color FFS was explored to characterize the complexation (association and dissociation) between Cy5-labeled oligonucleotides (Cy5-ONs) and FITC-labeled cationic liposomes (FITC-liposomes) in respectively buffer, cell lysate and the cytosol of Vero cells. In Hepes buffer the association of the Cy5-ONs to the FITC-liposomes could be clearly observed from the high peaks of Cy5- and FITC-fluorescence, which appeared simultaneously in the excitation volume. This was explained by the fact that in the complexed state many Cy5-ONs and FITC-liposomes are bound to each other and thus move together through the excitation volume thereby resulting in high fluorescence 'FITC/Cy5-peaks'. FFS measurements on FITC-liposome/Cy5-ONs complexes in cell lysate revealed that a minor part of the Cy5-ONs was released from the complexes. The major part of the Cy5-ONs remained in the complexes, which also seemed to aggregate in cell lysate. In agreement with the measurements in cell lysate, after microinjection of FITC-liposome/Cy5-ONs complexes in the cytosol of Vero cells a part of the Cy5-ONs was released (as Cy-ONs were detected by FFS in the nuclei) while the other part remained bound (as Cy5-peaks were frequently observed in the cytosol). As will be explained, the Cy5-peaks could be due both to Cy5-ONs clustered with cytosol components and Cy5-ONs still complexed to FITC-liposomes with quenched FITC-fluorescence.

Amphiphilic hydrogel nanoparticles.
Preparation, characterization, and preliminary assessment as new colloidal drug carriers.
Missirlis D, Tirelli N, Hubbell JA..
Langmuir.
[ expand abstract ]

Inverse emulsion photopolymerization of acrylated poly(ethylene glycol)-bl-poly(propylene glycol)-bl-poly(ethylene glycol) and poly(ethylene glycol) was successfully employed to prepare stable, cross-linked, amphiphilic nanoparticles. Even at low emulsifier concentrations (2%) and high water-to-hexane weight ratios (35/65), the stability of the inverse emulsion allowed for the formation of well-defined colloidal material. Inverse emulsion characteristics and polymerization conditions could be controlled to vary the size of the nanoparticles between 50 and 500 nm. The presence of hydrophobic nanodomains within these otherwise hydrophilic nanoparticles was verified by using pyrene as a microenvironmentally sensitive probe. The hydrophobic poly(propylene glycol)-rich domains appear to be suitable for incorporation of hydrophobic drugs, encapsulating Doxorubicin up to 9.8% (w/w). We believe that the complex nano-architecture of these materials makes them a potentially interesting colloidal drug delivery carrier system and that the method should be useful for a number of amphiphilic macromolecular precursors.

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Intravesical administration of small interfering RNA targeting PLK-1 successfully prevents the growth of bladder cancer.
Nogawa M, Yuasa T, Kimura S, Tanaka M, Kuroda J, Sato K, Yokota A, Segawa H, Toda Y, Kageyama S, Yoshiki T, Okada Y, Maekawa T.
[ expand abstract ]

The mainstay in the management of invasive bladder cancer continues to be radical cystectomy. With regard to improvement of quality of life, however, therapies that preserve the bladder are desirable. We investigated the use of intravesical PLK-1 small interfering RNA (siRNA) against bladder cancer. Patients with bladder cancers expressing high levels of PLK-1 have a poor prognosis compared with patients with low expression. Using siRNA/cationic liposomes, the expression of endogenous PLK-1 could be suppressed in bladder cancer cells in a time- and dose-dependent manner. As a consequence, PLK-1 functions were disrupted. Inhibition of bipolar spindle formation, accumulation of cyclin B1, reduced cell proliferation, and induction of apoptosis were observed. In order to determine the efficacy of the siRNA/liposomes in vivo, we established an orthotopic mouse model using a LUC-labeled bladder cancer cell line, UM-UC-3(LUC). PLK-1 siRNA was successfully transfected into the cells, reduced PLK-1 expression, and prevented the growth of bladder cancer in this mouse model. This is the first demonstration, to our knowledge, of inhibition of cancer growth in the murine bladder by intravesical siRNA/cationic liposomes. We believe intravesical siRNA instillation against bladder cancer will be useful as a therapeutic tool.

Self-assembled nanoparticles containing hydrophobically modified glycol chitosan for gene delivery.
Sang Yoo H, Eun Lee J, Chung H, Chan Kwon I, Young Jeong S.
J Control Release. 2005 Mar 2;103(1):235-43. Epub 2005 Jan 15.
[ expand abstract ]

A self-assembled nanoparticle was prepared using a hydrophobically modified glycol chitosan for gene delivery. A primary amine of glycol chitosan was modified with 5beta-cholanic acid to prepare a hydrophobically modified glycol chitosan (HGC). The modified chitosan spontaneously formed DNA nanoparticles by a hydrophobic interaction between HGC and hydrophobized DNA. As the HGC content increased, the encapsulation efficiencies of DNA increased while the size of HGC nanoparticles decreased. Upon increasing HGC contents, HGC nanoparticle became less cytotoxic. The increased HGC contents also facilitated endocytic uptakes of HGC nanoparticles by COS-1 cells, which were confirmed by a confocal microscopy. The HGC nanoparticles showed increasing in vitro transfection efficiencies in the presence serum. In vivo results also showed that the HGC nanoparticles had superior transfection efficiencies to naked DNA and a commercialized transfection agent. The HGC nanoparticles composed of hydrophobized DNA and hydrophobically modified glycol chitosan played a significant role in enhancing transfection efficiencies in vitro as well as in vivo.

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Physically bonded nanoparticle networks: a novel drug delivery system.
Xia X, Hu Z, Marquez M.
J Control Release. 2005 Mar 2;103(1):21-30. Epub 2004 Dec 19.
[ expand abstract ]

Monodispersed nanoparticles consisting of interpenetrating polymer networks (IPNs) of polyacrylic acid (PAAc) and poly(N-isopropylacrylamide) (PNIPAM) were prepared by a seed-and-feed method. The temperature-dependent viscosity measurement revealed that the IPN nanoparticle dispersions with polymer concentrations above 2.5 wt.% underwent an inverse thermoreversible gelation at about 33 degrees C. Dextran markers of various molecular weights as model macromolecular drugs were mixed with the IPN nanoparticle dispersion at room temperature. At body temperature, the dispersion became a gel. The drug release profiles were then measured using UV-Visible spectroscopy as a function of particle size and polymer concentration. The schematic structure of the nanoparticle network was proposed based on the experimental results. The drug delivery model presented here was significant because such a dispersion and a drug was mixed without chemical reaction at room temperature to form a drug delivery liquid. This liquid could be injected into a body to form in situ a gelled drug depot to release the drug slowly.

Therapeutic applications of polymeric artificial cells.
Chang TM.
Nat Rev Drug Discov. 2005 Mar;4(3):221-35.
[ expand abstract ]

Polymeric artificial cells have the potential to be used for a wide variety of therapeutic applications, such as the encapsulation of transplanted islet cells to treat diabetic patients. Recent advances in biotechnology, molecular biology, nanotechnology and polymer chemistry are now opening up further exciting possibilities in this field. However, it is also recognized that there are several key obstacles to overcome in bringing such approaches into routine clinical use. This review describes the historical development and principles behind polymeric artificial cells, the present state of the art in their therapeutic application, and the promises and challenges for the future.

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Preparation and characterization of methoxy poly(ethylene glycol)/poly(epsilon-caprolactone) amphiphilic block copolymeric nanospheres for tumor-specific folate-mediated targeting of anticancer drugs.
Park EK, Lee SB, Lee YM.
Biomaterials. 2005 Mar;26(9):1053-61.
[ expand abstract ]

Biodegradable methoxy poly(ethylene glycol)/poly(-caprolactone) (MPEG/PCL) amphiphilic block copolymer nanospheres coupled to folic acid have been designed to target a folate-binding protein that is overexpressed on the surface of many tumoral cells. For this purpose, hydroxy groups terminated on the MPEG/PCL copolymer were converted into primary amino groups, which were used to conjugate with the carboxylic group of folic acid. Nanospheres were prepared by the formation of micelles of the copolymer with or without the anticancer agent paclitaxel. Folate-mediated MPEG/PCL nanospheres were compared with hydroxyl- and amino-terminated nanospheres in terms of their size, surface characteristics, and drug-loading efficiency. Regardless of the type of terminal group, the MPEG/PCL nanospheres showed a narrow size distribution with an average diameter <80 nm without paclitaxel, and an average diameter of 115 nm when loaded with the drug. The results from zeta potential and X-ray photoelectron spectroscopy measurements revealed that the folate molecules were partially exposed, and were expressed on the surface of the nanospheres allowing folate receptor recognition. In in vitro, cytotoxicity tests, the nanospheres loaded with paclitaxel showed a higher cell viability than in cases where paclitaxel was absent. Thus, folate-mediated nanospheres composed of MPEG and PCL are potentially new drug carriers for tumor cell-selective targeting treatments.

Electrochemical antitumor drug sensitivity test for leukemia K562 cells at a carbon-nanotube-modified electrode.
Chen J, Du D, Yan F, Ju HX, Lian HZ.
Chemistry. 2005 Feb 18;11(5):1467-72.
[ expand abstract ]

The change in electrochemical behavior of tumor cells induced by antitumor drugs was detected by using a multiwall carbon nanotubes (MWNTs)-modified glass carbon electrode (GCE). Based on the changes observed, a simple, in vitro, electrochemical antitumor drug sensitivity test was developed. MWNTs promoted electron transfer between the electroactive centers of cells and the electrode. Leukemia K562 cells exhibited a well-defined anodic peak of guanine at +0.823 V at 50 mV s(-1). HPLC assay with ultraviolet detection was used to elucidate the reactant responsible for the electrochemical response of the tumor cells. The guanine content within the cytoplasm of each K562 cell was detected to be 920 amol. For the drug sensitivity tests, 5-fluorouracil (5-FU) and several clinical antitumor drugs, such as vincristine, adriamycin, and mitomycin C, were added to cell culture medium. As a result, the electrochemical responses of the K562 cells decreased significantly. The cytotoxicity curves and results obtained corresponded well with the results of MTT assays. In comparison to conventional methods, this electrochemical test is highly sensitive, accurate, inexpensive, and simple. The method proposed could be developed as a convenient means to study the sensitivity of tumor cells to antitumor drugs.

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Novel mechanism of hybrid liposomes-induced apoptosis in human tumor cells.
Matsumoto Y, Iwamoto Y, Matsushita T, Ueoka R.
Int J Cancer. 2005 Feb 7; [Epub ahead of print].
[ expand abstract ]

Hybrid liposomes can be prepared by simply ultrasonicating a mixture of vesicular and micellar molecules in a buffer solution. The physical properties of these liposomes, such as size, membrane fluidity, phase transition temperature and hydrophobicity can be controlled by changing the composition. Hybrid liposomes composed of dimyristoylphosphatidylcholine and polyoxyethylene (10) dodecyl ether were found to inhibit the growth of human promyelocytic leukemia (HL-60) cells without using any drugs. Induction of apoptosis by hybrid liposomes in HL-60 cells was verified on the basis of fluorescence microscopy and flow cytometry analysis, after fusion and accumulation of hybrid liposomes, which was revealed on the basis of microphysiometer. We elucidated the pathways of apoptosis induced by the hybrid liposomes. That is, hybrid liposomes fused and accumulated in tumor cell membranes, and the apoptosis signal first passed through mitochondria, caspase-9 and caspase-3, second through Fas, caspase-8, caspase-3 and then reached the nucleus. Hybrid liposomes themselves can induce apoptosis in human tumor cells along with high inhibitory effects on the growth of tumor cells.

Endostatin cDNA/Cationic liposome complexes as a promising therapy to prevent lung metastases in osteosarcoma: study in a human-like rat orthotopic tumor.
Dutour A, Monteil J, Paraf F, Charissoux JL, Kaletta C, Sauer B, Naujoks K, Rigaud M.
Mol Ther. 2005 Feb;11(2):311-9.
[ expand abstract ]

Antiangiogenesis or destruction of tumor neovessels is an effective strategy to prevent tumor growth. Endostatin, one of the many inhibitors of angiogenesis that have been discovered, has shown conflicting results in preclinical assays. We studied the therapeutic potential of lipid/DNA complexes consisting of cationic liposomes and an endostatin-coding plasmid (Endo cDNA/CLP) in an orthotopic osteosarcoma model in rats. Empty plasmid without the endostatin gene complexed with cationic liposomes served as control. Animals were treated intravenously three times a week starting on the day tumors were detectable by (18)FDG tomoscintigraphy. During treatment, tumor progression was followed by PET scan and angioscintigraphy, and the effects of antivascular therapy on primary tumor, metastases, and tumor vascular density were confirmed by histologic analysis. Our results demonstrate that therapy using Endo cDNA/CLP is associated with pronounced delay in tumor growth. Moreover, it effectively prevented the occurrence of lung metastases, the major reason for bad prognosis and death in osteosarcoma patients. This approach could be used as an adjuvant therapy for osteosarcoma.

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Growth inhibition of human pancreatic cancer cells by human interferon-beta gene combined with gemcitabine.
Endou M, Mizuno M, Nagata T, Tsukada K, Nakahara N, Tsuno T, Osawa H, Kuno T, Fujita M, Hatano M, Yoshida J.
Int J Mol Med. 2005 Feb;15(2):277-83.
[ expand abstract ]

We examined the anti-tumor effect of cationic multilamellar liposome containing human IFN-beta (huIFN-beta) gene against cultured human pancreatic cancer cells. We also evaluated the combined effect of huIFN-beta gene entrapped in liposomes and gemcitabine. Furthermore, we examined the anti-tumor mechanisms of the therapy, with emphasis on the Ras-related signal pathway. Three human pancreatic cancer cell lines (AsPc-1, MIAPaCa-2, and PANC-1) were used in this study. The growth inhibition together with the therapy were evaluated by WST-1 assay; the production of huIFN-beta protein was measured by ELISA; the cell cycle and apoptosis were analyzed using a FACScan flow cytometer; the protein levels of Son of sevenless (SOS-1) and Ras-GAP were measured by Western blotting; and the activation of Ras-GTP was evaluated by the immunoprecipitation method. As a result, we found that huIFN-beta gene entrapped in liposomes demonstrated a strong anti-tumor effect against human pancreatic cancer cells. The treatment that combined huIFN-beta gene entrapped in liposomes and gemcitabine was more effective than each treatment alone. Although gemcitabine remarkably reduced the level of SOS-1, the above combined therapy reduced the level of SOS-1 even more significantly. Both huIFN-beta gene entrapped in liposomes and the com-bination of huIFN-beta gene entrapped in liposomes and gemcitabine increased the level of Ras-GAP, and decreased the activity of Ras-GTP. These results suggest that this combination therapy can induce strong anti-tumor activity against human pancreatic cancer cells through the regulation of the Ras-related signal pathway.

Targeting human cancer cells with VEGF receptor-2-directed liposomes.
Rubio Demirovic A, Marty C, Console S, Zeisberger SM, Ruch C, Jaussi R, Schwendener RA, Ballmer-Hofer K.
Oncol Rep. 2005 Feb;13(2):319-24.
[ expand abstract ]

Antibodies are among the most versatile tools used today to characterize and target molecules in cells and in biological tissues. The development of phage display libraries encoding a large repertoire of single chain antibodies, scFv, allows the rapid and efficient isolation of antibodies specific for almost any type of molecule. A great advantage of such recombinant antibodies is the possibility to functionalize them by introducing new amino acid sequences. This leads to new features that would be difficult to introduce into naturally occurring antibody molecules. This approach has been successfully applied to create molecules with new biological activities, e.g. by generating chimeric scFv antibodies carrying sequences derived from other biomolecules such as blood clotting factors or enzymes. Here, we describe a new antibody isolated from an M13 phage library that recognizes vascular endothelial growth factor receptor 2, VEGFR-2. This antibody, scFvVR-2H9 was coupled to liposomes and used to specifically target VEGFR-2-expressing human cancer cells in culture.

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New liposome-encapsulated-polyoxometalates: synthesis and antitumoral activity.
Wang X, Li F, Liu S, Pope MT.
J Inorg Biochem. 2005 Feb;99(2):452-7.
[ expand abstract ]

Liposome-encapsulated polyoxometalates (abbreviated as LEP): incorporating K(6)SiW(11)TiO(40) {(SiW(11)Ti) LEP} have been synthesized and structurally characterized by elemental analysis, and IR, UV-Vis, and NMR spectroscopy. The particle sizes of these complexes ranged from 15 to 60 nm. The polyoxometalate retained the parent structure after encapsulation by the liposome. Based upon in vitro measurements with KB and HeLa cancer cells, the liposome-encapsulation enhances cell-membrane penetration, and the stability and antitumoral activity of the polyoxometalate. The toxicity of the polyoxometalate was reduced when LEPs were employed against HL-60 tumors in vivo.

Paclitaxel quantification in mouse plasma and tissues containing liposome-entrapped paclitaxel by liquid chromatography-tandem mass spectrometry: application to a pharmacokinetics study.
Guo W, Johnson JL, Khan S, Ahmad A, Ahmad I.
Anal Biochem. 2005 Jan 15;336(2):213-20.
[ expand abstract ]

A liquid chromatography-tandem mass spectrometry assay to quantify total paclitaxel in mouse plasma and tissue homogenates containing paclitaxel, Taxol, or liposome-entrapped paclitaxel-easy to use (LEP-ETU) was developed and validated. Docetaxel was used as the internal standard (IS). Liquid-liquid extraction with tert-butyl methyl ether was used for plasma sample preparation, and a one-step protein precipitation with acetonitrile containing 0.1% acetic acid was developed for tissue homogenates. Paclitaxel and IS are separated on a 50 x 2.1-mm C18 column and quantified using a triple-quadrupole mass spectrometer operating in positive ion electrospray multiple reaction monitoring mode, with a total run time of 3.5 min. The peak area of the m/z 854.4--> 286.2 transition of paclitaxel is measured versus that of the m/z 808.5--> 527.5 transition of IS to generate the standard curve. In plasma, the linear range is 0.2-500 ng/mL and could be extended by dilution to 100,000 ng/mL with acceptable precision and accuracy (< or = 15%). The lower limit of quantification is 0.5 ng/mL in tissue homogenates (10 ng/g tissue), and the standard curve is linear up to 1000 ng/mL, with precision and accuracy < or = 15%. This assay was used to support a pharmacokinetics and tissue distribution study of LEP-ETU in mice.

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PLGA:Poloxamer and PLGA:Poloxamine Blend Nanoparticles: New Carriers for Gene Delivery.
Csaba N, Caamano P, Sanchez A, Dominguez F, Alonso MJ.
Biomacromolecules. 2005 Jan 10;6(1):271-278.
[ expand abstract ]

The main objective of the present work was the development of new nanoparticulate carrier systems for the delivery of plasmid DNA. These new carriers consist of a blend matrix formed by a poly(lactic-co-glycolic acid) (PLGA) copolymer and polyoxyethylene derivatives. More specifically, we have prepared nanostructures with different PLGA:poloxamer and PLGA:poloxamine compositions by an optimized emulsification-solvent diffusion technique and studied the potential of these carriers for the encapsulation and controlled release of plasmid DNA. Depending on the particle composition, the encapsulation efficiency of the model plasmid pEGFP-C1 varied between 30% and 45%. All formulations provided continuous and controlled release of the plasmid with minimal burst effect. In addition, the release rate and duration was dependent on the composition of the particle matrix. Moreover, gel electrophoresis and cell culture (MCF-7 cell line) assays allowed us to confirm that the biologically active form of the plasmid was preserved during the particle preparation process and also during its release. Cell culture experiments also indicated that the new nanoparticles do not exhibit toxic effects on these cells at concentrations up to 5 mg/mL. Altogether, these results indicate that these composite nanostructures present a promising approach for the delivery of plasmid DNA.

In Vitro Gene Delivery Using Polyamidoamine Dendrimers with a Trimesyl Core.
Zhang XQ, Wang XL, Huang SW, Zhuo RX, Liu ZL, Mao HQ, Leong KW.
Biomacromolecules. 2005 Jan 10;6(1):341-350.
[ expand abstract ]

Polyamidoamine (PAMAM) dendrimer represents one of the most efficient polymeric gene carriers. To investigate the effect of the core structure and generation of dendrimers on the complex formation and transfection efficiency, a series of PAMAM dendrimers with a trimesyl core (DT) at different generations (DT4 to DT8) were developed as gene carriers and compared with the PAMAM dendrimers derived from pentaerythritol (DP) and inositol (DI). The minimal generation number of DTs at which the dendrimer has enough amino group density to effectively condense DNA was higher (generation 6) than those of DPs and DIs (generation 5). DTs of generation 6 or higher condensed DNA into complexes with an average diameter ranging from 100 to 300 nm, but the 4th and 5th generations of DT (DT4 and DT5) formed only a severe aggregate with DNA. Interestingly, the DT6/pDNA complex was determined to be much smaller (100-300 nm) than those prepared with DP5 or DI5 (>600 nm) at N/P ratios higher than 15. The optimal generation numbers at which the dendrimers showed the highest transgene expression in COS-7 cells were 5 for DPs and DIs but 6 for DTs. The DT6/pDNAcomplex with smaller size mediated higher transgene expression in COS-7 cells than those prepared with DP5 or DI5. The in vitro transfection efficiency of the DT dendrimers as evaluated in HeLa cells, COS-7 cells, and primary hepatocytes decreased in the order of DT6 > DT7 > DT8 > DT5 > DT4. The transfection mediated by DT6 was significantly inhibited by bafilomycin A1. The acid-base titration curve for DT6 showed high buffer capacity in the pH range from 5.5 to 6.4 (pK(a) approximately 6). This permits dendrimers to buffer the pH change in the endosomal compartment. However, the transfection efficiency mediated by DT6 decreased significantly in the presence of serum in both HeLa cells and COS-7 cells. The cytotoxicity of DTs evaluated in HeLa cells using the 3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide assay showed a trend of increasing toxicity with the polymer generations. The LD50 values of DT4 through DT8 were 628, 236, 79, 82, and 77 mug/mL, respectively, which were higher than that of poly(ethyleneimide) (18 mug/mL) and poly(l-lysine) (28 mug/mL) in the same assay. With a lower cytotoxicity and versatility for chemical conjugation, these PAMAM dendrimers with a DT core warrant further investigation for nonviral gene delivery.

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Encapsulation of Drug Nanoparticles in Self-Assembled Macromolecular Nanoshells.
Zahr AS, de Villiers M, Pishko MV.
Langmuir. 2005 Jan 4;21(1):403-410.
[ expand abstract ]

Layer-by-Layer (LbL) stepwise self-assembly of the polyelectrolytes poly(allylamine hydrochloride) and poly(styrenesulfonate) was used to create a macromolecular nanoshell around drug nanoparticles (approximately 150 nm in diameter). Dexamethasone, a steroid often used in conjugation with chemotherapy, was chosen as a model drug and was formulated into nanoparticles using a modified solvent-evaporation emulsification method. Measurement of the zeta potential (zeta-potential) after each polyelectrolyte layer was electrostatically added confirmed the successful addition of each layer. Additionally, data acquired from X-ray photon spectroscopy (XPS) indicated the presence of peaks representative of each physisorbed polyelectrolyte layer. Surface modification of the nanoshell was performed by covalently attaching poly(ethylene glycol) (PEG) with a molecular weight of 2000 to the outer surface of the nanoshell. Zeta potential measurements and XPS indicated the presence of PEG chains at the surface of the nanoshell. The polymeric nanoshell on the surface of the drug nanoparticle provides a template upon which surface modifications can be made to create a stealth or targeted drug delivery system.

Transmucosal macromolecular drug delivery.
Prego C, Garcia M, Torres D, Alonso MJ.
J Control Release. 2005 Jan 3;101(1-3):151-62.
[ expand abstract ]

Mucosal surfaces are the most common and convenient routes for delivering drugs to the body. However, macromolecular drugs such as peptides and proteins are unable to overcome the mucosal barriers and/or are degraded before reaching the blood stream. Among the approaches explored so far in order to optimize the transport of these macromolecules across mucosal barriers, the use of nanoparticulate carriers represents a challenging but promising strategy. The present paper aims to compare the characteristics and potential of nanostructures based on the mucoadhesive polysaccharide chitosan (CS). These are CS nanoparticles, CS-coated oil nanodroplets (nanocapsules) and CS-coated lipid nanoparticles. The characteristics and behavior of CS nanoparticles and CS-coated lipid nanoparticles already reported [A. Vila, A. Sanchez, M. Tobio, P. Calvo, M.J. Alonso, Design of biodegradable particles for protein delivery, J. Control. Rel. 78 (2002) 15-24; R. Fernandez-Urrusuno, P. Calvo, C. Remunan-Lopez, J.L. Vila-Jato, M.J. Alonso, Enhancement of nasal absorption of insulin using chitosan nanoparticles, Pharm. Res. 16 (1999) 1576-1581; M. Garcia-Fuentes, D. Torres, M.J. Alonso, New surface-modified lipid nanoparticles as delivery vehicles for salmon calcitonin (submitted for publication).] are compared with those of CS nanocapsules originally reported here. The three types of systems have a size in the nanometer range and a positive zeta potential that was attributed to the presence of CS on their surface. They showed an important capacity for the association of peptides such as insulin, salmon calcitonin and proteins, such as tetanus toxoid. Their mechanism of interaction with epithelia was investigated using the Caco-2 model cell line. The results showed that CS-coated systems caused a concentration-dependent reduction in the transepithelial resistance of the cell monolayer. Moreover, within the range of concentrations investigated, these systems were internalized in the monolayer in a concentration-dependent manner. This uptake was slightly enhanced by the presence of the CS coating but, as compared with previously published results [M. Garcia-Fuentes, C. Prego, D. Torres, M.J. Alonso, Triglyceride-chitosan nanostructures for oral calcitonin delivery: evaluation in the Caco-2 cell model and in vivo (submitted for publication)], highly dependent on the nature of the lipid core. Nevertheless, these differences in the uptake of the CS-coated systems (solid lipid core or oily core) by the Caco-2 cells did not have a consequence in the in vivo behaviour. Indeed, both CS-coated systems (nanocapsules and CS-coated nanoparticles) showed an important capacity to enhance the intestinal absorption of the model peptide, salmon calcitonin, as shown by the important and long-lasting decrease in the calcemia levels observed in rats.

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Development of a nanoprecipitation method intended for the entrapment of hydrophilic drugs into nanoparticles.
Bilati U, Allemann E, Doelker E.
Eur J Pharm Sci. 2005 Jan;24(1):67-75.
[ expand abstract ]

This study investigates formulation and process modifications to improve the versatility of the nanoprecipitation technique, particularly with respect to the encapsulation of hydrophilic drugs (e.g. proteins). More specifically, the principal objective was to explore the influence of such modifications on nanoparticle size. Selected parameters of the nanoprecipitation method, such as the solvent and the non-solvent nature, the solvent/non-solvent volume ratio and the polymer concentration, were varied so as to obtain polymeric nano-carriers. The feasibility of such a modified method was assessed and resulting unloaded nanoparticles were characterized with respect to their size and shape. It was shown that the mean particle size was closely dependent on the type of non-solvent selected. When alcohols were used, the final mean size increased in the sequence: methanol<ethanol<propanol. Surfactants added to the dispersing medium were usually unnecessary for final suspension stabilization. Changing the solvent/non-solvent volume ratio was also not a determinant factor for nanoparticle formation and their final characteristics, provided that the final mixture itself did not become a solvent for the polymer. A too high polymer concentration in the solvent, however, prevented nanoparticle formation. Both poly(lactic acid) (PLA) and poly(d,l-lactic-co-glycolic acid) (PLGA) could be used by accurately choosing the polymer solvent and in this respect, some non-toxic solvents with different dielectric constants were selected. The nanoparticles obtained ranged from about 85-560nm in size. The nanoparticle recovery step however needs further improvements, since bridges between particles which cause flocculation could be observed. Finally, the presented results demonstrate that the nanoprecipitation technique is more versatile and flexible than previously thought and that a wide range of parameters can be modified.

2004

Quantum dots and other nanoparticles: what can they offer to drug discovery?.
Ozkan M.
Drug Discov Today. 2004 Dec 15;9(24):1065-71
[ expand abstract ]

Nanocrystals (quantum dots) and other nanoparticles (gold colloids, magnetic bars, nanobars, dendrimers and nanoshells) have been receiving a lot of attention recently with their unique properties for potential use in drug discovery, bioengineering and therapeutics. In this review, structural, optical and biological assets of nanocrystals are summarized and their applications to drug discovery studies are discussed. Unique properties of these nanoparticles can offer new advancements in drug discovery.

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Folate-receptor-targeted delivery of doxorubicin nano-aggregates stabilized by doxorubicin-PEG-folate conjugate.
Yoo HS, Park TG.
J Control Release. 2004 Nov 24;100(2):247-56.
[ expand abstract ]

For folate-receptor-targeted anti-cancer therapy, doxorubicin aggregates in a nano-scale size were produced employing doxorubicin-polyethylene glycol-folate (DOX-PEG-FOL) conjugate. Doxorubicin and folate were respectively conjugated to alpha- and omega-terminal end group of a PEG chain. The conjugates assisted to form doxorubicin nano-aggregates with an average size of 200 nm in diameter when combined with an excess amount of deprotonated doxorubicin in an aqueous phase. Hydrophobically deprotonated doxorubicin molecules were aggregated within the core, while the DOX-PEG-FOL conjugates stabilized the aggregates with exposing folate moieties on the surface. The doxorubicin nano-aggregates showed a greater extent of intracellular uptake against folate-receptor-positive cancer cells than folate-receptor-negative cells, indicating that the cellular uptake occurred via a folate-receptor-mediated endocytosis mechanism. They also exhibited more potent cytotoxic effect on KB cells than free doxorubicin. In a human tumor xenograft nude mouse model, folate-targeted doxorubicin nano-aggregates significantly reduced the tumor volume compared to non-targeted doxorubicin aggregates or free doxorubicin. These results suggested that folate-targeted doxorubicin nano-aggregates could be a potentially useful delivery system for folate-receptor-positive cancer cells.

Controlled release of paclitaxel from microemulsion containing PLGA and evaluation of anti-tumor activity in vitro and in vivo.
Kang BK, Chon SK, Kim SH, Jeong SY, Kim MS, Cho SH, Lee HB, Khang G.
Int J Pharm. 2004 Nov 22;286(1-2):147-56.
[ expand abstract ]

The main objective of this study was to develop an optimal paclitaxel microemulsion prepared by self-microemulsifying drug delivery system (SMEDDS) which is a mixture of paclitaxel, tetraglycol, Cremophor ELP, and Labrafil 1944 and a paclitaxel microemulsion containing poly(D,L-lactide-co-glycolide) (PLGA) in order to offer controlled release of paclitaxel. To achieve this goal, paclitaxel and PLGA were dissolved by solubilizer like tetraglycol. There was not observed any change in molecular weight of PLGA after being solubilized by tetraglycol. The droplet size for all of the formulation of microemulsion was found in the range of 45-270nm by dynamic light scattering (DLS). It was observed that the droplet size of microemulsion without PLGA was smaller than that of microemulsion containing PLGA by transmission electron microscopy (TEM). The droplet of microemulsion containing PLGA was almost of spherical shape with smooth surface and there was no aggregation or adhesion among droplet of microemulsion by atomic force microscopy (AFM). The release behaviour of paclitaxel from microemulsion containing PLGA having various molecular weights (8K, 33K, and 90K) exhibited a biphasic pattern characterized by a fast initial release during the first 48h, followed by a slower and continuous release for 144h, in contrast that the release of paclitaxel from microemulsion without PLGA was finished during 24h. This result was identical with the result of anti-tumor activity in vitro of paclitaxel from microemulsion containing PLGA against human breast cancer cell line MCF7 and this formulation enhanced anti-tumor activity in vivo compared with microemulsion without PLGA against SKOV-3 human ovarian cancer cells bearing nude mice model.

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Efficacy of transferrin-conjugated paclitaxel-loaded nanoparticles in a murine model of prostate cancer.
Sahoo SK, Ma W, Labhasetwar V.
Int J Cancer. 2004 Nov 1;112(2):335-40
[ expand abstract ]

Chemotherapy remains the preferred choice of treatment for prostate cancer but modest drug response and significant toxicity by conventional methods of administration limit their efficacy. In our study, we determined the efficacy of paclitaxel (Tx)-loaded biodegradable nanoparticles (NPs) on tumor inhibition. We hypothesized that NPs following conjugation to transferrin (Tf) ligand (NPs-Tf) would enhance the therapeutic efficacy of the encapsulated drug. The antiproliferative activity of NPs was determined in human prostate cancer cell line (PC3) and their effect on tumor inhibition in a murine model of prostate cancer. NPs (approximately 220 nm in diameter, 5.4% w/w drug loading) under in vitro conditions exhibited sustained release of the encapsulated drug (60% release in 60 days). The IC50 (concentration of drug for 50% inhibition of cell growth) of the drug with Tf-conjugated NPs (Tx-NPs-Tf) was about 5-fold lower than that with unconjugated NPs (Tx-NPs) or drug in solution. Animals that received a single-dose intratumoral injection of Tx-NPs-Tf (Tx dose= 4 mg/kg) demonstrated complete tumor regression and greater survival rate than those that received either Tx-NPs or Tx-Cremophor EL formulation. In conclusion, sustained release NPs demonstrated greater antitumor activity following their conjugation to Tf ligand.

Size-dependent immunogenicity: therapeutic and protective properties of nano-vaccines against tumors.
Fifis T, Gamvrellis A, Crimeen-Irwin B, Pietersz GA, Li J, Mottram PL, McKenzie IF, Plebanski M.
J Immunol. 2004 Sep 1;173(5):3148-54.
[ expand abstract ]

Infection can protect against subsequent disease by induction of both humoral and cellular immunity, but inert protein-based vaccines are not as effective. In this study, we present a new vaccine design, with Ag covalently conjugated to solid core nano-beads of narrowly defined size (0.04-0.05 microm) that localize to dendritic cells (DEC205(+) CD40(+), CD86(+)) in draining lymph nodes, inducing high levels of IFN-gamma production (CD8 T cells: precursor frequencies 1/5000 to 1/1000) and high Ab titers in mice. Conjugation of Ag to these nano-beads induced responses that were significantly higher (2- to 10-fold) than those elicited by other bead sizes, and higher than a range of currently used adjuvants (alum, QuilA, monophosphoryl lipid A). Responses were comparable to CFA/IFA immunization for Abs and ex vivo peptide-pulsed dendritic cell immunization for CD8 T cells. A single dose of Ag-conjugated beads protected mice from tumors in two different model challenges and caused rapid clearance of established tumors in mice. Thus, a range of Ags conjugated to nano-beads was effective as immunogens in both therapeutic and prophylactic scenarios.

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Body distribution of nanoparticle-containing adriamycin injected into the hepatic artery of hepatoma-bearing rats.
Chen JH, Wang L, Ling R, Li Y, Wang Z, Yao Q, Ma Z.
Dig Dis Sci. 2004 Aug;49(7-8):1170-3.
[ expand abstract ]

The aim of the study was to investigate the body distribution of nanoparticle-containing adriamycin (NADR) injected into the hepatic artery of hepatoma-bearing rats. Thirty Walker-256 hepatoma-bearing rats were divided into two groups at random, with 15 rats in each. NADR and free adriamycin (FADR) were injected into the hepatic artery of animals on the seventh day after tumor implantation. At 1, 5, and 15 hr, after administration, five animals in each group were sacrificed and the ADR concentrations in the plasma, liver, heart, spleen, lungs, kidneys, and tumor were determined. The results showed that NADR substantially increased the ADR concentrations in liver, spleen, and tumor of rats compared to FADR, whereas the concentrations in plasma, heart, and lungs were significantly decreased. In conclusion, the body distribution of ADR can be modified by its encapsulation into nanoparticles and administration via the hepatic artery.

Preparation of magnetic polybutylcyanoacrylate nanospheres encapsulated with aclacinomycin A and its effect on gastric tumor.
Gao H, Wang JY, Shen XZ, Deng YH, Zhang W.
World J Gastroenterol.2004 Jul 15;10(14):2010-3.
[ expand abstract ]

AIM: To evaluate the effect of aclacinomycin A-loaded magnetic polybutylcyanoacrylate nanoparticles on gastric tumor growth in vivo and in vitro. METHODS: Magnetic polybutylcyanoacrylate (PBCA) nanospheres encapsulated with aclacinomycin A (MPNS-ACM) were prepared by interfacial polymerization. Particle size, shape and drug content were examined. Female BABL/c nude mice were implanted with MKN-45 gastric carcinoma tissues subcutaneously to establish human gastric carcinoma model. The mice were randomly divided into 5 groups of 6 each: ACM group (8 mg/kg bm); group of high dosage of MPNS-ACM (8 mg/kg bm); group of low dosage of MPNS-ACM (1.6 mg/kg bm); group of magnetic PBCA nanosphere (MPNS) and control group (normal saline). Magnets (2.5 T) were implanted into the tumor masses in all of the mice one day before the therapy. Above-mentioned drugs were administered intravenously to the mice of every group on the first day and sixth day. When the mice were sacrificed, tumor weight was measured, and the assay of granulocyte- macrophage colony forming-unit (CFU-GM) was performed on semi-solid culture. White blood cell, alanine aminotransferase and creatine were examined. 3-(4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to examine the viability of MKN-45 cells after incubation with different concentrations of ACM, MPNS and MPNS-ACM suspension respectively for 48 h. RESULTS: Content of ACM in MPNS-ACM was 12.0% and the average diameter of the particles was 210 nm. The inhibitory rates of ACM (8 mg/kg bm), high dosage of MPNS-ACM (8 mg/kg bm), low dosage of MPNS-ACM (1.6 mg/kg bm) and MPNS on human gastric carcinoma in nude mice were 22.63%, 52.55%, 30.66% and 10.22%, respectively. There was a significant decrease in the number of CFU-GM of bone marrow in ACM group compared with control group, whereas no obvious change was observed in that of the nanosphere groups. The values of 50% inhibition concentration (IC50) of ACM, MPNS and MPNS-ACM were 0.09, 97.78 and 1.07 microg/mL, respectively. CONCLUSION: The tumor inhibitory rate of MPNS-ACM was much higher than that of ACM under magnetic field and the inhibition on bone marrow was alleviated significantly compared with ACM group.

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Enhanced antitumor efficacy on hepatoma-bearing rats with adriamycin-loaded nanoparticles administered into hepatic artery.
Chen JH, Ling R, Yao Q, Wang L, Ma Z, Li Y, Wang Z, Xu H.
World J Gastroenterol. 2004 Jul 1;10(13):1989-91.
[ expand abstract ]

AIM: To investigate the antitumor activity of adriamycin (ADR) encapsulated in nanoparticles (NADR) and injected into the hepatic artery of hepatoma-bearing rats. METHODS: NADR was prepared by the interfacial polymerization method. Walker-256 carcinosarcomas were surgically implanted into the left liver lobes of 60 male Wistar rats, which were divided into 4 groups at random (15 rats per group). On the 7th day after implantation, normal saline (NS), free ADR (FADR), NADR, or ADR mixed with unloaded nanoparticles (ADR+NP) was respectively injected via the hepatic artery (i.a.) of rats in different groups. The dose of ADR in each formulation was 2.0 mg/kg body weight and the concentration was 1.0 mg/mL. Survival time, tumor enlargement ratio, and tumor necrosis degree were compared between each group. RESULTS: Compared with the rats that received NS i.a., the rats that received FADR or ADR+NP acquired apparent inhibition on tumor growth, as well as prolonged their life span. Further significant anticancer efficacy was observed in rats that received i.a. administration of NADR. Statistics indicated that NADR brought on a more significant tumor inhibition and more extensive tumor necrosis, as compared to FADR or ADR+NP. The mean tumor enlargement ratio on the 7th day after NADR i.a. was 1.106. The mean tumor-bearing survival time was 39.50 days. Prolonged life span ratio was 109.22% as compared with rats that accepted NS. CONCLUSION: Therapeutic effect of ADR on liver malignancy can be significantly enhanced by its nanopaticle formulation and administration via hepatic artery.

Photo-thermal tumor ablation in mice using near infrared-absorbing nanoparticles.
O'Neal DP, Hirsch LR, Halas NJ, Payne JD, West JL.
Cancer Lett. 2004 Jun 25;209(2):171-6.
[ expand abstract ]

The following study examines the feasibility of nanoshell-assisted photo-thermal therapy (NAPT). This technique takes advantage of the strong near infrared (NIR) absorption of nanoshells, a new class of gold nanoparticles with tunable optical absorptivities that can undergo passive extravasation from the abnormal tumor vasculature due to their nanoscale size. Tumors were grown in immune-competent mice by subcutaneous injection of murine colon carcinoma cells (CT26.WT). Polyethylene glycol (PEG) coated nanoshells (approximately 130 nm diameter) with peak optical absorption in the NIR were intravenously injected and allowed to circulate for 6 h. Tumors were then illuminated with a diode laser (808 nm, 4 W/cm2, 3 min). All such treated tumors abated and treated mice appeared healthy and tumor free >90 days later. Control animals and additional sham-treatment animals (laser treatment without nanoshell injection) were euthanized when tumors grew to a predetermined size, which occurred 6-19 days post-treatment. This simple, non-invasive procedure shows great promise as a technique for selective photo-thermal tumor ablation.

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Characterization of poly(D,L-lactic-co-glycolic acid) based nanoparticulate system for enhanced delivery of antigens to dendritic cells.
Elamanchili P, Diwan M, Cao M, Samuel J.
Vaccine.2004 Jun 23;22(19):2406-12.
[ expand abstract ]

Biodegradable nanoparticles made of poly(D,L-lactic acid-co-glycolic acid) (PLGA) copolymer were characterized for enhanced delivery of antigens to murine bone marrow derived dendritic cells (DCs) in vitro. PLGA nanoparticles were efficiently phagocytosed by the DCs (CD11c+, MHC class II+, CD86+) in culture, resulting in their intracellular localization. The efficiency of the uptake was influenced by the incubation time and nanoparticle concentration. DCs pulsed with PLGA nanoparticles containing an immunomodulator, monophosphoryl lipid A (MPLA), showed upregulation of surface expression of MHC class II and CD86 molecules. Delivery of a cancer-associated antigen (MUC1 mucin peptide: BLP25) and MPLA in PLGA nanoparticles was shown to be superior to their delivery in the soluble form for activation of naive T cells of normal and MUC1-transgenic mice. These results strongly suggest that PLGA nanoparticles provide an efficient vaccine delivery system for targeting DCs and the development of DC based cellular vaccines.

Chemotherapy of glioblastoma in rats using doxorubicin-loaded nanoparticles.
Steiniger SC, Kreuter J, Khalansky AS, Skidan IN, Bobruskin AI, Smirnova ZS, Severin SE, Uhl R, Kock M, Geiger KD, Gelperina SE.
Int J Cancer. 2004 May 1;109(5):759-67.
[ expand abstract ]

Glioblastomas belong to the most aggressive human cancers with short survival times. Due to the blood-brain barrier, they are mostly inaccessible to traditional chemotherapy. We have recently shown that doxorubicin bound to polysorbate-coated nanoparticles crossed the intact blood-brain barrier, thus reaching therapeutic concentrations in the brain. Here, we investigated the therapeutic potential of this formulation of doxorubicin in vivo using an animal model created by implantation of 101/8 glioblastoma tumor in rat brains. Groups of 5-8 glioblastoma-bearing rats (total n = 151) were subjected to 3 cycles of 1.5-2.5 mg/kg body weight of doxorubicin in different formulations, including doxorubicin bound to polysorbate-coated nanoparticles. The animals were analyzed for survival (% median increase of survival time, Kaplan-Meier). Preliminary histology including immunocytochemistry (glial fibrillary acidic protein, ezrin, proliferation and apoptosis) was also performed. Rats treated with doxorubicin bound to polysorbate-coated nanoparticles had significantly higher survival times compared with all other groups. Over 20% of the animals in this group showed a long-term remission. Preliminary histology confirmed lower tumor sizes and lower values for proliferation and apoptosis in this group. All groups of animals treated with polysorbate-containing formulations also had a slight inflammatory reaction to the tumor. There was no indication of neurotoxicity. Additionally, binding to nanoparticles may reduce the systemic toxicity of doxorubicin. This study showed that therapy with doxorubicin bound to nanoparticles offers a therapeutic potential for the treatment of human glioblastoma.

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Efficient cancer therapy with a nanobody-based conjugate.
Cortez-Retamozo V, Backmann N, Senter PD, Wernery U, De Baetselier P, Muyldermans S, Revets H.
Cancer Res. 2004 Apr 15;64(8):2853-7.
[ expand abstract ]

Nanobodies are the smallest fragments of naturally occurring single-domain antibodies that have evolved to be fully functional in the absence of a light chain. Nanobodies are strictly monomeric, very stable, and highly soluble entities. We identified a nanobody with subnanomolar affinity for the human tumor-associated carcinoembryonic antigen. This nanobody was conjugated to Enterobacter cloacae beta-lactamase, and its site-selective anticancer prodrug activation capacity was evaluated. The conjugate was readily purified in high yields without aggregation or loss of functionality of the constituents. In vitro experiments showed that the nanobody-enzyme conjugate effectively activated the release of phenylenediamine mustard from the cephalosporin nitrogen mustard prodrug 7-(4-carboxybutanamido) cephalosporin mustard at the surface of carcinoembryonic antigen-expressing LS174T cancer cells. In vivo studies demonstrated that the conjugate had an excellent biodistribution profile and induced regressions and cures of established tumor xenografts. The easy generation and manufacturing yield of nanobody-based conjugates together with their potent antitumor activity make nanobodies promising vehicles for new generation cancer therapeutics.

Enhanced cellular uptake of a triplex-forming oligonucleotide by nanoparticle formation in the presence of polypropylenimine dendrimers.
Santhakumaran LM, Thomas T, Thomas TJ.
Nucleic Acids Res.2004 Apr 15;32(7):2102-12. Print 2004.
[ expand abstract ]

We used polypropylenimine dendrimers for delivering a 31 nt triplex-forming oligonucleotide (ODN) in breast, prostate and ovarian cancer cell lines, using 32P-labeled ODN. Dendrimers enhanced the uptake of ODN by approximately 14-fold in MDA-MB-231 breast cancer cells, compared with control ODN uptake. Dendrimers exerted their effect in a concentration- and molecular weight-dependent manner, with generation 4 (G-4) dendrimer having maximum efficacy. A similar increase in ODN uptake was found with MCF-7 and SK-BR-3 (breast), LNCaP (prostate) and SK-OV-3 (ovarian) cancer cells. The dendrimers had no significant effect on cell viability at concentrations at which maximum ODN uptake occurred. [3H]Thymidine incorporation showed that complexing the ODN with G-4 significantly increased the growth-inhibitory effect of the ODN. Western blot analysis showed a significant 65% reduction of c-myc protein level in ODN-G-4 treated cells compared with that of ODN-treated/control cells. Gel electrophoretic analysis showed that ODN remained intact in cells even after 48 h of treatment. The hydrodynamic radii of nanoparticles formed from ODN in the presence of the dendrimers were in the range of 130-280 nm, as determined by dynamic laser light scattering. Taken together, our results indicate that polypropylenimine dendrimers might be useful vehicles for delivering therapeutic oligonucleotides in cancer cells.

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A novel antiangiogenesis therapy using an integrin antagonist or anti-Flk-1 antibody coated 90Y-labeled nanoparticles.
Li L, Wartchow CA, Danthi SN, Shen Z, Dechene N, Pease J, Choi HS, Doede T, Chu P, Ning S, Lee DY, Bednarski MD, Knox SJ.
Int J Radiat Oncol Biol Phys. 2004 Mar 15;58(4):1215-27.
[ expand abstract ]

PURPOSE: Integrin alpha(v)beta(3) and vascular endothelial growth factor receptor 2 (Flk-1) have been shown to be involved in tumor-induced angiogenesis. Selective targeting of upregulated alpha(v)beta(3) and Flk-1 on the neovasculature of tumors is a novel antiangiogenesis strategy for treating a wide variety of solid tumors. In the studies described here, we investigated the potential therapeutic efficacy of two three-component treatment regimens using two murine tumor models. METHODS AND MATERIALS: The treatment regimens used nanoparticle (NP) based targeting agents radiolabeled with (90)Y. The small molecule integrin antagonist (IA) 4-[2-(3,4,5,6-tetrahydropyrimidin-2-ylamino)ethoxy]-benzoyl-2-(5)-aminoeth ylsulfonylamino-beta-alanine, which binds to the integrin alpha(v)beta(3), and a monoclonal antibody against murine Flk-1 (anti-Flk-1 MAb) were used to target the NPs. Murine tumor models K1735-M2 (melanoma) and CT-26 (colon adenocarcinoma) were used to evaluate the treatment efficacy. RESULTS: In K1735-M2 and CT-26 tumors, a single treatment with IA-NP-(90)Y (14.2 microg/g IA, 5 or 6 microCi/g (90)Y) caused a significant tumor growth delay compared to untreated control tumors, as well as tumors treated with IA, IA-NP, and NP-(90)Y, respectively (p < 0.025, Wilcoxon test). In K1735-M2 tumors, a single treatment with anti-Flk-1 MAb-NP-(90)Y (0.36 microg/g anti-Flk-1 MAb, 5 microCi/g (90)Y) also caused a significant tumor growth delay (p < 0.05, Wilcoxon test) compared to untreated tumors, as well as tumors treated with anti-Flk-1 MAb, anti-Flk-1 MAb-NP, and conventional radioimmunotherapy with (90)Y-labeled anti-Flk mAb. Anti-CD31 staining showed a marked decrease in vessel density in tumors treated with anti-Flk-1 MAb-NP-(90)Y, which was associated with a high level of apoptotic death in these tumors, as shown by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. CONCLUSIONS: The present studies provide proof of principle that targeted radiotherapy works using different targeting agents on a nanoparticle, to target both the integrin alpha(v)beta(3) and the vascular endothelial growth factor receptor. These encouraging results demonstrate the potential therapeutic efficacy of the IA-NP-(90)Y and anti-Flk-1 MAb-NP-(90)Y complexes as novel therapeutic agents for the treatment of a variety of tumor types.

Gold glyconanoparticles as new tools in antiadhesive therapy.
Rojo J, Diaz V, de la Fuente JM, Segura I, Barrientos AG, Riese HH, Bernad A, Penades S.
Chembiochem. 2004 Mar 5;5(3):291-7.
[ expand abstract ]

Gold glyconanoparticles (GNPs) have been prepared as new multivalent tools that mimic glycosphingolipids on the cell surface. GNPs are highly soluble under physiological conditions, stable against enzymatic degradation and nontoxic. Thereby GNPs open up a novel promising multivalent platform for biological applications. It has recently been demonstrated that specific tumor-associated carbohydrate antigens (glycosphingolipids and glycoproteins) are involved in the initial step of tumor spreading. A mouse melanoma model was selected to test glyconanoparticles as possible inhibitors of experimental lung metastasis. A carbohydrate-carbohydrate interaction is proposed as the first recognition step for this process. Glyconanoparticles presenting lactose (lacto-GNPs) have been used successfully to significantly reduce the progression of experimental metastasis. This result shows for the first time a clear biological effect of lacto-GNPs, demonstrating the potential application of this glyconanotechnology in biological processes.

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Potential of gene therapy for the treatment of pituitary tumors.
Goya RG, Sarkar DK, Brown OA, Herenu CB.
Curr Gene Ther. 2004 Mar;4(1):79-87.
[ expand abstract ]

Pituitary adenomas constitute the most frequent neuroendocrine pathology, comprising up to 15% of primary intracranial tumors. Current therapies for pituitary tumors include surgery and radiotherapy, as well as pharmacological approaches for some types. Although all of these approaches have shown a significant degree of success, they are not devoid of unwanted side effects, and in most cases do not offer a permanent cure. Gene therapy-the transfer of genetic material for therapeutic purposes-has undergone an explosive development in the last few years. Within this context, the development of gene therapy approaches for the treatment of pituitary tumors emerges as a promising area of research. We begin by presenting a brief account of the genesis of prolactinomas, with particular emphasis on how estradiol induces prolactinomas in animals. In so doing, we discuss the role of each of the recently discovered growth inhibitory and growth stimulatory substances and their interactions in estrogen action. We also evaluate the cell-cell communication that may govern these growth factor interactions and subsequently promote the growth and survival of prolactinomas. Current research efforts to implement gene therapy in pituitary tumors include the treatment of experimental prolactinomas or somatomammotropic tumors with adenoviral vector-mediated transfer of the suicide gene for the herpes simplex type 1 (HSV1) thymidine kinase, which converts the prodrug ganciclovir into a toxic metabolite. In some cases, the suicide transgene has been placed under the control of pituitary cell-type specific promoters, like the human prolactin or human growth hormone promoters. Also, regulatable adenoviral vector systems are being assessed in gene therapy approaches for experimental pituitary tumors. In a different type of approach, an adenoviral vector, encoding the human retinoblastoma suppressor oncogene, has been successfully used to rescue the phenotype of spontaneous pituitary tumors of the pars intermedia in mice. We close the article by discussing the future of molecular therapies. We point out that although, gene therapy represents a key step in the development of molecular medicine, it has inherent limitations. As a consequence, it is our view that at some point, genetic therapies will have to move from exogenous gene transfer (i.e. gene therapy) to endogenous gene repair. This approach will call for radically new technologies, such as nanotechnology, whose present state of development is outlined.

Efficacy of lytic peptide-bound magnetite nanoparticles in destroying breast cancer cells.
Kumar CS, Leuschner C, Doomes EE, Henry L, Juban M, Hormes J.
J Nanosci Nanotechnol. 2004 Mar;4(3):245-9.
[ expand abstract ]

A 23-amino-acid synthetic lytic peptide (Hecate) was covalently linked to magnetite nanoparticles and the lytic peptide-bound nanoparticles were characterized by X-ray absorption near-edge structure spectroscopy, transmission electron microscopy, and electron diffraction. Investigation of magnetic properties with a superconducting quantum interference device (SQUID) magnetometer has shown a reduction in the saturation magnetization (Ms) of magnetite nanoparticles after binding with lytic peptide. An in vitro cell culture assay with breast cancer cell lines MDA-MB-435S revealed that the lytic peptide-bound magnetite nanoparticles were therapeutically active.

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Nanoparticles of biodegradable polymers for clinical administration of paclitaxel.
Feng SS, Mu L, Win KY, Huang G.
Curr Med Chem. 2004 Feb;11(4):413-24.
[ expand abstract ]

Paclitaxel is one of the best antineoplastic drugs found from nature in the past decades, which has been found effective against a wide spectrum of cancers including ovarian cancer, breast cancer, small and non small cell lung cancer, colon cancer, head and neck cancer, multiple myeloma, melanoma, and Kaposi's sarcoma. Like many other anticancer drugs, it has difficulties in clinical administration due to its poor solubility in water and most pharmaceutical reagents. In its current clinical application, an adjuvant called Cremophor EL has to be employed, which has been found to be responsible for many serious side effects. Nanoparticles of biodegradable polymers can provide an ideal solution to such an adjuvant problem and realize a controlled and targeted delivery of the drug with better efficacy and less side effects. With further development, such as particle size optimization and surface coating, nanoparticle formulation of paclitaxel can promote a new concept of chemotherapy to realize its full efficacy and to improve quality of life of the patients, which includes personalized chemotherapy, local chemotherapy, sustained chemotherapy, oral chemotherapy, chemotherapy across the blood-brain barrier, chemotherapy across the microcirculation barrier, etc. The present research proposes a novel formulation for fabrication of nanoparticles of poly(lactic-co-glycolic acid) (PLGA) by a modified solvent extraction/evaporation technique, in which natural emulsifiers, such as phospholipids, cholesterol and vitamin E TPGS are creatively applied to achieve high drug encapsulation efficiency, desired drug released kinetics, high cell uptake and high cytotoxicity. The nanoparticles composed of various recipes and manufactured under various conditions were characterized by laser light scattering (LLS) for size and size distribution, scanning electron microscopy (SEM) and atomic force microscopy (AFM) for morphological properties, X-ray photoelectron spectroscopy (XPS) and Fourier Transformation Infrared Spectroscopy (FTIR) for surface chemistry, zeta-potential for surface charge, and differential scanning calorimetry (DSC) for the thermogram properties. The drug encapsulation efficiency and the drug release kinetics under in vitro conditions were measured by high performance liquid chromatography (HPLC). It was found that these natural emulsifiers have great advantages for nanoparticle formulation of paclitaxel over the traditional macromolecular emulsifiers, such as polyvinyl alcohol (PVA). Nanoparticles of desired small size and narrow size distribution can be obtained. The drug encapsulation efficiency can be achieved as high as 100 %. The released kinetics can be made under control. The HT-29 cancer cell line experiment showed that after 24 hours of incubation, the cell mortality caused by the drug administered by such nanoparticle formulation could be more than 13 times higher than that caused by the free drug under similar conditions.

Critical determinants in PLGA/PLA nanoparticle-mediated gene expression.
Prabha S, Labhasetwar V.
Pharm Res. 2004 Feb;21(2):354-64.
[ expand abstract ]

PURPOSE: The aim of the study was to determine the critical determinants in nanoparticle-mediated gene transfection. It was hypothesized that different formulation parameters could affect the nanoparticle characteristics and hence its gene transfection. METHODS: Nanoparticles encapsulating plasmid DNA encoding for firefly luciferase were formulated using polylactide (PLA) and poly (D,L-lactide-co-glycolide) (PLGA) polymers of different compositions and molecular weights. A multiple-emulsion solvent-evaporation method with polyvinyl alcohol (PVA) as an emulsifier was used to formulate DNA-loaded nanoparticles. Gene expression of nanoparticles was determined in breast cancer (MCF-7) and prostate cancer (PC-3) cell lines. RESULTS: Nanoparticles formulated using PLGA polymer demonstrated greater gene transfection than those formulated using PLA polymer, and this was attributed to the higher DNA release from PLGA nanoparticles. Higher-molecular-weight PLGA resulted in the formation of nanoparticles with higher DNA loading, which demonstrated higher gene expression than those formulated with lower-molecular-weight PLGA. In addition, the nanoparticles with lower amount of surface-associated PVA demonstrated higher gene transfection in both the cell lines. Higher gene transfection with these nanoparticles was attributed to their higher intracellular uptake and cytoplasmic levels. Further study demonstrated that the molecular weight and the degree of hydrolyzation of PVA used as an emulsifier also affect the gene expression of nanoparticles. CONCLUSIONS: Results thus demonstrate that the DNA loading in nanoparticles and its release, and the surface-associated PVA influencing the intracellular uptake and endolysosomal escape of nanoparticles, are some of the critical determinants in nanoparticle-mediated gene transfection.

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2003

Nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance.
Hirsch LR, Stafford RJ, Bankson JA, Sershen SR, Rivera B, Price RE, Hazle JD, Halas NJ, West JL.
Proc Natl Acad Sci U S A. 2003 Nov 11;100(23):13549-54. Epub 2003 Nov 03.
[ expand abstract ]

Metal nanoshells are a class of nanoparticles with tunable optical resonances. In this article, an application of this technology to thermal ablative therapy for cancer is described. By tuning the nanoshells to strongly absorb light in the near infrared, where optical transmission through tissue is optimal, a distribution of nanoshells at depth in tissue can be used to deliver a therapeutic dose of heat by using moderately low exposures of extracorporeally applied near-infrared (NIR) light. Human breast carcinoma cells incubated with nanoshells in vitro were found to have undergone photothermally induced morbidity on exposure to NIR light (820 nm, 35 W/cm2), as determined by using a fluorescent viability stain. Cells without nanoshells displayed no loss in viability after the same periods and conditions of NIR illumination. Likewise, in vivo studies under magnetic resonance guidance revealed that exposure to low doses of NIR light (820 nm, 4 W/cm2) in solid tumors treated with metal nanoshells reached average maximum temperatures capable of inducing irreversible tissue damage (DeltaT = 37.4 +/- 6.6 degrees C) within 4-6 min. Controls treated without nanoshells demonstrated significantly lower average temperatures on exposure to NIR light (DeltaT < 10 degrees C). These findings demonstrated good correlation with histological findings. Tissues heated above the thermal damage threshold displayed coagulation, cell shrinkage, and loss of nuclear staining, which are indicators of irreversible thermal damage. Control tissues appeared undamaged.

Heat shock protein 70 gene therapy combined with hyperthermia using magnetic nanoparticles.
Ito A, Matsuoka F, Honda H, Kobayashi T.
Cancer Gene Ther. 2003 Dec;10(12):918-25.
[ expand abstract ]

Heat shock proteins (HSPs) are recognized as significant participants in immune reactions. We previously reported that expression of HSP70 in response to hyperthermia, produced using our original magnetite cationic liposomes (MCLs), induces antitumor immunity. In the present study, we examine whether the antitumor immunity induced by hyperthermia is enhanced by hsp70 gene transfer. A human hsp70 gene mediated by cationic liposomes was injected into a B16 melanoma nodule in C57BL/6 mice in situ. At 24 hours after the injection of the hsp70 gene, MCLs were injected into melanoma nodules in C57BL/6 mice, which were subjected to an alternating magnetic field for 30 minutes. The temperature at the tumor reached 43 degrees C and was maintained by controlling the magnetic field intensity. The combined treatment strongly arrested tumor growth over a 30-day period, and complete regression of tumors was observed in 30% (3/10) of mice. Systemic antitumor immunity was induced in the cured mice. This study demonstrates that this novel therapeutic strategy combining the use of hsp70 gene therapy and hyperthermia using MCLs may be applicable to patients with advanced malignancies.

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Nanoparticles for the delivery of genes and drugs to human hepatocytes.
Yamada T, Iwasaki Y, Tada H, Iwabuki H, Chuah MK, VandenDriessche T, Fukuda H, Kondo A, Ueda M, Seno M, Tanizawa K, Kuroda S.
Nat Biotechnol.2003 Aug;21(8):885-90.
[ expand abstract ]

Hepatitis B virus envelope L particles form hollow nanoparticles displaying a peptide that is indispensable for liver-specific infection by hepatitis B virus in humans. Here we demonstrate the use of L particles for the efficient and specific transfer of a gene or drug into human hepatocytes both in culture and in a mouse xenograft model. In this model, intravenous injection of L particles carrying the gene for green fluorescent protein (GFP) or a fluorescent dye resulted in observable fluorescence only in human hepatocellular carcinomas but not in other human carcinomas or in mouse tissues. When the gene encoding human clotting factor IX was transferred into the xenograft model using L particles, factor IX was produced at levels relevant to the treatment of hemophilia B. The yeast-derived L particle is free of viral genomes, highly specific to human liver cells and able to accommodate drugs as well as genes. These advantages should facilitate targeted delivery of genes and drugs to the human liver.

Colloidal anticancer drugs bioavailabilities in oral administration models.
Moutardier V, Tosini F, Vlieghe P, Cara L, Delpero JR, Clerc T.
Int J Pharm. 2003 Jul 9;260(1):23-38.
[ expand abstract ]

Liposomes have been prepared with a polymerised core. Drug release and gastrointestinal (GI) degradation of liposomes with this polymerised core was slightly less important than those of classical liposomes. Vincristine, 5-fluorouracil (5-FU), and methotrexate (MTX) have been incorporated into the liposomes, and studies carried out using the differentiated cell lines Caco-2 and TC7, and with 150 histologically normal sections of human colon. Encapsulation of the drugs in liposomes had variable effects, depending on the test system and the drug used. For 5-FU and MTX calculated to be in a therapeutic range, liposomal formulation enhanced drug permeation, but not for the other drugs tested. In the excised human colon model, the treatment history of the patients can affect bioavailability: pre-operative radiation increased the drug tissue uptake. Transmucosal transport of ions was modified by prior chemotherapy. These results should be taken into account in the design of oral anticancer treatments both at the level of nutritional and pharmacological considerations.

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Ceramic-based nanoparticles entrapping water-insoluble photosensitizing anticancer drugs: a novel drug-carrier system for photodynamic therapy.
Roy I, Ohulchanskyy TY, Pudavar HE, Bergey EJ, Oseroff AR, Morgan J, Dougherty TJ, Prasad PN.
J Am Chem Soc. 2003 Jul 2;125(26):7860-5.
[ expand abstract ]

A novel nanoparticle-based drug carrier for photodynamic therapy is reported which can provide stable aqueous dispersion of hydrophobic photosensitizers, yet preserve the key step of photogeneration of singlet oxygen, necessary for photodynamic action. A multidisciplinary approach is utilized which involves (i) nanochemistry in micellar cavity to produce these carriers, (ii) spectroscopy to confirm singlet oxygen production, and (iii) in vitro studies using tumor cells to investigate drug-carrier uptake and destruction of cancer cells by photodynamic action. Ultrafine organically modified silica-based nanoparticles (diameter approximately 30 nm), entrapping water-insoluble photosensitizing anticancer drug 2-devinyl-2-(1-hexyloxyethyl) pyropheophorbide, have been synthesized in the nonpolar core of micelles by hydrolysis of triethoxyvinylsilane. The resulting drug-doped nanoparticles are spherical, highly monodispersed, and stable in aqueous system. The entrapped drug is more fluorescent in aqueous medium than the free drug, permitting use of fluorescence bioimaging studies. Irradiation of the photosensitizing drug entrapped in nanoparticles with light of suitable wavelength results in efficient generation of singlet oxygen, which is made possible by the inherent porosity of the nanoparticles. In vitro studies have demonstrated the active uptake of drug-doped nanoparticles into the cytosol of tumor cells. Significant damage to such impregnated tumor cells was observed upon irradiation with light of wavelength 650 nm. Thus, the potential of using ceramic-based nanoparticles as drug carriers for photodynamic therapy has been demonstrated.

Encapsulation of p-THPP into nanoparticles: cellular uptake, subcellular localization and effect of serum on photodynamic activity.
Konan YN, Chevallier J, Gurny R, Allemann E.
Photochem Photobiol. 2003 Jun;77(6):638-44.
[ expand abstract ]

The cellular uptake, localization and efflux of meso-tetra-(4-hydroxyphenyl)porphyrin (p-THPP)-loaded nanoparticles have been studied in EMT-6 tumor cells. The effect of blood serum on photocytotoxicity has also been evaluated. Sub-130 nm nanoparticles based on poly(D,L-lactide-co-glycolide) (PLGA) (50:50 PLGA and 75:25 PLGA) and poly(D,L-lactide) (PLA) have been examined in comparison with free p-THPP. For all formulations tested, uptake of photosensitizer into cells was dependent on concentration, time and temperature. All nanoparticulate formulations accumulated within the cells to a greater extent relative to free drug. Indeed, the fluorescence intensities measured on EMT-6 cells treated with p-THPP-loaded nanoparticulate formulations were at least two-fold higher than those obtained with free dye. Furthermore, the highest accumulation level was found with PLGA nanoparticles. Fluorescence microscopy revealed that endocytosis is a major intracellular sequestration mechanism of these p-THPP formulations and that these were localized into early and late endosomes. The efflux study performed on both nonirradiated and irradiated cells indicated that free and p-THPP-loaded nanoparticles gradually escaped from EMT-6 cells as a function of time. This was more pronounced when cells were treated with nanoparticles and irradiated, reflecting important photodamage. It was also found that regardless of the nanoparticulate formulations tested, p-THPP photocytotoxicity was influenced by the concentration of the serum.

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Selective cell targeting with light-absorbing microparticles and nanoparticles.
Pitsillides CM, Joe EK, Wei X, Anderson RR, Lin CP.
Biophys J. 2003 Jun;84(6):4023-32.
[ expand abstract ]

We describe a new method for selective cell targeting based on the use of light-absorbing microparticles and nanoparticles that are heated by short laser pulses to create highly localized cell damage. The method is closely related to chromophore-assisted laser inactivation and photodynamic therapy, but is driven solely by light absorption, without the need for photochemical intermediates (particularly singlet oxygen). The mechanism of light-particle interaction was investigated by nanosecond time-resolved microscopy and by thermal modeling. The extent of light-induced damage was investigated by cell lethality, by cell membrane permeability, and by protein inactivation. Strong particle size dependence was found for these interactions. A technique based on light to target endogenous particles is already being exploited to treat pigmented cells in dermatology and ophthalmology. With exogenous particles, phamacokinetics and biodistribution studies are needed before the method can be evaluated against photodynamic therapy for cancer treatment. However, particles are unique, unlike photosensitizers, in that they can remain stable and inert in cells for extended periods. Thus they may be particularly useful for prelabeling cells in engineered tissue before implantation. Subsequent irradiation with laser pulses will allow control of the implanted cells (inactivation or modulation) in a noninvasive manner.

Magnetic drug targeting--biodistribution of the magnetic carrier and the chemotherapeutic agent mitoxantrone after locoregional cancer treatment.
Alexiou C, Jurgons R, Schmid RJ, Bergemann C, Henke J, Erhardt W, Huenges E, Parak F.
J Drug Target. 2003 Apr;11(3):139-49.
[ expand abstract ]

Magnetic Drug Targeting means the specific delivery of chemotherapeutic agents to their desired targets, e.g. tumors, by using magnetic nanoparticles (ferrofluids) bound to these agents and an external magnetic field which is focused on the tumor. This type of target directed drug injection attempts to concentrate a pharmacologic agent by enhancing its efficacy while simultaneously minimizing deleterious side effects. In previous studies, we have been able to demonstrate the efficacy of this type of localized intraarterial chemotherapy in VX2 squamous cell carcinoma among rabbits [Alexiou, C., Arnold, W., Klein, R.J., Parak, F.G., Hulin, P., Bergemann, C., Erhardt, W., Wagenpfeil, S. and Lubbe, A.S. "Locoregional cancer treatment with Magnetic Drug Targeting", Cancer Res. 60 (2000) 6641-6648]. In the present investigation, we have studied the biodistribution of ferrofluids and chemotherapeutic agent by measuring the amount in the tumor, peritumoral area, various organs and body fluids (e.g. blood and urine), with and without Magnetic Drug Targeting. We compared results to that of administering a chemotherapeutic agent soley. An external magnetic field was directed toward the tumor for 60 min. Biodistribution of ferrofluids in the tumor was investigated using histological cross sections and measured semi-quantitatively using 123I-labeled nanoparticles and quantitatively by the use of radioactive 59Fe-ferrofluids. Mitoxantrone was quantitatively measured using HPLC-analysis. The strength of the external magnetic field was 0.6 Tesla (permanent magnet) in the 123iodine study and 1.7 Tesla (electromagnet) in the 59Fe-study and HPLC-analysis. The concentration of the ferrofluids (FFs) in the tumor region i.e. the tumor tissue and the surrounding area, which was under the influence of an external magnetic field, was found to be much higher than in the absence of one. In contrast to systemic chemotherapy, a much higher concentration of mitoxantrone in the tumor and the peritumoral area (region surrounding the tumor < or = 1 cm), by using only 50% and 20% of the normal dose was seen. Thus, the higher concentration of mitoxantrone could explain the therapeutic efficacy of Magnetic Drug Targeting in treatment of VX2 squamous cell carcinoma in rabbits in our previous studies with the advantage of no adverse clinical side effects.

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A novel controlled release formulation for the anticancer drug paclitaxel (Taxol): PLGA nanoparticles containing vitamin E TPGS.
Mu L, Feng SS.
J Control Release. 2003 Jan 9;86(1):33-48.
[ expand abstract ]

Paclitaxel (Taxol) is one of the best antineoplastic drugs found from nature in the past decades. Like many other anticancer drugs, there are difficulties in its clinical administration due to its poor solubility. Therefore an adjuvant called Cremophor EL has to be employed, but this has been found to cause serious side-effects. However, nanoparticles of biodegradable polymers can provide an ideal solution to the adjuvant problem and realise a controlled and targeted delivery of the drug with better efficacy and fewer side-effects. The present research proposes a novel formulation for fabrication of nanoparticles of biodegradable polymers containing d-alpha-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS or TPGS) to replace the current method of clinical administration and, with further modification, to provide an innovative solution for oral chemotherapy. In the modified solvent extraction/evaporation technique employed in this research, the emulsifier/stabiliser/additive and the matrix material can play a key role in determining the morphological, physicochemical and pharmaceutical properties of the produced nanoparticles. We found that vitamin E TPGS could be a novel surfactant as well as a matrix material when blended with other biodegradable polymers. The nanoparticles composed of various formulations and manufactured under various conditions were characterised by laser light scattering (LLS) for size and size distribution, scanning electron microscopy (SEM) and atomic force microscopy (AFM) for morphological properties, X-ray photoelectron spectroscopy (XPS) for surface chemistry and differential scanning calorimetry (DSC) for thermogram properties. The drug encapsulation efficiency (EE) and the drug release kinetics under in vitro conditions were measured by high performance liquid chromatography (HPLC). It was concluded that vitamin E TPGS has great advantages for the manufacture of polymeric nanoparticles for controlled release of paclitaxel and other anti-cancer drugs. Nanoparticles of nanometer size with narrow distribution can be obtained. A drug encapsulation efficiency as high as 100% can be achieved and the release kinetics can be controlled.

Integrin-mediated targeting of drug delivery to irradiated tumor blood vessels.
Hallahan D, Geng L, Qu S, Scarfone C, Giorgio T, Donnelly E, Gao X, Clanton J.
Cancer Cell. 2003 Jan;3(1):63-74.
[ expand abstract ]

The objective of this study was to target drug delivery to radiation-induced neoantigens, which include activated receptors within the tumor vasculature. These responses include posttranslational changes in pre-existing proteins, which can be discovered by phage-displayed peptide libraries administered to mice bearing irradiated tumors. Phage-displayed peptides recovered from irradiated tumors included the amino acid sequence RGDGSSV. This peptide binds to integrins within the tumor microvasculature. Immunohistochemical staining of irradiated tumors showed accumulation of fibrinogen receptor alpha(2b)beta(3) integrin. We studied tumor targeting efficiency of ligands to radiation-induced alpha(2b)beta(3). Radiopharmaceuticals were localized to irradiated tumors by use of alpha(2b)beta(3) ligands conjugated to nanoparticles and liposomes. Fibrinogen-conjugated nanoparticles bind to the radiation-activated receptor, obliterate tumor blood flow, and significantly increase regression and growth delay in irradiated tumors. Radiation-guided drug delivery to tumor blood vessels is a novel paradigm for targeted drug delivery.

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Antitumor properties of irinotecan-containing nanoparticles prepared using poly(DL-lactic acid) and poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol).
Onishi H, Machida Y, Machida Y.
Biol Pharm Bull. 2003 Jan;26(1):116-9.
[ expand abstract ]

Irinotecan-containing nanoparticles (NP) were prepared by coprecipitation with addition of water to acetone solution of poly(DL-lactic acid), poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) and irinotecan, and subsequent evaporation of organic solvent. NP were purified by gel filtration and used for experiments after condensation by evaporation. The obtained NP showed the drug content of 4.5% (w/w) and the mean particle diameter of 118 nm with the particle diameter distribution between 80-210 nm. When the antitumor effect was examined at a repeated dose of 20 mg irinotecan eq/kg for 3 d (3 x 20 mg/kg) using mice bearing Sarcoma 180 subcutaneously, only NP suppressed tumor growth significantly. After i.v. injection in rats, NP maintained irinotecan plasma concentration longer than CPT-11 aqueous solution. The present nanoparticle formation is suggested as a possibly useful dosage form of irinotecan against solid tumor.

Nanoparticle-mediated gene delivery to tumour neovasculature.
Reynolds AR, Moein Moghimi S, Hodivala-Dilke K.
Trends Mol Med. 2003 Jan;9(1):2-4.
[ expand abstract ]

The alpha(v)beta(3) integrin is a potential pharmacological target for anti-angiogenic therapy. A recent report describes the use of alpha(v)beta(3)-targeted nanoparticles to deliver a gene to tumour vasculature selectively. This resulted in substantial tumour regression in several experimental mouse tumour models. Hence, this approach has great potential for the treatment of human cancer.

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Cellular uptake and concentrations of tamoxifen upon administration in poly(epsilon-caprolactone) nanoparticles.
Chawla JS, Amiji MM.
AAPS PharmSci. 2003;5(1):E3.
[ expand abstract ]

PURPOSE: In an attempt to increase the local concentration of tamoxifen in estrogen receptor positive breast cancer cells, we have prepared and characterized poly(epsilon-caprolactone) (PCL) nanoparticle formulation. METHODS: PCL (mol wt 14,800 daltons) nanoparticles were prepared by the solvent displacement method in acetone-water system in the presence of Pluronic F- 68. PCL nanoparticles, labeled with rhodamine123, were incubated with MCF-7 estrogen receptor positive breast cancer cells to determine uptake, intracellular distribution, and localization as a function of time. Intracellular drug concentrations over a specified period of time using different initial doses were examined using tritiated [3H]-tamoxifen. RESULTS: A significant fraction of the administered rhodamine123-loaded PCL nanoparticles was found in the perinuclear region of the MCF-7 cells, where estrogen receptors are also localized, after 1 hour of incubation. Measurements of the intracellular concentrations revealed that most of the administered nanoparticle dose was internalized within the first 30 minutes of incubation, and the uptake followed saturable transport kinetics. CONCLUSION: Results of this study show that PCL nanoparticles were rapidly internalized in MCF-7 cells and intracellular tamoxifen concentrations followed a saturable process. This approach may provide better therapeutic benefit by delivering the drug locally, near the tumor cells, for a longer period of time.

Biodegradable nanoparticle mediated antigen delivery to human cord blood derived dendritic cells for induction of primary T cell responses.
Diwan M, Elamanchili P, Lane H, Gainer A, Samuel J.
J Drug Target. 2003;11(8-10):495-507.
[ expand abstract ]

Dendritic cells (DCs) in the peripheral tissues act as sentinels of the immune system. They detect and capture pathogens entering the body and present their antigens to T cells to trigger responses directed towards elimination of the pathogen. The induction of peripheral tolerance against self and certain foreign antigens is also believed to be mediated through DCs. The outcome of any immune response is largely controlled by the microenvironment of antigen capture, processing and presentation by DCs. The "context" of antigen delivery to DCs will directly influence the microenvironment of antigen presentation and hence the regulation of immune responses. We report here preliminary investigations describing the formulation of a pharmaceutically acceptable, biodegradable, and strategic nanoparticulate delivery system, and its application for efficient antigen loading of DCs to achieve antigen specific T cell activation. "Pathogen-mimicking" nanoparticles capable of interacting with DCs were fabricated by incorporating monophosphoryl lipid A (MPLA; toll-like receptor (TLR) 4 ligand) or CpG ODN (seq #2006; TLR9 ligand) in biodegradable copolymer, poly(D,L,-lactic-co-glycolic acid) (PLGA). The uptake of PLGA nanoparticles by human umbilical cord blood derived DCs (DCs propagated from CD34 progenitors) was conclusively demonstrated by scanning electron microscopy (SEM), fluorescence activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM). Cell phenotype at day 12 of cultures was determined as immature DC using specific cell surface markers, i.e. CD11c (approximately 90%), MHC-II (approximately 70%), CD86 (approximately 20%), CD83 (approximately 5%), CD80 (approximately 40%), CD40 (approximately 40%), and CCR7 (approximately 5%). Tetanus toxoid (TT), a model antigen, was encapsulated in nanoparticles along with an immunomodulator, i.e. either MPLA or CpG ODN. DCs pulsed with various antigen formulations were co-cultured with autologous naive T cells at various cell ratios (DC: T cells were 1:5-20). The DCs pulsed with TT and MPLA together in nanoparticles induced significantly higher T cell proliferation (P<0.05) as compared to when DCs pulsed with TT and MPLA in solution were employed. A similar trend was observed when CpG ODN was used instead of MPLA in the TT nanoparticles. This strategy of antigen delivery to DCs was then tested with a cancer vaccine candidate, a MUC1 lipopeptide. The T cell proliferation observed in the presence of nanoparticulate MUC1 and MPLA pulsed-DCs was much higher than DCs pulsed with soluble antigen (P<0.0005). These results indicate that PLGA nanoparticles mimicking certain features of pathogens are efficient delivery systems for targeting vaccine antigens to DCs and activation of potent T cell responses.

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2002

Biodegradable poly(epsilon -caprolactone) nanoparticles for tumor-targeted delivery of tamoxifen.
Chawla JS, Amiji MM.
Int J Pharm. 2002 Dec 5;249(1-2):127-38
[ expand abstract ]

To increase the local concentration of tamoxifen in estrogen receptor (ER) positive breast cancer, we have developed and characterized nanoparticle formulation using poly(epsilon -caprolactone) (PCL). The nanoparticles were prepared by solvent displacement method using acetone-water system. Particle size analysis, scanning electron microscopy, zeta potential measurements, and differential scanning calorimetry (DSC) were used for nanoparticle characterization. Biodegradation studies were performed in the presence and absence of Pseudomonas lipase in phosphate-buffered saline (PBS, pH 7.4) at 37 degrees C. Tamoxifen loading over different concentrations was analyzed by high-performance liquid chromatography (HPLC) and the optimum loading concentration was determined. In vitro release studies were performed in 0.5% (w/v) sodium lauryl sulfate (SLS) containing PBS at 37 degrees C. Cellular uptake and distribution of fluorescent-labeled nanoparticles was examined in MCF-7 breast cancer cells. SEM micrographs and Coulter analysis showed nanoparticles with spherical shape and uniform size distribution (250-300 nm), respectively. Zeta potential analysis revealed a positive surface charge of +25 mV on the tamoxifen-loaded formulation. Being hydrophobic crystalline polyester, PCL did not degrade in PBS alone, but the degradation was enhanced by the presence of lipase. The maximum tamoxifen loading efficiency was 64%. Initial burst release of tamoxifen was observed, probably due to significant surface presence of the drug on the nanoparticles. A large fraction of the administered nanoparticle dose was taken up by MCF-7 cells through non-specific endocytosis. The nanoparticles were found in the perinuclear region after 1 h. Results of the study suggest that nanoparticle formulations of selective ER modulators, like tamoxifen, would provide increased therapeutic benefit by delivering the drug in the vicinity of the ER.

Poly(ethylene glycol)-coated hexadecylcyanoacrylate nanospheres display a combined effect for brain tumor targeting.
Brigger I, Morizet J, Aubert G, Chacun H, Terrier-Lacombe MJ, Couvreur P, Vassal G.
J Pharmacol Exp Ther. 2002 Dec;303(3):928-36.
[ expand abstract ]

The aim of the present study was to evaluate the tumor accumulation of radiolabeled long-circulating poly(ethylene glycol) (PEG)-coated hexadecylcyanoacrylate nanospheres and non-PEG-coated hexadecylcyanoacrylate nanospheres (used as control), after intravenous injection in Fischer rats bearing intracerebrally well established 9L gliosarcoma. Both types of nanospheres showed an accumulation with a retention effect in the 9L tumor. However, long-circulating nanospheres concentrated 3.1 times higher in the gliosarcoma, compared with non-PEG-coated nanospheres. The tumor-to-brain ratio of pegylated nanospheres was found to be 11, which was in accordance with the ratios reported for other carriers tested for brain tumor targeting such as long-circulating liposomes or labels for magnetic resonance imaging. In addition, a 4- to 8-fold higher accumulation of the PEG-coated carriers was observed in normal brain regions, when compared with control nanospheres. Using a simplified pharmacokinetic model, two different mechanisms were proposed to explain this higher concentration of PEG-coated nanospheres in a tumoral brain. 1) in the 9L tumor, the preferential accumulation of pegylated nanospheres was attributable to their slower plasma clearance, relative to control nanospheres. Diffusion/convection was the proposed mechanism for extravasation of the nanospheres in the 9L interstitium, across the altered blood-brain barrier. 2) In addition, PEG-coated nanospheres displayed an affinity with the brain endothelial cells (normal brain region), which may not be considered as the result of a simple diffusion/convection process. The exact underlying mechanism of such affinity deserves further investigation, since it was observed to be as important as specific interactions described for immunoliposomes with the blood-brain barrier.

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Paclitaxel-loaded PLGA nanoparticles: preparation, physicochemical characterization and in vitro anti-tumoral activity.
Fonseca C, Simoes S, Gaspar R.
J Control Release. 2002 Oct 4;83(2):273-286.
[ expand abstract ]

The main objective of this study was to develop a polymeric drug delivery system for paclitaxel, intended to be intravenously administered, capable of improving the therapeutic index of the drug and devoid of the adverse effects of Cremophor EL. To achieve this goal paclitaxel (Ptx)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (Ptx-PLGA-Nps) were prepared by the interfacial deposition method. The influence of different experimental parameters on the incorporation efficiency of paclitaxel in the nanoparticles was evaluated. Our results demonstrate that the incorporation efficiency of paclitaxel in nanoparticles was mostly affected by the method of preparation of the organic phase and also by the organic phase/aqueous phase ratio. Our data indicate that the methodology of preparation allowed the formation of spherical nanometric (<200 nm), homogeneous and negatively charged particles which are suitable for intravenous administration. The release behaviour of paclitaxel from the developed Nps exhibited a biphasic pattern characterised by an initial fast release during the first 24 h, followed by a slower and continuous release. The in vitro anti-tumoral activity of Ptx-PLGA-Nps developed in this work was assessed using a human small cell lung cancer cell line (NCI-H69 SCLC) and compared to the in vitro anti-tumoral activity of the commercial formulation Taxol. The influence of Cremophor EL on cell viability was also investigated. Exposure of NCI-H69 cells to 25 microg/ml Taxol resulted in a steep decrease in cell viability. Our results demonstrate that incorporation of Ptx in nanoparticles strongly enhances the cytotoxic effect of the drug as compared to Taxol, this effect being more relevant for prolonged incubation times.

A study on the preparation and anti-tumor efficacy of bovine serum albumin nanospheres containing 5-fluorouracil.
Santhi K, Dhanaraj SA, Joseph V, Ponnusankar S, Suresh B.
Drug Dev Ind Pharm. 2002 Oct;28(9):1171-9.
[ expand abstract ]

The therapeutic profile of many anti-cancer drugs has been improved by their modified distribution through a colloidal carrier system. Hence, bovine serum albumin nanospheres containing 5-fluorouracil were prepared by pH-coacervation methods. To select the most suitable cryoprotector for the formulated nanosphere system, a study on the effect of cryoprotectors in the prevention of particle agglomeration was done. Using glucose and mannitol at various concentrations during freeze drying, glucose at a concentration of 5% was observed to be relatively more effective in the prevention of particle agglomeration than the other cryoprotectors. The carrier capacity was determined through the drug-to-albumin ratio. The particle size of all the drug-loaded batches was analyzed before and after freeze drying. The batch of nanospheres with uniform size distribution, and highest drug loading, was used for other subsequent studies. The effect of surfactant in drug loading was estimated through various concentrations of sodium lauryl sulfate, and it was observed that the surfactant has no influence on drug loading at the selected concentrations. The batch of nanospheres with highest drug loading was evaluated for its in-vitro release, and the drug release was found to be in a bi-phasic pattern. To evaluate the efficacy of 5-fluorouracil-loaded nanospheres against cancer cells, an in vitro cytotoxicity study was carried out using HEp-2 cell lines. The nanosphere-bound drug was observed to produce a better cytotoxic effect than the free drug. The anti-tumor efficacy of drug-loaded nanosphere was investigated in DLA tumor-induced mice models, and the percentage tumor inhibition was relatively higher in animals treated with nanosphere-bound drug than with free drug.

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Tumor accumulation of gadolinium in lipid-nanoparticles intravenously injected for neutron-capture therapy of cancer.
Watanabe T, Ichikawa H, Fukumori Y.
Eur J Pharm Biopharm. 2002 Sep;54(2):119-24.
[ expand abstract ]

Gadolinium-incorporating lipid-nanoemulsions (Gd-nanoLE) for neutron-capture therapy were prepared. As a more convenient administration route than the intraperitoneal (i.p.) injection previously reported, the intravenous (i.v.) injections in tumor-bearing hamsters were carried out at an administration volume of 1 ml, which was the maximum tolerable injection volume of an i.v. injection and half that of an i.p. injection. When the standard-Gd-nanoLE of 1.5 mgGd/ml was administered, the absolute bioavailability in the i.p. injection was 57%, probably resulting from incomplete absorption from the peritoneal cavity into the blood stream. The biodistribution data revealed that the i.v. injection had three advantages over the i.p. injection, namely, a faster and higher accumulation of Gd-nano LE, and a more extended retention time in the tumor. Two i.v. injections of the standard-Gd-nanoLE with a 24h interval doubled the tumor accumulation of Gd, resulting in 49.7 microg Gd/g wet tumor 12h after administration. By using a twofold Gd-enriched formulation (High-Gd-nanoLE) of 3.0 mgGd/ml in the repeated administration schedule, the accumulation was doubled again, reaching 101 microg Gd/g wet tumor. This level was comparable to the maximum level in the single i.p. injection previously reported. These results demonstrated that i.v. injection could be an alternative to i.p. injection as an administration route.

Antiangiogenics meet nanotechnology.
Ruoslahti E.
Cancer Cell. 2002 Aug;2(2):97-8.
[ expand abstract ]

A mutant Raf-1 gene loaded onto nanoparticles, delivered to tumor vasculature with an integrin binding compound, provides effective antiangiogenic gene therapy in mice.

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Long-circulating poly(ethylene glycol)-modified gelatin nanoparticles for intracellular delivery.
Kaul G, Amiji M.
Pharm Res. 2002 Jul;19(7):1061-7.
[ expand abstract ]

PURPOSE: The objective of this study was to develop and characterize long-circulating, biodegradable, and biocompatible nanoparticulate formulation as an intracellular delivery vehicle. METHODS: Poly(ethylene glycol) (PEG)-modified gelatin was synthesized by reacting Type-B gelatin with PEG-epoxide. The nanoparticles, prepared by pH and temperature controlled ethanol-water solvent displacement technique, were characterized for mean size, size distribution, and surface morphology. Electron spectroscopy for chemical analysis (ESCA) was used to confirm the surface presence of PEG chains. In vitro release of tetramethylrhodamine-labeled dextran (TMR-dextran, Mol. wt. 10,000 daltons) from the nanoparticle formulations was examined in PBS, with and without 0.2-mg/ml protease, at 37 degrees C. Relative cytotoxicity profile of control and PEGylated gelatin was evaluated in BT-20 a human breast cancer cell line. The nanoparticles were incubated with BT-20 cells to determine uptake and cellular distribution using confocal microscopy. RESULTS: Gelatin and PEGylated gelatin nanoparticles were found to be spherical in shape with a smooth surface in a size range of 200-500 nm and a unimodal size distribution. ESCA results showed an increase in the ether carbon (-C-O-) peak in the PEGylated gelatin nanoparticles due to the presence of PEG chains. The presence of PEG chains decreased the percent release of TMR-dextran in the presence of proteolytic enzyme due to steric repulsion. Cytotoxicity assays indicated that both gelatin and PEGylated gelatin were completely non-toxic to the cells. A large fraction of the administered control gelatin and PEGylated gelatin nanoparticles were found to be concentrated in the perinuclear region of the BT-20 cells after 12 hours indicating possible vesicular transport through initial uptake by endocytosis and endosomal processing. CONCLUSION: The results of this study show that PEGylation of gelatin may prove beneficial as long-circulating delivery system in vivo. Additionally, the nanoparticles could encapsulate hydrophilic macromolecules and are internalized by tumor cells.

Tumor regression by targeted gene delivery to the neovasculature.
Hood JD, Bednarski M, Frausto R, Guccione S, Reisfeld RA, Xiang R, Cheresh DA.
Science. 2002 Jun 28;296(5577):2404-7.
[ expand abstract ]

Efforts to influence the biology of blood vessels by gene delivery have been hampered by a lack of targeting vectors specific for endothelial cells in diseased tissues. Here we show that a cationic nanoparticle (NP) coupled to an integrin alphavbeta3-targeting ligand can deliver genes selectively to angiogenic blood vessels in tumor-bearing mice. The therapeutic efficacy of this approach was tested by generating NPs conjugated to a mutant Raf gene, ATPmu-Raf, which blocks endothelial signaling and angiogenesis in response to multiple growth factors. Systemic injection of the NP into mice resulted in apoptosis of the tumor-associated endothelium, ultimately leading to tumor cell apoptosis and sustained regression of established primary and metastatic tumors.

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Self-assembly of a virus-mimicking nanostructure system for efficient tumor-targeted gene delivery.
Xu L, Frederik P, Pirollo KF, Tang WH, Rait A, Xiang LM, Huang W, Cruz I, Yin Y, Chang EH.
Hum Gene Ther. 2002 Feb 10;13(3):469-81.
[ expand abstract ]

Molecular therapy, including gene therapy, is a promising strategy for the treatment of human disease. However, delivery of molecular therapeutics efficiently and specifically to the target tissue remains a significant challenge. A human transferrin (Tf)-targeted cationic liposome-DNA complex, Tf-lipoplex, has shown high gene transfer efficiency and efficacy with human head and neck cancer in vitro and in vivo (Xu, L., Pirollo, K.F., Tang, W.H., Rait, A., and Chang, E.H. Hum. Gene Ther. 1999;10:2941-2952). Here we explore the structure, size, formation process, and structure-function relationships of Tf-lipoplex. We have observed Tf-lipoplex to have a highly compact structure, with a relatively uniform size of 50-90 nm. This nanostructure is novel in that it resembles a virus particle with a dense core enveloped by a membrane coated with Tf molecules spiking the surface. More importantly, compared with unliganded lipoplex, Tf-lipoplex shows enhanced stability, improved in vivo gene transfer efficiency, and long-term efficacy for systemic p53 gene therapy of human prostate cancer when used in combination with conventional radiotherapy. On the basis of our observations, we propose a multistep self-assembly process and Tf-facilitated DNA cocondensation model that may provide an explanation for the resultant small size and effectiveness of our nanostructural Tf-lipoplex system.

Nanocapsules: lipid-coated aggregates of cisplatin with high cytotoxicity.
Burger KN, Staffhorst RW, de Vijlder HC, Velinova MJ, Bomans PH, Frederik PM, de Kruijff B.
Nat Med. 2002 Jan;8(1):81-4.
[ expand abstract ]

Cisplatin is one of the most widely used agents in the treatment of solid tumors, but its clinical utility is limited by toxicity. The development of less toxic, liposomal formulations of cisplatin has been hampered by the low water solubility and low lipophilicity of cisplatin, resulting in very low encapsulation efficiencies. We describe a novel method allowing the efficient encapsulation of cisplatin in a lipid formulation; it is based on repeated freezing and thawing of a concentrated solution of cisplatin in the presence of negatively charged phospholipids. The method is unique in that it generates nanocapsules, which are small aggregates of cisplatin covered by a single lipid bilayer. The nanocapsules have an unprecedented drug-to-lipid ratio and an in vitro cytotoxicity up to 1000-fold higher than the free drug. Analysis of the mechanism of nanocapsule formation suggests that the method may be generalized to other drugs showing low water solubility and lipophilicity.

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