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

Nanotechnologies in Advanced Imaging

2007  2006   2005   2004   2003   2002

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2007

Compact cysteine-coated CdSe(ZnCdS) quantum dots for in vivo applications.
Liu W, Choi HS, Zimmer JP, Tanaka E, Frangioni JV, Bawendi M.
J Am Chem Soc. 2007 Nov 28;129(47):14530-1.
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We have developed a versatile nanoparticle construct using a compact cysteine coating on a CdSe(ZnCdS) core(shell) nanocrystal (QD-Cys) that is biologically compatible, exceptionally compact, highly fluorescent, and easily functionalized. The small hydrodynamic diameter of QD-Cys ( approximately 6 nm) allows for renal clearance of these nanoparticles in rat models. Moreover, the ability to directly conjugate to QD-Cys opens up the possibility of functionalized nanocrystals for in vivo targeted imaging, in which small targeting molecules can be appended to QD-Cys, and unbound QDs can be rapidly cleared to achieve high signal/noise ratios and to reduce background toxicity.

InGaP@ZnS-Enriched Chitosan Nanoparticles: A Versatile Fluorescent Probe for Deep-Tissue Imaging.
Sandros MG, Behrendt M, Maysinger D, Tabrizian M.
Advanced Functional Materials. 2007;17(18):3724-30.
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InGaP QDs overcoated with several monolayers of ZnS are covalently bound to chitosan to address the challenges of developing highly biologically stable and fluorescent nanoparticle probes for deep-tissue imaging. Transmission electron microscopy images reveal that the average diameter of these luminescent nanoparticles is approximately 29 nm, and they contain multiple InGaP@ZnS QDs that have an average diameter between 4 and 5 nm. These new InGaP@ZnS-chitosan nanoparticles emit near the near IR region at 670 nm and are able to penetrate three times deeper into tissue (e.g., even through a mouse skull) while revealing a higher uptake efficiency into PC12 cells with a robust signal. Additionally, a cell viability assay demonstrates that these new fluorescent nanoparticles have good biocompatibility and stability with PC12 cells and neural cells. As a result, these near-IR-emitting nanoparticles can be used for real-time and deep-tissue examination of diverse specimens, such as lymphatic organs, kidneys, hearts, and brains, while leaving the tissue intact.

Folate-mediated tumor cell uptake of quantum dots entrapped in lipid nanoparticles.
Schroeder JE, Shweky I, Shmeeda H, Banin U, Gabizon A.
J Control Release. 2007 Dec 4;124(1-2):28-34.
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Quantum dots (QDs) are fluorescent semiconductor nanocrystals with superior optical properties compared to organic dyes currently undergoing rapid development for biological applications, particularly in fluorescence imaging. The folate receptor, overexpressed in a broad spectrum of malignant tumors, is an attractive target for selective delivery of imaging agents to tumor cells. This study examines nanoparticles containing QDs entrapped in a lipid shell, and post-loaded with a folate-lipid conjugate for targeting to mouse and human tumor cells expressing the folate receptor. Hydrophobic QDs were mixed with 1,2 dipalmitoyl-sn-glycero-3 phosphocholine and methoxy-polyethylene-glycol-distearoyl-phosphatidyl-ethanolamine (mPEG-DSPE) generating a nanoparticle referred to as lipodot, with a mean diameter size of approximately 100 nm. Folate-derivatized PEG-DSPE was post-loaded into the lipodots at 0.5% lipid molar concentration. Mouse J6456 lymphoma cells (J6456-FR) and human head and neck KB cancer cells (KB-FR), up-regulated for their folate receptors, were incubated with folate-targeted and non-targeted lipodots in vitro. Using fluorescence microscopy, it was found that only folate-targeted lipodots were taken up by tumor cells. Confocal depth scanning showed substantial internalization. Confirming the specificity of folate-targeted lipodots, binding and internalization were inhibited by free folate, and no uptake was found in a folate-receptor negative cell line. Selective binding and uptake of folate-targeted lipodots by J6456-FR cells was also observed in vivo after intra-peritoneal injection in mice bearing ascitic J6456-FR tumors based on FACS analysis and confocal imaging of harvested cells from the peritoneal cavity. Folate-targeted lipodots represent an attractive approach for tumor cell labeling both in vitro and in vivo.

Preparation and characterization of poly(lipid)-coated, fluorophore-doped silica nanoparticles for biolabeling and cellular imaging.
Senarath-Yapa MD, Phimphivong S, Coym JW, Wirth MJ, Aspinwall CA, Saavedra SS.
Langmuir. 2007 Dec 4;23(25):12624-33.
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The fabrication, characterization, and implementation of poly(lipid)-coated, highly luminescent silica nanoparticles as fluorescent probes for labeling of cultured cells are described. The core of the probe is a sol-gel-derived silica nanoparticle, 65-100 nm in diameter, in which up to several thousand dye molecules are encapsulated (Lian, W.; et al. Anal. Biochem. 2004, 334, 135-144). The core is coated with a membrane composed of bis-sorbylphosphatidylcholine, a synthetic polymerizable lipid that is chemically cross-linked to enhance the environmental and chemical stability of the membrane relative to a fluid lipid membrane. The poly(lipid) coating has two major functions: (i) to reduce nonspecific interactions, based on the inherently biocompatible properties of the phosphorylcholine headgroup, and (ii) to permit functionalization of the particle, by doping the coating with lipids bearing chemically reactive or bioactive headgroups. Both functions are demonstrated: (i) Nonspecific adsorption of dissolved proteins to bare silica nanoparticles and of bare nanoparticles to cultured cells is significantly reduced by application of the poly(lipid) coating. (ii) Functionalization of poly(lipid)-coated nanoparticles with a biotin-conjugated lipid creates a probe that can be used to target both dissolved protein receptors as well as receptors on the membranes of cultured cells. Measurements performed on single nanoparticles bound to planar supported lipid bilayers verify that the emission intensity of these probes is significantly greater than that of single protein molecules labeled with several fluorophores.

Temperature-responsive magnetite/PEO-PPO-PEO block copolymer nanoparticles for controlled drug targeting delivery.
Chen S, Li Y, Guo C, Wang J, Ma J, Liang X, Yang LR, Liu HZ.
Langmuir. 2007 Dec 4;23(25):12669-76.
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In this study, temperature-responsive magnetite/polymer nanoparticles were developed from iron oxide nanoparticles and poly(ethyleneimine)-modified poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) block copolymer. The particles were characterized by TEM, XRD, DLS, VSM, FTIR, and TGA. A typical product has an approximately 20 nm magnetite core and an approximately 40 nm hydrodynamic diameter with a narrow size distribution and is superparamagnetic with large saturation magnetization (51.34 emu/g) at room temperature. The most attractive feature of the nanoparticles is their temperature-responsive volume-transition property. DLS results indicated that their average hydrodynamic diameter underwent a sharp decrease from 45 to 25 nm while evaluating the temperature from 20 to 35 degrees C. The temperature-dependent evolution of the C-O stretching band in the FTIR spectra of the aqueous nanoparticles solution revealed that thermo-induced self-assembly of the immobilized block copolymers occurred on the magnetite solid surfaces, which is accompanied by a conformational change from a fully extended state to a highly coiled state of the copolymer. Consequently, the copolymer shell could act as a temperature-controlled "gate" for the transit of guest substance. The uptake and release of both hydrophobic and hydrophilic model drugs were well controlled by switching the transient opening and closing of the polymer shell at different temperatures. A sustained release of about 3 days was achieved in simulated human body conditions. In primary mouse experiments, drug-entrapped magnetic nanoparticles showed good biocompatibility and effective therapy for spinal cord damage. Such intelligent magnetic nanoparticles are attractive candidates for widespread biomedical applications, particularly in controlled drug-targeting delivery.

High cytotoxicity and resistant-cell reversal of novel paclitaxel loaded micelles by enhancing the molecular-target delivery of the drug
You J, Hu FQ, Du YZ, Yuan H, Ye BF.
Nanotechnology. 2007 Dec 12;18:495101.
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Many antitumor drugs, such as paclitaxel (PTX), are widely used in cancer chemotherapy. However, their clinical use is limited by systemic toxicity, rapid blood clearance, and the occurrence of resistance. To increase the therapeutic index of these drugs, the antitumor drug PTX was encapsulated in novel micelles with glycolipid-like structure, which were formed by stearate grafted chitosan oligosaccharide in aqueous medium. The micelles could load the poorly soluble antitumor drug (PTX) with high entrapment efficiency and drug loading. PTX release was retarded as a result of the encapsulation of the micelles. PTX loaded micelles present excellent internalization into tumor cells as well as resistant cells and subsequently reside in cytoplasm, which results in increased intracellular accumulation of PTX in its molecular-target site. Consequently, cytotoxicity of PTX loaded micelles was improved sharply and resistant cells were reversed. In conclusion, high cytotoxicity can be obtained and resistant cells can be reversed by enhancing PTX's molecular-target delivery and accumulation via the encapsulation of the micelles. The present micelles are a promising carrier candidate for effective therapy of antitumor drugs with the target molecule in cytoplasm.

Bimodal Paramagnetic and Fluorescent Liposomes for Cellular and Tumor Magnetic Resonance Imaging.
Kamaly N, Kalber T, Ahmad A, Oliver MH, So PW, Herlihy AH, Bell JD, Jorgensen MR, Miller AD.
Bioconjug Chem. 2008 Jan 16;19(1):118-129.
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A novel bimodal fluorescent and paramagnetic liposome is described for cellular labeling. In this study, we show the synthesis of a novel gadolinium lipid, Gd.DOTA.DSA, designed for liposomal cell labeling and tumor imaging. Liposome formulations consisting of this lipid were optimized in order to allow for maximum cellular entry, and the optimized formulation was used to label HeLa cells in vitro. The efficiency of this novel bimodal Gd-liposome formulation for cell labeling was demonstrated using both fluorescence microscopy and magnetic resonance imaging (MRI). The uptake of Gd-liposomes into cells induced a marked reduction in their MRI T 1 relaxation times. Fluorescence microscopy provided concomitant proof of uptake and revealed liposome internalization into the cell cytosol. The optimized formulation was also found to exhibit minimal cytotoxicity and was shown to have capacity for plasmid DNA (pDNA) transfection. A further second novel neutral bimodal Gd-liposome is described for the labeling of xenograft tumors in vivo utilizing the enhanced permeation and retention effect (EPR). Balb/c nude mice were inoculated with IGROV-1 cells, and the resulting tumor was imaged by MRI using these in vivo Gd-liposomes formulated with low charge and a poly(ethylene glycol) (PEG) calyx for long systemic circulation. These Gd-liposomes which were less than 100 nm in size were shown to accumulate in tumor tissue by MRI, and this was also verified by fluorescence microscopy of histology samples. Our in vivo tumor imaging results demonstrate the effectiveness of MRI to observe passive targeting of long-term circulating liposomes to tumors in real time, and allow for MRI directed therapy, wherein the delivery of therapeutic genes and drugs to tumor sites can be monitored while therapeutic effects on tumor mass and/or size may be simultaneously observed, quantitated, and correlated.

Multifunctional magneto-polymeric nanohybrids for targeted detection and synergistic therapeutic effects on breast cancer.
Yang J, Lee CH, Ko HJ, Suh JS, Yoon HG, Lee K, Huh YM, Haam S.
Angew Chem Int Ed Engl. 2007;46(46):8836-9.
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No abstract available. (Citation link)

Oligonucleotide-coated metallic nanoparticles as a flexible platform for molecular imaging agents.
Nitin N, Javier DJ, Richards-Kortum R.
Bioconjug Chem. 2007 Nov-Dec;18(6):2090-6.
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Targeted metallic nanoparticles have shown promise as contrast agents for molecular imaging. To obtain molecular specificity, the nanoparticle surface must be appropriately functionalized with probe molecules that will bind to biomarkers of interest. The aim of this study was to develop and characterize a flexible approach to generate molecular imaging agents based on gold nanoparticles conjugated to a diverse range of probe molecules. We present two complementary oligonucleotide-based approaches to develop gold nanoparticle contrast agents which can be functionalized with a variety of biomolecules ranging from small molecules, to peptides, to antibodies. The size, biocompatibility, and protein concentration per nanoparticle are characterized for the two oligonucleotide-based approaches; the results are compared to contrast agents prepared using adsorption of proteins on gold nanoparticles by electrostatic interaction. Contrast agents prepared from oligonucleotide-functionalized nanoparticles are significantly smaller in size and more stable than contrast agents prepared by adsorption of proteins on gold nanoparticles. We demonstrate the flexibility of the oligonucleotide-based approach by preparing contrast agents conjugated to folate, EGF peptide, and anti-EGFR antibodies. Reflectance images of cancer cell lines labeled with functionalized contrast agents show significantly increased image contrast which is specific for the target biomarker. To demonstrate the modularity of this new bioconjugation approach, we use it to conjugate both fluorophore and anti-EGFR antibodies to metal nanoparticles, yielding a contrast agent which can be probed with multiple imaging modalities. This novel bioconjugation approach can be used to prepare contrast agents targeted with biomolecules that span a diverse range of sizes; at the same time, the bioconjugation method can be adapted to develop multimodal contrast agents for molecular imaging without changing the coating design or material.

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.

Hybrid nanoparticles for magnetic resonance imaging of target-specific viral gene delivery.
Huh YM, Lee ES, Lee JH, Jun YW, Kim PH, Yun CO, Kim JH, Suh JS, Cheon J.
Advanced Materials. 2007;19(20):3109-12.
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No abstract available. (Citation link)

Conjugated polymer dots for multiphoton fluorescence imaging.
Wu C, Szymanski C, Cain Z, McNeill J.
J Am Chem Soc. 2007 Oct 31;129(43):12904-5.
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We report on the two-photon excited fluorescence of conjugated polymer dots (CPdots). As a new class of two-photon fluorescent probes, CPdots exhibit two-photon action cross sections as high as 2.0 × 105 GM, to our knowledge, the largest reported thus far for a nanoparticle. The cross section values are 3-4 orders of magnitude higher than those of conventional fluorescent dyes and an order of magnitude higher than those of inorganic quantum dots. Single particle fluorescence imaging was achieved using relatively low laser power.

Ultrasmall mixed ferrite colloids as multidimensional magnetic resonance imaging, cell labeling, and cell sorting agents.
Groman EV, Bouchard JC, Reinhardt CP, Vaccaro DE.
Bioconjug Chem. 2007 Nov-Dec;18(6):1763-71.
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One area that has been overlooked in the evolution of magnetic nanoparticle technology is the possibility of introducing informational atoms into the iron oxide core of the coated colloid. Introduction of suitable atoms into the iron oxide core offers an opportunity to produce a quantifiable probe, thereby adding one or more dimensions to the magnetic colloid's informational status. Lanthanide-doped iron oxide nanoparticles have been synthesized to introduce informational atoms through the formation of colloidal mixed ferrites. These colloids are designated ultrasmall mixed ferrite iron oxides (USMIOs). USMIOs containing 5 mol % europium exhibit superparamagnetic behavior with an induced magnetization of 56 emu/g Fe at 1.5 T, a powder X-ray diffraction pattern congruent with magnetite, and R1 and R2 relaxivity values of 15.4 (mM s) (-1) and 33.9 (mM s) (-1), respectively, in aqueous solution at 37 degrees C and 0.47 T. USMIO can be detected by five physical methods, combining the magnetic resonance imaging (MRI) qualities of iron with the sensitive and quantitative detection of lanthanide metals by neutron activation analysis (NA), time-resolved fluorescence (TRF), X-ray fluorescence, along with detection by electron microscopy (EM). In addition to quantitative detection using neutron activation analysis, the presence of lanthanides in the iron oxide matrix confers attractive optical properties for long-term multilabeling studies with europium and terbium. These USMIOs offer high photostability, a narrow emission band, and a broad absorption band combining the high sensitivity of time-resolved fluorescence with the high spatial resolution of MRI. USMIO nanoparticles are prepared through modifications of traditional magnetite-based iron oxide colloid synthetic methods. A 5 mol % substitution of ferric iron with trivalent europium yielded a colloid with nearly identical magnetic, physical, and chemical characteristics to its magnetite colloid parent.

Cancer cells assemble and align gold nanorods conjugated to antibodies to produce highly enhanced, sharp, and polarized surface Raman spectra: a potential cancer diagnostic marker.
Huang X, El-Sayed IH, Qian W, El-Sayed MA.
Nano Lett. 2007 Jun;7(6):1591-7.
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Human oral cancer cells are found to assemble and align gold nanorods conjugated to anti-epidermal growth factor receptor (anti-EGFR) antibodies. Immnoconjugated gold nanorods and nanospheres were shown previously to exhibit strong Rayleigh (Mie) scattering useful for imaging. In the present letter, molecules near the nanorods on the cancer cells are found to give a Raman spectrum that is greatly enhanced (due to the high surface plasmon field of the nanorod assembly in which their extended surface plasmon fields overlap), sharp (due to a homogeneous environment), and polarized (due to anisotropic alignments). These observed properties can be used as diagnostic signatures for cancer cells.

microPET-based biodistribution of quantum dots in living mice.
Schipper ML, Cheng Z, Lee SW, Bentolila LA, Iyer G, Rao J, Chen X, Wu AM, Weiss S, Gambhir SS.
J Nucl Med. 2007 Sep;48(9):1511-8.
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This study evaluates the quantitative biodistribution of commercially available CdSe quantum dots (QD) in mice. METHODS: (64)Cu-Labeled 800- or 525-nm emission wavelength QD (21- or 12-nm diameter), with or without 2,000 MW (molecular weight) polyethylene glycol (PEG), were injected intravenously into mice (5.55 MBq/25 pmol QD) and studied using well counting or by serial microPET and region-of-interest analysis. RESULTS: Both methods show rapid uptake by the liver (27.4-38.9 %ID/g) (%ID/g is percentage injected dose per gram tissue) and spleen (8.0-12.4 %ID/g). Size has no influence on biodistribution within the range tested here. Pegylated QD have slightly slower uptake into liver and spleen (6 vs. 2 min) and show additional low-level bone uptake (6.5-6.9 %ID/g). No evidence of clearance from these organs was observed. CONCLUSION: Rapid reticuloendothelial system clearance of QD will require modification of QD for optimal utility in imaging living subjects. Formal quantitative biodistribution/imaging studies will be helpful in studying many types of nanoparticles, including quantum dots.

Biodegradable nanoparticles for targeted ultrasound imaging of breast cancer cells in vitro.
Liu J, Li J, Rosol TJ, Pan X, Voorhees JL.
Phys Med Biol. 2007 Aug 21;52(16):4739-47.
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Disease-specific enhanced imaging through a targeted agent promises to improve the specificity of medical ultrasound. Nanoparticles may provide unique advantages for targeted ultrasound imaging due to their novel physical and surface properties. In this study, we examined a nanoparticle agent developed from a biodegradable polymer, polylactic acid (PLA). The nanoparticles (mean diameter = 250 nm) were surface conjugated to an anti-Her2 antibody (i.e., Herceptin) for specific binding to breast cancer cells that overexpress Her2 receptors. We examined the targeting specificity and the resultant ultrasound enhancement in Her2-positive and negative cells. Flow cytometry and confocal imaging were used to assess the nanoparticle-cell binding. Her2-positive cells demonstrated substantial staining after incubation with nanoparticle/antibody conjugates, while minimal staining was found in Her2-negative cells, indicating receptor-specific binding of the conjugated PLA nanoparticles. In high-resolution ultrasound B-mode images, the average gray scale of the Her2-positive cells was consistently and significantly higher after nanoparticle treatment (133 +/- 4 in treated cells versus 109 +/- 4 in control, p < 0.001, n = 5), while no difference was detected in the cells that did not overexpress the receptors (117 +/- 3 in treated cells versus 118 +/- 5 in control). In conclusion, the feasibility of using targeted nanoparticles to enhance ultrasonic images was demonstrated in vitro. This may be a promising approach to target cancer biomarkers for site-specific ultrasound imaging.

Use of lanthanide-grafted inorganic nanoparticles as effective contrast agents for cellular uptake imaging.
Voisin P, Ribot EJ, Miraux S, Bouzier-Sore AK, Lahitte JF, Bouchaud V, Mornet S, Thiaudière E, Franconi JM, Raison L, Labrugère C, Delville MH.
Bioconjug Chem. 2007 Jul-Aug;18(4):1053-63.
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The improvement of commonly used Gd3+ -based MRI agents requires the design of new systems with optimized in vivo efficacy, pharmacokinetic properties, and specificity. To design these contrast agents, two parameters are usually considered: increasing the number of coordinated water molecules or increasing the rotational correlation time by increasing molecular weight and size. This has been achieved by noncovalent or covalent binding of low-molecular weight Gd3+ chelates to macromolecules or polymers. The grafting of these high-spin paramagnetic gadolinium chelates on metal oxide nanoparticles (SiO2, Al2O3) is proposed. This new synthetic strategy presents at least two main advantages: (1) a high T1-relaxivity for MRI with a 275% increase of the MRI signal and (2) the ability of nanoparticles to be internalized in cells. Results indicate that these new contrast agents lead to a huge reconcentration of Gd3+ paramagnetic species inside microglial cells. This reconcentration phenomenon gives rise to high signal-to-noise ratios on MR images of cells after particle internalization, from 1.4 to 3.75, using Al2O3 or SiO2 particles, respectively. The properties of these new particles will be further used to get new insight into gene therapy against glioma, using microglial cells as vehicles to simultaneously transport a suicide gene and contrast agents. Since microglia are chemoattracted to brain tumors, the presence of these new contrast agents inside the cells will lead to a better MRI determination of the in vivo location, shape, and borders of the tumors. These Gd3+-loaded microglia can therefore provide effective localization of tumors by MRI before applying any therapeutic treatment. The rate of carcinoma remission following a suicide gene strategy is also possible.

On-off regulation of 19F magnetic resonance signals based on pH-sensitive PEGylated nanogels for potential tumor-specific smart 19F MRI probes.
Oishi M, Sumitani S, Nagasaki Y.
Bioconjug Chem. 2007 Sep-Oct;18(5):1379-82.
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The pH-sensitive PEGylated nanogels constructed from tethered PEG chains and a polyamine gel core containing 19F compounds showed remarkable on-off regulation of 19F MR (magnetic resonance) signals in response to the extracellular pH (6.5) of the tumor environment, even in the presence of 90% fetal bovine serum, due to the increase in the molecular motion of the 19F compounds through the hydrophilic-hydrophobic (volume-phase) transition of the polyamine gel core. Eventually, an appreciably enhanced 19F MR signal at an extremely low 19F compound concentration (approximately 55 microM) was achieved, demonstrating the utility of these nanogels as solid tumor-specific smart 19F MRI probes.

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.
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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.

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.
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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.

Radiopaque iodinated polymeric nanoparticles for X-ray imaging applications.
Galperin A, Margel D, Baniel J, Dank G, Biton H, Margel S.
Biomaterials. 2007 Oct;28(30):4461-8.
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Iodinated radiopaque polymeric nanoparticles of sizes ranging between 30 and 350 nm were formed by emulsion polymerization of the monomer 2-methacryloyloxyethyl(2,3,5-triiodobenzoate) in the presence of sodium dodecyl sulfate as surfactant and potassium persulfate as initiator. The influence of various polymerization parameters, e.g., monomer, initiator and surfactant concentrations on the molecular weight, polymerization yield, size and size distribution of the particles was elucidated. Characterization of these iodinated nanoparticles was accomplished by routine methods such as FTIR, 1H NMR, TEM, TGA, DSC, GPC and light scattering. These polymeric nanoparticles are composed of ca. 58% by weight iodine, and are therefore expected to possess significant radiopaque nature. In vitro radiopacity of the iodinated nanoparticles of 30.6+/-5.0 nm diameter, dispersed in water and in the dry state, was demonstrated with a CT scanner. In vivo CT-imaging performed in a dog model by intravenous administration of the uniform 30.6+/-5.0 nm diameter radiopaque nanoparticles dispersed in saline demonstrated significant enhanced visibility of lymph nodes, liver, kidney and spleen. These results indicate that these nanoparticles may be useful as new efficient contrast agents for X-ray imaging applications.

Self-Assembling Nanoparticles Image Tumor Cells.
Kim JS, Rieter WJ, Taylor KML, An H, Lin W, Lin W.
J. Am. Chem. Soc. 2007 July 25;129(29):8962-63.
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A layer-by-layer (LbL) polyelectrolyte deposition strategy is used to prepare multifunctional nanoparticles (MFNPs) with multimodal imaging capabilities. Alternating treatment of hybrid silica nanoparticles (NP0) containing a luminescent [Ru(bpy)3]Cl2 core and anionic monolayer coating of the Gd-(siloxylpropyl)diethylenetriamine tetraacetate (Gd-DTTA) complex with cationic Gd(III)-DOTA oligomer 1 and anionic poly(styrene sulfonate) (PSS) led to the deposition of multilayers of 1 and PSS via electrostatic interactions. This LbL deposition technique offers a superb strategy for the assembly of hybrid nanoparticles with imbedded luminophores and very high MR relaxivities. The PSS-terminated multilayered nanoparticles can be noncovalently functionalized with targeting peptides that carry positive charges under physiological conditions via electrostatic interactions to lead to cancer-specific MFNPs for optical and MR imaging of HT-29 human colon cancer cells. The generality of this approach should allow the design of imaging and/or therapeutic MFNPs that can specifically target a wide range of diseased cells.

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.
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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.

Nanoprobes with near-infrared persistent luminescence for in vivo imaging.
le Masne de Chermont Q, Chanéac C, Seguin J, Pellé F, Maîtrejean S, Jolivet JP, Gourier D, Bessodes M, Scherman D.
Proc Natl Acad Sci U S A. 2007 May 29;104(22):9266-71.
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Fluorescence is increasingly used for in vivo imaging and has provided remarkable results. Yet this technique presents several limitations, especially due to tissue autofluorescence under external illumination and weak tissue penetration of low wavelength excitation light. We have developed an alternative optical imaging technique by using persistent luminescent nanoparticles suitable for small animal imaging. These nanoparticles can be excited before injection, and their in vivo distribution can be followed in real-time for more than 1 h without the need for any external illumination source. Chemical modification of the nanoparticles' surface led to lung or liver targeting or to long-lasting blood circulation. Tumor mass could also be identified on a mouse model.

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.

In vivo imaging of siRNA delivery and silencing in tumors.
Medarova Z, Pham W, Farrar C, Petkova V, Moore A.
Nat Med. 2007 Mar;13(3):372-7.
[ expand abstract ]

With the increased potential of RNA interference (RNAi) as a therapeutic strategy, new noninvasive methods for detection of siRNA delivery and silencing are urgently needed. Here we describe the development of dual-purpose probes for in vivo transfer of siRNA and the simultaneous imaging of its accumulation in tumors by high-resolution magnetic resonance imaging (MRI) and near-infrared in vivo optical imaging (NIRF). These probes consisted of magnetic nanoparticles labeled with a near-infrared dye and covalently linked to siRNA molecules specific for model or therapeutic targets. Additionally, these nanoparticles were modified with a membrane translocation peptide for intracellular delivery. We show the feasibility of in vivo tracking of tumor uptake of these probes by MRI and optical imaging in two separate tumor models. We also used proof-of-principle optical imaging to corroborate the efficiency of the silencing process. These studies represent the first step toward the advancement of siRNA delivery and imaging strategies, essential for cancer therapeutic product development and optimization.

Hybrid gadolinium oxide nanoparticles: multimodal contrast agents for in vivo imaging.
Bridot JL, Faure AC, Laurent S, Rivière C, Billotey C, Hiba B, Janier M, Josserand V, Coll JL, Elst LV, Muller R, Roux S, Perriat P, Tillement O.
J Am Chem Soc. 2007 Apr 25;129(16):5076-84.
[ expand abstract ]

Luminescent hybrid nanoparticles with a paramagnetic Gd2O3 core were applied as contrast agents for both in vivo fluorescence and magnetic resonance imaging. These hybrid particles were obtained by encapsulating Gd2O3 cores within a polysiloxane shell which carries organic fluorophores and carboxylated PEG covalently tethered to the inorganic network. Longitudinal proton relaxivities of these particles are higher than the positive contrast agents like Gd-DOTA which are commonly used for clinical magnetic resonance imaging. Moreover these particles can be followed up by fluorescence imaging. This study revealed that these particles suited for dual modality imaging freely circulate in the blood vessels without undesirable accumulation in lungs and liver.

Simultaneous two-color spectral fluorescence lymphangiography with near infrared quantum dots to map two lymphatic flows from the breast and the upper extremity.
Hama Y, Koyama Y, Urano Y, Choyke PL, Kobayashi H.
Breast Cancer Res Treat. 2007 May;103(1):23-8.
[ expand abstract ]

Due to their small size and poor access, the lymphatic function has been difficult to study in vivo. Especially difficult is the mapping of lymphatic drainage from two basins into the same node. Quantum dots can be used to perform multicolor images with high fluorescent intensity and are of a nano-size size suitable for lymphatic imaging via direct interstitial injection. Here we show simultaneous two-color in vivo wavelength-resolved spectral fluorescence lymphangiography using two near infrared quantum dots with different emission spectra, which allow non-invasive and simultaneous visualization of two separate lymphatic flows draining the breast and the upper extremity and variations in the drainage patterns and the water sheds within the axillary node. Two-color spectral fluorescence lymphangiography can provide insight into mechanisms of drainage from different lymphatic basins that may lead to sentinel lymph nodes detection of the breast cancer as well as prevention of complications such as lymphedema of the arm.

Ligand conjugated low-density lipoprotein nanoparticles for enhanced optical cancer imaging in vivo.
Chen J, Corbin IR, Li H, Cao W, Glickson JD, Zheng G.
J. Am. Chem. Soc. 2007 May 9;129(18):5798-99.
[ expand abstract ]

LDL particles are high-capacity nanocarriers with precisely controlled size and are naturally biocompatible, biodegradable, and nonimmunogenic. However, their utilities as drug carriers are limited by the narrow purview of LDL receptor-positive tumors. Here, we synthsized a ligand-conjugated, NIR-labeled LDL that enables the first in vivo demonstration of rerouting LDL from LDL receptors to selected alternate receptors, thus drastically expanding the range of using LDL particles as nanocarriers for in vivo cancer imaging and treatment.

A paramagnetic nanoprobe to detect tumor cell death using magnetic resonance imaging.
Neves AA, Krishnan AS, Kettunen MI, Hu DE, Backer MM, Davletov B, Brindle KM.
Nano Lett. 2007 May;7(5):1419-23.
[ expand abstract ]

A 110 kDa (ca. 5 nm in diameter) bivalent paramagnetic nanoprobe for detecting cell death using magnetic resonance imaging (MRI) is described, in which two biotinylated C2A domains of the protein synaptotagmin-I were complexed with a single avidin molecule, which had been labeled with gadolinium chelates. This nanoprobe bound with high affinity and specificity to the phosphatidylserine exposed by dying cells and was demonstrated to allow MRI detection of apoptotic tumor cells in vitro.

Imaging of Vx-2 rabbit tumors with alpha(nu)beta3-integrin-targeted 111In nanoparticles.
Hu G, Lijowski M, Zhang H, Partlow KC, Caruthers SD, Kiefer G, Gulyas G, Athey P, Scott MJ, Wickline SA, Lanza GM.
Int J Cancer. 2007 May 1;120(9):1951-7.
[ expand abstract ]

Earlier tumor detection can improve 5-year survival of patients, particularly among those presenting with cancers less than 1 cm in diameter. alpha(nu)beta(3)-Targeted (111)In nanoparticles (NP) were developed and studied for detection of tumor angiogenesis. Studies were conducted in New Zealand white rabbits implanted 12 days earlier with Vx-2 tumor. alpha(nu)beta(3)-Targeted (111)In/NP bearing approximately 10 (111)In/NP vs. approximately 1 (111)In/NP nuclide payloads were compared to nontargeted radiolabeled control particles. In vivo competitive binding studies were used to assess ligand-targeting specificity. alpha(nu)beta(3)-Integrin-targeted NP with approximately 10 (111)In/NP provided better (p < 0.05) tumor-to-muscle ratio contrast (6.3 +/- 0.2) than approximately 1 (111)In/NP (5.1 +/- 0.1) or nontargeted particles with approximately 10 (111)In/NP (3.7 +/- 0.1) over the initial 2-hr postinjection. At 18 hr, mean tumor activity in rabbits receiving alpha(nu)beta(3)-integrin-targeted NP was 4-fold higher than the nontargeted control. Specificity of the NP for the tumor neovasculature was supported by in vivo competition studies and by fluorescence microscopy of alpha(nu)beta(3)-targeted fluorescent-labeled NP. Biodistribution studies revealed that the primary clearance organ in rabbits as a %ID/g tissue was the spleen. Circulatory half-life (t(1/2)beta) was estimated to be approximately 5 hr using a 2-compartment model. alpha(nu)beta(3)-Targeted (111)In perfluorocarbon NP may provide a clinically useful tool for sensitively detecting angiogenesis in nascent tumors, particularly in combination with secondary high-resolution imaging modalities, such as MRI.

Quantum Rod Bioconjugates as Targeted Probes for Confocal and Two-Photon Fluorescence Imaging of Cancer Cells.
Yong KT, Qian J, Roy I, Lee HH, Bergey EJ, Tramposch KM, He S, Swihart MT, Maitra A, Prasad PN.
Nano Lett. 2007 Feb 9; [Epub ahead of print].
[ expand abstract ]

Live cell imaging using CdSe/CdS/ZnS quantum rods (QRs) as targeted optical probes is reported. The QRs, synthesized in organic media using a binary surfactant mixture, were dispersed in aqueous media using mercaptoundecanoic acid (MUA) and lysine. Transferrin (Tf) was linked to the QRs to produce QR-Tf bioconjugates that were used for targeted in vitro delivery to a human cancer cell line. Confocal and two-photon imaging were used to confirm receptor-mediated uptake of QR-Tf conjugates into the HeLa cells, which overexpress the transferrin receptor (TfR). Uptake was not observed with QRs that lacked Tf functionalization or with cells that were presaturated with free Tf and then treated with Tf-functionalized QRs.

Spatial dose distributions in solid tumors from (186)Re transported by liposomes using HS radiochromic media.
Medina LA, Goins B, Rodriguez-Villafuerte M, Bao A, Martinez-Davalos A, Awasthi V, Galvan OO, Santoyo C, Phillips WT, Brandan ME.
Eur J Nucl Med Mol Imaging. 2007 Feb 8; [Epub ahead of print].
[ expand abstract ]

PURPOSE: A procedure for the measurement of spatial dose rate distribution of beta particles emitted by (186)Re-liposomes in tumoral tissue, using HS GafChromic films, is presented. METHODS: HNSCC xenografts were intratumorally injected with 3.7 or 11.1 MBq of (186)Re-liposomes, and planar gamma camera images were acquired to determine the liposome retention in the tumor. After imaging, rats were sacrificed and tumors were excised and processed in slices; HS film sections were placed between slices and the tumor lobe was reassembled. Tumors and films were kept in the dark at 4 degrees C for 18 h. After irradiation, films were removed and response was read using a transmission scanner. Films were analyzed to determine two-dimensional spatial dose rate distributions and cumulative dose volume histograms. Dose rate distributions were quantified using a (60)Co calibration curve, the (186)Re physical half-life, and a perturbation factor that takes into account the effect of the film protective layer. RESULTS: Dose rate distributions are highly heterogeneous with maximal dose rates about 0.4 Gy h(-1) in tumors injected with 3.7 MBq and 1.3 Gy h(-1) in tumors injected with 11.1 MBq. Dose volume histograms showed dose distributed in more than 95% and 80% of the tumor when injected with the lower and the higher activity, respectively. CONCLUSION: The described procedures and techniques have shown the potential and utility of HS GafChromic film for determination of dose rate distributions in solid tumors injected intratumorally with (186)Re-liposomes. The film's structure and the liposomes' biodistribution must be taken into account to obtain quantitative dose measurements.

19F magnetic resonance imaging for stem/progenitor cell tracking with multiple unique perfluorocarbon nanobeacons.
Partlow KC, Chen J, Brant JA, Neubauer AM, Meyerrose TE, Creer MH, Nolta JA, Caruthers SD, Lanza GM, Wickline SA.
FASEB J. 2007 Feb 6; [Epub ahead of print].
[ expand abstract ]

MRI has been employed to elucidate the migratory behavior of stem/progenitor cells noninvasively in vivo with traditional proton ((1)H) imaging of iron oxide nanoparticle-labeled cells. Alternatively, we demonstrate that fluorine ((19)F) MRI of cells labeled with different types of liquid perfluorocarbon (PFC) nanoparticles produces unique and sensitive cell markers distinct from any tissue background signal. To define the utility for cell tracking, mononuclear cells harvested from human umbilical cord blood were grown under proendothelial conditions and labeled with nanoparticles composed of two distinct PFC cores (perfluorooctylbromide and perfluoro-15-crown-5 ether). The sensitivity for detecting and imaging labeled cells was defined on 11.7T (research) and 1.5T (clinical) scanners. Stem/progenitor cells (CD34(+)CD133(+)CD31(+)) readily internalized PFC nanoparticles without aid of adjunctive labeling techniques, and cells remained functional in vivo. PFC-labeled cells exhibited distinct (19)F signals and were readily detected after both local and intravenous injection. PFC nanoparticles provide an unequivocal and unique signature for stem/progenitor cells, enable spatial cell localization with (19)F MRI, and permit quantification and detection of multiple fluorine signatures via (19)F MR spectroscopy. This method should facilitate longitudinal investigation of cellular events in vivo for multiple cell types simultaneously.

Fluorescence Analysis with Quantum Dot Probes for Hepatoma Under One- and Two-Photon Excitation.
Yu X, Chen L, Deng Y, Li K, Wang Q, Li Y, Xiao S, Zhou L, Luo X, Liu J, Pang D.
J Fluoresc. 2007 Feb 6; [Epub ahead of print].
[ expand abstract ]

A new class of fluorescent probe produced by conjugating semiconductor quantum dots (QDs) with protein molecule is proposed as an alternative to conventional organic labels. However the fluorescence characteristics of the QD bioconjugates are not clear while they are excitied with one- or two-photon laser pulse. We synthesized specific immunofluorescent probes by linking QDs to alpha fetoprotein (AFP) antibody for specific binding alpha-fetoprotein -an important marker for hepatocellular carcinoma cell lines, and archived specific fluorescence detection with the QDs-Anti-AFP in nude mice. Then, we have analyzed the fluorescence characteristics of QDs-Anti-AFP and original QDs both under one- and two-photon excitations. The results demonstrated that QDs-Anti-AFP's fluorescent spectral and lifetime haven't varied much from that of original QDs. Moreover, QDs-Anti-AFP have exhibited higher fluorescence efficiency than QDs under two-photon examination.

Moving in the right direction-Nanoimaging in cancer cell motility and metastasis.
Soon L, Braet F, Condeelis J.
Microsc Res Tech. 2007 Feb 5; [Epub ahead of print].
[ expand abstract ]

Although genetic and protein manipulations have been the cornerstone for the study and understanding of biological processes for many decades, complimentary nanoscale observations have only more recently been achieved in the live-imaging mode. It is at the nano measurement level that events such as protein-protein interactions, enzymatic conversions, and single-molecule stochastic behavior take place. Therefore, nanoscale observations allow us to reinterpret knowledge from large-scale or bulk techniques and gain new insight into molecular events that has cellular, tissue, and organismal phenotypic manifestations. This review identifies pertinent questions relating to the sensing and directional component of cancer cell chemotaxis and discusses the platforms that provide insight into the molecular events related to cell motility. The study of cell motility at the molecular imaging level often necessitates the use of devices such as microinjection, microfluidics, in vivo/intravital and in vitro chemotaxis assays, as well as fluorescence methods like uncaging and FRET. The micro- and nanofabricated devices that facilitate these techniques and their incorporation to specialized microscopes such as the multiphoton, AFM, and TIR-FM, for high-resolution imaging comprise the nanoplatforms used to explore the mechanisms of carcinogenesis. In real-time observations, within a milieu of physiological protein concentrations, true states of dynamic and kinetic fluxes can be monitored.

Imaging of Vx-2 rabbit tumors with alpha(nu)beta(3)-integrin-targeted (111)In nanoparticles.
Hu G, Lijowski M, Zhang H, Partlow KC, Caruthers SD, Kiefer G, Gulyas G, Athey P, Scott MJ, Wickline SA, Lanza GM.
Int J Cancer. 2007 Feb 2; [Epub ahead of print].
[ expand abstract ]

Earlier tumor detection can improve 5-year survival of patients, particularly among those presenting with cancers less than 1 cm in diameter. alpha(nu)beta(3)-Targeted (111)In nanoparticles (NP) were developed and studied for detection of tumor angiogenesis. Studies were conducted in New Zealand white rabbits implanted 12 days earlier with Vx-2 tumor. alpha(nu)beta(3)-Targeted (111)In/NP bearing approximately approximately 10 (111)In/NP vs. approximately approximately 1 (111)In/NP nuclide payloads were compared to nontargeted radiolabeled control particles. In vivo competitive binding studies were used to assess ligand-targeting specificity. alpha(nu)beta(3)-Integrin-targeted NP with approximately approximately 10 (111)In/NP provided better (p < 0.05) tumor-to-muscle ratio contrast (6.3 +/- 0.2) than approximately approximately 1 (111)In/NP (5.1 +/- 0.1) or nontargeted particles with approximately approximately 10 (111)In/NP (3.7 +/- 0.1) over the initial 2-hr postinjection. At 18 hr, mean tumor activity in rabbits receiving alpha(nu)beta(3)-integrin-targeted NP was 4-fold higher than the nontargeted control. Specificity of the NP for the tumor neovasculature was supported by in vivo competition studies and by fluorescence microscopy of alpha(nu)beta(3)-targeted fluorescent-labeled NP. Biodistribution studies revealed that the primary clearance organ in rabbits as a %ID/g tissue was the spleen. Circulatory half-life (t(1/) (2) (beta)) was estimated to be approximately approximately 5 hr using a 2-compartment model. alpha(nu)beta(3)-Targeted (111)In perfluorocarbon NP may provide a clinically useful tool for sensitively detecting angiogenesis in nascent tumors, particularly in combination with secondary high-resolution imaging modalities, such as MRI.

In vivo tumor targeting and radionuclide imaging with self-assembled nanoparticles: mechanisms, key factors, and their implications.
Cho YW, Park SA, Han TH, Son DH, Park JS, Oh SJ, Moon DH, Cho KJ, Ahn CH, Byun Y, Kim IS, Kwon IC, Kim SY.
Biomaterials. 2007 Feb;28(6):1236-47.
[ expand abstract ]

The development of more selective delivery systems for cancer diagnosis and chemotherapy is one of the most important goals of current anticancer research. The purpose of this study is to evaluate various self-assembled nanoparticles as candidates to shuttle radionuclide and/or drugs into tumors and to investigate the mechanisms underlying the tumor targeting with self-assembled nanoparticles. By combining different hydrophobic moieties and hydrophilic polymer backbones, various self-assembled nanoparticles were prepared, and their in vivo distributions in tumor-bearing mice were studied by radionuclide imaging. One type of nanoparticles (fluorescein isothiocyanate-conjugated glycol chitosan (FGC) nanoparticles) exhibited highly selective tumoral localization. Scintigraphic images obtained 1 day after the intravenous injection of FGC nanoparticles clearly delineated the tumor against adjacent tissues. The mechanisms underlying the tumor targeting with self-assembled nanoparticles were investigated in terms of the physicochemical properties of nanoparticles and tumor microenvironments. FGC nanoparticles were preferentially localized in perivascular regions, implying their extravasation to tumors through the hyperpermeable tumor vasculature. The magnitude and pattern of tumoral distribution of self-assembled nanoparticles were influenced by several key factors--(i) in vivo colloidal stability: nanoparticles should maintain their intact nanostructures in vivo for a long period of time, (ii) particle size, (iii) intracellular uptake of nanoparticle: fast cellular uptake greatly facilitates the tumor targeting, (iv) tumor angiogenesis: pathological angiogenesis permits access of nanoparticles to tumors. We believe that this work can provide insight for the engineering of nanoparticles and be extended to cancer therapy and diagnosis, so as to deliver multiple therapeutic agents and imaging probes at high local concentrations.

MR lymphangiography using dendrimer-based contrast agents: A comparison at 1.5T and 3.0T.
Hama Y, Bernardo M, Regino CA, Koyama Y, Brechbiel MW, Krishna MC, Choyke PL, Kobayashi H.
Magn Reson Med. 2007 Feb;57(2):431-6.
[ expand abstract ]

Most macromolecular contrast agents (CAs) show lower r(1) and higher r(2) relaxivities at 3.0T than at 1.5T. MR lymphangiography in mice using a macromolecular G6 dendrimer-based CA was serially performed and compared at both 1.5T and 3.0T. The r(1) and r(2) relaxivities of the G6 CA were 25 and 78/s/mM at 1.5T and 17 and 82/s/mM at 3.0T, respectively. The lymph node (LN)-to-fat ratios (LN signal intensity (SI)/fat SI) of T(1)-weighted 3D-fast spoiled gradient-echo (3D-FSPGR) were 3.2 +/- 0.4 (mean +/- standard deviation (SD)) at 1.5T and 2.7 +/- 0.3 at 3.0T (P = 0.021), and the LN-to-fat ratios of T(2)/T(1)-weighted 3D-fast imaging employing steady-state acquisition with phase cycling (3D-FIESTA-C) were 1.8 +/- 0.2 at 1.5T and 1.2 +/- 0.4 at 3.0T (P = 0.003). Although 3D-FSPGR successfully delineated the LNs at both 1.5T and 3.0T, 3D-FIESTA-C at 3.0T failed to visualize the LNs. Magn Reson Med 57:431-436, 2007.

Surface modification of magnetite nanoparticles using lactobionic acid and their interaction with hepatocytes.
Kamruzzaman Selim KM, Ha YS, Kim SJ, Chang Y, Kim TJ, Ho Lee G, Kang IK.
Biomaterials. 2007 Feb;28(4):710-6.
[ expand abstract ]

In the current study, superparamagnetic magnetite nanoparticles were surface-modified with lactobionic acid (LA) to improve their intracellular uptake and ability to target hepatocytes. Maltotrionic acid (MA)-modified nanoparticles were also synthesized as a control. Cell culture experiment showed that LA-modified nanoparticles were internalized into hepatocytes and atomic absorption spectrometer (AAS) measurement indicated that the uptake amount of LA-modified magnetite into hepatocytes was higher than that of unmodified and MA-modified nanoparticles. LA-modified nanoparticle solution was injected in rabbit and the magnetic resonance (MR) images obtained showed that LA-coated nanoparticles were selectively accumulated onto the hepatocytes. This result demonstrates that the LA-modified magnetite nanoparticles have a great potential to be used as contrast agent for liver diagnosis.

Sentinel Lymph Node Imaging Using Quantum Dots in Mouse Tumor Models.
Ballou B, Ernst LA, Andreko S, Harper T, Fitzpatrick JA, Waggoner AS, Bruchez MP.
Bioconjug Chem. 2007 Jan 31; [Epub ahead of print].
[ expand abstract ]

We demonstrate that quantum dots injected into two model tumors rapidly migrate to sentinel lymph nodes. PEG-coated quantum dots having terminal carboxyl, amino, or methoxyl groups all migrated from the tumor to surrounding lymph nodes similarly. Passage from the tumor through lymphatics to adjacent nodes could be visualized dynamically through the skin; at least two nodes could usually be defined. Imaging during necropsy confirmed confinement of the quantum dots to the lymphatic system and demonstrated easy tagging of sentinel lymph nodes for pathology. Examination of the sentinel nodes identified by quantum dot localization showed that at least some contained metastatic tumor foci.

Nanoshell Magnetic Resonance Imaging Contrast Agents.
Su CH, Sheu HS, Lin CY, Huang CC, Lo YW, Pu YC, Weng JC, Shieh DB, Chen JH, Yeh CS.
J Am Chem Soc. 2007 Jan 31; [Epub ahead of print].
[ expand abstract ]

Nanocontrast agents have great potential in magnetic resonance (MR) molecular imaging applications for clinical diagnosis. We synthesized Au3Cu1 (gold and copper) nanoshells that showed a promising MR contrast effect. For in vitro MR images, the large proton r1 relaxivities brightened T1-weighted images. As for the proton-dephasing effect in T2, Au3Cu1 lightened MR images at the low concentration of 0.125 mg mL-1 (3.84 x 10-7 mM), and then the signal continuously decreased as the concentration increased. For in vivo MR imaging, Au3Cu1 nanocontrast agents enhanced the contrast of blood vessels and suggested their potential use in MR angiography as blood-pool agents. We propose that (1) the cooperativity originating from the form of the nanoparticles and (2) the large surface area coordinated to water from their porous hollow morphology are important for efficient relaxivity. In a cytotoxicity and animal survival assay, Au3Cu1 nanocontrast agents showed a dose-dependent toxic effect: the viability rate of experimental mice reached 83% at a dose of 20 mg kg-1 and as much as 100% at 2 mg kg-1.

Gum Arabic as a Phytochemical Construct for the Stabilization of Gold Nanoparticles: In Vivo Pharmacokinetics and X-ray-Contrast-Imaging Studies.
Kattumuri V, Katti K, Bhaskaran S, Boote EJ, Casteel SW, Fent GM, Robertson DJ, Chandrasekhar M, Kannan R, Katti KV.
Small. 2007 Jan 29;3(2):333-341 [Epub ahead of print].
[ expand abstract ]

Gold nanoparticles (AuNPs) have exceptional stability against oxidation and therefore will play a significant role in the advancement of clinically useful diagnostic and therapeutic nanomedicines. Despite the huge potential for a new generation of AuNP-based nanomedicinal products, nontoxic AuNP constructs and formulations that can be readily administered site-specifically through the intravenous mode, for diagnostic imaging by computed tomography (CT) or for therapy via various modalities, are still rare. Herein, we report results encompassing: 1) the synthesis and stabilization of AuNPs within the nontoxic phytochemical gum-arabic matrix (GA-AuNPs); 2) detailed in vitro analysis and in vivo pharmacokinetics studies of GA-AuNPs in pigs to gain insight into the organ-specific localization of this new generation of AuNP vector, and 3) X-ray CT contrast measurements of GA-AuNP vectors for potential utility in molecular imaging. Our results demonstrate that naturally occurring GA can be used as a nontoxic phytochemical construct in the production of readily administrable biocompatible AuNPs for diagnostic and therapeutic applications in nanomedicine.

Optical and Magnetic Resonance Imaging of Cell Death and Platelet Activation Using Annexin A5-Functionalized Quantum Dots.
Prinzen L, Miserus RJ, Dirksen A, Hackeng TM, Deckers N, Bitsch NJ, Megens RT, Douma K, Heemskerk JW, Kooi ME, Frederik PM, Slaaf DW, Zandvoort MA, Reutelingsperger CP.
Nano Lett. 2007 Jan 10;7(1):93-100.
[ expand abstract ]

A quantum-dot-based nanoparticle is presented, allowing visualization of cell death and activated platelets with fluorescence imaging and MRI. The particle exhibits intense fluorescence and a large MR relaxivity (r1) of 3000-4500 mM-1 s-1 per nanoparticle due to a newly designed construct increasing the gadolinium-DTPA load. The nanoparticle is suitable for both anatomic and subcellular imaging of structures in the vessel wall and is a promising bimodal contrast agent for future in vivo imaging studies.

Magnetic resonance imaging of temperature-sensitive liposome release: drug dose painting and antitumor effects.
Ponce AM, Viglianti BL, Yu D, Yarmolenko PS, Michelich CR, Woo J, Bally MB, Dewhirst MW.
J Natl Cancer Inst. 2007 Jan 3;99(1):53-63.
[ expand abstract ]

BACKGROUND: In preclinical studies, lysolipid-based temperature-sensitive liposomes (LTSLs) containing chemotherapy drugs administered in combination with local hyperthermia have been found to increase tumor drug concentrations and improve antitumor efficacy of the drugs. We used a novel magnetic resonance imaging (MRI) method to measure the temporal and spatial patterns of drug delivery in a rat fibrosarcoma model during treatment with LTSLs containing doxorubicin and an MRI contrast agent (manganese) (Dox/Mn-LTSLs) administered at different times with respect to hyperthermia. METHODS: Rats bearing 10- to 12-mm fibrosarcomas (n = 6-7 per group) were treated with Dox/Mn-LTSLs (at a dose of 5 mg doxorubicin/kg body weight) before and/or during 60 minutes of local tumor hyperthermia administered via a catheter inserted at the center of the tumor. Drug distribution was monitored continuously via MRI. Magnetic resonance changes were used to calculate intratumoral doxorubicin concentrations throughout treatment. Tumors were monitored until they reached five times their volume on the day of treatment or 60 days. Doxorubicin concentrations and times for tumors to reach five times their volume on the day of treatment were analyzed using the Kruskal-Wallis test and the Kaplan-Meier product-limit method, respectively. All statistical tests were two-sided. RESULTS: Administration of Dox/Mn-LTSLs before, during, and both before and during hyperthermia yielded central, peripheral, and uniform drug distributions, respectively. Doxorubicin accumulated more quickly and reached higher concentrations in the tumor when Dox/Mn-LTSLs were administered during hyperthermia than when administered before hyperthermia (rate: 9.8 versus 1.8 microg/min, difference = 8.0 microg/min, 95% confidence interval [CI] = 6.8 to 12.8 microg/min, P = .003; concentration: 15.1 versus 8.0 ng/mg, difference = 7.1 ng/mg, 95% CI = 3.6 to 10.6 ng/mg, P = .028). LTSL administered during hyperthermia also yielded the greatest antitumor effect, with a median time for tumors to reach five times their volume on the day of treatment of 34 days (95% CI = 30 days to infinity) compared with 18.5 days (95% CI = 16 to 23 days) for LTSL before hyperthermia and 22.5 days (95% CI = 15 to 25 days) for LTSL before and during hyperthermia. CONCLUSIONS: In this rat fibrosarcoma model, LTSLs were most effective when delivered during hyperthermia, which resulted in a peripheral drug distribution.

Artificially engineered magnetic nanoparticles for ultra-sensitive molecular imaging.
Lee JH, Huh YM, Jun YW, Seo JW, Jang JT, Song HT, Kim S, Cho EJ, Yoon HG, Suh JS, Cheon J.
Nat Med. 2007 Jan;13(1):95-9.
[ expand abstract ]

Successful development of ultra-sensitive molecular imaging nanoprobes for the detection of targeted biological objects is a challenging task. Although magnetic nanoprobes have the potential to perform such a role, the results from probes that are currently available have been far from optimal. Here we used artificial engineering approaches to develop innovative magnetic nanoprobes, through a process that involved the systematic evaluation of the magnetic spin, size and type of spinel metal ferrites. These magnetism-engineered iron oxide (MEIO) nanoprobes, when conjugated with antibodies, showed enhanced magnetic resonance imaging (MRI) sensitivity for the detection of cancer markers compared with probes currently available. Also, we successfully visualized small tumors implanted in a mouse. Such high-performance, nanotechnology-based molecular probes could enhance the ability to visualize other biological events critical to diagnostics and therapeutics.

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2006

Targeting of cancer cells with ferrimagnetic ferritin cage nanoparticles.
Uchida M, Flenniken ML, Allen M, Willits DA, Crowley BE, Brumfield S, Willis AF, Jackiw L, Jutila M, Young MJ, Douglas T.
J Am Chem Soc. 2006 Dec 27;128(51):16626-33.
[ expand abstract ]

Protein cage architectures such as virus capsids and ferritins are versatile nanoscale platforms amenable to both genetic and chemical modification. Incorporation of multiple functionalities within these nanometer-sized protein architectures demonstrate their potential to serve as functional nanomaterials with applications in medical imaging and therapy. In the present study, we synthesized an iron oxide (magnetite) nanoparticle within the interior cavity of a genetically engineered human H-chain ferritin (HFn). A cell-specific targeting peptide, RGD-4C which binds alphavbeta3 integrins upregulated on tumor vasculature, was genetically incorporated on the exterior surface of HFn. Both magnetite-containing and fluorescently labeled RGD4C-Fn cages bound C32 melanoma cells in vitro. Together these results demonstrate the capability of a genetically modified protein cage architecture to serve as a multifunctional nanoscale container for simultaneous iron oxide loading and cell-specific targeting.

Accumulation of liposome with Sialyl Lewis X to inflammation and tumor region: Application to in vivo bio-imaging.
Hirai M, Minematsu H, Kondo N, Oie K, Igarashi K, Yamazaki N.
Biochem Biophys Res Commun. 2006 Dec 19; [Epub ahead of print].
[ expand abstract ]

We prepared the liposome binding Sialyl Lewis X (SLX) on the surface in order to specifically and efficiently deliver substances (fluorescent materials, chemical substances, proteins, genes, etc.) to inflammation or tumor regions. The liposome with SLX (SLX-Lipo-Cy5.5), in which fluorescent substance Cy5.5 was included, was administered intravenously to arthritis or Ehrlich Ascites Tumor (EAT) bearing mouse, and the accumulation of liposome was observed using two types of in vivo fluorescent imaging equipment. The result was that the accumulation of SLX-Lipo-Cy5.5 to inflammation or tumor regions was significantly higher than the control liposome without sugar chain (Lipo-Cy5.5) at 24 and 48h after administration. In addition, it was confirmed that this accumulation showed a shift of liposome from blood vessels to the surrounding tissues. Thus, it was proven that this liposome is useful not only as an in vivo bio-imaging reagent but also as a drug delivery system (DDS).

Dual-Mode Nanoparticle Probes for High-Performance Magnetic Resonance and Fluorescence Imaging of Neuroblastoma.
Lee JH, Jun YW, Yeon SI, Shin JS, Cheon J.
Angew Chem Int Ed Engl. 2006 Dec 11;45(48):8160-8162.
[ expand abstract ]

No abstract available

Recent advances in iron oxide nanocrystal technology for medical imaging.
Corot C, Robert P, Idee JM, Port M.
Adv Drug Deliv Rev. 2006 Dec 1;58(14):1471-504.
[ expand abstract ]

Superparamagnetic iron oxide particles (SPIO and USPIO) have a variety of applications in molecular and cellular imaging. Most of the recent research has concerned cellular imaging with imaging of in vivo macrophage activity. According to the iron oxide nanoparticle composition and size which influence their biodistribution, several clinical applications are possible: detection liver metastases, metastatic lymph nodes, inflammatory and/or degenerative diseases. USPIO are investigated as blood pool agents with T1 weighted sequence for angiography, tumour permeability and tumour blood volume or steady-state cerebral blood volume and vessel size index measurements using T2() weighted sequences. Stem cell migration and immune cell trafficking, as well as targeted iron oxide nanoparticles for molecular imaging studies, are at the stage of proof of concept, mainly in animal models.

Cervical lymph node metastases: MR imaging of gadofluorine M and monocrystalline iron oxide nanoparticle-47 in a rabbit model of head and neck cancer.
Choi SH, Han MH, Moon WK, Son KR, Won JK, Kim JH, Kwon BJ, Na DG, Weinmann HJ, Chang KH.
Radiology. 2006 Dec;241(3):753-62.
[ expand abstract ]

PURPOSE: To prospectively compare the accuracy of gadofluorine M with that of monocrystalline iron oxide nanoparticle (MION)-47 for the depiction of cervical lymph node metastases at magnetic resonance (MR) imaging in a rabbit model of head and neck cancer by using histologic analysis as the reference standard. MATERIALS AND METHODS: Experiments were approved by the animal care committee. VX2 carcinomas were implanted in both ears of 11 rabbits 4 weeks before MR imaging. T2-weighted, T2*-weighted, and T1-weighted MR images were acquired, and sequential T1-weighted MR imaging was performed immediately and 30 minutes after administration of gadofluorine M (0.05 mmol gadolinium per kilogram body weight). T2-weighted and T2*-weighted MR imaging were performed 24 hours after administration of MION-47 (2.6 mg iron per kilogram body weight). Gadofluorine M- and MION-47-enhanced MR imaging were performed separately and independently by two radiologists who had no knowledge of histopathologic results, and the presence of metastases in lymph nodes was evaluated. A receiver operating characteristic analysis was conducted to compare the diagnostic value of gadofluorine M- and MION-47-enhanced MR imaging. RESULTS: Metastases were confirmed in 20 of 77 lymph nodes at histopathologic analysis. The area under the curve was significantly greater for gadofluorine M-enhanced MR imaging (0.997 and 0.981 for readers 1 and 2, respectively) than for MION-47-enhanced MR imaging (0.889 and 0.846 for readers 1 and 2, respectively). For gadofluorine M-enhanced MR imaging, sensitivity was 100% for both readers and specificity was 89.5% for reader 1 and 87.7% for reader 2. For MION-47-enhanced MR imaging, sensitivity was 80.0% for both readers and specificity was 75.4% for reader 1 and 71.9% for reader 2. CONCLUSION: Gadofluorine M-enhanced MR imaging has higher accuracy for depicting lymph node metastases than does MION-47-enhanced MR imaging.

Comparison of lymphotropic nanoparticle-enhanced MRI sequences in patients with various primary cancers.
Saksena M, Harisinghani M, Hahn P, Kim J, Saokar A, King B, Weissleder R.
AJR Am J Roentgenol. 2006 Dec;187(6):W582-8.
[ expand abstract ]

OBJECTIVE: This study was performed to empirically evaluate T2-weighted fast spin-echo, moderately T2*-weighted gradient-refocused echo (GRE), and heavily T2*-weighted GRE sequences to determine which sequence is the most effective for nodal characterization on lymphotropic nanoparticle-enhanced MRI (LNMRI). MATERIALS AND METHODS: The study included 65 patients who had proven primary cancer and were scheduled for either surgical lymph node dissection or imaging-guided lymph node biopsy. All patients underwent LNMRI using T2-weighted fast spin-echo, moderately T2*-weighted GRE, and heavily T2*-weighted GRE sequences. Unequivocal correlation of histopathology and MRI could be made in 140 nodes and only these were included in the analysis. Two blinded reviewers performed qualitative analysis of the nodes. Alternative free-response receiver operating characteristic (ROC) curves with a continuous rating scale were plotted for each sequence for both reviewers and the diagnostic accuracy of fast spin-echo T2-weighted and GRE T2*-weighted images were compared by calculating the area under the curve (A(Z)). A two-tailed Student's t test was performed to test the significance (p < 0.05) of the differences between the ROC curves derived from the three sequences. RESULTS: Irrespective of reviewer experience, T2*-weighted sequences showed better nodal characterization when compared with T2-weighted sequences. For both reviewers, there was a statistically significant difference between the A(Z) for T2- and the two T2*-weighted sequences (p < 0.05). Neither reviewer showed a statistically significant difference between the two T2*-weighted sequences. CONCLUSION: GRE T2*-weighted sequences are superior for nodal characterization on LNMRI to fast spin-echo T2-weighted sequences. Imaging protocols for LNMRI should include fast spin-echo T2-weighted imaging for anatomic localization, but characterization of nodes should be based on their appearance on contrast-enhanced T2*-weighted images. The T2*-weighted images acquired with dual TE values, one of which is intermediate and the other longer, improve nodal characterization.

Targeted PARACEST nanoparticle contrast agent for the detection of fibrin.
Winter PM, Cai K, Chen J, Adair CR, Kiefer GE, Athey PS, Gaffney PJ, Buff CE, Robertson JD, Caruthers SD, Wickline SA, Lanza GM.
Magn Reson Med. 2006 Dec;56(6):1384-8.
[ expand abstract ]

A lipid-encapsulated perfluorocarbon nanoparticle molecular imaging contrast agent that utilizes a paramagnetic chemical exchange saturation transfer (PARACEST) chelate is presented. PARACEST agents are ideally suited for molecular imaging applications because one can switch the contrast on and off at will simply by adjusting the pulse sequence parameters. This obviates the need for pre- and postinjection images to define contrast agent binding. Spectroscopy (4.7T) of PARACEST nanoparticles revealed a bound water peak at 52 ppm, in agreement with results from the water-soluble chelate. Imaging of control nanoparticles showed no appreciable contrast, while PARACEST nanoparticles produced >10% signal enhancement. PARACEST nanoparticles were targeted to clots via antifibrin antibodies and produced a contrast-to-noise ratio (CNR) of 10 at the clot surface.

Emerging concepts in molecular MRI.
Sosnovik DE, Weissleder R.
Curr Opin Biotechnol. 2006 Nov 23; [Epub ahead of print].
[ expand abstract ]

Molecular magnetic resonance imaging (MRI) offers the potential to image some events at the cellular and subcellular level and many significant advances have recently been witnessed in this field. The introduction of targeted MR contrast agents has enabled the imaging of sparsely expressed biological targets in vivo. Furthermore, high-throughput screens of nanoparticle libraries have identified nanoparticles that act as novel contrast agents and which can be targeted with enhanced diagnostic specificity and range. Another class of magnetic nanoparticles have also been designed to image dynamic events; these act as 'switches' and could be used in vitro, and potentially in vivo, as biosensors. Other specialized MR probes have been developed to image enzyme activity in vivo. Lastly, the use of chemical exchange and off-resonance techniques have been developed, adding another dimension to the broad capabilities of molecular MRI and offering the potential of multispectral imaging. These and other advances in molecular MRI offer great promise for the future and have significant potential for clinical translation.

Vascular targeted nanoparticles for imaging and treatment of brain tumors.
Reddy GR, Bhojani MS, McConville P, Moody J, Moffat BA, Hall DE, Kim G, Koo YE, Woolliscroft MJ, Sugai JV, Johnson TD, Philbert MA, Kopelman R, Rehemtulla A, Ross BD.
Clin Cancer Res. 2006 Nov 15;12(22):6677-86.
[ expand abstract ]

PURPOSE: Development of new therapeutic drug delivery systems is an area of significant research interest. The ability to directly target a therapeutic agent to a tumor site would minimize systemic drug exposure, thus providing the potential for increasing the therapeutic index. EXPERIMENTAL DESIGN: Photodynamic therapy (PDT) involves the uptake of a sensitizer by the cancer cells followed by photoirradiation to activate the sensitizer. PDT using Photofrin has certain disadvantages that include prolonged cutaneous photosensitization. Delivery of nanoparticles encapsulated with photodynamic agent specifically to a tumor site could potentially overcome the drawbacks of systemic therapy. In this study, we have developed a multifunctional polymeric nanoparticle consisting of a surface-localized tumor vasculature targeting F3 peptide and encapsulated PDT and imaging agents. RESULTS: The nanoparticles specifically bound to the surface of MDA-435 cells in vitro and were internalized conferring photosensitivity to the cells. Significant magnetic resonance imaging contrast enhancement was achieved in i.c. rat 9L gliomas following i.v. nanoparticle administration. Serial magnetic resonance imaging was used for determination of pharmacokinetics and distribution of nanoparticles within the tumor. Treatment of glioma-bearing rats with targeted nanoparticles followed by PDT showed a significant improvement in survival rate when compared with animals who received PDT after administration of nontargeted nanoparticles or systemic Photofrin. CONCLUSIONS: This study reveals the versatility and efficacy of the multifunctional nanoparticle for the targeted detection and treatment of cancer.

Real-time Imaging and Quantification of Brain Delivery of Liposomes.
Krauze MT, Forsayeth J, Park JW, Bankiewicz KS.
Pharm Res. 2006 Nov;23(11):2493-504.
[ expand abstract ]

The surgical delivery of therapeutic agents into the parenchyma of the brain is problematic because it has been virtually impossible to know with any certainty where infused material is going, and how much to infuse. We have started to use liposomes loaded with Gadoteridol (GDL) as a tracer that allows us to follow infusions in real-time on magnetic resonance imaging (MRI). MRI allows precise tracking and measurement of liposomes loaded with markers and therapeutics. This review provides an overview of real-time delivery of liposomes to the central nervous system (CNS), and discusses the technical aspects of delivery, liposomes as colloidal systems of delivery, real-time distribution of liposomes in CNS, and quantification of liposome distribution. Our data suggests that real-time monitoring of liposomal drug infusion is likely to improve outcomes of clinical trials where convection-enhanced delivery (CED) is being used to target drugs to specific brain structures through limitation of systemic toxicity and reduction of side effects. This review is a summary of work done by our group over the past four years.

One-Pot Synthesis of PEGylated Ultrasmall Iron-Oxide Nanoparticles and Their in Vivo Evaluation as Magnetic Resonance Imaging Contrast Agents.
Lutz JF, Stiller S, Hoth A, Kaufner L, Pison U, Cartier R.
Biomacromolecules. 2006 Nov;7(11):3132-8.
[ expand abstract ]

A well-defined copolymer poly(oligo(ethylene glycol) methacrylate-co-methacrylic acid) P(OEGMA-co-MAA) was studied as a novel water-soluble biocompatible coating for superparamagnetic iron oxide nanoparticles. This copolymer was prepared via a two-step procedure: a well-defined precursor poly(oligo(ethylene glycol) methacrylate-co-tert-butyl methacrylate), P(OEGMA-co-tBMA) (M(n) = 17300 g mol(-1); M(w)/M(n) = 1.22), was first synthesized by atom-transfer radical polymerization in the presence of the catalyst system copper(I) chloride/2,2'-bipyridyl and subsequently selectively hydrolyzed in acidic conditions. The resulting P(OEGMA-co-MAA) was directly utilized as a polymeric stabilizer in the nanoparticle synthesis. Four batches of ultrasmall PEGylated magnetite nanoparticles (i.e., with an average diameter below 30 nm) were prepared via aqueous coprecipitation of iron salts in the presence of variable amounts of P(OEGMA-co-MAA). The diameter of the nanoparticles could be easily tuned in the range 10-25 nm by varying the initial copolymer concentration. Moreover, the formed PEGylated ferrofluids exhibited a long-term colloidal stability in physiological buffer and could therefore be studied in vivo by magnetic resonance (MR) imaging. Intravenous injection into rats showed no detectable signal in the liver within the first 2 h. Maximum liver accumulation was found after 6 h, suggesting a prolongated circulation of the nanoparticles in the bloodstream as compared to conventional MR imaging contrast agents.

Cervical Lymph Node Metastases: MR Imaging of Gadofluorine M and Monocrystalline Iron Oxide Nanoparticle-47 in a Rabbit Model of Head and Neck Cancer.
Choi SH, Han MH, Moon WK, Son KR, Won JK, Kim JH, Kwon BJ, Na DG, Weinmann HJ, Chang KH.
Radiology. 2006 Oct 10; [Epub ahead of print] .
[ expand abstract ]

Purpose: To prospectively compare the accuracy of gadofluorine M with that of monocrystalline iron oxide nanoparticle (MION)-47 for the depiction of cervical lymph node metastases at magnetic resonance (MR) imaging in a rabbit model of head and neck cancer by using histologic analysis as the reference standard. Materials and Methods: Experiments were approved by the animal care committee. VX2 carcinomas were implanted in both ears of 11 rabbits 4 weeks before MR imaging. T2-weighted, T2*-weighted, and T1-weighted MR images were acquired, and sequential T1-weighted MR imaging was performed immediately and 30 minutes after administration of gadofluorine M (0.05 mmol gadolinium per kilogram body weight). T2-weighted and T2*-weighted MR imaging were performed 24 hours after administration of MION-47 (2.6 mg iron per kilogram body weight). Gadofluorine M- and MION-47-enhanced MR imaging were performed separately and independently by two radiologists who had no knowledge of histopathologic results, and the presence of metastases in lymph nodes was evaluated. A receiver operating characteristic analysis was conducted to compare the diagnostic value of gadofluorine M- and MION-47-enhanced MR imaging. Results: Metastases were confirmed in 20 of 77 lymph nodes at histopathologic analysis. The area under the curve was significantly greater for gadofluorine M-enhanced MR imaging (0.997 and 0.981 for readers 1 and 2, respectively) than for MION-47-enhanced MR imaging (0.889 and 0.846 for readers 1 and 2, respectively). For gadofluorine M-enhanced MR imaging, sensitivity was 100% for both readers and specificity was 89.5% for reader 1 and 87.7% for reader 2. For MION-47-enhanced MR imaging, sensitivity was 80.0% for both readers and specificity was 75.4% for reader 1 and 71.9% for reader 2. Conclusion: Gadofluorine M-enhanced MR imaging has higher accuracy for depicting lymph node metastases than does MION-47-enhanced MR imaging.

Calcium-sensitive MRI contrast agents based on superparamagnetic iron oxide nanoparticles and calmodulin.
Atanasijevic T, Shusteff M, Fam P, Jasanoff A.
Proc Natl Acad Sci U S A. 2006 Oct 3;103(40):14707-12.
[ expand abstract ]

We describe a family of calcium indicators for magnetic resonance imaging (MRI), formed by combining a powerful iron oxide nanoparticle-based contrast mechanism with the versatile calcium-sensing protein calmodulin and its targets. Calcium-dependent protein-protein interactions drive particle clustering and produce up to 5-fold changes in T2 relaxivity, an indication of the sensors' potency. A variant based on conjugates of wild-type calmodulin and the peptide M13 reports concentration changes near 1 muM Ca(2+), suitable for detection of elevated intracellular calcium levels. The midpoint and cooperativity of the response can be tuned by mutating the protein domains that actuate the sensor. Robust MRI signal changes are achieved even at nanomolar particle concentrations (<1 muM in calmodulin) that are unlikely to buffer calcium levels. When combined with technologies for cellular delivery of nanoparticulate agents, these sensors and their derivatives may be useful for functional molecular imaging of biological signaling networks in live, opaque specimens.

Noninvasive vascular cell adhesion molecule-1 imaging identifies inflammatory activation of cells in atherosclerosis.
Nahrendorf M, Jaffer FA, Kelly KA, Sosnovik DE, Aikawa E, Libby P, Weissleder R.
Circulation. 2006 Oct 3;114(14):1504-11.
[ expand abstract ]

BACKGROUND: Noninvasive imaging of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) may identify early stages of inflammation in atherosclerosis. We hypothesized that a novel, second-generation VCAM-1-targeted agent with enhanced affinity had sufficient sensitivity to enable real-time detection of VCAM-1 expression in experimental atherosclerosis in vivo, to quantify pharmacotherapy-induced reductions in VCAM-1 expression, and to identify activated cells in human plaques. METHODS AND RESULTS: In vivo phage display in apolipoprotein E-deficient mice identified a linear peptide affinity ligand, VHPKQHR, homologous to very late antigen-4, a known ligand for VCAM-1. This peptide was developed into a multivalent agent detectable by MRI and optical imaging (denoted VINP-28 for VCAM-1 internalizing nanoparticle 28, with 20 times higher affinity than previously reported for VNP). In vitro, VINP-28 targeted all cell types expressing VCAM-1. In vivo, MRI and optical imaging in apolipoprotein E-deficient mice (n=28) after injection with VINP-28 or saline revealed signal enhancement in the aortic root of mice receiving VINP-28 (P<0.05). VINP-28 colocalized with endothelial cells and other VCAM-1-expressing cells, eg, macrophages, and was spatially distinct compared with untargeted control nanoparticles. Atheromata of atorvastatin-treated mice showed reduced VINP-28 deposition and VCAM-1 expression. VINP-28 enhanced early lesions in juvenile mice and resected human carotid artery plaques. CONCLUSIONS: VINP-28 allows noninvasive imaging of VCAM-1-expressing endothelial cells and macrophages in atherosclerosis and spatial monitoring of anti-VCAM-1 pharmacotherapy in vivo and identifies inflammatory cells in human atheromata. This clinically translatable agent could noninvasively detect inflammation in early, subclinical atherosclerosis.

Off-resonance saturation as a means of generating contrast with superparamagnetic nanoparticles.
Zurkiya O, Hu X.
Magn Reson Med. 2006 Oct;56(4):726-32.
[ expand abstract ]

This work demonstrates an alternative approach, termed off-resonance saturation (ORS), for generating contrast that is sensitive to superparamagnetic particles. This method leads to a calculated contrast that increases with superparamagnetic nanoparticle concentration. Experimental data demonstrate that in the presence of superparamagnetic particles, an off-resonance effect exists that is distinct from the magnetization transfer (MT) effect and is highly dependent on diffusion. Data show that the dependence on water diffusion becomes most significant at rates of 0.5 x 10(-9) m(2)/s and slower. We investigated the dependence of the off-resonance effect on off-resonance frequency and particle concentration. The data suggest a useful frequency offset range of 500 Hz < |Deltaomega| < 1500 Hz at 3T. This approach may be especially useful in organs and diseases in which diffusion may be altered by pathologies.

Simultaneous two-color spectral fluorescence lymphangiography with near infrared quantum dots to map two lymphatic flows from the breast and the upper extremity.
Hama Y, Koyama Y, Urano Y, Choyke PL, Kobayashi H.
Breast Cancer Res Treat. 2006 Sep 21; [Epub ahead of print] .
[ expand abstract ]

Due to their small size and poor access, the lymphatic function has been difficult to study in vivo. Especially difficult is the mapping of lymphatic drainage from two basins into the same node. Quantum dots can be used to perform multicolor images with high fluorescent intensity and are of a nano-size size suitable for lymphatic imaging via direct interstitial injection. Here we show simultaneous two-color in vivo wavelength-resolved spectral fluorescence lymphangiography using two near infrared quantum dots with different emission spectra, which allow non-invasive and simultaneous visualization of two separate lymphatic flows draining the breast and the upper extremity and variations in the drainage patterns and the water sheds within the axillary node. Two-color spectral fluorescence lymphangiography can provide insight into mechanisms of drainage from different lymphatic basins that may lead to sentinel lymph nodes detection of the breast cancer as well as prevention of complications such as lymphedema of the arm.

MAGfect: a novel liposome formulation for MRI labelling and visualization of cells.
Oliver M, Ahmad A, Kamaly N, Perouzel E, Caussin A, Keller M, Herlihy A, Bell J, Miller AD, Jorgensen MR.
Org Biomol Chem. 2006 Sep 21;4(18):3489-97.
[ expand abstract ]

Cellular entry of imaging probes, such as contrast agents for magnetic resonance imaging (MRI), is a key requirement for many molecular imaging studies, particularly imaging intracellular events and cell tracking. Here, we describe the successful development and in vitro analysis of MAGfect, a novel liposome formulation containing a lipidic gadolinium contrast agent for MRI, Gd-DOTA-Chol , designed to enter and label cells. Liposome formulation and cell incubation time were optimised for maximum cellular uptake of the imaging probe in a variety of cell lines. MRI analysis of cells incubated with MAGfect showed them to be highly MRI active. This formulation was examined further for cytotoxicity, cell viability and mechanism of cell labelling. One of the key advantages of using MAGfect as a labelling vehicle arises from its potential for additional functions, such as concomitant drug or gene delivery and fluorescent labelling. The gadolinium liposome was found to be an effective vehicle for transport of plasmid DNA (pDNA) into cells and expression levels were comparable to the commercial transfection agent Trojenetrade mark.

Lymphotrophic nanoparticle enhanced MR imaging (LNMRI) for lymph node imaging.
Saokar A, Braschi M, Harisinghani MG.
Abdom Imaging. 2006 Sep 15; [Epub ahead of print] .
[ expand abstract ]

The accurate staging of lymph nodes in various primary tumors continues to pose a major diagnostic challenge. Following the detection of lymph nodes, the next arduous task is the characterization of nodes into benign and malignant categories. Cross-sectional modalities like CT and MRI rely on nodal size as the primary yardstick for differentiating benign from malignant lymph nodes. Other parameters such as the nodal shape and contours, the intrinsic architecture of the node, its level of enhancement may also provide useful information. In comparison, the emerging modality of PET relies on the metabolic activity for nodal characterization. Despite their widespread use for detecting and staging the primary tumors, cross-sectional modalities are limited in their ability to characterize lymph nodes as there is considerable overlap in the imaging features of benign and malignant lymph nodes. PET imaging combined with CT overcomes some of these disadvantages of cross-sectional imaging by offering functional information on tissue activity. The ability of PET to detect malignant nodes is however largely dependant on the activity of the primary tumor and also on the size of the affected node. Primary prostate cancer, for example, is not very 18-FDG avid, and hence PET cannot be reliably used for nodal staging and detection of small malignant nodes. There are few emerging techniques that have proven to be of value in nodal staging and will eventually provide a robust non-invasive way to stage lymph nodes. Lymphotrophic nanoparticle enhanced MRI (LNMRI) is one such technique that has proven to be beneficial in overcoming some of the imaging handicaps and when available will prove to be a very useful adjunct for staging of lymph nodes. The ensuing articles provide an overview of nodal imaging as it stands today and also a preview of the newer LNMRI technique. We hope that this feature section will enhance the understanding of nodal staging with imaging.

Real-time Imaging and Quantification of Brain Delivery of Liposomes.
Krauze MT, Forsayeth J, Park JW, Bankiewicz KS.
Pharm Res. 2006 Sep 14; [Epub ahead of print] .
[ expand abstract ]

The surgical delivery of therapeutic agents into the parenchyma of the brain is problematic because it has been virtually impossible to know with any certainty where infused material is going, and how much to infuse. We have started to use liposomes loaded with Gadoteridol (GDL) as a tracer that allows us to follow infusions in real-time on magnetic resonance imaging (MRI). MRI allows precise tracking and measurement of liposomes loaded with markers and therapeutics. This review provides an overview of real-time delivery of liposomes to the central nervous system (CNS), and discusses the technical aspects of delivery, liposomes as colloidal systems of delivery, real-time distribution of liposomes in CNS, and quantification of liposome distribution. Our data suggests that real-time monitoring of liposomal drug infusion is likely to improve outcomes of clinical trials where convection-enhanced delivery (CED) is being used to target drugs to specific brain structures through limitation of systemic toxicity and reduction of side effects. This review is a summary of work done by our group over the past four years.

Folic acid-PEG conjugated superparamagnetic nanoparticles for targeted cellular uptake and detection by MRI.
Sun C, Sze R, Zhang M.
J Biomed Mater Res A. 2006 Sep 1;78(3):550-7.
[ expand abstract ]

We report the development and in vitro study of a nanoconjugate serving as a targeted magnetic resonance imaging (MRI) contrast enhancement agent for detection of cancer cells overexpressing the folate receptor. The nanoconjugate was synthesized by coating superparamagnetic iron oxide nanoparticles with covalently bound bifunctional poly(ethylene glycol) (PEG), followed by conjugation with folic acid (FA). The specificity of the nanoconjugate targeting cancerous cells was demonstrated by comparative intracellular uptake of the nanoconjugate and PEG-/dextran-coated nanoparticles by human adenocarcinoma HeLa cells. Preferential targeting to cancerous cells was studied by comparing the uptake of the nanoconjugate by HeLa cells and by non-FR expressing osteosarcoma MG-63 cells. Uptake of the nanoconjugate by HeLa cells after 4 h incubation was found to be a 12-fold higher than that of PEG- or dextran-coated nanoparticles as quantified by inductively coupled plasma spectroscopy. A significant negative contrast enhancement was observed with magnetic resonance (MR) phantom imaging for HeLa cells over MG-63 cells, when both were cultured with the nanoconjugate. Specificity of the nanoconjugate for folate receptors was also verified with a competitive inhibition assay, in which HeLa cells were incubated with both NP-PEG-FA and free FA. The bifunctional PEG used has amide linkages within the PEG chains that can form interchain hydrogen bonding, leading to improved stability of the PEG coating. Self-assembled PEG can be controlled at the molecular level and are suitable for nanoscale coatings.

In vitro and in vivo imaging studies of a new endohedral metallofullerene nanoparticle.
Fatouros PP, Corwin FD, Chen ZJ, Broaddus WC, Tatum JL, Kettenmann B, Ge Z, Gibson HW, Russ JL, Leonard AP, Duchamp JC, Dorn HC.
Radiology. 2006 Sep;240(3):756-64.
[ expand abstract ]

PURPOSE: To evaluate the effectiveness of a functionalized trimetallic nitride endohedral metallofullerene nanoparticle as a magnetic resonance (MR) imaging proton relaxation agent and to follow its distribution for in vitro agarose gel infusions and in vivo infusions in rat brain. MATERIALS AND METHODS: The animal study was approved by the animal care and use committee. Gd(3)N@C(80) was functionalized with poly(ethylene glycol) units, and the carbon cage was hydroxylated to provide improved water solubility and biodistribution. Relaxation rate measurements (R1 = 1/T1 and R2 = 1/T2) of water solutions of this contrast agent were conducted at 0.35-, 2.4-, and 9.4-T MR imaging. Images of contrast agent distributions were produced following infusions in six agarose gel samples at 2.4 T and from direct brain infusions into normal and tumor-bearing rat brain at 2.4 T. The relaxivity of a control functionalized lutetium agent, Lu(3)N@C(80), was also determined. RESULTS: Water hydrogen MR imaging relaxivity (r1) for this metallofullerene nanoparticle was markedly higher than that for commercial agents (eg, gadodiamide); r1 values of 102, 143, and 32 L . mmol(-1) . sec(-1) were measured at 0.35, 2.4, and 9.4 T, respectively. In studies of in vitro agarose gel infusion, the use of functionalized Gd(3)N@C(80) at concentrations an order of magnitude lower resulted in equivalent visualization in comparison with commercial agents. Comparable contrast enhancement was obtained with direct infusions of 0.013 mmol/L of Gd(3)N@C(80) and 0.50 mmol/L of gadodiamide in live normal rat brain. Elapsed-time studies demonstrated lower diffusion rates for Gd(3)N@C(80) relative to gadodiamide in live normal rat brain tissue. Functionalized metallofullerenes directly infused into a tumor-bearing brain provided an improved tumor delineation in comparison with the intravenously injected conventional Gd(3+) chelate. A control lutetium functionalized Lu(3)N@C(80) nanoparticle exhibited very low MR imaging relaxivity. CONCLUSION: The new functionalized trimetallic nitride endohedral metallofullerene species Gd(3)N@C(80)[DiPEG5000(OH)(x)] is an effective proton relaxation agent, as demonstrated with in vitro relaxivity and MR imaging studies, in infusion experiments with agarose gel and in vivo rat brain studies simulating clinical conditions of direct intraparenchymal drug delivery for the treatment of brain tumors.

Characterization of digital waveforms using thermodynamic analogs: detection of contrast-targeted tissue in vivo.
Hughes MS, Marsh JN, Zhang H, Woodson AK, Allen JS, Lacy EK, Carradine C, Lanza GM, Wickline SA.
IEEE Trans Ultrason Ferroelectr Freq Control. 2006 Sep;53(9):1609-16.
[ expand abstract ]

We describe characterization of backscatter from tumor tissue targeted with a nanoparticle-based ultrasound contrast agent in vivo using analogs of thermodynamic quantities. We apply these waveform characteristics to detection of tumor neovasculature in tumors implanted in athymic nude mice, which were imaged using a research ultrasound scanner over a 2-hour period after injection of alpha upsilon beta3-targeted perfluorocarbon nanoparticles. Images were constructed from backscattered ultrasound using two different approaches: fundamental B-mode imaging and a signal receiver based on a thermodynamic analog (H(C)). The study shows that the thermodynamic analog is capable of detecting differences in backscattered signals that are not apparent with the B-mode approach.

Focal disruption of the blood-brain barrier due to 260-kHz ultrasound bursts: a method for molecular imaging and targeted drug delivery.
Hynynen K, McDannold N, Vykhodtseva N, Raymond S, Weissleder R, Jolesz FA, Sheikov N.
J Neurosurg. 2006 Sep;105(3):445-54.
[ expand abstract ]

OBJECT: The goal of this study was to explore the feasibility of using low-frequency magnetic resonance (MR) image-guided focused ultrasound as a noninvasive method for the temporary disruption of the blood-brain barrier (BBB) at targeted locations. METHODS: Rabbits were placed inside a clinical 1.5-tesla MR imaging unit, and sites in their brains were targeted for 20-second burst sonications (frequency 260 kHz). The peak pressure amplitude during the burst varied between 0.1 and 0.9 MPa. Each sonication was performed after an intravenous injection of an ultrasound contrast agent (Optison). The disruption of the BBB was evaluated with the aid of an injection of an MR imaging contrast agent (MAG-NEVIST). Additional tests involving the use of MION-47, a 20-nm magnetic nanoparticle contrast agent, were also performed. The animals were killed at different time points between 3 minutes and 5 weeks postsonication, after which light or electron microscopic evaluation was performed. The threshold for BBB disruption was approximately 0.2 MPa. More than 80% of the brain sites sonicated showed BBB disruption when the pressure amplitude was 0.3 MPa; at 0.4 MPa, this percentage was greater than 90%. Tissue necrosis, ischemia, and apoptosis were not found in tissue in which the pressure amplitude was less than 0.4 MPa; however, in a few areas of brain tissue erythrocytes were identified outside blood vessels following exposures of 0.4 MPa or higher. Survival experiments did not show any long-term adverse events. CONCLUSIONS: These results demonstrate that low-frequency ultrasound bursts can induce local, reversible disruption of the BBB without undesired long-term effects. This technique offers a potential noninvasive method for targeted drug delivery in the brain aided by a relatively simple low-frequency device.

Off-resonance saturation as a means of generating contrast with superparamagnetic nanoparticles.
Zurkiya O, Hu X.
Magn Reson Med. 2006 Aug 29; [Epub ahead of print] .
[ expand abstract ]

This work demonstrates an alternative approach, termed off-resonance saturation (ORS), for generating contrast that is sensitive to superparamagnetic particles. This method leads to a calculated contrast that increases with superparamagnetic nanoparticle concentration. Experimental data demonstrate that in the presence of superparamagnetic particles, an off-resonance effect exists that is distinct from the magnetization transfer (MT) effect and is highly dependent on diffusion. Data show that the dependence on water diffusion becomes most significant at rates of 0.5 x 10(-9) m(2)/s and slower. We investigated the dependence of the off-resonance effect on off-resonance frequency and particle concentration. The data suggest a useful frequency offset range of 500 Hz < |Deltaomega| < 1500 Hz at 3T. This approach may be especially useful in organs and diseases in which diffusion may be altered by pathologies.

In Vitro and in Vivo Imaging Studies of a New Endohedral Metallofullerene Nanoparticle.
Fatouros PP, Corwin FD, Chen ZJ, Broaddus WC, Tatum JL, Kettenmann B, Ge Z, Gibson HW, Russ JL, Leonard AP, Duchamp JC, Dorn HC.
Radiology. 2006 Jul 12; [Epub ahead of print].
[ expand abstract ]

Purpose: To evaluate the effectiveness of a functionalized trimetallic nitride endohedral metallofullerene nanoparticle as a magnetic resonance (MR) imaging proton relaxation agent and to follow its distribution for in vitro agarose gel infusions and in vivo infusions in rat brain. Materials and Methods: The animal study was approved by the animal care and use committee. Gd(3)N@C(80) was functionalized with poly(ethylene glycol) units, and the carbon cage was hydroxylated to provide improved water solubility and biodistribution. Relaxation rate measurements (R1 = 1/T1 and R2 = 1/T2) of water solutions of this contrast agent were conducted at 0.35-, 2.4-, and 9.4-T MR imaging. Images of contrast agent distributions were produced following infusions in six agarose gel samples at 2.4 T and from direct brain infusions into normal and tumor-bearing rat brain at 2.4 T. The relaxivity of a control functionalized lutetium agent, Lu(3)N@C(80), was also determined. Results: Water hydrogen MR imaging relaxivity (r1) for this metallofullerene nanoparticle was markedly higher than that for commercial agents (eg, gadodiamide); r1 values of 102, 143, and 32 L . mmol(-1) . sec(-1) were measured at 0.35, 2.4, and 9.4 T, respectively. In studies of in vitro agarose gel infusion, the use of functionalized Gd(3)N@C(80) at concentrations an order of magnitude lower resulted in equivalent visualization in comparison with commercial agents. Comparable contrast enhancement was obtained with direct infusions of 0.013 mmol/L of Gd(3)N@C(80) and 0.50 mmol/L of gadodiamide in live normal rat brain. Elapsed-time studies demonstrated lower diffusion rates for Gd(3)N@C(80) relative to gadodiamide in live normal rat brain tissue. Functionalized metallofullerenes directly infused into a tumor-bearing brain provided an improved tumor delineation in comparison with the intravenously injected conventional Gd(3+) chelate. A control lutetium functionalized Lu(3)N@C(80) nanoparticle exhibited very low MR imaging relaxivity. Conclusion: The new functionalized trimetallic nitride endohedral metallofullerene species Gd(3)N@C(80)[DiPEG5000(OH)(x)] is an effective proton relaxation agent, as demonstrated with in vitro relaxivity and MR imaging studies, in infusion experiments with agarose gel and in vivo rat brain studies simulating clinical conditions of direct intraparenchymal drug delivery for the treatment of brain tumors.

Dendrimer-based nanoprobe for dual modality magnetic resonance and fluorescence imaging.
Talanov VS, Regino CA, Kobayashi H, Bernardo M, Choyke PL, Brechbiel MW.
Nano Lett. 2006 Jul;6(7):1459-63.
[ expand abstract ]

A novel PAMAM dendrimer-based nanoprobe for dual magnetic resonance and fluorescence imaging modalities was synthesized. Fluorescence studies revealed that Gd(III) complexation to the probe has no effect on the quantum yield; however, increases in the dye content resulted in partial quenching. The potential of the new nanoprobe, G6-(Cy5.5)(1.25)(1B4M-Gd)(145), as a dual modality imaging agent was demonstrated in vivo by the efficient visualization of sentinel lymph nodes in mice by both MRI and fluorescence imaging modalities.

18F-FET PET for planning of thermotherapy using magnetic nanoparticles in recurrent glioblastoma.
Plotkin M, Gneveckow U, Meier-Hauff K, Amthauer H, Feussner A, Denecke T, Gutberlet M, Jordan A, Felix R, Wust P.
Int J Hyperthermia. 2006 Jun;22(4):319-325.
[ expand abstract ]

Purpose: Thermotherapy using magnetic nanoparticles (nano cancer therapy) is a new concept of local tumour therapy, which is based on controlled heating of intra-tumoural injected magnetic nanoparticles. The aim of this study was to evaluate the usefulness of PET with a recently introduced amino acid tracer O-(2-[18F]fluoroethyl)-]L-tyrosine (FET) for targeting the nanoparticles implantation.Materials and methods: Eleven patients with glioblastoma recurrences underwent MR and FET-PET imaging for planning of the nano cancer therapy. Thereafter, the gross tumour volumes (GTV) were defined, taking into consideration the results of both imaging tools.Results: The MRI-based mean GTV was 24.3 cm3 (range 2.5-59.7) and the PET-based mean GTV 31.9 cm3 (range 5.2-77.9). On the average the MRI identified an additional 8.9 +/- 4.7 cm3 and the FET-PET scan-an additional 16.5 +/- 15.2 cm3 outside of the common GTV (15.4 +/- 11.0 cm3). The mean final GTV accounted to 33.8 cm3 (range, 5.2-77.9). The additional information of FET-PET led to an increase in GTV by 22-286% in eight patients and to a decrease of 23% and 26%, respectively, in two patients. In one patient, the final GTV was defined on the basis of MRI data only.Conclusions: FET-PET adds important information on the actual tumour volume in recurrent glioblastomas and is highly valuable for defining the target volume for the nano cancer therapy.

Lymphotropic nanoparticle enhanced MR imaging (LNMRI) technique for lymph node imaging.
Saksena MA, Saokar A, Harisinghani MG.
Eur J Radiol. 2006 Jun;58(3):367-74; Epub 2006 Feb 10.
[ expand abstract ]

Accurate nodal staging is important in the management of any primary malignancy. The presence of nodal metastases has both therapeutic and prognostic implications. Lymphotropic nanoparticles are a new class of MRI contrast agents, which are promising in detecting minimal metastatic nodal disease particularly in normal sized lymph nodes. This paper discusses the technique and interpretation of lymphotropic nanoparticle enhanced MRI (LNMRI) and reviews the various trials evaluating nodal staging with ferumoxtran-10 enhanced MRI.

Folic acid-PEG conjugated superparamagnetic nanoparticles for targeted cellular uptake and detection by MRI.
Sun C, Sze R, Zhang M.
J Biomed Mater Res A. 2006 May 30; [Epub ahead of print] .
[ expand abstract ]

We report the development and in vitro study of a nanoconjugate serving as a targeted magnetic resonance imaging (MRI) contrast enhancement agent for detection of cancer cells overexpressing the folate receptor. The nanoconjugate was synthesized by coating superparamagnetic iron oxide nanoparticles with covalently bound bifunctional poly(ethylene glycol) (PEG), followed by conjugation with folic acid (FA). The specificity of the nanoconjugate targeting cancerous cells was demonstrated by comparative intracellular uptake of the nanoconjugate and PEG-/dextran-coated nanoparticles by human adenocarcinoma HeLa cells. Preferential targeting to cancerous cells was studied by comparing the uptake of the nanoconjugate by HeLa cells and by non-FR expressing osteosarcoma MG-63 cells. Uptake of the nanoconjugate by HeLa cells after 4 h incubation was found to be a 12-fold higher than that of PEG- or dextran-coated nanoparticles as quantified by inductively coupled plasma spectroscopy. A significant negative contrast enhancement was observed with magnetic resonance (MR) phantom imaging for HeLa cells over MG-63 cells, when both were cultured with the nanoconjugate. Specificity of the nanoconjugate for folate receptors was also verified with a competitive inhibition assay, in which HeLa cells were incubated with both NP-PEG-FA and free FA. The bifunctional PEG used has amide linkages within the PEG chains that can form interchain hydrogen bonding, leading to improved stability of the PEG coating. Self-assembled PEG can be controlled at the molecular level and are suitable for nanoscale coatings.

Nanoparticles as image enhancing agents for ultrasonography.
Liu J, Levine AL, Mattoon JS, Yamaguchi M, Lee RJ, Pan X, Rosol TJ.
Phys Med Biol. 2006 May 7;51(9):2179-89; Epub 2006 Apr 10.
[ expand abstract ]

Nanoparticles have drawn great attention as targeted imaging and/or therapeutic agents. The small size of the nanoparticles allows them to target cells that are beyond capillary vasculature, such as cancer cells. We investigated the effect of solid nanoparticles for enhancing ultrasonic grey scale images in tissue phantoms and mouse livers in vivo. Silica nanospheres (100 nm) were dispersed in agarose at 1-2.5% mass concentration and imaged by a high-resolution ultrasound imaging system (transducer centre frequency: 30 MHz). Polystyrene particles of different sizes (500-3000 nm) and concentrations (0.13-0.75% mass) were similarly dispersed in agarose and imaged. Mice were injected intravenously with nanoparticle suspensions in saline. B-mode images of the livers were acquired at different time points after particle injection. An automated computer program was used to quantify the grey scale changes. Ultrasonic reflections were observed from nanoparticle suspensions in agarose gels. The image brightness, i.e., mean grey scale level, increased with particle size and concentration. The mean grey scale of mouse livers also increased following particle administration. These results indicated that it is feasible to use solid nanoparticles as contrast enhancing agents for ultrasonic imaging.

Lymphotrophic nanoparticle enhanced MR imaging (LNMRI) for lymph node imaging.
Saokar A, Braschi M, Harisinghani M.
Abdom Imaging. 2006 May 6; [Epub ahead of print] .
[ expand abstract ]

Nodal staging is an integral part of the pretreatment staging of any patient with malignancy and has therapeutic and prognostic implications. Currently used imaging techniques used for nodal evaluation are limited in accuracy because they rely on size criteria for the detection of metastases. This has led to the emergence of lymphotropic nanoparticle enhanced magnetic resonance imaging as a promising tool for nodal characterization. This article reviews the properties of lymphotropic iron oxide nanoparticles and the technique, image interpretation, and initial clinical experience with lymphotropic nanoparticle enhanced magnetic resonance imaging.

Toward the emergence of nanoneurosurgery: part II--nanomedicine: diagnostics and imaging at the nanoscale level.
Leary SP, Liu CY, Apuzzo ML.
Neurosurgery. 2006 May;58(5):805-23; discussion 805-23.
[ 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 (58). This article reviews and discusses the applications of nanotechnology to biological systems that will undoubtedly transform the foundations of disease diagnosis, treatment, and prevention in the future. Specific attention is given to developments in diagnostics and imaging at the nanoscale level. The use of nanoparticles and nanomaterials as biodetection agents for deoxyribonucleic acid and proteins is presented. In addition, nanodevices, such as nanowires, nanotubes, and nanocantilevers, can be combined with nanoarrays and nanofluidics to create integrated and automated nanodetection platforms. Molecular imaging modalities based on quantum dots and magnetic nanoparticles are also discussed. This technology has been extended to the imaging of intracranial neoplasms. Further innovation within these disciplines will form the basis for the development of mature nanomedicine. The final article of the series will focus on additional advancements in nanomedicine, namely nanotherapy and nanosurgery, and will cover the innovations that will lead to the eventual realization of nanoneurosurgery.

Liposome-enhanced MRI of neointimal lesions in the ApoE-KO mouse.
Mulder WJ, Douma K, Koning GA, van Zandvoort MA, Lutgens E, Daemen MJ, Nicolay K, Strijkers GJ.
Magn Reson Med. 2006 May;55(5):1170-4.
[ expand abstract ]

Conventional high-resolution MRI is capable of detecting lipid-rich atherosclerotic plaques in both human atherosclerosis and animal models of atherosclerosis. In this study we induced neointimal lesions in ApoE-KO mice by placing a constrictive collar around the right carotid artery. The model was imaged with conventional multispectral MRI, and the thickened wall could not be distinguished from surrounding tissue. We then tested paramagnetic liposomes (mean size=90 nm) for their ability to improve MRI visualization of induced thickening, using Gd-DTPA as a control. T1-weighted (T1-w), black-blood MRI of the neck area of the mice was performed before and 15 min, 45 min, and 24 hr after intravenous injection of either paramagnetic liposomes or Gd-DTPA. The collared vessel wall of mice that were injected with liposomes showed a pronounced signal enhancement of approximately 100% immediately after injection, which was sustained largely until 24 hr postinjection. In contrast, the vessel wall of all controls (left carotid artery and animals injected with Gd-DTPA) did not show significant contrast enhancement at those time points. This study demonstrates that intimal thickening in ApoE-KO mice can be effectively detected by contrast-enhanced (CE)-MRI upon injection of paramagnetic liposomes. Copyright (c) 2006 Wiley-Liss, Inc.

Dynamic imaging with MRI contrast agents: quantitative considerations.
Shapiro MG, Atanasijevic T, Faas H, Westmeyer GG, Jasanoff A.
Magn Reson Imaging. 2006 May;24(4):449-62; Epub 2006 Mar 20.
[ expand abstract ]

Time-resolved MRI has had enormous impact in cognitive science and may become a significant tool in basic biological research with the application of new molecular imaging agents. In this paper, we examine the temporal characteristics of MRI contrast agents that could be used in dynamic studies. We consider "smart" T1 contrast agents, T2 agents based on reversible aggregation of superparamagnetic nanoparticles and sensors that produce changes in saturation transfer effects (chemical exchange saturation transfer, CEST). We discuss response properties of several agents with reference to available experimental data, and we develop a new theoretical model that predicts the response rates and relaxivity changes of aggregation-based sensors. We also perform calculations to define the extent to which constraints on temporal resolution are imposed by the imaging methods themselves. Our analysis confirms that some small T1 agents may be compatible with MRI temporal resolution on the order of 100 ms. Nanoparticle aggregation T2 sensors are applicable at much lower concentrations, but are likely to respond on a single second or slower timescale. CEST agents work at high concentrations and temporal resolutions of 1-10 s, limited by a requirement for long presaturation periods in the MRI pulse sequence.

Polymeric nano/microcapsules of liquid perfluorocarbons for ultrasonic imaging: physical characterization.
Pisani E, Tsapis N, Paris J, Nicolas V, Cattel L, Fattal E.
Langmuir. 2006 Apr 25;22(9):4397-402.
[ expand abstract ]

Ultrasonic imaging is a widely available, noninvasive, and cost-effective diagnostic modality, but vessels smaller than 200 mum in diameter are impossible to visualize. Commercial ultrasound contrast agents (UCAs), consisting of encapsulated gas microbubbles injected intravenously, enable only a qualitative visualization of the microvascularization for a short period of time since they are rather unstable. In a strategy to develop more stable UCAs, we designed a process to obtain nano/microcapsules with a single core of liquid perfluorocarbons within a biodegradable polymeric shell of homogeneous thickness. The polymer shell should improve the stability of the capsules as compared to UCAs stabilized by a monomolecular layer, while the acoustic impedance of the perfluorocarbons should ensure their echogenicity. These capsules have been optimized to encapsulate several liquid perfluorocarbons: perfluorohexane, perfluorodecalin, and perfluorooctyl bromide. The system is rather versatile: the mean size of the capsules can be adjusted between 70 nm and 25 mum and the thickness-to-radius ratio (T/R) can be easily modulated by simply modifying the polymer-to-perfluorocarbon ratio. T/R does not depend on the size of the capsules and is between 0.2 and 0.6. The dependence of the echogenic properties of the capsules with their size and their T/R has yet to be studied experimentally before this system can be evaluated in vivo.

Calculated Absorption and Scattering Properties of Gold Nanoparticles of Different Size, Shape, and Composition: Applications in Biological Imaging and Biomedicine.
Jain PK, Lee KS, El-Sayed IH, El-Sayed MA.
J Phys Chem B Condens Matter Mater Surf Interfaces Biophys. 2006 Apr 13;110(14):7238-7248.
[ expand abstract ]

The selection of nanoparticles for achieving efficient contrast for biological and cell imaging applications, as well as for photothermal therapeutic applications, is based on the optical properties of the nanoparticles. We use Mie theory and discrete dipole approximation method to calculate absorption and scattering efficiencies and optical resonance wavelengths for three commonly used classes of nanoparticles: gold nanospheres, silica-gold nanoshells, and gold nanorods. The calculated spectra clearly reflect the well-known dependence of nanoparticle optical properties viz. the resonance wavelength, the extinction cross-section, and the ratio of scattering to absorption, on the nanoparticle dimensions. A systematic quantitative study of the various trends is presented. By increasing the size of gold nanospheres from 20 to 80 nm, the magnitude of extinction as well as the relative contribution of scattering to the extinction rapidly increases. Gold nanospheres in the size range commonly employed ( approximately 40 nm) show an absorption cross-section 5 orders higher than conventional absorbing dyes, while the magnitude of light scattering by 80-nm gold nanospheres is 5 orders higher than the light emission from strongly fluorescing dyes. The variation in the plasmon wavelength maximum of nanospheres, i.e., from approximately 520 to 550 nm, is however too limited to be useful for in vivo applications. Gold nanoshells are found to have optical cross-sections comparable to and even higher than the nanospheres. Additionally, their optical resonances lie favorably in the near-infrared region. The resonance wavelength can be rapidly increased by either increasing the total nanoshell size or increasing the ratio of the core-to-shell radius. The total extinction of nanoshells shows a linear dependence on their total size, however, it is independent of the core/shell radius ratio. The relative scattering contribution to the extinction can be rapidly increased by increasing the nanoshell size or decreasing the ratio of the core/shell radius. Gold nanorods show optical cross-sections comparable to nanospheres and nanoshells, however, at much smaller effective size. Their optical resonance can be linearly tuned across the near-infrared region by changing either the effective size or the aspect ratio of the nanorods. The total extinction as well as the relative scattering contribution increases rapidly with the effective size, however, they are independent of the aspect ratio. To compare the effectiveness of nanoparticles of different sizes for real biomedical applications, size-normalized optical cross-sections or per micron coefficients are calculated. Gold nanorods show per micron absorption and scattering coefficients that are an order of magnitude higher than those for nanoshells and nanospheres. While nanorods with a higher aspect ratio along with a smaller effective radius are the best photoabsorbing nanoparticles, the highest scattering contrast for imaging applications is obtained from nanorods of high aspect ratio with a larger effective radius.

Peptide-Labeled Near-Infrared Quantum Dots for Imaging Tumor Vasculature in Living Subjects.
Cai W, Shin DW, Chen K, Gheysens O, Cao Q, Wang SX, Gambhir SS, Chen X.
Nano Lett. 2006 Apr 12;6(4):669-676.
[ expand abstract ]

We report the in vivo targeting and imaging of tumor vasculature using arginine-glycine-aspartic acid (RGD) peptide-labeled quantum dots (QDs). Athymic nude mice bearing subcutaneous U87MG human glioblastoma tumors were administered QD705-RGD intravenously. The tumor fluorescence intensity reached maximum at 6 h postinjection with good contrast. The results reported here open up new perspectives for integrin-targeted near-infrared optical imaging and may aid in cancer detection and management including imaging-guided surgery.

Delivery of gadolinium-labeled nanoparticles to the sentinel lymph node: Comparison of the sentinel node visualization and estimations of intra-nodal gadolinium concentration by the magnetic resonance imaging.
Kobayashi H, Kawamoto S, Bernardo M, Brechbiel MW, Knopp MV, Choyke PL.
J Control Release. 2006 Apr 10;111(3):343-51; [Epub 2006 Feb 21].
[ expand abstract ]

Sentinel node imaging is commonly performed prior to surgery for breast cancer and melanoma. While current methods are based on radio-lymphoscintigraphy, MR lymphangiography (MRL) offers the benefits of better spatial resolution without ionizing radiation. However, the optimal nanoparticle for imaging the sentinel nodes remains unclear. Gadolinium-labeled (Gd) contrast agents ranging in diameter from <1 to 12 nm were evaluated to determine which size provides the most rapid and most concentrated delivery of contrast agent to the lymph nodes in a mouse model of lymphatic metastases. Specifically, PAMAM-G2, -G4, -G6 and -G8, and DAB-G5 Gd-dendrimer agents, as well as Gadomer-17 and Gd-DTPA, were compared. Among these agents, the G6 Gd dendrimer depicted the lymphatics and lymph nodes with the highest peak concentrations and this occurred 24-36 min post-injection (p<0.01; all except G8). Based on ex vivo concentration phantoms, high accumulations of Gd(III) ions occurred within lymph nodes (1.7-4.4 mM Gd/270-680 ppm Gd) with high target to background ratios (>100). These concentrations are sufficient to contemplate the use of Gd-neutron capture therapy of regional lymph nodes. Thus, when injected interstitially, the PAMAM-G6 Gd dendrimer not only provides excellent opacification of sentinel lymph nodes, but also provides the potential for targeted therapy of sentinel lymph nodes.

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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A new bioimaging carrier for fluorescent quantum dots: phospholipid nanoemulsion mimicking natural lipoprotein core.
Liu S, Lee CM, Wang S, Lu DR.
Drug Deliv. 2006 Mar-Apr;13(2):159-64.
[ expand abstract ]

Fluorescent quantum dots (semiconductor nanocrystals) have the potential to revolutionize biological imaging, but their use has been limited by difficulties in obtaining quantum dots that are water soluble and biocompatible. The objectives of our research were to develop a methodology for encapsulation of cadnium-selenium (CdSe) quantum dots (QDs) in phospholipid nanoemulsion that mimics the natural lipoprotein core and to study their interactions with cultured non-small cell lung cancer cells (NSCLC). We found that CdSe QDs can be efficiently encapsulated in the phospholipid nanoemulsion. The QD nanoemulsion has a particle size approximately 80 nm and appears physically stable. The QD nanoemulsion interacts well with cells. The intensity of cellular fluorescence imaging increases with the cell incubation time, indicating more QDs were taken up by the cells, respectively. Two types of fluorescence microscopies confirm that QDs are primarily localized in the cytoplasm but not in the nucleus of the cells.

In vitro demonstration using 19F magnetic resonance to augment molecular imaging with paramagnetic perfluorocarbon nanoparticles at 1.5 Tesla.
Caruthers SD, Neubauer AM, Hockett FD, Lamerichs R, Winter PM, Scott MJ, Gaffney PJ, Wickline SA, Lanza GM.
Invest Radiol. 2006 Mar;41(3):305-12.
[ expand abstract ]

OBJECTIVES: This study explored the use of F spectroscopy and imaging with targeted perfluorocarbon nanoparticles for the simultaneous identification of multiple bio-signatures at 1.5 T. MATERIALS AND METHODS: Two nanoparticle emulsions with perfluoro-15-crown-5-ether (CE) or perfluorooctylbromide (PFOB) cores were targeted in vitro to fibrin clot phantoms (n=12) in 4 progressive ratios using biotin-avidin interactions. The CE nanoparticles incorporated gadolinium. Fluorine images were acquired using steady-state gradient-echo techniques; spectra using volume-selective and nonselective sampling. RESULTS: On conventional T1-weighted imaging, clots with CE nanoparticles enhanced as expected, with intensity decreasing monotonically with CE concentration. All clots were visualized using wide bandwidth fluorine imaging, while restricted bandwidth excitation permitted independent imaging of CE or PFOB nanoparticles. Furthermore, F imaging and spectroscopy allowed visual and quantitative confirmation of relative perfluorocarbon nanoparticle distributions. CONCLUSIONS: F MRI/S molecular imaging of perfluorocarbon nanoparticles in vitro suggests that noninvasive phenotypic characterization of pathologic bio-signatures is feasible at clinical field strengths.

Microfluidic technology for PET radiochemistry.
Gillies JM, Prenant C, Chimon GN, Smethurst GJ, Dekker BA, Zweit J.
Appl Radiat Isot. 2006 Mar;64(3):333-6.
[ expand abstract ]

This paper describes the first application of a microfabricated reaction system to positron emission tomography (PET) radiochemistry. We have applied microfluidic technology to synthesise PET radiopharmaceuticals using (18)F and (124)I as labels for fluorodeoxyglucose (FDG) and Annexin-V, respectively. These reactions involved established methods of nucleophilic substitution on a mannose triflate precursor and direct iodination of the protein using iodogen as an oxidant. This has demonstrated a proof of principle of using microfluidic technology to radiochemical reactions involving low and high molecular weight compounds. Using microfluidic reactions, [(18)F]FDG was synthesised with a 50% incorporation of the available F-18 radioactivity in a very short time of 4s. The radiolabelling efficiency of (124)I Annexin-V was 40% after 1min reaction time. Chromatographic analysis showed that such reaction yields are comparable to conventional methods, but in a much shorter time. The yields can be further improved with more optimisation of the microfluidic device itself and its fluid mixing profiles. This demonstrates the potential for this technology to have an impact on rapid and simpler radiopharmaceutical synthesis using short and medium half-life radionuclides.

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Microfluidic reactor for the radiosynthesis of PET radiotracers.
Gillies JM, Prenant C, Chimon GN, Smethurst GJ, Perrie W, Hamblett I, Dekker B, Zweit J.
Appl Radiat Isot. 2006 Mar;64(3):325-32.
[ expand abstract ]

Here we show the first application of a microfabricated reaction system to PET radiochemistry, we term "microfluidic PET". The short half-life of the positron emitting isotopes and the trace chemical quantities used in radiolabelling make PET radiochemistry amenable to miniaturisation. Microfluidic technologies are capable of controlling and transferring tiny quantities of liquids which allow chemical and biochemical assays to be integrated and carried out on a small scale. Such technologies provide distinct advantages over current methods of PET radiochemical synthesis. To demonstrate "proof of principle" we have investigated the radiohalogenation of small and large molecular weight molecules using the microfluidic device. These reactions involved the direct radioiodination of the apoptosis marker Annexin V using iodine-124, the indirect radioiodination of the anti-cancer drug doxorubicin from a tin-butyl precursor and the radiosynthesis of 2-[(18)F]FDG from a mannose triflate precursor and fluorine-18 and hence provide a test bed for microfluidic reactions. We demonstrate the rapid radioiodination of the protein Annexin V (40% radiochemical yield within 1min) and the rapid radiofluorination of 2-[(18)F]FDG (60% radiochemical yield within 4s) using a polymer microreactor chip. Chromatographic analysis showed that the labelling efficiency of the unoptimised microfluidic chip is comparable to conventional PET radiolabelling reactions.

Re: Regional lymph node staging using lymphotropic nanoparticle enhanced magnetic resonance imaging with ferumoxtran-10 in patients with penile cancer.
Kroon BK, Horenblas S.
J Urol. 2006 Mar;175(3 Pt 1):1174.
[ expand abstract ]

No abstract available

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Advances in fluorescence imaging with quantum dot bio-probes.
Pinaud F, Michalet X, Bentolila LA, Tsay JM, Doose S, Li JJ, Iyer G, Weiss S.
Biomaterials. 2006 Mar;27(9):1679-87.
[ expand abstract ]

After much effort in surface chemistry development and optimization by several groups, fluorescent semiconductor nanocrystals probes, also known as quantum dots or qdots, are now entering the realm of biological applications with much to offer to biologists. The road to success has been paved with hurdles but from these efforts has stemmed a multitude of original surface chemistries that scientists in the biological fields can draw from for their specific biological applications. The ability to easily modulate the chemical nature of qdot surfaces by employing one or more of the recently developed qdot coatings, together with their exceptional photophysics have been key elements for qdots to acquire a status of revolutionary fluorescent bio-probes. Indeed, the unique properties of qdots not only give biologists the opportunity to explore advanced imaging techniques such as single molecule or lifetime imaging but also to revisit traditional fluorescence imaging methodologies and extract yet unobserved or inaccessible information in vitro or in vivo.

A fluorescent nanosensor for apoptotic cells.
Quinti L, Weissleder R, Tung CH.
Nano Lett. 2006 Mar;6(3):488-90.
[ expand abstract ]

A biocompatible surface-functionalized nanoparticle was designed to sense phosphatidylserine exposed on apoptotic cells. We conjugated synthetic artificial phosphatidylserine binding ligands in a multivalent fashion onto magnetofluorescent nanoparticles. Our results show that (1) the synthetic nanoparticles bind to apoptotic cells, (2) there is excellent correlation with annexin V staining by microscopy, and (3) FACS analysis with nanoparticles allows the measurement of therapeutic apoptosis induction. The described nanomaterials should be useful for a variety of biomedical applications including in vivo imaging of apoptosis.

Self-illuminating quantum dot conjugates for in vivo imaging.
So MK, Xu C, Loening AM, Gambhir SS, Rao J.
Nat Biotechnol. 2006 Mar;24(3):339-43.
[ expand abstract ]

Fluorescent semiconductor quantum dots hold great potential for molecular imaging in vivo. However, the utility of existing quantum dots for in vivo imaging is limited because they require excitation from external illumination sources to fluoresce, which results in a strong autofluorescence background and a paucity of excitation light at nonsuperficial locations. Here we present quantum dot conjugates that luminesce by bioluminescence resonance energy transfer in the absence of external excitation. The conjugates are prepared by coupling carboxylate-presenting quantum dots to a mutant of the bioluminescent protein Renilla reniformis luciferase. We show that the conjugates emit long-wavelength (from red to near-infrared) bioluminescent light in cells and in animals, even in deep tissues, and are suitable for multiplexed in vivo imaging. Compared with existing quantum dots, self-illuminating quantum dot conjugates have greatly enhanced sensitivity in small animal imaging, with an in vivo signal-to-background ratio of > 10(3) for 5 pmol of conjugate.

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Sentinel lymph node mapping of the gastrointestinal tract by using invisible light.
Soltesz EG, Kim S, Kim SW, Laurence RG, De Grand AM, Parungo CP, Cohn LH, Bawendi MG, Frangioni JV.
Ann Surg Oncol. 2006 Mar;13(3):386-96.
[ expand abstract ]

BACKGROUND: Because many gastrointestinal (GI) tumors spread by way of lymphatics, histological assessment of the first draining lymph nodes has both prognostic and therapeutic significance. However, sentinel lymph node mapping of the GI tract by using available techniques is limited by unpredictable drainage patterns, high background signal, and the inability to image lymphatic tracers relative to surgical anatomy in real time. Our goal was to develop a method for patient-specific intraoperative sentinel lymph node mapping of the GI tract by using invisible near-infrared light. METHODS: We developed an intraoperative near-infrared fluorescence imaging system that simultaneously displays surgical anatomy and otherwise invisible near-infrared fluorescence images of the surgical field. Near-infrared fluorescent quantum dots were injected intraparenchymally into the stomach, small bowel, and colon, and draining lymphatic channels and sentinel lymph nodes were visualized. Dissection was performed under real-time image guidance. RESULTS: In 10 adult pigs, we demonstrated that 200 pmol of quantum dots quickly and accurately map lymphatic drainage and sentinel lymph nodes. Injection into the mid jejunum and colon results in fluorescence of a single lymph node at the root of the bowel mesentery. Injection into the stomach resulted in identification of a retrogastric node. Histological analysis in all cases confirmed the presence of nodal tissue. CONCLUSIONS: We report the use of invisible near-infrared light for intraoperative sentinel lymph node mapping of the GI tract. This technology overcomes the limitations of currently available methods, permits patient-specific imaging of lymphatic flow and sentinel nodes, and provides highly sensitive, real-time image-guided dissection.

Multi-functional polymeric nanoparticles for tumour-targeted drug delivery.
van Vlerken LE, Amiji MM.
Expert Opin Drug Deliv. 2006 Mar;3(2):205-216.
[ expand abstract ]

The use of nanoparticles as drug delivery vehicles for anticancer therapeutics has great potential to revolutionise the future of cancer therapy. As tumour architecture causes nanoparticles to preferentially accumulate at the tumour site, their use as drug delivery vectors results in the localisation of a greater amount of the drug load at the tumour site; thus improving cancer therapy and reducing the harmful nonspecific side effects of chemotherapeutics. In addition, formulation of these nanoparticles with imaging contrast agents provides a very efficient system for cancer diagnostics. Given the exhaustive possibilities available to polymeric nanoparticle chemistry, research has quickly been directed at multi-functional nanoparticles, combining tumour targeting, tumour therapy and tumour imaging in an all-in-one system, providing a useful multi-modal approach in the battle against cancer. This review will discuss the properties of nanoparticles that allow for such multiple functionality, as well as recent scientific advances in the area of multi-functional nanoparticles for cancer therapeutics.

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Simultaneous sustained release of fludarabine monophosphate and Gd-DTPA from an interstitial liposome depot in rats: potential for indirect monitoring of drug release by magnetic resonance imaging.
Port RE, Schuster C, Port CR, Bachert P.
Cancer Chemother Pharmacol. 2006 Feb 28; [Epub ahead of print] .
[ expand abstract ]

Introduction: Cytostatic depot preparations are interstitially administered for local chemotherapy and prevention of tumor recurrence. It would be of interest to monitor in patients as to when, to what extent, and exactly where, the drug is actually released. Liposomes containing a hydrophilic cytostatic and a hydrophilic contrast agent might be expected to release both agents simultaneously. If so, then drug release could be indirectly followed by monitoring contrast enhancement at the injection site. Methods: Multivesicular liposomes containing the antimetabolite fludarabine monophosphate and the magnetic resonance imaging (MRI) contrast agent Gd-DTPA were subcutaneously injected in rats and both agents were monitored at the injection site for 6 weeks by (19)F nuclear magnetic resonance spectroscopy (MRS) in vivo and contrast-enhanced (1)H MRI (T (1w ) 3D FLASH), respectively, in a 1.5-T whole-body tomograph. The MRS and MRI data were analyzed simultaneously by pharmacokinetic modeling using NONMEM. Results: During an initial lag time, the amount of drug at the injection site stayed constant while the contrast-enhanced depot volume expanded beyond the volume injected. Drug amount and depot volume then decreased in parallel. Lag time and elimination half-life were 9 and 6 days, respectively, in three animals, and were about 50% shorter in another animal where the depot split into sub-depots. Conclusion: The preliminary data in rats suggest that simultaneous release of a hydrophilic cytostatic and a hydrophilic contrast agent from an interstitial depot can be achieved by encapsulation in liposomes. Thus, there seems to be a potential for indirect drug monitoring through imaging.

Delivery of gadolinium-labeled nanoparticles to the sentinel lymph node: Comparison of the sentinel node visualization and estimations of intra-nodal gadolinium concentration by the magnetic resonance imaging.
Kobayashi H, Kawamoto S, Bernardo M, Brechbiel MW, Knopp MV, Choyke PL.
J Control Release. 2006 Feb 18; [Epub ahead of print] .
[ expand abstract ]

Sentinel node imaging is commonly performed prior to surgery for breast cancer and melanoma. While current methods are based on radio-lymphoscintigraphy, MR lymphangiography (MRL) offers the benefits of better spatial resolution without ionizing radiation. However, the optimal nanoparticle for imaging the sentinel nodes remains unclear. Gadolinium-labeled (Gd) contrast agents ranging in diameter from <1 to 12 nm were evaluated to determine which size provides the most rapid and most concentrated delivery of contrast agent to the lymph nodes in a mouse model of lymphatic metastases. Specifically, PAMAM-G2, -G4, -G6 and -G8, and DAB-G5 Gd-dendrimer agents, as well as Gadomer-17 and Gd-DTPA, were compared. Among these agents, the G6 Gd dendrimer depicted the lymphatics and lymph nodes with the highest peak concentrations and this occurred 24-36 min post-injection (p<0.01; all except G8). Based on ex vivo concentration phantoms, high accumulations of Gd(III) ions occurred within lymph nodes (1.7-4.4 mM Gd/270-680 ppm Gd) with high target to background ratios (>100). These concentrations are sufficient to contemplate the use of Gd-neutron capture therapy of regional lymph nodes. Thus, when injected interstitially, the PAMAM-G6 Gd dendrimer not only provides excellent opacification of sentinel lymph nodes, but also provides the potential for targeted therapy of sentinel lymph nodes.

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Superparamagnetic Iron Oxide Nanoparticle Probes for Molecular Imaging.
Thorek DL, Chen AK, Czupryna J, Tsourkas A.
Ann Biomed Eng. 2006 Feb 16; [Epub ahead of print] .
[ expand abstract ]

The field of molecular imaging has recently seen rapid advances in the development of novel contrast agents and the implementation of insightful approaches to monitor biological processes non-invasively. In particular, superparamagnetic iron oxide nanoparticles (SPIO) have demonstrated their utility as an important tool for enhancing magnetic resonance contrast, allowing researchers to monitor not only anatomical changes, but physiological and molecular changes as well. Applications have ranged from detecting inflammatory diseases via the accumulation of non-targeted SPIO in infiltrating macrophages to the specific identification of cell surface markers expressed on tumors. In this article, we attempt to illustrate the broad utility of SPIO in molecular imaging, including some of the recent developments, such as the transformation of SPIO into an activatable probe termed the magnetic relaxation switch.

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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Iridium-complex modified CdSe/ZnS quantum dots; a conceptual design for bifunctionality toward imaging and photosensitization.
Hsieh JM, Ho ML, Wu PW, Chou PT, Tsai TT, Chi Y.
Chem Commun (Camb). 2006 Feb 14;(6):615-7.
[ expand abstract ]

We report the design and synthesis of Ir-complex functionalized CdSe/ZnS quantum dots (QDs), in which the QD plays a key role in imaging, while the Ir-complex acts as a sensitizer to produce singlet oxygen; this conceptual design presents a novel scheme in both bio-imaging and photodynamic therapy.

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Lymphotropic nanoparticle enhanced MR imaging (LNMRI) technique for lymph node imaging.
Saksena MA, Saokar A, Harisinghani MG.
Eur J Radiol. 2006 Feb 9; [Epub ahead of print] .
[ expand abstract ]

Accurate nodal staging is important in the management of any primary malignancy. The presence of nodal metastases has both therapeutic and prognostic implications. Lymphotropic nanoparticles are a new class of MRI contrast agents, which are promising in detecting minimal metastatic nodal disease particularly in normal sized lymph nodes. This paper discusses the technique and interpretation of lymphotropic nanoparticle enhanced MRI (LNMRI) and reviews the various trials evaluating nodal staging with ferumoxtran-10 enhanced MRI.

Controlled Clustering of Superparamagnetic Nanoparticles Using Block Copolymers: Design of New Contrast Agents for Magnetic Resonance Imaging.
Berret JF, Schonbeck N, Gazeau F, El Kharrat D, Sandre O, Vacher A, Airiau M.
J Am Chem Soc. 2006 Feb 8;128(5):1755-1761.
[ expand abstract ]

When polyelectrolyte-neutral block copolymers are mixed in aqueous solutions with oppositely charged species, stable complexes are found to form spontaneously. The mechanism is based on electrostatics and on the compensation between the opposite charges. Electrostatic complexes exhibit a core-shell microstructure. In the core, the polyelectrolyte blocks and the oppositely charged species are tightly bound and form a dense coacervate microphase. The shell is made of the neutral chains and surrounds the core. In this paper, we report on the structural and magnetic properties of such complexes made from 6.3 nm diameter superparamagnetic nanoparticles (maghemite gamma-Fe(2)O(3)) and cationic-neutral copolymers. The copolymers investigated are poly(trimethylammonium ethylacrylate methyl sulfate)-b-poly(acrylamide), with molecular weights 5000-b-30000 g mol(-)(1) and 110000-b-30000 g mol(-)(1). The mixed copolymer-nanoparticle aggregates were characterized by a combination of light scattering and cryo-transmission electron microscopy. Their hydrodynamic diameters were found in the range 70-150 nm, and their aggregation numbers (number of nanoparticles per aggregate) from tens to hundreds. In addition, Magnetic Resonance Spin-Echo measurements show that the complexes have a better contrast in Magnetic Resonance Imaging than single nanoparticles and that these complexes could be used for biomedical applications.

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Theoretical predictions of harmonic generation from submicron ultrasound contrast agents for nonlinear biomedical ultrasound imaging.
Zheng H, Mukdadi O, Shandas R.
Phys Med Biol. 2006 Feb 7;51(3):557-73.
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Submicron ultrasound contrast agents have aroused attention for their significant promise in ultrasonic contrast/molecular imaging, targeted therapy and echo particle imaging velocimetry. However, nonlinear acoustic properties of submicron encapsulated gas bubbles for ultrasonic applications are still not clearly understood. In this paper, nonlinear acoustic emission characteristics from submicron bubbles were examined using a numerical study. The modified RP equation incorporating viscosity, acoustic radiation, thermal effects and encapsulated shell was used to study single bubble dynamics. Further, a size integration method, shown previously to be useful in prediction of backscatter spectra from groups of bubbles, was applied to analyse response from a bubble population. We show that bubbles with radii (200-500 nm) produce significant subharmonic and ultraharmonic components of the backscatter spectrum, while smaller bubbles (<200 nm) provide substantial second harmonic components. Additionally, nanoscale bubbles (<100 nm) produce very low backscatter amplitudes and thus may not be useful with the use of current ultrasound technology. Analysing optimal ultrasound driving pressures and bubbles size ranges for maximal subharmonic and ultraharmonic signals showed that sub and ultraharmonic mode nonlinear imaging methods may be potentially competitive for larger size bubbles (>200 nm) in providing proper contrast-to-tissue signal ratios.

Engineering Luminescent Quantum Dots for In Vivo Molecular and Cellular Imaging.
Smith AM, Ruan G, Rhyner MN, Nie S.
Ann Biomed Eng. 2006 Feb 1;:1-12 .
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Semiconductor quantum dots are luminescent nanoparticles that are under intensive development for use as a new class of optical imaging contrast agents. Their novel properties such as optical tunability, improved photostability, and multicolor light emission have opened new opportunities for imaging living cells and in vivo animal models at unprecedented sensitivity and spatial resolution. Combined with biomolecular engineering strategies for tailoring the particle surfaces at the molecular level, bioconjugated quantum dot probes are well suited for imaging single-molecule dynamics in living cells, for monitoring protein-protein interactions within specific intracellular locations, and for detecting diseased sites and organs in deep tissue. In this article, we describe the engineering principles for preparing high-quality quantum dots and for conjugating the dots to biomolecular ligands. We also discuss recent advances in using quantum dots for in vivo molecular and cellular imaging.

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Evaluation of Gd(III)DTPA-terminated poly(propylene imine) dendrimers as contrast agents for MR imaging.
Langereis S, de Lussanet QG, van Genderen MH, Meijer EW, Beets-Tan RG, Griffioen AW, van Engelshoven JM, Backes WH.
NMR Biomed. 2006 Feb;19(1):133-41.
[ expand abstract ]

Different generations of Gd(III)DTPA-terminated poly(propylene imine) dendrimers {G1 [n = 4 Gd(III) ions per molecule], G3 (n = 16) and G5 (n = 64)} and reference Gd(III)DTPA complex [G0 (n = 1)] were characterized in terms of (i) longitudinal (r(1)) and transverse (r(2)) relaxivities in mouse blood plasma, (ii) concentration detection limits in vitro and (iii) in vivo contrast-enhanced MR imaging (CE-MRI) in mice at 1.5 T. Serial and dynamic CE-MRI were performed to monitor the distribution of MRI contrast agent in the heart, arteries, renal system, liver, spleen, bladder and tumor periphery. The relaxivities increased non-linearly with molecular weight (for G0 ionic r(1) = 8.1 mM(-1) s(-1) and ionic r(2) = 8.6 mM(-1) s(-1) to G5 19.3 and 25.0, respectively). The minimal detectable dendrimer concentration was more than two orders of magnitude lower for G5 (8.1 x 10(-8) M) than for G0 (3.1 x 10(-5) M). Sub-millimeter-sized blood vessels were well visualized with serial CE-MRI with each contrast agent. Dynamic CE-MRI showed timely renal clearance for all contrast agents, but a stronger and a prolonged blood signal enhancement for the higher generations of the dendritic contrast agent. Moreover, G0 and G1 showed a rapid tumor wash-in and wash-out, whereas G3 and G5 displayed a more gradual and prolonged tumor wash-in. In conclusion, both G0 and dendritic contrast agents G1, G3 and G5 are well suited for non-tissue-specific MRI of sub-millimeter-sized blood vessels and evaluating tumor microcirculatory characteristics in mice. Higher generations of dendritic contrast agents display lower concentration detection limits, which suggests their future use for molecular imaging. Copyright (c) 2006 John Wiley & Sons, Ltd.

A Liposomal Nanoscale Contrast Agent for Preclinical CT in Mice.
Mukundan S Jr, Ghaghada KB, Badea CT, Kao CY, Hedlund LW, Provenzale JM, Johnson GA, Chen E, Bellamkonda RV, Annapragada A.
AJR Am J Roentgenol. 2006 Feb;186(2):300-7.
[ expand abstract ]

OBJECTIVE: The goal of this study was to determine if an iodinated, liposomal contrast agent could be used for high-resolution, micro-CT of low-contrast, small-size vessels in a murine model. MATERIALS AND METHODS: A second-generation, liposomal blood pool contrast agent encapsulating a high concentration of iodine (83-105 mg I/mL) was evaluated. A total of five mice weighing between 20 and 28 g were infused with equivalent volume doses (500 muL of contrast agent/25 g of mouse weight) and imaged with our micro-CT system for intervals of up to 240 min postinfusion. The animals were anesthetized, mechanically ventilated, and vital signs monitored allowing for simultaneous cardiac and respiratory gating of image acquisition. RESULTS: Initial enhancement of about 900 H in the aorta was obtained, which decreased to a plateau level of approximately 800 H after 2 hr. Excellent contrast discrimination was shown between the myocardium and cardiac blood pool (650-700 H). No significant nephrogram was identified, indicating the absence of renal clearance of the agent. CONCLUSION: The liposomal-based iodinated contrast agent shows long residence time in the blood pool, very high attenuation within submillimeter vessels, and no significant renal clearance rendering it an effective contrast agent for murine vascular imaging using a micro-CT scanner.

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Lipid-based nanoparticles for contrast-enhanced MRI and molecular imaging.
Mulder WJ, Strijkers GJ, van Tilborg GA, Griffioen AW, Nicolay K.
NMR Biomed. 2006 Feb;19(1):142-64.
[ expand abstract ]

In the field of MR imaging and especially in the emerging field of cellular and molecular MR imaging, flexible strategies to synthesize contrast agents that can be manipulated in terms of size and composition and that can be easily conjugated with targeting ligands are required. Furthermore, the relaxivity of the contrast agents, especially for molecular imaging applications, should be very high to deal with the low sensitivity of MRI. Lipid-based nanoparticles, such as liposomes or micelles, have been used extensively in recent decades as drug carrier vehicles. A relatively new and promising application of lipidic nanoparticles is their use as multimodal MR contrast agents. Lipids are amphiphilic molecules with both a hydrophobic and a hydrophilic part, which spontaneously assemble into aggregates in an aqueous environment. In these aggregates, the amphiphiles are arranged such that the hydrophobic parts cluster together and the hydrophilic parts face the water. In the low concentration regime, a wide variety of structures can be formed, ranging from spherical micelles to disks or liposomes. Furthermore, a monolayer of lipids can serve as a shell to enclose a hydrophobic core. Hydrophobic iron oxide particles, quantum dots or perfluorocarbon emulsions can be solubilized using this approach. MR-detectable and fluorescent amphiphilic molecules can easily be incorporated in lipidic nanoparticles. Furthermore, targeting ligands can be conjugated to lipidic particles by incorporating lipids with a functional moiety to allow a specific interaction with molecular markers and to achieve accumulation of the particles at disease sites. In this review, an overview of different lipidic nanoparticles for use in MRI is given, with the main emphasis on Gd-based contrast agents. The mechanisms of particle formation, conjugation strategies and applications in the field of contrast-enhanced, cellular and molecular MRI are discussed. Copyright (c) 2006 John Wiley & Sons, Ltd.

An X-ray computed tomography imaging agent based on long-circulating bismuth sulphide nanoparticles.
Rabin O, Manuel Perez J, Grimm J, Wojtkiewicz G, Weissleder R.
Nat Mater. 2006 Feb;5(2):118-22.
[ expand abstract ]

Nanomaterials have become increasingly important in the development of new molecular probes for in vivo imaging, both experimentally and clinically. Nanoparticulate imaging probes have included semiconductor quantum dots, magnetic and magnetofluorescent nanoparticles, gold nanoparticles and nanoshells, among others. However, the use of nanomaterials for one of the most common imaging techniques, computed tomography (CT), has remained unexplored. Current CT contrast agents are based on small iodinated molecules. They are effective in absorbing X-rays, but non-specific distribution and rapid pharmacokinetics have rather limited their microvascular and targeting performance. Here we propose the use of a polymer-coated Bi(2)S(3) nanoparticle preparation as an injectable CT imaging agent. This preparation demonstrates excellent stability at high concentrations (0.25 M Bi(3+)), high X-ray absorption (fivefold better than iodine), very long circulation times (>2 h) in vivo and an efficacy/safety profile comparable to or better than iodinated imaging agents. We show the utility of these polymer-coated Bi(2)S(3) nanoparticles for enhanced in vivo imaging of the vasculature, the liver and lymph nodes in mice. These nanoparticles and their bioconjugates are expected to become an important adjunct to in vivo imaging of molecular targets and pathological conditions.

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Sentinel Lymph Node Mapping of the Gastrointestinal Tract by Using Invisible Light.
Soltesz EG, Kim S, Kim SW, Laurence RG, De Grand AM, Parungo CP, Cohn LH, Bawendi MG, Frangioni JV.
Ann Surg Oncol. 2006 Jan 31; .
[ expand abstract ]

BACKGROUND: Because many gastrointestinal (GI) tumors spread by way of lymphatics, histological assessment of the first draining lymph nodes has both prognostic and therapeutic significance. However, sentinel lymph node mapping of the GI tract by using available techniques is limited by unpredictable drainage patterns, high background signal, and the inability to image lymphatic tracers relative to surgical anatomy in real time. Our goal was to develop a method for patient-specific intraoperative sentinel lymph node mapping of the GI tract by using invisible near-infrared light. METHODS: We developed an intraoperative near-infrared fluorescence imaging system that simultaneously displays surgical anatomy and otherwise invisible near-infrared fluorescence images of the surgical field. Near-infrared fluorescent quantum dots were injected intraparenchymally into the stomach, small bowel, and colon, and draining lymphatic channels and sentinel lymph nodes were visualized. Dissection was performed under real-time image guidance. RESULTS: In 10 adult pigs, we demonstrated that 200 pmol of quantum dots quickly and accurately map lymphatic drainage and sentinel lymph nodes. Injection into the mid jejunum and colon results in fluorescence of a single lymph node at the root of the bowel mesentery. Injection into the stomach resulted in identification of a retrogastric node. Histological analysis in all cases confirmed the presence of nodal tissue. CONCLUSIONS: We report the use of invisible near-infrared light for intraoperative sentinel lymph node mapping of the GI tract. This technology overcomes the limitations of currently available methods, permits patient-specific imaging of lymphatic flow and sentinel nodes, and provides highly sensitive, real-time image-guided dissection.

Directing energy flow through quantum dots: towards nanoscale sensing.
Willard DM, Mutschler T, Yu M, Jung J, Van Orden A.
Anal Bioanal Chem. 2006 Jan 27;:1-8 .
[ expand abstract ]

Nanoscale sensors can be created when an expected energetic pathway is created and then that pathway is either initiated or disrupted by a specific binding event. Constructing the sensor on the nanoscale could lead to greater sensitivity and lower limits of detection. To this end, quantum dots (QDs) can be considered prime candidates for the active components. Relative to organic chromophores, QDs have tunable spectral properties, show less susceptibility to photobleaching, have similar brightness, and have been shown to display electro-optical properties. In this review, we discuss recent articles that incorporate QDs into directed energy flow systems, some with the goal of building new and more powerful sensors and others that could lead to more powerful sensors.

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Quantum Dots with a Paramagnetic Coating as a Bimodal Molecular Imaging Probe.
Mulder WJ, Koole R, Brandwijk RJ, Storm G, Chin PT, Strijkers GJ, de Mello Donega C, Nicolay K, Griffioen AW.
Nano Lett. 2006 Jan 11;6(1):1-6.
[ expand abstract ]

MRI detectable and targeted quantum dots were developed. To that aim, quantum dots were coated with paramagnetic and pegylated lipids, which resulted in a relaxivity, r(1), of nearly 2000 mM(-1)s(-1) per quantum dot. The quantum dots were functionalized by covalently linking alphavbeta3-specific RGD peptides, and the specificity was assessed and confirmed on cultured endothelial cells. The bimodal character, the high relaxivity, and the specificity of this nanoparticulate probe make it an excellent contrast agent for molecular imaging purposes.

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Surfactant-stabilized contrast agent on the nanoscale for diagnostic ultrasound imaging.
Wheatley MA, Forsberg F, Dube N, Patel M, Oeffinger BE.
Ultrasound Med Biol. 2006 Jan;32(1):83-93.
[ expand abstract ]

Ultrasound contrast agents (CA) are generally micron-sized stabilized gas bubbles, injected IV. However, to penetrate beyond the vasculature and accumulate in targets such as tumors, CA must be an order of magnitude smaller. We describe a method of achieving nanometer-sized, surfactant-stabilized CA by differential centrifugation. High g force was shown to destroy bubble integrity. Optimal conditions (300 rpm for 3 min) produced an agent with a mean diameter of 450 nm, which gave 25.5 dB enhancement in vitro at a dose of 10 muL/mL, with a 13 min half-life. In vivo, the CA produced excellent power Doppler and grey-scale pulse inversion harmonic images at low acoustic power when administered. In vivo dose-response curves obtained in three rabbits showed enhancement between 20 and 25 dB for dosages above 0.025 mL/kg. These results encourage further investigation of the possible diagnostic and therapeutic benefits of using nanoparticles as CA, including passive targeting and accumulation in tumors.

Diagnostic precision of nanoparticle-enhanced MRI for lymph-node metastases: a meta-analysis.
Will O, Purkayastha S, Chan C, Athanasiou T, Darzi AW, Gedroyc W, Tekkis PP.
Lancet Oncol. 2006 Jan;7(1):52-60.
[ expand abstract ]

BACKGROUND: At present, there is no accepted, ideal imaging modality or technique for diagnosis of lymph-node metastases. We aimed to assess the diagnostic precision of MRI with ferumoxtran-10-an ultrasmall superparamagnetic iron-oxide nanoparticle used as a contrast agent for diagnosis of lymph-node metastases, compared with that of unenhanced MRI and final histological diagnosis. METHODS: We did a meta-analysis of prospective studies that compared MRI, with and without ferumoxtran-10, with histological diagnosis after surgery or biopsy. Sensitivity, specificity, and diagnostic odds ratio (DOR) were calculated for every study; summary receiver operating characteristic (ROC) and subgroup analyses were done; and study quality and heterogeneity were assessed. Metaregression analysis was used to analyse the effect of ferumoxtran-10 in diagnostic precision of MRI. FINDINGS: Summary ROC curve analysis for per-lymph-node data showed an overall sensitivity of 0.88 (95% CI 0.85-0.91) and overall specificity of 0.96 (0.95-0.97) for ferumoxtran-10-enhanced MRI. Overall weighted area under the curve for ferumoxtran-10-enhanced MRI was 0.96 (SE 0.01), DOR 123.05 (95% CI 5.93-256.93). Unenhanced MRI had less overall sensitivity (0.63 [0.57-0.69]) and specificity (0.93 [0.91-0.94]), with an overall weighted area under the ROC curve of 0.84 (SE 0.11) and DOR of 26.75 (95% CI 8.48-84.42). Significant heterogeneity was noted for studies reporting enhanced MRI and unenhanced MRI. Metaregression analysis confirmed the significant effect of ferumoxtran-10 in the diagnostic precision of MRI (p=0.001). INTERPRETATION: Ferumoxtran-10-enhanced MRI is sensitive and specific in detection of lymph-node metastases for various tumours. It offers higher diagnostic precision than does unenhanced MRI for detection of lymph-node metastases, and allows functional and anatomical definition when used as an imaging modality.

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Evolving and experimental technologies in medical imaging.
Wolbarst AB, Hendee WR.
Radiology. 2006 Jan;238(1):16-39.
[ expand abstract ]

Medical images are created by detecting radiation probes transmitted through or emitted or scattered by the body. The radiation, modulated through interactions with tissues, yields patterns that provide anatomic and/or physiologic information. X-rays, gamma rays, radiofrequency signals, and ultrasound waves are the standard probes, but others like visible and infrared light, microwaves, terahertz rays, and intrinsic and applied electric and magnetic fields are being explored. Some of the younger technologies, such as molecular imaging, may enhance existing imaging modalities; however, they also, in combination with nanotechnology, biotechnology, bioinformatics, and new forms of computational hardware and software, may well lead to novel approaches to clinical imaging. This review provides a brief overview of the current state of image-based diagnostic medicine and offers comments on the directions in which some of its subfields may be heading. RSNA, 2006.

2005

Multistep synthesis of a radiolabeled imaging probe using integrated microfluidics.
Lee CC, Sui G, Elizarov A, Shu CJ, Shin YS, Dooley AN, Huang J, Daridon A, Wyatt P, Stout D, Kolb HC, Witte ON, Satyamurthy N, Heath JR, Phelps ME, Quake SR, Tseng HR.
Science. 2005 Dec 16;310(5755):1793-6.
[ expand abstract ]

Microreactor technology has shown potential for optimizing synthetic efficiency, particularly in preparing sensitive compounds. We achieved the synthesis of an [(18)F]fluoride-radiolabeled molecular imaging probe, 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG), in an integrated microfluidic device. Five sequential processes-[18F]fluoride concentration, water evaporation, radiofluorination, solvent exchange, and hydrolytic deprotection-proceeded with high radio-chemical yield and purity and with shorter synthesis time relative to conventional automated synthesis. Multiple doses of [18F]FDG for positron emission tomography imaging studies in mice were prepared. These results, which constitute a proof of principle for automated multistep syntheses at the nanogram to microgram scale, could be generalized to a range of radiolabeled substrates

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Nano-sized MRI contrast agents with dendrimer cores.
Kobayashi H, Brechbiel MW.
Adv Drug Deliv Rev. 2005 Dec 14;57(15):2271-86.
[ expand abstract ]

Gadolinium-based MRI contrast agents (CAs) can be effective at a approximately 100-fold lower concentration of Gadolinium ions in comparison to the concentration of Iodine atoms required for CT imaging. Therefore, a number of dendrimer based macromolecular MRI CAs of various sizes and properties prepared employing relatively simple chemistry are readily available that can provide sufficient contrast enhancement for various applications. Molecules up to 20 nm in diameter behave differently in the body depending on their size. Even if these molecules possess similar chemical properties, small changes in size can greatly impact their pharmacokinetics. Changes in molecular size up to 15 nm in diameter altered permeability across the vascular wall, excretion route, and recognition by the reticuloendothelial system. Smaller sized polyamidoamine (PAMAM) dendrimer-based contrast agents, i.e., less than 3 nm in diameter, easily "leak" across the vascular wall resulting in rapid perfusion throughout the body. Contrast agents 3-6 nm in diameter were quickly excreted through the kidney indicating these agents to be potentially suitable as functional renal contrast agents. Contrast agents 7-12 nm in diameter were retained in circulation and were better suited for use as blood pool contrast agents. Hydrophobic variants of CAs formed with polypropylenimine diaminobutane (DAB) dendrimer cores quickly accumulated in the liver and potentially have use as liver contrast agents. Larger hydrophilic agents have suitable characteristics for lymphatic imaging. Finally, contrast agents conjugated with either monoclonal antibodies or with avidin are able to function as tumor-specific contrast agents and might also be employed as either gadolinium neutron capture therapy or in conjunction with radioimmunotherapy.

Quantum dots to monitor RNAi delivery and improve gene silencing.
Chen AA, Derfus AM, Khetani SR, Bhatia SN.
Nucleic Acids Res. 2005 Dec 13;33(22):e190.
[ expand abstract ]

A critical issue in using RNA interference for identifying genotype/phenotype correlations is the uniformity of gene silencing within a cell population. Variations in transfection efficiency, delivery-induced cytotoxicity and 'off target' effects at high siRNA concentrations can confound the interpretation of functional studies. To address this problem, we have developed a novel method of monitoring siRNA delivery that combines unmodified siRNA with seminconductor quantum dots (QDs) as multi color biological probes. We co-transfected siRNA with QDs using standard transfection techniques, thereby leveraging the photostable fluorescent nanoparticles to track delivery of nucleic acid, sort cells by degree of transfection and purify homogenously-silenced subpopulations. Compared to alternative RNAi tracking methods (co-delivery of reporter plasmids and end-labeling the siRNA), QDs exhibit superior photostability and tunable optical properties for an extensive selection of non-overlapping colors. Thus this simple, modular system can be extended toward multiplexed gene knockdown studies, as demonstrated in a two color proof-of-principle study with two biological targets. When the method was applied to investigate the functional role of T-cadherin (T-cad) in cell-cell communication, a subpopulation of highly silenced cells obtained by QD labeling was required to observe significant downstream effects of gene knockdown.

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Development of water-soluble metallofullerenes as X-ray contrast media.
Miyamoto A, Okimoto H, Shinohara H, Shibamoto Y.
Eur Radiol. 2005 Dec 13;1-4 [Epub ahead of print].
[ expand abstract ]

Fullerenes are a new carbonic allotrope having a cage structure. We investigated whether fullerenes containing one or two atoms of heavy metals could be an X-ray contrast material with little adverse effects. One or two atoms of dysprosium (Dy), erbium (Er), gadolinium (Gd), europium (Eu) and lutetium (Lu) were encapsulated into fullerene (C(82)), which was synthesized as a polyhydroxyl form (e.g., Gd@C(82)(OH)n, n=40, Gd - fullerenols). They were dissolved in water at maximum soluble concentrations and subjected to CT number analysis. The CT numbers of the solutions were measured using a 4- or 16-row multidetector CT scanner. The CT number of the water-soluble metallofullerenes were 56.0 HU for Dy@C(82)(OH)(40), 111.5 HU for Er@C(82)(OH)(40), 58.4 HU for Gd@C(82)(OH)(40), 100.9 HU for Eu@C(82)(OH)(40) and 23.3 HU for Lu(2)@C(82)(OH)(40). The CT numbers of the metallofullerenes investigated in the present study were not high enough to be used in the clinic in place of iodinated contrast materials. However, if nanotechnology progresses in the near future, it may prove to have a possibility as an X-ray contrast material.

Real-time visualization and characterization of liposomal delivery into the monkey brain by magnetic resonance imaging.
Krauze MT, McKnight TR, Yamashita Y, Bringas J, Noble CO, Saito R, Geletneky K, Forsayeth J, Berger MS, Jackson P, Park JW, Bankiewicz KS.
Brain Res Brain Res Protoc. 2005 Dec;16(1-3):20-6.
[ expand abstract ]

Liposomes loaded with Gadoteridol, in combination with convection-enhanced delivery (CED), offer an excellent option to monitor CNS delivery of therapeutic compounds with MRI. In previous studies, we investigated possible clinical applications of liposomes to the treatment of brain tumors. In this study, up to 700 mul of Gadoteridol/rhodamine-loaded liposomes were distributed in putamen, corona radiata and brainstem of non-human primates. Distribution was monitored by real-time MRI throughout infusion procedures and allowed accurate calculation of volume of distribution within anatomical structures. We found that different regions of the brain gave various volumes of distribution when infused with the same volume of liposome. Based on these findings, distinct distribution pathways within infused structures can be predicted. This work underlines the importance of monitoring drug delivery to CNS and enables accurate delivery of drug-loaded liposomes to specific brain regions with a standard MRI procedure. Findings presented in this manuscript may allow for modeling of parameters used for direct delivery of therapeutics into various regions of the brain.

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MR molecular imaging and fluorescence microscopy for identification of activated tumor endothelium using a bimodal lipidic nanoparticle.
Mulder WJ, Strijkers GJ, Habets JW, Bleeker EJ, van der Schaft DW, Storm G, Koning GA, Griffioen AW, Nicolay K.
FASEB J. 2005 Dec;19(14):2008-10.
[ expand abstract ]

In oncological research, there is a great need for imaging techniques that specifically identify angiogenic blood vessels in tumors on the basis of differences in the expression level of biomolecular markers. In the angiogenic cascade, different cell surface receptors, including the alphavbeta3-integrin, are strongly expressed on activated endothelial cells. In the present study, we aimed to image angiogenesis by detecting the expression of alphavbeta3 in tumor bearing mice with a combination of magnetic resonance imaging (MRI) and fluorescence microscopy. To that end, we prepared MR-detectable and fluorescent liposomes, which carry approximately 700 alphavbeta3-specific RGD peptides per liposome. RGD competition experiments and RAD-conjugated liposomes were used as controls for specificity. In vivo, both RAD liposomes and RGD liposomes gave rise to signal increase on T1-weighted MR images. It was established by the use of ex vivo fluorescence microscopy that RGD liposomes and RAD liposomes accumulated in the tumor by different mechanisms. RGD liposomes were specifically associated with activated tumor endothelium, while RAD liposomes were located in the extravascular compartment. This study demonstrates that MR molecular imaging of angiogenesis is feasible by using a targeted contrast agent specific for the alphavbeta3-integrin, and that the multimodality imaging approach gave insight into the exact mechanism of accumulation in the tumor.

Fluorescent nanocrystals for use in early cervical cancer detection.
Nida DL, Rahman MS, Carlson KD, Richards-Kortum R, Follen M.
Gynecol Oncol. 2005 Dec;99(3 Suppl):S89-94.
[ expand abstract ]

BACKGROUND.: Quantum dots (qdots) are a promising alternative to organic fluorophores for biological imaging. Advantages of quantum dots over organic fluorophores include broad excitation coupled with narrow, tunable emission, high resistance to chemical and metabolic degradation, a higher photobleaching threshold and finally the ability to be modified with a targeting ligand. These many properties allow quantum dots to be used in conjunction with optical detection methods for imaging. METHODS.: We are investigating the use of quantum dots to detect precancerous biomarkers. We have directly targeted epidermal growth factor receptors with quantum dots conjugated to anti-EGFR antibodies. RESULTS.: Compared to appropriate controls, we do see specific labeling of EGF receptors. CONCLUSIONS.: Quantum dots provide a promising alternative to conventional organic dyes for biological imaging. Combined with optical imaging technologies, quantum dots can help visualize changes in cervical cancer at the molecular level. This ability may alert health care providers to the need for intervention before a cancer can metastasize.

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Optical imaging of cervical pre-cancers with structured illumination: An integrated approach.
Rahman M, Abd-El-Barr M, Mack V, Tkaczyk T, Sokolov K, Richards-Kortum R, Descour M.
Gynecol Oncol. 2005 Dec;99(3 Suppl):S112-5.
[ expand abstract ]

OBJECTIVE.: Structured illumination microscopy is an inexpensive alternative to confocal microscopy that allows optical sectioning at a sub-cellular resolution. However, its application in imaging biological tissue has been limited by inadequate contrast present in them especially in reflectance imaging. Novel, optically active contrast agents like gold nanoparticles and quantum dots targeted against biomarkers of cancer can be integrated with structured illumination to image both the morphological and biochemical changes associated with epithelial pre-cancers. METHODS.: We modified the optical path of a widefield microscope to implement structured illumination both in reflectance and fluorescence modes. For imaging, we used 25-nm-diameter gold nanoparticles and CdSe quantum dots for reflectance and fluorescence imaging, respectively, to label three-dimensional tissue constructs of SiHa cervical cancer cells. Contrast agents were targeted against the epidermal growth factor receptor (EGFR) using an anti-EGFR monoclonal antibody. Agents targeted with a non-specific IgG antibody served as a control to monitor non-specific labeling. RESULTS.: Our result shows that optically sectioned images taken with structured illumination are very comparable to those obtained using confocal microscopy. Moreover, images of three-dimensional cultures stained with the anti-EGFR agents show significantly more image intensity than those stained with the IgG targeted control. CONCLUSION.: Our findings suggest that the combination of novel optical contrast agents and structured illumination can differentiate neoplastic cells which overexpress EGFR from normal cells in intact tissue. Combining structured illumination microscopy with novel contrast agents can potentially provide a powerful and inexpensive tool to aid in the detection of cervical pre-cancers.

Gadolinium-loaded liposomes allow for real-time magnetic resonance imaging of convection-enhanced delivery in the primate brain.
Saito R, Krauze MT, Bringas JR, Noble C, McKnight TR, Jackson P, Wendland MF, Mamot C, Drummond DC, Kirpotin DB, Hong K, Berger MS, Park JW, Bankiewicz KS.
Exp Neurol. 2005 Dec;196(2):381-9.
[ expand abstract ]

Drug delivery to brain tumors has long posed a major challenge. Convection-enhanced delivery (CED) has been developed as a drug delivery strategy to overcome this difficulty. Ideally, direct visualization of the tissue distribution of drugs infused by CED would assure successful delivery of therapeutic agents to the brain tumor while minimizing exposure of the normal brain. We previously developed a magnetic resonance imaging (MRI)-based method to visualize the distribution of liposomal agents after CED in rodent brains. In the present study, CED of liposomes was further examined in the non-human primate brain (n = 6). Liposomes containing Gadoteridol, DiI-DS, and rhodamine were infused in corona radiata, putamen nucleus, and brain stem. Volume of distribution was analyzed for all delivery locations by histology and MR imaging. Real-time MRI monitoring of liposomes containing gadolinium allowed direct visualization of a robust distribution. MRI of liposomal gadolinium was highly accurate at determining tissue distribution, as confirmed by comparison with histological results from concomitant administration of fluorescent liposomes. Linear correlation for liposomal infusions between infusion volume and distribution volume was established in all targeted locations. We conclude that an integrated strategy combining liposome/nanoparticle technology, CED, and MRI may provide new opportunities for the treatment of brain tumors. Our ability to directly monitor and to control local delivery of liposomal drugs will most likely result in greater clinical efficacy when using CED in management of patients.

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The molecular analysis of breast cancer utilizing targeted nanoparticle based ultrasound contrast agents.
Sakamoto JH, Smith BR, Xie B, Rokhlin SI, Lee SC, Ferrari M.
Technol Cancer Res Treat. 2005 Dec;4(6):627-36.
[ expand abstract ]

This study was structured to challenge the hypothesis that nano-sized particulates could be molecularly targeted and bound to the prognostic and predictive HER-2/neu cell membrane receptor to elicit detectable changes in ultrasound response from human breast cancer cells. SKBR-3 human breast cancer cells were enlisted to test the efficacy of the particle conjugation strategy used in this study and ultimately, to provide conclusive remarks regarding the validity of the stated hypothesis. A characterization-mode ultrasound (CMUS) system was used to apply a continuum mechanics based, two-step inversion algorithm to reconstruct the mechanical material properties of four cell/agarose test conditions upon three independent test samples. The four test conditions include: Herceptin(R) conjugated iron oxide nanoparticles bound to cells (HER-con), Herceptin(R) bound to cells (HER), iso-type matched antibody conjugated iron oxide nanoparticles bound to cells (ISO-con), and Cold Flow Buffer mixed with agarose (CFB). The statistical analysis of these ultrasound results supported the ability to differentiate between HER-2/neu positive SKBR-3 cells that have been successfully tagged with Herceptin(R) conjugated iron oxide particles to those that have not demonstrated particle binding. These findings serve as promising proof-of-concept data for the development of a quantitative histopathologic evaluation tool directed towards both in situ and in vivo applications. The ultimate goal of this research is to exploit the molecular expression of the HER-2/neu protein to offer rapid, quantitative ultrasound information concerning the malignancy rating of human breast tissue employing tumor targeting nanoparticle based ultrasound contrast agents. When fully developed, this could potentially help 32,000-63,000 women efficiently find their proper treatment strategy to fight and win their battle against breast cancer.

Fluorescent nanoparticle probes for cancer imaging.
Santra S, Dutta D, Walter GA, Moudgil BM.
Technol Cancer Res Treat. 2005 Dec;4(6):593-602.
[ expand abstract ]

Optical imaging technique has strong potential for sensitive cancer diagnosis, particularly at the early stage of cancer development. This is a sensitive, non-invasive, non-ionizing (clinically safe) and relatively inexpensive technique. Cancer imaging with optical technique however greatly relies upon the use of sensitive and stable optical probes. Unlike the traditional organic fluorescent probes, fluorescent nanoparticle probes such as dye-doped nanoparticles and quantum dots (Qdots) are bright and photostable. Fluorescent nanoparticle probes are shown to be very effective for sensitive cancer imaging with greater success in the cellular level. However, cancer imaging in an in vivo setup has been recently realized. There are several challenges in developing fluorescent nanoparticle probes for in vivo cancer imaging applications. In this review, we will discuss various aspects of nanoparticle design, synthesis, surface functionalization for bioconjugation and cancer cell targeting. A brief overview of in vivo cancer imaging with Qdots will also be presented.

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Aqueous dispersions of magnetite nanoparticles with NH3+ surfaces for magnetic manipulations of biomolecules and MRI contrast agents.
Shieh DB, Cheng FY, Su CH, Yeh CS, Wu MT, Wu YN, Tsai CY, Wu CL, Chen DH, Chou CH.
Biomaterials. 2005 Dec;26(34):7183-91.
[ expand abstract ]

In the current study, amine surface modified iron-oxide nanoparticles of 6 nm diameter without polymer coating were fabricated in an aqueous solution by organic acid modification as an adherent following chemical coprecipitation. Structure and the superparamagnetic property of magnetite nanoparticles were characterized by selected area electron diffraction (SAED) and superconducting quantum interference measurement device (SQUID). X-ray photoelectron spectrometer (XPS) and zeta potential measurements revealed cationic surface mostly decorated with terminal -NH(3)(+). This feature enables them to function as a magnetic carrier for nucleotides via electrostatic interaction. In addition, Fe(3)O(4)/trypsin conjugates with well-preserved functional activity was demonstrated. The nanoparticles displayed excellent in vitro biocompatibility. The NMR and the in vitro MRI measurements showed significantly reduced water proton relaxation times of both T(1) and T(2). Significantly reduced T(2) and T(2)*-weighted signal intensity were observed in a 1.5 T clinical MR imager. In vivo imaging contrast effect showed a fast and prolonged inverse contrast effect in the liver that lasted for more than 1 week. In addition, it was found that the spherical Fe(3)O(4) assembled as rod-like configuration through an aging process in aqueous solution at room temperature. Interestingly, TEM observation of the liver tissue revealed the rod-like shape but not the spherical-type nanoparticles being taken up by the Kupffer cells 120 h after tail vein infusion. Combining these results, we have demonstrated the potential applications of the newly synthesized magnetite nanoparticles in a broad spectrum of biomedical applications.

Imaging of small nanoparticle-containing objects by finite-element-based photoacoustic tomography.
Yuan Z, Wu C, Zhao H, Jiang H.
Opt Lett. 2005 Nov 15;30(22):3054-6.
[ expand abstract ]

We present semiquantitative photoacoustic images of small nanoparticle-containing objects having a wide range of contrast levels relative to the background. The images are obtained by a finite-element reconstruction algorithm that is based on the Helmholtz-like photoacoustic wave equation in the frequency domain. Our reconstruction approach is an iterative Newton method coupled with combined Marquardt and Tikhonov regularizations that can extract the spatial distribution of relative optical absorption property in heterogeneous media. We demonstrate experimental images in single- and multiple-object configurations with a circular scanning photoacoustic tomographic system. The results obtained show that millimeter-size nanoparticle-containing objects can be clearly detected in terms of position, size, and relative optical properties.

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Nanosphere Induced Gene Expression in Human Dendritic Cells.
Matsusaki M, Larsson K, Akagi T, Lindstedt M, Akashi M, Borrebaeck CA.
Nano Lett. 2005 Nov 9;5(11):2168-2173.
[ expand abstract ]

The molecular mechanisms of nanosphere-induced mucosal immunization are important to decipher, since this can form the basis for novel approaches in, e.g., nasal vaccination. In this study, we have investigated the effect of nanospheres as antigen carriers on immature human dendritic cells. The results clearly indicate that tetanus toxoid immobilized nanospheres have a direct effect on human monocyte derived dendritic cells and induce a specific transcriptional profile involving genes crucial for phagocytosis and a protective immune response.

Paramagnetic liposomes containing amphiphilic bisamide derivatives of Gd-DTPA with aromatic side chain groups as possible contrast agents for magnetic resonance imaging.
Parac-Vogt TN, Kimpe K, Laurent S, Pierart C, Elst LV, Muller RN, Binnemans K.
Eur Biophys J. 2005 Oct 11;:1-9 [Epub ahead of print].
[ expand abstract ]

Three amphiphilic DTPA bisamide derivatives containing long-chain phenylalanine esters (with 14, 16 and 18 carbon atoms in the alkyl chain) were synthesized and their corresponding gadolinium(III) complexes were prepared. The attempts to form paramagnetic micelles carrying the gadolinium(III) complexes yielded unstable or polydisperse micelles implying that the presence of the bulky aromatic side groups in the amphiphilic Gd-DTPA bisamide complexes results in an inefficient packing of the paramagnetic complex into micelles. All complexes were efficiently incorporated into liposomes consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), yielding stable and monodisperse paramagnetic liposomes. All liposomes had a comparable size, typically between 120 and 160 nm. As a result of the reduced mobility of the gadolinium(III) complexes, solutions of these supramolecular structures show a higher relaxivity than solutions of Gd-DTPA. However, the relaxivity gain is lower compared to compounds consisting of purely aliphatic chains of the same length, most likely due to the less efficient packing or increased local mobility of the gadolinium(III) complex. In the case of the Gd-DTPA bisamide complex with 18 carbon atoms, the immobilization inside the liposomal structure is less effective, probably because the aliphatic chains of the complex are longer than the alkyl chains of the DPPC host, resulting in a relatively high local mobility. The paramagnetic liposomes containing the Gd-DTPA bisamide complexes with 14 carbon atoms showed the highest relaxivity because the optimal length match between the hydrophobic chains of the DPPC and the ligand allowed very efficient packing of the paramagnetic complex into the liposome.

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MR molecular imaging and fluorescence microscopy for identification of activated tumor endothelium using a bimodal lipidic nanoparticle.
Mulder WJ, Strijkers GJ, Habets JW, Bleeker EJ, van der Schaft DW, Storm G, Koning GA, Griffioen AW, Nicolay K.
FASEB J. 2005 Oct 4; [Epub ahead of print].
[ expand abstract ]

In oncological research, there is a great need for imaging techniques that specifically identify angiogenic blood vessels in tumors on the basis of differences in the expression level of biomolecular markers. In the angiogenic cascade, different cell surface receptors, including the alphavbeta3-integrin, are strongly expressed on activated endothelial cells. In the present study, we aimed to image angiogenesis by detecting the expression of alphavbeta3 in tumor bearing mice with a combination of magnetic resonance imaging (MRI) and fluorescence microscopy. To that end, we prepared MR-detectable and fluorescent liposomes, which carry ~700 alphavbeta3-specific RGD peptides per liposome. RGD competition experiments and RAD-conjugated liposomes were used as controls for specificity. In vivo, both RAD liposomes and RGD liposomes gave rise to signal increase on T1-weighted MR images. It was established by the use of ex vivo fluorescence microscopy that RGD liposomes and RAD liposomes accumulated in the tumor by different mechanisms. RGD liposomes were specifically associated with activated tumor endothelium, while RAD liposomes were located in the extravascular compartment. This study demonstrates that MR molecular imaging of angiogenesis is feasible by using a targeted contrast agent specific for the alphavbeta3-integrin, and that the multimodality imaging approach gave insight into the exact mechanism of accumulation in the tumor.

Paramagnetic viral nanoparticles as potential high-relaxivity magnetic resonance contrast agents.
Allen M, Bulte JW, Liepold L, Basu G, Zywicke HA, Frank JA, Young M, Douglas T.
Magn Reson Med. 2005 Oct;54(4):807-12.
[ expand abstract ]

In order to compensate for the inherent high threshold of detectability of MR contrast agents, there has been an active interest in the development of paramagnetic nanoparticles incorporating high payloads of Gd(3+) with high molecular relaxivities. Toward this end, the protein cage of Cowpea chlorotic mottle virus (CCMV), having 180 metal binding sites, is being explored. In vivo CCMV binds Ca(2+) at specific metal binding sites; however, Gd(3+) can also bind at these sites. Using fluorescence resonance energy transfer we have characterized the binding affinity of Gd(3+) to the metal binding sites by competition experiments with Tb(3+). The measured dissociation constant (K(d)) for Gd(3+) bound to the virus is 31 muM. The T(1) and T(2) relaxivities of solvent water protons in the presence of Gd(3+)-bound CCMV were 202 and 376 mM(-1) s(-1), respectively, at 61 MHz Larmor frequency. The unusually high relaxivity values of the Gd(3+)-CCMV are largely a result of the nanoparticle virus size and the large number of Gd(3+) ions bound to the virus. These preliminary results should encourage further investigations into the use of viral protein cages as a new platform for MR contrast agents. Magn Reson Med, 2005. Published 2005 Wiley-Liss, Inc.

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Semiconductor nanocrystals for biological imaging.
Fu A, Gu W, Larabell C, Alivisatos AP.
Curr Opin Neurobiol. 2005 Oct;15(5):568-75.
[ expand abstract ]

Conventional organic fluorophores suffer from poor photo stability, narrow absorption spectra and broad emission spectra. Semiconductor nanocrystals, however, are highly photo-stable with broad absorption spectra and narrow size-tunable emission spectra. Recent advances in the synthesis of these materials have resulted in the generation of bright, sensitive, extremely photo-stable and biocompatible semiconductor fluorophores. Commercial availability facilitates their application in a variety of unprecedented biological experiments, including multiplexed cellular imaging, long-term in vitro and in vivo labeling, deep tissue structure mapping and single particle investigation of dynamic cellular processes. Semiconductor nanocrystals are one of the first examples of nanotechnology enabling a new class of biomedical applications.

An exploratory study of ferumoxtran-10 nanoparticles as a blood-brain barrier imaging agent targeting phagocytic cells in CNS inflammatory lesions.
Manninger SP, Muldoon LL, Nesbit G, Murillo T, Jacobs PM, Neuwelt EA.
AJNR Am J Neuroradiol. 2005 Oct;26(9):2290-300.
[ expand abstract ]

BACKGROUND AND PURPOSE: Iron oxide-based contrast agents have been investigated as more specific MR imaging agents for central nervous system (CNS) inflammation. Ferumoxtran-10 is a virus-size nanoparticle, taken up by reactive cells, that allows visualization of the phagocytic components of CNS lesions. Ferumoxtran-10 was compared with standard gadolinium-enhanced MR images in this exploratory trial to assess its potential in evaluation of CNS lesions with inflammatory aspects, including lymphoma, multiple sclerosis (MS), acute disseminated encephalomyelitis (ADEM), and vascular lesions. METHODS: Twenty-three patients with different types of intracranial "inflammatory" lesions underwent standard brain MR with and without gadolinium, followed an average of 10 days later by a ferumoxtran-10 scan. Patients were imaged 24 hours after infusion of 2.6 mg/kg ferumoxtran-10. All MR images were evaluated subjectively by 4 investigators for a difference in enhancement patterns, which could be useful in differential diagnoses. RESULTS: In 5 cases, (one ADEM, 2 stroke, one cavernous venous vascular malformation, one primary central nervous lymphoma) the ferumoxtran-10 scan showed higher signal intensity, larger area of enhancement, or new enhancing areas compared with gadolinium. Most MS patients showed less enhancement with ferumoxtran-10 than with gadolinium. CONCLUSION: Ferumoxtran-10 showed different enhancement patterns in a variety of CNS lesions with inflammatory components in comparison to gadolinium. The impact of timing and therapy need further evaluation to better assess ferumoxtran-10 in addition to gadolinium as contrast agents for use in diagnosis and monitoring therapy in patients with CNS inflammatory lesions.

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Gadolinium-loaded liposomes allow for real-time magnetic resonance imaging of convection-enhanced delivery in the primate brain.
Saito R, Krauze MT, Bringas JR, Noble C, McKnight TR, Jackson P, Wendland MF, Mamot C, Drummond DC, Kirpotin DB, Hong K, Berger MS, Park JW, Bankiewicz KS.
Exp Neurol. 2005 Sep 27; [Epub ahead of print].
[ expand abstract ]

Drug delivery to brain tumors has long posed a major challenge. Convection-enhanced delivery (CED) has been developed as a drug delivery strategy to overcome this difficulty. Ideally, direct visualization of the tissue distribution of drugs infused by CED would assure successful delivery of therapeutic agents to the brain tumor while minimizing exposure of the normal brain. We previously developed a magnetic resonance imaging (MRI)-based method to visualize the distribution of liposomal agents after CED in rodent brains. In the present study, CED of liposomes was further examined in the non-human primate brain (n = 6). Liposomes containing Gadoteridol, DiI-DS, and rhodamine were infused in corona radiata, putamen nucleus, and brain stem. Volume of distribution was analyzed for all delivery locations by histology and MR imaging. Real-time MRI monitoring of liposomes containing gadolinium allowed direct visualization of a robust distribution. MRI of liposomal gadolinium was highly accurate at determining tissue distribution, as confirmed by comparison with histological results from concomitant administration of fluorescent liposomes. Linear correlation for liposomal infusions between infusion volume and distribution volume was established in all targeted locations. We conclude that an integrated strategy combining liposome/nanoparticle technology, CED, and MRI may provide new opportunities for the treatment of brain tumors. Our ability to directly monitor and to control local delivery of liposomal drugs will most likely result in greater clinical efficacy when using CED in management of patients.

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Real-time visualization and characterization of liposomal delivery into the monkey brain by magnetic resonance imaging.
Krauze MT, McKnight TR, Yamashita Y, Bringas J, Noble CO, Saito R, Geletneky K, Forsayeth J, Berger MS, Jackson P, Park JW, Bankiewicz KS.
Brain Res Brain Res Protoc. 2005 Sep 20; [Epub ahead of print].
[ expand abstract ]

Liposomes loaded with Gadoteridol, in combination with convection-enhanced delivery (CED), offer an excellent option to monitor CNS delivery of therapeutic compounds with MRI. In previous studies, we investigated possible clinical applications of liposomes to the treatment of brain tumors. In this study, up to 700 mul of Gadoteridol/rhodamine-loaded liposomes were distributed in putamen, corona radiata and brainstem of non-human primates. Distribution was monitored by real-time MRI throughout infusion procedures and allowed accurate calculation of volume of distribution within anatomical structures. We found that different regions of the brain gave various volumes of distribution when infused with the same volume of liposome. Based on these findings, distinct distribution pathways within infused structures can be predicted. This work underlines the importance of monitoring drug delivery to CNS and enables accurate delivery of drug-loaded liposomes to specific brain regions with a standard MRI procedure. Findings presented in this manuscript may allow for modeling of parameters used for direct delivery of therapeutics into various regions of the brain.

Paramagnetic viral nanoparticles as potential high-relaxivity magnetic resonance contrast agents.
Allen M, Bulte JW, Liepold L, Basu G, Zywicke HA, Frank JA, Young M, Douglas T.
Magn Reson Med. 2005 Sep 9; [Epub ahead of print].
[ expand abstract ]

In order to compensate for the inherent high threshold of detectability of MR contrast agents, there has been an active interest in the development of paramagnetic nanoparticles incorporating high payloads of Gd(3+) with high molecular relaxivities. Toward this end, the protein cage of Cowpea chlorotic mottle virus (CCMV), having 180 metal binding sites, is being explored. In vivo CCMV binds Ca(2+) at specific metal binding sites; however, Gd(3+) can also bind at these sites. Using fluorescence resonance energy transfer we have characterized the binding affinity of Gd(3+) to the metal binding sites by competition experiments with Tb(3+). The measured dissociation constant (K(d)) for Gd(3+) bound to the virus is 31 muM. The T(1) and T(2) relaxivities of solvent water protons in the presence of Gd(3+)-bound CCMV were 202 and 376 mM(-1) s(-1), respectively, at 61 MHz Larmor frequency. The unusually high relaxivity values of the Gd(3+)-CCMV are largely a result of the nanoparticle virus size and the large number of Gd(3+) ions bound to the virus. These preliminary results should encourage further investigations into the use of viral protein cages as a new platform for MR contrast agents.

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Relaxivity of liposomal paramagnetic MRI contrast agents.
Strijkers GJ, Mulder WJ, van Heeswijk RB, Frederik PM, Bomans P, Magusin PC, Nicolay K.
MAGMA. 2005 Sep 9; [Epub ahead of print].
[ expand abstract ]

Paramagnetic liposomes, spherical particles formed by a lipid bilayer, are able to accommodate a high payload of Gd-containing lipid and therefore can serve as a highly potent magnetic resonance imaging contrast agent. In this paper the relaxation properties of paramagnetic liposomes were studied as a function of composition, temperature and magnetic field strength. The pegylated liposomes with a diameter of approximately 100 nm were designed for favorable pharmacokinetic properties in vivo. The proton relaxivity, i.e. the T(1) relaxation rate per mmol of Gd(III) ions, of liposomes with unsaturated DOPC phospholipids was higher than those with saturated DSPC lipids. Addition of cholesterol was essential to obtain monodisperse liposomes and led to a further, although smaller, increase of the relaxivity. Nuclear magnetic relaxation dispersion measurements showed that the relaxivity was limited by water exchange. These results show that these paramagnetic liposomes are very effective contrast agents, making them excellent candidates for many applications in magnetic resonance imaging.

Optical imaging of cervical pre-cancers with structured illumination: An integrated approach.
Rahman M, Abd-El-Barr M, Mack V, Tkaczyk T, Sokolov K, Richards-Kortum R, Descour M.
Gynecol Oncol. 2005 Sep 8; [Epub ahead of print].
[ expand abstract ]

OBJECTIVE.: Structured illumination microscopy is an inexpensive alternative to confocal microscopy that allows optical sectioning at a sub-cellular resolution. However, its application in imaging biological tissue has been limited by inadequate contrast present in them especially in reflectance imaging. Novel, optically active contrast agents like gold nanoparticles and quantum dots targeted against biomarkers of cancer can be integrated with structured illumination to image both the morphological and biochemical changes associated with epithelial pre-cancers. METHODS.: We modified the optical path of a widefield microscope to implement structured illumination both in reflectance and fluorescence modes. For imaging, we used 25-nm-diameter gold nanoparticles and CdSe quantum dots for reflectance and fluorescence imaging, respectively, to label three-dimensional tissue constructs of SiHa cervical cancer cells. Contrast agents were targeted against the epidermal growth factor receptor (EGFR) using an anti-EGFR monoclonal antibody. Agents targeted with a non-specific IgG antibody served as a control to monitor non-specific labeling. RESULTS.: Our result shows that optically sectioned images taken with structured illumination are very comparable to those obtained using confocal microscopy. Moreover, images of three-dimensional cultures stained with the anti-EGFR agents show significantly more image intensity than those stained with the IgG targeted control. CONCLUSION.: Our findings suggest that the combination of novel optical contrast agents and structured illumination can differentiate neoplastic cells which overexpress EGFR from normal cells in intact tissue. Combining structured illumination microscopy with novel contrast agents can potentially provide a powerful and inexpensive tool to aid in the detection of cervical pre-cancers.

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In vivo magnetic resonance detection of cancer by using multifunctional magnetic nanocrystals.
Huh YM, Jun YW, Song HT, Kim S, Choi JS, Lee JH, Yoon S, Kim KS, Shin JS, Suh JS, Cheon J.
J Am Chem Soc. 2005 Sep 7;127(35):12387-91.
[ expand abstract ]

The unique properties of magnetic nanocrystals provide them with high potential as key probes and vectors in the next generation of biomedical applications. Although superparamagnetic iron oxide nanocrystals have been extensively studied as excellent magnetic resonance imaging (MRI) probes for various cell trafficking, gene expression, and cancer diagnosis, further development of in vivo MRI applications has been very limited. Here, we describe in vivo diagnosis of cancer, utilizing a well-defined magnetic nanocrystal probe system with multiple capabilities, such as small size, strong magnetism, high biocompatibility, and the possession of active functionality for desired receptors. Our magnetic nanocrystals are conjugated to a cancer-targeting antibody, Herceptin, and subsequent utilization of these conjugates as MRI probes has been successfully demonstrated for the monitoring of in vivo selective targeting events of human cancer cells implanted in live mice. Further conjugation of these nanocrystal probes with fluorescent dye-labeled antibodies enables both in vitro and ex vivo optical detection of cancer as well as in vivo MRI, which are potentially applicable for an advanced multimodal detection system. Our study finds that high performance in vivo MR diagnosis of cancer is achievable by utilizing improved and multifunctional material properties of iron oxide nanocrystal probes.

Magnetic resonance imaging of cardiomyocyte apoptosis with a novel magneto-optical nanoparticle.
Sosnovik DE, Schellenberger EA, Nahrendorf M, Novikov MS, Matsui T, Dai G, Reynolds F, Grazette L, Rosenzweig A, Weissleder R, Josephson L.
Magn Reson Med. 2005 Sep;54(3):718-24.
[ expand abstract ]

The ability to image cardiomyocyte apoptosis in vivo with high-resolution MRI could facilitate the development of novel cardioprotective therapies. The sensitivity of the novel nanoparticle AnxCLIO-Cy5.5 for cardiomyocyte apoptosis was thus compared in vitro to that of annexin V-FITC and showed a high degree of colocalization. MRI was then performed, following transient coronary artery (LAD) occlusion, in five mice given AnxCLIO-Cy5.5 and in four mice given an identical dose (2 mg Fe/kg) of CLIO-Cy5.5. MR signal intensity and myocardial T2* were evaluated, in vivo, in hypokinetic regions of myocardium in the LAD distribution. Ex vivo fluorescence imaging was performed to confirm the in vivo findings. Myocardial T2* was significantly lower in the mice given AnxCLIO-Cy5.5 (8.1 versus 13.2 ms, P<0.01), and fluorescence target to background ratio was significantly higher (2.1 versus 1.1, P<0.01). This study thus demonstrates the feasibility of obtaining high-resolution MR images of cardiomyocyte apoptosis in vivo with the novel nanoparticle, AnxCLIO-Cy5.5.

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Developing implantable optical biosensors.
Ziegler KJ.
Trends Biotechnol. 2005 Sep;23(9):440-4.
[ expand abstract ]

Nanobiotechnologists are developing devices that can measure specific enzymes and proteins. These devices are expected to detect single enzyme or protein molecules accurately, providing highly sensitive biosensing applications. A recent study by Strano and co-workers shows that single-walled carbon nanotubes (SWNTs) hold great promise as implantable biosensors. Although most researchers have focused on substrate-oriented biosensors, Strano and colleagues have shown that the inherent fluorescent properties of suspended individual SWNTs can be used for solution-phase beta-d-glucose sensing.

Fluorescent nanocrystals for use in early cervical cancer detection.
Nida DL, Rahman MS, Carlson KD, Richards-Kortum R, Follen M.
Gynecol Oncol. 2005 Aug 31; [Epub ahead of print].
[ expand abstract ]

BACKGROUND.: Quantum dots (qdots) are a promising alternative to organic fluorophores for biological imaging. Advantages of quantum dots over organic fluorophores include broad excitation coupled with narrow, tunable emission, high resistance to chemical and metabolic degradation, a higher photobleaching threshold and finally the ability to be modified with a targeting ligand. These many properties allow quantum dots to be used in conjunction with optical detection methods for imaging. METHODS.: We are investigating the use of quantum dots to detect precancerous biomarkers. We have directly targeted epidermal growth factor receptors with quantum dots conjugated to anti-EGFR antibodies. RESULTS.: Compared to appropriate controls, we do see specific labeling of EGF receptors. CONCLUSIONS.: Quantum dots provide a promising alternative to conventional organic dyes for biological imaging. Combined with optical imaging technologies, quantum dots can help visualize changes in cervical cancer at the molecular level. This ability may alert health care providers to the need for intervention before a cancer can metastasize.

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Superparamagnetic gadonanotubes are high-performance MRI contrast agents.
Sitharaman B, Kissell KR, Hartman KB, Tran LA, Baikalov A, Rusakova I, Sun Y, Khant HA, Ludtke SJ, Chiu W, Laus S, Toth E, Helm L, Merbach AE, Wilson LJ.
Chem Commun (Camb). 2005 Aug 21;(31):3915-7.
[ expand abstract ]

We report the nanoscale loading and confinement of aquated Gd3+n-ion clusters within ultra-short single-walled carbon nanotubes (US-tubes); these Gd3+n@US-tube species are linear superparamagnetic molecular magnets with Magnetic Resonance Imaging (MRI) efficacies 40 to 90 times larger than any Gd3+-based contrast agent (CA) in current clinical use.

Folate-receptor-mediated delivery of InP quantum dots for bioimaging using confocal and two-photon microscopy.
Bharali DJ, Lucey DW, Jayakumar H, Pudavar HE, Prasad PN.
J Am Chem Soc. 2005 Aug 17;127(32):11364-71.
[ expand abstract ]

A novel method for the synthesis of highly monodispersed hydrophillic InP-ZnS nanocrystals and their use as luminescence probes for live cell imaging is reported. Hydrophobic InP-ZnS nanocrystals are prepared by a new method that yields high-quality, luminescent core-shell nanocrystals within 6-8 h of total reaction time. Then by carefully manipulating the surface of these passivated nanocrystals, aqueous dispersions of folate-conjugated nanocrystals (folate-QDs) with high photostability are prepared. By use of confocal microscopy, we demonstrate the receptor-mediated delivery of folic acid conjugated quantum dots into folate-receptor-positive cell lines such as KB cells. These folate-QDs tend to accumulate in multi-vescicular bodies of KB cells after 6 h of incubation. Receptor-mediated delivery was confirmed by comparison with the uptake of these particles in folate-receptor-negative cell lines such as A549. Efficient two-photon excitation of these particles and two-photon imaging using these particles are also demonstrated. The use of these InP-ZnS nanoparticles and their efficient two-photon excitation can be potentially useful for deep tissue imaging for future in vivo studies.

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Nanoimaging with a compact extreme-ultraviolet laser.
Vaschenko G, Brizuela F, Brewer C, Grisham M, Mancini H, Menoni CS, Marconi MC, Rocca JJ, Chao W, Liddle JA, Anderson EH, Attwood DT, Vinogradov AV, Artioukov IA, Pershyn YP, Kondratenko VV.
Opt Lett. 2005 Aug 15;30(16):2095-7.
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Images with a spatial resolution of 120-150 nm were obtained with 46.9 nm light from a compact capillary-discharge laser by use of the combination of a Sc-Si multilayer-coated Schwarzschild condenser and a free-standing imaging zone plate. The results are relevant to the development of compact extreme-ultraviolet laser-based imaging tools for nanoscience and nanotechnology.

Magnetic resonance imaging of cardiomyocyte apoptosis with a novel magneto-optical nanoparticle.
Sosnovik DE, Schellenberger EA, Nahrendorf M, Novikov MS, Matsui T, Dai G, Reynolds F, Grazette L, Rosenzweig A, Weissleder R, Josephson L.
Magn Reson Med. 2005 Aug 5; [Epub ahead of print].
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The ability to image cardiomyocyte apoptosis in vivo with high-resolution MRI could facilitate the development of novel cardioprotective therapies. The sensitivity of the novel nanoparticle AnxCLIO-Cy5.5 for cardiomyocyte apoptosis was thus compared in vitro to that of annexin V-FITC and showed a high degree of colocalization. MRI was then performed, following transient coronary artery (LAD) occlusion, in five mice given AnxCLIO-Cy5.5 and in four mice given an identical dose (2 mg Fe/kg) of CLIO-Cy5.5. MR signal intensity and myocardial T(2)* were evaluated, in vivo, in hypokinetic regions of myocardium in the LAD distribution. Ex vivo fluorescence imaging was performed to confirm the in vivo findings. Myocardial T(2)* was significantly lower in the mice given AnxCLIO-Cy5.5 (8.1 versus 13.2 ms, P < 0.01), and fluorescence target to background ratio was significantly higher (2.1 versus 1.1, P < 0.01). This study thus demonstrates the feasibility of obtaining high-resolution MR images of cardiomyocyte apoptosis in vivo with the novel nanoparticle, AnxCLIO-Cy5.5. Magn Reson Med, 2005. (c) 2005 Wiley-Liss, Inc.

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Generation of superparamagnetic liposomes revealed as highly efficient MRI contrast agents for in vivo imaging.
Martina MS, Fortin JP, Menager C, Clement O, Barratt G, Grabielle-Madelmont C, Gazeau F, Cabuil V, Lesieur S.
J Am Chem Soc. 2005 Aug 3;127(30):10676-85.
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Maghemite (gamma-Fe2O3) nanocrystals stable at neutral pH and in isotonic aqueous media were synthesized and encapsulated within large unilamellar vesicles of egg phosphatidylcholine (EPC) and distearoyl-SN-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-2000] (DSPE-PEG(2000), 5 mol %), formed by film hydration coupled with sequential extrusion. The nonentrapped particles were removed by flash gel exclusion chromatography. The magnetic-fluid-loaded liposomes (MFLs) were homogeneous in size (195 +/- 33 hydrodynamic diameters from quasi-elastic light scattering). Iron loading was varied from 35 up to 167 Fe(III)/lipid mol %. Physical and superparamagnetic characteristics of the iron oxide particles were preserved after liposome encapsulation as shown by cryogenic transmission electron microscopy and magnetization curve recording. In biological media, MFLs were highly stable and avoided ferrofluid flocculation while being nontoxic toward the J774 macrophage cell line. Moreover, steric stabilization ensured by PEG-surface-grafting significantly reduced liposome association with the macrophages. The ratios of the transversal (r2) and longitudinal (r1) magnetic resonance (MR) relaxivities of water protons in MFL dispersions (6 < r2/r1 < 18) ranked them among the best T2 contrast agents, the higher iron loading the better the T2 contrast enhancement. Magnetophoresis demonstrated the possible guidance of MFLs by applying a magnetic field gradient. Mouse MR imaging assessed MFLs efficiency as contrast agents in vivo: MR angiography performed 24 h after intravenous injection of the contrast agent provided the first direct evidence of the stealthiness of PEG-ylated magnetic-fluid-loaded liposomes

Optical and MRI Multifunctional Nanoprobe for Targeting Gliomas.
Veiseh O, Sun C, Gunn J, Kohler N, Gabikian P, Lee D, Bhattarai N, Ellenbogen R, Sze R, Hallahan A, Olson J, Zhang M.
Nano Lett. 2005 Jun 8;5(6):1003-1008.
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A multifunctional nanoprobe capable of targeting glioma cells, detectable by both magnetic resonance imaging and fluorescence microscopy, was developed. The nanoprobe was synthesized by coating iron oxide nanoparticles with covalently bound bifunctional poly(ethylene glycol) (PEG) polymer, which were subsequently functionalized with chlorotoxin and the near-infrared fluorescing molecule Cy5.5. Both MR imaging and fluorescence microscopy showed significant preferential uptake of the nanoparticle conjugates by glioma cells. Such a nanoprobe could potentially be used to image resections of glioma brain tumors in real time and to correlate preoperative diagnostic images with intraoperative pathology at cellular-level resolution

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Sentinel lymph node mapping of the pleural space.
Parungo CP, Colson YL, Kim SW, Kim S, Cohn LH, Bawendi MG, Frangioni JV.
Chest. 2005 May;127(5):1799-804.
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STUDY OBJECTIVES: Although the sentinel lymph node (SLN) concept has traditionally been applied to solid organs, we hypothesized that the pleural space might drain into a specific SLN group. The identification of such a nodal group could assist in the staging and treatment of pleural-based diseases, such as mesothelioma, or other lung cancers with visceral pleural invasion. The purpose of this study was to determine whether the pleural space has an SLN group. DESIGN: Sixteen rats underwent right or left pleural space injection of a novel lymph tracer, quantum dots (QDs), which have a hydrodynamic diameter of 15 nm and fluoresce in the near-infrared (NIR) spectrum. Nodal uptake of the entire thorax was imaged with a custom system that simultaneously acquired color video, NIR fluorescence of the QDs, and a merged picture of the two in real-time. Six pigs underwent right or left pleural space injection of QDs and similar imaging. MEASUREMENTS AND RESULTS: In the rat, the QDs drained solely to the highest superior mediastinal lymph node group, corresponding to lymph node station 1, according the regional lymph node classification for lung of the American Joint Committee on Cancer. In one rat, the injection of QDs in the left pleural space resulted in migration to the contralateral station 1 lymph node group. The injection of QDs in the right or left pleural space of the pig resulted in migration solely to the ipsilateral highest superior mediastinal lymph node group. CONCLUSIONS: NIR fluorescence imaging in two species demonstrated that the highest superior mediastinal lymph nodes of station 1 are the SLNs of the pleural space. This study also provides intraoperative feasibility and proof of the concept for identifying lymph nodes communicating with the pleural space on a patient-specific basis, in real-time, and with high sensitivity.

Diagnostic performance of nanoparticle-enhanced magnetic resonance imaging in the diagnosis of lymph node metastases in patients with endometrial and cervical cancer.
Rockall AG, Sohaib SA, Harisinghani MG, Babar SA, Singh N, Jeyarajah AR, Oram DH, Jacobs IJ, Shepherd JH, Reznek RH.
J Clin Oncol. 2005 Apr 20;23(12):2813-21.
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PURPOSE: Lymph node metastases affect management and prognosis of patients with gynecologic malignancies. Preoperative nodal assessment with computed tomography or magnetic resonance imaging (MRI) is inaccurate. A new lymph node-specific contrast agent, ferumoxtran-10, composed of ultrasmall particles of iron oxide (USPIO), may enhance the detection of lymph node metastases independent of node size. Our aim was to compare the diagnostic performance of MRI with USPIO against standard size criteria. METHODS: Forty-four patients with endometrial (n = 15) or cervical (n = 29) cancer were included. MRI was performed before and after administration of USPIO. Two independent observers viewed the MR images before lymph node sampling. Lymph node metastases were predicted using size criteria and USPIO criteria. Lymph node sampling was performed in all patients. RESULTS: Lymph node sampling provided 768 pelvic or para-aortic nodes for pathology, of which 335 were correlated on MRI; 17 malignant nodes were found in 11 of 44 patients (25%). On a node-by-node basis, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) by size criteria were 29%*, 99%, 56%, and 96%, and by USPIO criteria (reader 1/reader 2) were 93%/82%* (*P =.008/.004), 97%/97%, 61%/59%, and 100%/99%, respectively (where [*] indicates the statistical difference of P = x/x between the two results marked by the asterisk). On a patient-by-patient basis, sensitivity, specificity, PPV, and NPV by size criteria were 27%*, 94%, 60%, and 79%, and by USPIO criteria (reader 1/reader 2) were 100%/91%* (*P =.031/.06), 94%/87%, 82%/71%, and 100%/96%, respectively. The kappa statistic was 0.93. CONCLUSION: Lymph node characterization with USPIO increases the sensitivity of MRI in the prediction of lymph node metastases, with no loss of specificity. This may greatly improve preoperative treatment planning.

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Design, synthesis, physical and chemical characterisation, and biological interactions of lectin-targeted latex nanoparticles bearing Gd-DTPA chelates: an exploration of magnetic resonance molecular imaging (MRMI).
Paschkunova-Martic I, Kremser C, Mistlberger K, Shcherbakova N, Dietrich H, Talasz H, Zou Y, Hugl B, Galanski M, Solder E, Pfaller K, Holiner I, Buchberger W, Keppler B, Debbage P.
Histochem Cell Biol. 2005 Apr 12; [Epub ahead of print].
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The physical and chemical parameters involved in the design and synthesis of biospecifically targeted nanoparticulate contrast media for magnetic resonance molecular imaging (MRMI) were explored in this pilot investigation. Latex nanoparticles 100, 400 and 900 nm in diameter were doubly derivatised, first with tomato lectin and then with gadolinium(III)-diethylenetriamine pentaacetic acid (Gd-chelates) to target them to epithelial and endothelial glycocalyceal N-glycans and to generate contrast enhancement in magnetic resonance imaging (MRI). After intravenous injection into mice, human placental cotyledons or human Vena saphena magna, contrasty images of the vascular structures were obtained in 1.5 T MRI with spatial resolution 0.1 mm in the imaging plane and 0.6 mm in the z axis, persisting >60 min and resistant to washing out by buffer rinses. Ultrastructural analysis of the nanoparticles revealed the targeting groups at the nanoparticle surfaces and the distribution of the Gd-chelates within the nanoparticles and enabled counts for use in determining relaxivity. The relaxivity values revealed were extremely high, accounting for the strong MR signals observed. Occasionally, nanoparticles larger than 100 nm were seen in close spatial association with disrupted regions of cell membrane or of collagen fibrils in the extracellular matrix. The data suggest that 100-nm nanoparticles generate adequate contrast for MRMI and cause least disruption to endothelial cell surfaces.

Dendrimer modified magnetite nanoparticles for protein immobilization.
Pan BF, Gao F, Gu HC.
J Colloid Interface Sci. 2005 Apr 1;284(1):1-6.
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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.

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Intraoperative identification of esophageal sentinel lymph nodes with near-infrared fluorescence imaging.
Parungo CP, Ohnishi S, Kim SW, Kim S, Laurence RG, Soltesz EG, Chen FY, Colson YL, Cohn LH, Bawendi MG, Frangioni JV.
J Thorac Cardiovasc Surg. 2005 Apr;129(4):844-50.
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Objective In esophageal cancer, selective removal of involved lymph nodes could improve survival and limit complications from extended lymphadenectomy. Mapping with vital blue dyes or technetium Tc-99m often fails to identify intrathoracic sentinel lymph nodes. Our purpose was to develop an intraoperative method for identifying sentinel lymph nodes of the esophagus with high-sensitivity near-infrared fluorescence imaging. Methods Six Yorkshire pigs underwent thoracotomy and received submucosal, esophageal injection of quantum dots, a novel near-infrared fluorescent lymph tracer designed for retention in sentinel lymph nodes. Six additional pigs underwent thoracotomy and received submucosal esophageal injection of CW800 conjugated to human serum albumin, another novel lymph tracer designed for uptake into distant lymph nodes. Finally, 6 pigs received submucosal injection of the fluorophore-conjugated albumin with an endoscopic needle through an esophagascope. These lymph tracers fluoresce in the near-infrared, permitting visualization of migration to sentinel lymph nodes with a custom intraoperative imaging system. Results Injection of the near-infrared fluorescent lymph tracers into the esophagus revealed communicating lymph nodes within 5 minutes of injection. In all 6 pigs that received quantum dot injection, only a single sentinel lymph node was identified. Among pigs that received fluorophore-conjugated albumin injection, in 5 of 12 a single sentinel lymph node was revealed, but in 7 of 12 two sentinel lymph nodes were identified. There was no dominant pattern in the appearance of the sentinel lymph nodes either cranial or caudal to the injection site. Conclusion Near-infrared fluorescence imaging of sentinel lymph nodes is a novel and reliable intraoperative technique with the power to assist with identification and resection of esophageal sentinel lymph nodes.

A biomagnetic system for in vivo cancer imaging.
Flynn ER, Bryant HC.
Phys Med Biol. 2005 Mar 21;50(6):1273-93.
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An array of highly sensitive biomagnetic sensors of the superconducting quantum interference detector (SQUID) type can identify disease in vivo by detecting and imaging microscopic amounts of nanoparticles. We describe in detail procedures and parameters necessary for implementation of in vivo detection through the use of antibody-labelled magnetic nanoparticles as well as methods of determining magnetic nanoparticle properties. We discuss the weak field magnetic sensor SQUID system, the method of generating the magnetic polarization pulse to align the magnetic moments of the nanoparticles, and the measurement techniques to measure their magnetic remanence fields following this pulsed field. We compare these results to theoretical calculations and predict optimal properties of nanoparticles for in vivo detection.

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Rotational Dynamics Account for pH-Dependent Relaxivities of PAMAM Dendrimeric, Gd-Based Potential MRI Contrast Agents.
Laus S, Sour A, Ruloff R, Toth E, Merbach AE.
Chemistry. 2005 Mar 17; [Epub ahead of print].
[ expand abstract ]

The EPTPA(5-) chelate, which ensures fast water exchange in Gd(III) complexes, has been coupled to three different generations (5, 7, and 9) of polyamidoamine (PAMAM) dendrimers through benzylthiourea linkages (H(5)EPTPA=ethylenepropylenetriamine-N,N,N',N'',N''-pentaacetic acid). The proton relaxivities measured at pH 7.4 for the dendrimer complexes G5-(GdEPTPA)(111), G7-(GdEPTPA)(253) and G9-(GdEPTPA)(1157) decrease with increasing temperature, indicating that, for the first time for dendrimers, slow water exchange does not limit relaxivity. At a given field and temperature, the relaxivity increases from G5 to G7, and then slightly decreases for G9 (r(1)=20.5, 28.3 and 27.9 mM(-1) s(-1), respectively, at 37 degrees C, 30 MHz). The relaxivities show a strong and reversible pH dependency for all three dendrimer complexes. This originates from the pH-dependent rotational dynamics of the dendrimer skeleton, which was evidenced by a combined variable-temperature and multiple-field (17)O NMR and (1)H relaxivity study performed at pH 6.0 and 9.9 on G5-(GdEPTPA)(111). The longitudinal (17)O and (1)H relaxation rates of the dendrimeric complex are strongly pH-dependent, whereas they are not for the [Gd(EPTPA)(H(2)O)](2-) monomer chelate. The longitudinal (17)O and (1)H relaxation rates have been analysed by the Lipari-Szabo spectral density functions and correlation times have been calculated for the global motion of the entire macromolecule (tau(gO)) and the local motion of the Gd(III) chelates on the surface (tau(lO)), correlated by means of an order parameter S(2). The dendrimer complex G5-(GdEPTPA)(111) has a considerably higher tau(gO) under acidic than under basic conditions (${\tau {{298\hfill \atop {\rm gO}\hfill}}}$=4040 ps and 2950 ps, respectively), while local motions are less influenced by pH (${\tau {{298\hfill \atop {\rm lO}\hfill}}}$=150 and 125 ps). The order parameter, characterizing the rigidity of the macromolecule, is also higher at pH 6.0 than at pH 9.9 (S(2)=0.43 vs 0.36, respectively). The pH dependence of the global correlation time can be related to the protonation of the tertiary amine groups in the PAMAM skeleton, which leads to an expanded and more rigid dendrimeric structure at lower pH. The increase of tau(gO) with decreasing pH is responsible for the pH dependent proton relaxivities. The water exchange rate on G5-(GdEPTPA)(111) (${k{{298\hfill \atop {\rm ex}\hfill}}}$=150x10(6) s(-1)) shows no significant pH dependency and is similar to the one measured for the monomer [Gd(EPTPA)(H(2)O)](2-). The proton relaxivity of G5-(GdEPTPA)(111) is mainly limited by the important flexibility of the dendrimer structure, and to a small extent, by a faster than optimal water exchange rate.

Detection of vascular adhesion molecule-1 expression using a novel multimodal nanoparticle.
Kelly KA, Allport JR, Tsourkas A, Shinde-Patil VR, Josephson L, Weissleder R.
Circ Res. 2005 Feb 18;96(3):327-36. Epub 2005 Jan 13.
[ expand abstract ]

Endothelial vascular adhesion molecule-1 (VCAM-1) is a critical component of the leukocyte-endothelial adhesion cascade, and its strict temporal and spatial regulation make it an ideal target for imaging and therapy. The goal of this study was to develop novel VCAM-1-targeted imaging agents detectable by MRI and fluorescence imaging using phage display-derived peptide sequences and multimodal nanoparticles (NPs). We hypothesized that VCAM-1-mediated cell internalization of phage display-selected peptides could be harnessed as an amplification strategy to chaperone and trap imaging agents inside VCAM-1-expressing cells, thus improving target-to-background ratios. To accomplish our goal, iterative phage display was performed on murine endothelium under physiological flow conditions to identify a family of VCAM-1-mediated cell-internalizing peptides. One specific sequence, containing the VHSPNKK motif that has homology to the alpha-chain of very late antigen (a known ligand for VCAM-1), was shown to bind VCAM-1 and block leukocyte-endothelial interactions. Compared with VCAM-1 monoclonal antibody, the peptide showed 12-fold higher target-to-background ratios. A VHSPNKK-modified magnetofluorescent NP (VNP) showed high affinity for endothelial cells expressing VCAM-1 but surprisingly low affinity for macrophages. In contrast, a control NP without VCAM-1-targeting sequences showed no affinity for endothelial cells. In vivo, VNP successfully identified VCAM-1-expressing endothelial cells in a murine tumor necrosis factor-alpha-induced inflammatory model and colocalized with VCAM-1-expressing cells in atherosclerotic lesions present in cholesterol-fed apolipoprotein E apoE-/- mice. These results indicate that: (1) small peptide sequences can significantly alter targeting of NPs, (2) the used amplification strategy of internalization results in high target-to-background ratios, and (3) this technology is useful for in vivo imaging of endothelial markers.

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Quantum dots for live cells, in vivo imaging, and diagnostics.
Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, Li JJ, Sundaresan G, Wu AM, Gambhir SS, Weiss S.
Science. 2005 Jan 28;307(5709):538-44.
[ expand abstract ]

Research on fluorescent semiconductor nanocrystals (also known as quantum dots or qdots) has evolved over the past two decades from electronic materials science to biological applications. We review current approaches to the synthesis, solubilization, and functionalization of qdots and their applications to cell and animal biology. Recent examples of their experimental use include the observation of diffusion of individual glycine receptors in living neurons and the identification of lymph nodes in live animals by near-infrared emission during surgery. The new generations of qdots have far-reaching potential for the study of intracellular processes at the single-molecule level, high-resolution cellular imaging, long-term in vivo observation of cell trafficking, tumor targeting, and diagnostics.

Real-time multiple-particle tracking: applications to drug and gene delivery.
Suh J, Dawson M, Hanes J.
Adv Drug Deliv Rev. 2005 Jan 2;57(1):63-78.
[ expand abstract ]

Complex biological environments, such as the cell cytoplasm or the mucus lining the airways of the lungs, can pose significant barriers to efficient therapeutic drug and gene delivery. Biological barriers are particularly important in controlled drug delivery applications that utilize a large carrier particle, such as a liposome or a polymer micro- or nanosphere. The dynamic transport of particulate drug and gene delivery vehicles through these barriers is poorly understood, having been primarily studied with static methods in the past. Recently, the transport of synthetic drug and gene carriers has been investigated quantitatively with real-time particle tracking technology, providing new insight into particle behavior in complex biological environments that is guiding rational improvements in particle design. This review briefly highlights basic principles of particle tracking and its application to elucidate important phenomena that limit effective particulate drug and gene delivery.

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Intraoperative sentinel lymph node mapping of the lung using near-infrared fluorescent quantum dots.
Soltesz EG, Kim S, Laurence RG, DeGrand AM, Parungo CP, Dor DM, Cohn LH, Bawendi MG, Frangioni JV, Mihaljevic T.
Ann Thorac Surg. 2005 Jan;79(1):269-77; discussion 269-77.
[ expand abstract ]

BACKGROUND: The presence of lymph node metastases is an important prognostic marker with regard to non-small-cell lung cancer (NSCLC). Assessment of the sentinel lymph node (SLN) for the presence of tumor may improve staging. Our objective was to develop an optical noninvasive imaging tool that would permit intraoperative SLN mapping and provide real-time visual feedback for image-guided localization and resection. METHODS: Invisible near-infrared (NIR) light penetrates relatively deeply into tissue and background autofluorescence is low. We have developed a NIR fluorescence imaging system that simultaneously displays color video and NIR images of the surgical field. We recently engineered 15 nm nonradioactive NIR fluorescent quantum dots (QDs) as optimal lymphotrophic optical probes. The introduction of these QDs into lung tissue allows real-time visualization of draining lymphatic channels and nodes. RESULTS: In 12 Yorkshire pigs (mean weight 35 kg) we demonstrated that 200 pmol of NIR QDs injected into lobar parenchyma accurately maps lymphatic drainage and the SLN. All SLNs were strongly fluorescent and easily visualized within 5 minutes of injection. In 14 separate injections QDs localized to a mediastinal node, whereas in 2 injections QDs localized to a hilar intraparenchymal node. Histologic analysis in all cases confirmed the presence of nodal tissue. CONCLUSIONS: We report a highly sensitive rapid technique for SLN mapping of the lung. This technique permits precise real-time imaging and therefore overcomes many limitations of currently available techniques.

2004

Semiconductor quantum dots as contrast agents for whole animal imaging.
Jiang W, Papa E, Fischer H, Mardyani S, Chan WC.
Trends Biotechnol. 2004 Dec;22(12):607-9.
[ expand abstract ]

Recent developments in quantum dot (QD) technology have paved the way for using QDs as optical contrast agents for in vivo imaging. Pioneering papers showed the use of QDs as luminescent contrast agents for imaging cancer and guiding cancer surgery. The possible future use of QDs for clinical applications is expected to have a significant impact, however many challenges in this field have yet to be overcome.

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In vivo cancer targeting and imaging with semiconductor quantum dots.
Gao X, Cui Y, Levenson RM, Chung LW, Nie S.
Nat Biotechnol. 2004 Aug;22(8):969-76. Epub 2004 Jul 18.
[ expand abstract ]

We describe the development of multifunctional nanoparticle probes based on semiconductor quantum dots (QDs) for cancer targeting and imaging in living animals. The structural design involves encapsulating luminescent QDs with an ABC triblock copolymer and linking this amphiphilic polymer to tumor-targeting ligands and drug-delivery functionalities. In vivo targeting studies of human prostate cancer growing in nude mice indicate that the QD probes accumulate at tumors both by the enhanced permeability and retention of tumor sites and by antibody binding to cancer-specific cell surface biomarkers. Using both subcutaneous injection of QD-tagged cancer cells and systemic injection of multifunctional QD probes, we have achieved sensitive and multicolor fluorescence imaging of cancer cells under in vivo conditions. We have also integrated a whole-body macro-illumination system with wavelength-resolved spectral imaging for efficient background removal and precise delineation of weak spectral signatures. These results raise new possibilities for ultrasensitive and multiplexed imaging of molecular targets in vivo.

Luminescent nanoparticle probes for bioimaging.
Santra S, Xu J, Wang K, Tan W.
J Nanosci Nanotechnol. 2004 Jul;4(6):590-9.
[ expand abstract ]

Bioimaging with luminescent nanoparticle probes have recently attracted widespread interest in biology and medicine. In comparison with commonly used organic dyes, luminescent nanoparticles are better in terms of photostability and sensitivity. These optical features of nanoparticle probes are critical for real time tracking and monitoring of biological events in the cellular level, which may not be accomplished using regular fluorescent dyes. Nanoparticle probes are also shown highly suitable for immunoassay and other diagnostic and therapeutic applications. In this article, we describe a variety of optical nanoparticle probes such as quantum dots, metal nanoparticles, dye-doped nanoparticles etc. for bioimaging applications.

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Molecular imaging applications in nanomedicine.
Li KC, Pandit SD, Guccione S, Bednarski MD.
Biomed Microdevices. 2004 Jun;6(2):113-6.
[ expand abstract ]

The purpose of this article is to explore how molecular imaging techniques can be used as useful adjunts in the development of "nanomedicine" and in personalizing treatment of patients. The discussion focuses on in vivo applications at the whole organism level even though imaging can also play an important role in research at the cellular and subcellular level.

Lymphatic drainage imaging of breast cancer in mice by micro-magnetic resonance lymphangiography using a nano-size paramagnetic contrast agent.
Kobayashi H, Kawamoto S, Sakai Y, Choyke PL, Star RA, Brechbiel MW, Sato N, Tagaya Y, Morris JC, Waldmann TA.
J Natl Cancer Inst.2004 May 5;96(9):703-8.
[ expand abstract ]

BACKGROUND: The presence of lymph node metastases is an important factor in breast cancer patient prognosis. Therefore, the precise identification of sentinel lymph nodes in these patients is critical. Improving current magnetic resonance (MR) imaging methods using a newly synthesized nano-size paramagnetic molecule, G6, as a contrast agent, provides an attractive means toward attaining this goal. METHODS: A four-dimensional method of micro-MR lymphangiography using G6 (9 nm/240 kd) was developed to visualize the lymphatic ducts and lymph nodes draining mouse mammary tumors over time. The ability of micro-MR lymphangiography with the G6 contrast agent to visualize lymphatic drainage of normal mouse mammary tissue was compared with that of the conventional MR contrast agent, Gd-[DTPA]-dimeglumine (<1 kd). Lymphatic drainage in spontaneous and xenografted breast tumor models was visualized using the G6 contrast agent. RESULTS: Draining lymphatic ducts and lymph nodes were clearly visualized in the mammary tissue of normal mice and in spontaneous and xenografted breast tumor models after a direct mammary gland or peritumoral injection of G6. Gd-[DTPA]-dime-glumine, by contrast, failed to depict lymphatic flow from the mammary tissue in normal mice using the same method. Micro-MR lymphangiography using the G6 contrast agent revealed the absence of filling in the metastatic foci of affected lymph nodes. CONCLUSIONS: The superior temporal and spatial resolution of micro-MR lymphangiography using the contrast agent G6 may facilitate the study of tumor lymphatic drainage and lymphatic metastasis in both experimental animals and clinical medicine. In addition, this may be a powerful new method for sentinel lymph node localization in human breast cancer.

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Nanoshell-enabled photonics-based imaging and therapy of cancer.
Loo C, Lin A, Hirsch L, Lee MH, Barton J, Halas N, West J, Drezek R.
Technol Cancer Res Treat. 2004 Feb;3(1):33-40.
[ expand abstract ]

Metal nanoshells are a novel type of composite spherical nanoparticle consisting of a dielectric core covered by a thin metallic shell which is typically gold. Nanoshells possess highly favorable optical and chemical properties for biomedical imaging and therapeutic applications. By varying the relative the dimensions of the core and the shell, the optical resonance of these nanoparticles can be precisely and systematically varied over a broad region ranging from the near-UV to the mid-infrared. This range includes the near-infrared (NIR) wavelength region where tissue transmissivity peaks. In addition to spectral tunability, nanoshells offer other advantages over conventional organic dyes including improved optical properties and reduced susceptibility to chemical/thermal denaturation. Furthermore, the same conjugation protocols used to bind biomolecules to gold colloid are easily modified for nanoshells. In this article, we first review the synthesis of gold nanoshells and illustrate how the core/shell ratio and overall size of a nanoshell influences its scattering and absorption properties. We then describe several examples of nanoshell-based diagnostic and therapeutic approaches including the development of nanoshell bioconjugates for molecular imaging, the use of scattering nanoshells as contrast agents for optical coherence tomography (OCT), and the use of absorbing nanoshells in NIR thermal therapy of tumors.

2003

A multimodal nanoparticle for preoperative magnetic resonance imaging and intraoperative optical brain tumor delineation.
Kircher MF, Mahmood U, King RS, Weissleder R, Josephson L.
Cancer Res.2003 Dec 1;63(23):8122-5.
[ expand abstract ]

The determination of brain tumor margins both during the presurgical planning phase and during surgical resection has long been a challenging task in the therapy of brain tumor patients. Using a model of gliosarcoma with stably green fluorescence protein-expressing 9L glioma cells, we explored a multimodal (near-infrared fluorescent and magnetic) nanoparticle as a preoperative magnetic resonance imaging contrast agent and intraoperative optical probe. Key features of nanoparticle metabolism, namely intracellular sequestration by microglia and the combined optical and magnetic properties of the probe, allowed delineation of brain tumors both by preoperative magnetic resonance imaging and by intraoperative optical imaging. This prototypical multimodal nanoparticle has unique properties that may allow radiologists and neurosurgeons to see the same probe in the same cells and may offer a new approach for obtaining tumor margins.

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In vivo high resolution three-dimensional imaging of antigen-specific cytotoxic T-lymphocyte trafficking to tumors.
Kircher MF, Allport JR, Graves EE, Love V, Josephson L, Lichtman AH, Weissleder R.
Cancer Res. 2003 Oct 15;63(20):6838-46.
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Magnetic resonance imaging (MRI) allows noninvasive and three-dimensional visualization of whole organisms over time, and, therefore, would be ideally suited to monitor cell trafficking in vivo. Until now, systemically injected cells had been difficult to visualize by MRI because of relatively inefficient labeling methods. We developed a novel, biocompatible, and physiologically inert nanoparticle (highly derivatized cross-linked iron oxide nanoparticle; CLIO-HD) for highly efficient intracellular labeling of a variety of cell types that now allows in vivo MRI tracking of systemically injected cells at near single-cell resolution. CD8+ cytotoxic T lymphocytes labeled with CLIO-HD were detectable via MRI with a detection threshold of 2 cells/voxel in vitro and approximately 3 cells/voxel in vivo in live mice. Using B16-OVA melanoma and CLIO-HD-labeled OVA-specific CD8+ T cells, we have demonstrated for the first time high resolution imaging of T-cell recruitment to intact tumors in vivo. We have revealed the extensive three-dimensional spatial heterogeneity of T-cell recruitment to target tumors and demonstrated a temporal regulation of T-cell recruitment within the tumor. Significantly, our data indicate that serial administrations of CD8+ T cells appear to home to different intratumoral locations, and may, therefore, provide a more effective treatment regimen than a single bolus administration. Together, these results demonstrate that CLIO-HD is uniquely suited for quantitative repetitive MRI of adoptively transferred cells and that this approach may be particularly useful for evaluating novel cell-based therapies in vivo.

In vivo near-infrared fluorescence imaging.
Frangioni JV.
Curr Opin Chem Biol. 2003 Oct;7(5):626-34.
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Photon penetration into living tissue is highly dependent on the absorption and scattering properties of tissue components. The near-infrared region of the spectrum offers certain advantages for photon penetration, and both organic and inorganic fluorescence contrast agents are now available for chemical conjugation to targeting molecules. This review focuses on those parameters that affect image signal and background during in vivo imaging with near-infrared light and exogenous contrast agents. Recent examples of in vivo near-infrared fluorescence imaging of animals and humans are presented, including imaging of normal and diseased vasculature, tissue perfusion, protease activity, hydroxyapatite and cancer.

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A novel polyacrylamide magnetic nanoparticle contrast agent for molecular imaging using MRI.
Moffat BA, Reddy GR, McConville P, Hall DE, Chenevert TL, Kopelman RR, Philbert M, Weissleder R, Rehemtulla A, Ross BD.
Mol Imaging. 2003 Oct;2(4):324-32.
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A novel polyacrylamide superparamagnetic iron oxide nanoparticle platform is described which has been synthetically prepared such that multiple crystals of iron oxide are encapsulated within a single polyacrylamide matrix (PolyAcrylamide Magnetic [PAM] nanoparticles). This formulation provides for an extremely large T2 and T2* relaxivity of between 620 and 1140 sec(-1) mM(-1). Administration of PAM nanoparticles into rats bearing orthotopic 9L gliomas allowed quantitative pharmacokinetic analysis of the uptake of nanoparticles in the vasculature, brain, and glioma. Addition of polyethylene glycol of varying sizes (0.6, 2, and 10 kDa) to the surface of the PAM nanoparticles resulted in an increase in plasma half-life and affected tumor uptake and retention of the nanoparticles as quantified by changes in tissue contrast using MRI. The flexible formulation of these nanoparticles suggests that future modifications could be accomplished allowing for their use as a targeted molecular imaging contrast agent and/or therapeutic platform for multiple indications.

Molecular imaging of angiogenesis in nascent Vx-2 rabbit tumors using a novel alpha(nu)beta3-targeted nanoparticle and 1.5 tesla magnetic resonance imaging.
Winter PM, Caruthers SD, Kassner A, Harris TD, Chinen LK, Allen JS, Lacy EK, Zhang H, Robertson JD, Wickline SA, Lanza GM.
Cancer Res. 2003 Sep 15;63(18):5838-43.
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Early noninvasive detection and characterization of solid tumors and their supporting neovasculature is a fundamental prerequisite for effective therapeutic intervention, particularly antiangiogenic treatment regimens. Emerging molecular imaging techniques now allow recognition of early biochemical, physiological, and anatomical changes before manifestation of gross pathological changes. Although new tumor, vascular, extracellular matrix, and lymphatic biomarkers continue to be discovered, the alpha(nu)beta(3)-integrin remains an attractive biochemical epitope that is highly expressed on activated neovascular endothelial cells and essentially absent on mature quiescent cells. In this study, we report the first in vivo use of a magnetic resonance (MR) molecular imaging nanoparticle to sensitively detect and spatially characterize neovascularity induced by implantation of the rabbit Vx-2 tumor using a common clinical field strength (1.5T). New Zealand White rabbits (2 kg) 12 days after implantation of fresh Vx-2 tumors (2 x 2 x 2 mm(3)) were randomized into one of three treatment groups: (a) alpha(nu)beta(3)-targeted, paramagnetic formulation; (b) nontargeted, paramagnetic formulation; and (c) alpha(nu)beta(3)-targeted nonparamagnetic nanoparticles followed by (2 h) the alpha(nu)beta(3)-targeted, paramagnetic formulation to competitively block magnetic resonance imaging (MRI) signal enhancement. After i.v. systemic injection (0.5 ml of nanoparticles/kg), dynamic T(1)-weighted MRI was used to spatially and temporally determine nanoparticle deposition in the tumor and adjacent tissues, including skeletal muscle. At 2-h postinjection, alpha(nu)beta(3)-targeted paramagnetic nanoparticles increased MRI signal by 126% in asymmetrically distributed regions primarily in the periphery of the tumor. Similar increases in MR contrast were also observed within the walls of some vessels proximate to the tumor. Despite their relatively large size, nanoparticles penetrated into the leaky tumor neovasculature but did not appreciably migrate into the interstitium, leading to a 56% increase in MR signal at 2 h. Pretargeting of the alpha(nu)beta(3)-integrin with nonparamagnetic nanoparticles competitively blocked the specific binding of alpha(nu)beta(3)-targeted paramagnetic nanoparticles, decreasing the MR signal enhancement (50%) to a level attributable to local extravasation. The MR signal of adjacent hindlimb muscle or contralateral control tissues was unchanged by either the alpha(nu)beta(3)-targeted or control paramagnetic agents. Immunohistochemistry of alpha(nu)beta(3)-integrin corroborated the extent and asymmetric distribution of neovascularity observed by MRI. These studies demonstrate the potential of this targeted molecular imaging agent to detect and characterize (both biochemically and morphologically) early angiogenesis induced by minute solid tumors with a clinical 1.5 Tesla MRI scanner, facilitating the localization of nascent cancers or metastases, as well as providing tools to phenotypically categorize and segment patient populations for therapy and to longitudinally follow the effectiveness of antitumor treatment regimens.

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Linking radiation oncology and imaging through molecular biology (or now that therapy and diagnosis have separated, it's time to get together again!).
Radiology. 2003 Jul;228(1):29-35.
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Among the areas defined by the National Cancer Institute as "Extraordinary Opportunities for Research Investment" that are highly relevant to the technology-oriented disciplines within the broad field of radiology are cancer imaging, defining the signatures (ie, underlying molecular features) of cancer cells, and molecular targets of prevention and treatment. In molecular target credentialing, a specific molecular target is imaged, the molecular signature is defined, a treatment is given, and the effect of the intervention on the image findings and the signature is then evaluated. Such an approach is used to validate the proposed target as a legitimate one for cancer therapy or prevention and to provide the opportunity to ultimately individualize therapy on the basis of both the initial characteristics of the tumor and the tumor's response to an intervention. Therapeutic radiation is focused biology (ie, radiation produces molecular events in the irradiated tissue). Radiation can (a) kill cancer cells by itself, (b) be combined with cytotoxic or cytostatic drugs, and (c) serve to initiate radiation-inducible molecular targets that are amenable to treatment with drugs and/or biologic therapies. Focused biology can be anatomically confined with various types of external beams and with brachytherapy, and it can be used systemically with targeted radioisotopes. These new paradigms link diagnostic imaging, radiation therapy, and nuclear medicine in unique ways by way of basic biology. It is timely to develop new collaborative research, training, and education agendas by building on one another's expertise and adopting new fields of microtechnology, nanotechnology, and mathematical analysis and optimization.

Noninvasive detection of clinically occult lymph-node metastases in prostate cancer.
Carella M, Volinia S, Gasparini P, J Nephrol 2003 Jul-Aug;16(4):597-602., Coleman CN, Radiology 2003 Jul;228(1):29-35., Harisinghani MG, Barentsz J, Hahn PF, Deserno WM, Tabatabaei S, van de Kaa CH, de la Rosette J.
N Engl J Med.2003 Jun 19;348(25):2491-9.
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BACKGROUND: Accurate detection of lymph-node metastases in prostate cancer is an essential component of the approach to treatment. We investigated whether highly lymphotropic superparamagnetic nanoparticles, which gain access to lymph nodes by means of interstitial-lymphatic fluid transport, could be used in conjunction with high-resolution magnetic resonance imaging (MRI) to reveal small nodal metastases. METHODS: Eighty patients with presurgical clinical stage T1, T2, or T3 prostate cancer who underwent surgical lymph-node resection or biopsy were enrolled. All patients were examined by MRI before and 24 hours after the intravenous administration of lymphotropic superparamagnetic nanoparticles (2.6 mg of iron per kilogram of body weight). The imaging results were correlated with histopathological findings. RESULTS: Of the 334 lymph nodes that underwent resection or biopsy, 63 (18.9 percent) from 33 patients (41 percent) had histopathologically detected metastases. Of these 63 nodes, 45 (71.4 percent) did not fulfill the usual imaging criteria for malignancy. MRI with lymphotropic superparamagnetic nanoparticles correctly identified all patients with nodal metastases, and a node-by-node analysis had a significantly higher sensitivity than conventional MRI (90.5 percent vs. 35.4 percent, P<0.001) or nomograms. CONCLUSIONS: High-resolution MRI with magnetic nanoparticles allows the detection of small and otherwise undetectable lymph-node metastases in patients with prostate cancer.

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2002

Scaling down imaging: molecular mapping of cancer in mice.
Weissleder R.
Nat Rev Cancer. 2002 Jan;2(1):11-8.
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The development of miniaturized imaging equipment and reporter probes has improved our ability to study animal models of disease, such as transgenic and knockout mice. These technologies can now be used to continuously monitor in vivo tumour development, the effects of therapeutics on individual populations of cells, or even specific molecules. If these techniques prove effective in mice, they might be translated into the clinic in the future, where they could be used to non-invasively detect and monitor treatment of human cancers.