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

Environment, Health and Safety

2007  2006   2005

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2007

Quantification of Fullerenes by LC/ESI-MS and Its Application to in Vivo Toxicity Assays.
Isaacson CW, Usenko CY, Tanguay RL, Field JA.
Anal Chem. 2007 Dec 1;79(23):9091-7.
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With production and use of carbon nanoparticles increasing, it is imperative that the toxicity of these materials be determined; yet such testing requires specific and selective analytical methodologies that do not yet exist. Quantitative liquid-liquid extraction was coupled with liquid chromatography/electrospray ionization mass spectrometry for the quantitative determination of fullerenes from C60 to C98. Isotopically enriched, 13C60, was used as an internal standard. The method was applied to determine the loss of C60 from exposure water solution and uptake of C60 by embryonic zebrafish. The average recovery of C60 from zebrafish embryo extracts and 1% DMSO in aqueous-exposure solutions was 90 and 93%, respectively, and precision, as indicated by the relative standard deviation, was 2 and 7%, respectively. The method quantification limit was 0.40 mug/L and the detection limit was 0.02 mug/L. During the toxicological assay, loss of C60 due to sorption to test vials resulted in the reduction of exposure-solution concentrations over 6 h to less than 50% of the initial concentration. Time-course experiments indicated embryo uptake increased over course of the 12-h exposure. A lethal concentration that caused 50% mortality was determined to be 130 mug/L and was associated with a zebrafish embryo concentration, LD50, of 0.079 mug/g of embryo.

Adsorbed proteins influence the biological activity and molecular targeting of nanomaterials.
Dutta D, Sundaram SK, Teeguarden JG, Riley BJ, Fifield LS, Jacobs JM, Addleman SR, Kaysen GA, Moudgil BM, Weber TJ.
Toxicol Sci. 2007 Nov;100(1):303-15.
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The possible combination of specific physicochemical properties operating at unique sites of action within cells and tissues has led to considerable uncertainty surrounding nanomaterial toxic potential. We have investigated the importance of proteins adsorbed onto the surface of two distinct classes of nanomaterials (single-walled carbon nanotubes [SWCNTs]; 10-nm amorphous silica) in guiding nanomaterial uptake or toxicity in the RAW 264.7 macrophage-like model. Albumin was identified as the major fetal bovine or human serum/plasma protein adsorbed onto SWCNTs, while a distinct protein adsorption profile was observed when plasma from the Nagase analbuminemic rat was used. Damaged or structurally altered albumin is rapidly cleared from systemic circulation by scavenger receptors. We observed that SWCNTs inhibited the induction of cyclooxygenase-2 (Cox-2) by lipopolysaccharide (LPS; 1 ng/ml, 6 h) and this anti-inflammatory response was inhibited by fucoidan (scavenger receptor antagonist). Fucoidan also reduced the uptake of fluorescent SWCNTs (Alexa647). Precoating SWCNTs with a nonionic surfactant (Pluronic F127) inhibited albumin adsorption and anti-inflammatory properties. Albumin-coated SWCNTs reduced LPS-mediated Cox-2 induction under serum-free conditions. SWCNTs did not reduce binding of LPS(Alexa488) to RAW 264.7 cells. The profile of proteins adsorbed onto amorphous silica particles (50-1000 nm) was qualitatively different, relative to SWCNTs, and precoating amorphous silica with Pluronic F127 dramatically reduced the adsorption of serum proteins and toxicity. Collectively, these observations suggest an important role for adsorbed proteins in modulating the uptake and toxicity of SWCNTs and nano-sized amorphous silica.

Direct imaging of single-walled carbon nanotubes in cells.
Porter AA, Gass M, Muller K, Skepper JN, Midgley PA, Welland M.
Nature Nanotechnology. 2007;2(11):713–717.
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The development of single-walled carbon nanotubes for various biomedical applications is an area of great promise. However, the contradictory data on the toxic effects of single-walled carbon nanotubes1, 2, 3, 4, 5, 6, 7, 8, 9, 10 highlight the need for alternative ways to study their uptake and cytotoxic effects in cells. Single-walled carbon nanotubes have been shown to be acutely toxic1, 2, 3 in a number of types of cells, but the direct observation of cellular uptake of single-walled carbon nanotubes has not been demonstrated previously due to difficulties in discriminating carbon-based nanotubes from carbon-rich cell structures. Here we use transmission electron microscopy and confocal microscopy to image the translocation of single-walled carbon nanotubes into cells in both stained and unstained human cells. The nanotubes were seen to enter the cytoplasm and localize within the cell nucleus, causing cell mortality in a dose-dependent manner.

Imaging and tracking of tat peptide-conjugated quantum dots in living cells: new insights into nanoparticle uptake, intracellular transport, and vesicle shedding.
Ruan G, Agrawal A, Marcus AI, Nie S.
J Am Chem Soc. 2007 Nov 28;129(47):14759-66.
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We report the use of Tat peptide-conjugated quantum dots (Tat-QDs) to examine the complex behavior of nanoparticle probes in live cells, a topic that is of considerable current interest in developing advanced nanoparticle agents for molecular and cellular imaging. Dynamic confocal imaging studies indicate that the peptide-conjugated QDs are internalized by macropinocytosis, a fluid-phase endocytosis process triggered by Tat-QD binding to negatively charged cell membranes. The internalized Tat-QDs are tethered to the inner vesicle surfaces and are trapped in cytoplasmic organelles. The QD loaded vesicles are found to be actively transported by molecular machines (such as dyneins) along microtubule tracks. The destination of this active transport is an asymmetric perinuclear region (outside the cell nucleus) known as the microtubule organizing center (MTOC). We also find that Tat-QDs strongly bind to cellular membrane structures such as filopodia and that large QD-containing vesicles are released from the tips of filopodia by vesicle shedding. These results provide new insights into the mechanisms of Tat peptide-mediated delivery as well as toward the design of functionalized nanoparticles for molecular imaging and targeted therapy.

Single-walled carbon nanotubes in the intact organism: near-IR imaging and biocompatibility studies in Drosophila.
Leeuw TK, Reith RM, Simonette RA, Harden ME, Cherukuri P, Tsyboulski DA, Beckingham KM, Weisman RB.
Nano Lett. 2007 Sep;7(9):2650-4.
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The ability of near-infrared fluorescence imaging to detect single-walled carbon nanotubes (SWNTs) in organisms and biological tissues has been explored using Drosophila melanogaster (fruit flies). Drosophila larvae were raised on food containing approximately 10 ppm of disaggregated SWNTs. Their viability and growth were not reduced by nanotube ingestion. Near-IR nanotube fluorescence was imaged from intact living larvae, and individual nanotubes in dissected tissue specimens were imaged, structurally identified, and counted to estimate a biodistribution.

Renal clearance of quantum dots.
Soo Choi H, Liu W, Misra P, Tanaka E, Zimmer JP, Itty Ipe B, Bawendi MG, Frangioni JV.
Nat Biotechnol. 2007 Oct;25(10):1165-70.
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The field of nanotechnology holds great promise for the diagnosis and treatment of human disease. However, the size and charge of most nanoparticles preclude their efficient clearance from the body as intact nanoparticles. Without such clearance or their biodegradation into biologically benign components, toxicity is potentially amplified and radiological imaging is hindered. Using intravenously administered quantum dots in rodents as a model system, we have precisely defined the requirements for renal filtration and urinary excretion of inorganic, metal-containing nanoparticles. Zwitterionic or neutral organic coatings prevented adsorption of serum proteins, which otherwise increased hydrodynamic diameter by >15 nm and prevented renal excretion. A final hydrodynamic diameter <5.5 nm resulted in rapid and efficient urinary excretion and elimination of quantum dots from the body. This study provides a foundation for the design and development of biologically targeted nanoparticles for biomedical applications.

Long-term toxicity of holmium-loaded poly(L-lactic acid) microspheres in rats.
Zielhuis SW, Nijsen JF, Seppenwoolde JH, Bakker CJ, Krijger GC, Dullens HF, Zonnenberg BA, van Rijk PP, Hennink WE, van het Schip AD.
Biomaterials. 2007 Nov;28(31):4591-9.
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The aim of this study was to get insight into the toxic effects of holmium-166-loaded poly(L-lactic acid) microspheres (Ho-PLLA-MS) which have very interesting features for treatment of liver malignancies. Acute, mid- and long-term effects were studied in healthy Wistar rats by evaluating clinical, biochemical and tissue response. Rats were divided into four treatment groups: sham, decayed neutron-irradiated Ho-PLLA-MS, non-irradiated Ho-PLLA-MS and PLLA-MS. After implantation of the microspheres into the liver of the rats, the animals were monitored (body weight, temperature and liver enzymes) for a period of 14-18 months. Some of the rats that received previously neutron-irradiated Ho-PLLA-MS were periodically scanned with magnetic resonance imaging (MRI) to see if holmium was released from the microspheres. After sacrifice, the liver tissue was histologically evaluated. Bone tissue was subjected to neutron-activation analysis in order to examine whether accumulation of released holmium in the bone had occurred. No measurable clinical and biochemical toxic effects were observed in any of the treatment groups. Furthermore, histological analyses of liver tissue samples only showed signs of a slight chronic inflammation and no significant differences in the tissue reaction between rats of the different treatment groups could be observed. The non-irradiated PLLA-MS and Ho-PLLA-MS stayed intact during the study. In contrast, 14 months after administration, the neutron-irradiated Ho-PLLA-MS was not completely spherical anymore, indicating that degradation had started. However, the holmium loading had not been released as was illustrated with MRI and affirmed by neutron-activation analysis of bone tissue. In conclusion, neutron-irradiated Ho-PLLA-MS does not provoke any toxic reaction and can be applied safely in vivo.

Characterization of the size, shape, and state of dispersion of nanoparticles for toxicological studies
Powers KW, Palazuelos M, Moudgil BM, Roberts SM.
Nanotoxicology. 2007 Mar 1;1(1):42–51.
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This paper describes the issues relating to the measurement of nanoparticle size, shape and dispersion when evaluating the toxicity of nanoparticles. Complete characterization of these materials includes much more than size, size distribution and shape; nonetheless, these attributes are usually the essential foundation. The measurement of particle size, particularly at scales of 100 nm or less, can be challenging under the best of conditions. Measurements that are routine in the laboratory setting become even more difficult when made under the physiological conditions relevant to toxicity studies, where the environment of the particles can be quite complex. Passive and active cellular responses, as well as the presence of a variety of nano-scale biological structures, often complicate the collection and interpretation of size and shape data. In this paper, we highlight several of the common issues faced when characterizing nanoparticles for toxicity testing and suggest general protocols to address these problems. Keywords: Nanoparticles; size; shape; dispersion; characterization; toxicity

Kinetic analysis of nanoparticulate polyelectrolyte complex interactions with endothelial cells.
Hartig SM, Greene RR, Carlesso G, Higginbotham JN, Khan WN, Prokop A, Davidson JM.
Biomaterials. 2007 Sep;28(26):3843-55.
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A non-toxic, nanoparticulate polyelectrolyte complex (PEC) drug delivery system was formulated to maintain suitable physicochemical properties at physiological pH. Toxicity, binding, and internalization were evaluated in relevant microvascular endothelial cells. PEC were non-toxic, as indicated by cell proliferation studies and propidium iodide staining. Inhibitor studies revealed that PEC were bound, in part, via heparan sulfate proteoglycans and internalized through macropinocytosis. A novel, flow cytometric, Scatchard protocol was established and showed that PEC, in the absence of surface modification, bind cells non-specifically with positive cooperativity, as seen by graphical transformations.

Migration of intradermally injected quantum dots to sentinel organs in mice.
Gopee NV, Roberts DW, Webb P, Cozart CR, Siitonen PH, Warbritton AR, Yu WW, Colvin VL, Walker NJ, Howard PC.
Toxicol Sci. 2007 Jul;98(1):249-57.
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Topical exposure to nanoscale materials is likely from a variety of sources including sunscreens and cosmetics. Because the in vivo disposition of nanoscale materials is not well understood, we have evaluated the distribution of quantum dots (QDs) following intradermal injection into female SKH-1 hairless mice as a model system for determining tissue localization following intradermal infiltration. The QD (CdSe core, CdS capped, poly[ethylene glycol] coated, 37 nm diameter, 621 nm fluorescence emission) were injected intradermally (ID) on the right dorsal flank. Within minutes following intradermal injection, the highly UV fluorescent QD could be observed moving from the injection sites apparently through the lymphatic duct system to regional lymph nodes. Residual fluorescent QD remained at the site of injection until necropsy at 24 h. Quantification of cadmium and selenium levels after 0, 4, 8, 12, or 24 h in multiple tissues, using inductively coupled plasma mass spectrometry (ICP-MS), showed a time-dependent loss of cadmium from the injection site, and accumulation in the liver, regional draining lymph nodes, kidney, spleen, and hepatic lymph node. Fluorescence microscopy corroborated the ICP-MS results regarding the tissue distribution of QD. The results indicated that (1) ID injected nanoscale QD remained as a deposit in skin and penetrated the surrounding viable subcutis, (2) QD were distributed to draining lymph nodes through the sc lymphatics and to the liver and other organs, and (3) sentinel organs are effective locations for monitoring transdermal penetration of nanoscale materials into animals.

Analysis of fullerene-based nanomaterial in serum matrix by CE.
Chan KC, Patri AK, Veenstra TD, McNeil SE, Issaq HJ.
Electrophoresis. 2007 May;28(10):1518-24.
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With the increasing interest in using nanoparticles as vehicles for drug delivery and image contrast agents, there is a need to develop assays for their detection and quantitation in complex matrices to facilitate monitoring their biodistribution. In this study, we developed a CE approach for the analysis of two nanoparticles: carboxyfullerene (C3) and dendrofullerene (DF1) in both standard solutions and a serum matrix. These highly soluble, charged C(60) derivatives were characterized by CZE using either a bare or dynamically coated fused-silica capillaries. The resolution of both nanoparticles was slightly lower with the coated capillary; however, their migration times were faster. While separation of the DF1 nanoparticles using MEKC resulted in a greater number of observable peaks, the peak profile of C3 was basically unchanged regardless of whether SDS micelles were added to the running buffers or not. The MEKC and/or CZE assays were then used to quantitate the C3 and DF1 nanoparticles in spiked human serum samples. The quantitation of the nanoparticles was linear from 0-500 microg/mL with detection limits ranging from 0.5 to 6 microg/mL.

Nanotechnology Safety Concerns Revisited.
Stern, ST, McNeil, SE
Toxicol. Sci. 2007 Jul 4
[ view article ]

Multivariate toxicity screening of liposomal formulations on a human buccal cell line.
Smistad G, Jacobsen J, Sande SA.
Int J Pharm. 2007 Feb 7;330(1-2):14-22.
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The influence of various formulation factors on the in vitro cellular toxicity of liposomes on human buccal cells (TR146), were studied by using the concept of statistical experimental design and multivariate evaluation. The factors investigated were the type of main phospholipid (egg-PC, DMPC, DPPC), lipid concentration, the type of charge, liposome size, and amount and nature of the charged component (diacyl-PA, diacyl-PG, diacyl-PS, stearylamine (SA), diacyl-TAP) in the liposomes. Both full factorial design and D-optimal designs were created. Several significant main factors and interactions were revealed. Positively charged liposomes were shown to be toxic. The toxicity of negatively charged liposomes was relatively low. Diacyl-TAP was less toxic than SA, and DPPC was less toxic than DMPC. Low level of positively charged component was favourable and essential when using egg-PC as the main lipid. The amount of negatively charged component, the liposome size, and the total lipid concentration did not affect the toxicity within the experimental room. DPPC appeared to be a good candidate when formulating both positively and negatively charged liposomes with low cellular toxicity. The concept of statistical experimental design and multivariate evaluation was shown to be a useful approach in cell toxicity screening studies.

New Method for Delivering a Hydrophobic Drug for Photodynamic Therapy Using Pure Nanocrystal Form of the Drug.
Baba K, Pudavar HE, Roy I, Ohulchanskyy TY, Chen Y, Pandey RK, Prasad PN.
Mol Pharm. 2007 Feb 1; [Epub ahead of print].
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A carrier-free method for delivery of a hydrophobic drug in its pure form, using nanocrystals (nanosized crystals), is proposed. To demonstrate this technique, nanocrystals of a hydrophobic photosensitizing anticancer drug, 2-devinyl-2-(1-hexyloxyethyl)pyropheophorbide (HPPH), have been synthesized using the reprecipitation method. The resulting drug nanocrystals were monodispersed and stable in aqueous dispersion, without the necessity of an additional stabilizer (surfactant). As shown by confocal microscopy, these pure drug nanocrystals were taken up by the cancer cells with high avidity. Though the fluorescence and photodynamic activity of the drug were substantially quenched in the form of nanocrystals in aqueous suspension, both these characteristics were recovered under in vitro and in vivo conditions. This recovery of drug activity and fluorescence is possibly due to the interaction of nanocrystals with serum albumin, resulting in conversion of the drug nanocrystals into the molecular form. This was confirmed by demonstrating similar recovery in presence of fetal bovine serum (FBS) or bovine serum albumin (BSA). Under similar treatment conditions, the HPPH in nanocrystal form or in 1% Tween-80/water formulation showed comparable in vitro and in vivo efficacy. Keywords: Nanocrystals; reprecipitation method; photosensitizers; photodynamic therapy; singlet oxygen; drug delivery.

Nanotechnology approaches for drug and small molecule delivery across the blood brain barrier.
Silva GA.
Surg Neurol. 2007 Feb;67(2):113-6.
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Nanotechnology involves the design, synthesis, and characterization of materials and devices that have a functional organization in at least one dimension on the nanometer (ie, one billionth of a meter) scale. One area in which nanotechnology may have a significant clinical impact in neuroscience is the selective transport and delivery of drugs and other small molecules across the blood brain barrier that cannot cross otherwise. Using a variety of nanoparticles composed of different chemical compositions, different groups are exploring proof-of-concept approaches for the delivery of different antineoplastic drugs, oligonucleotides, genes, and magnetic resonance imaging contrast agents. This review discusses some of the main technical challenges associated with the development of nanotechnologies for delivery across the blood brain barrier and summarizes ongoing work.

Cytotoxic effects of aggregated nanomaterials.
Soto K, Garza KM, Murr LE.
Acta Biomater. 2007 Jan 31; [Epub ahead of print].
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This study deals with cytotoxicity assays performed on an array of commercially manufactured inorganic nanoparticulate materials, including Ag, TiO(2), Fe(2)O(3), Al(2)O(3), ZrO(2), Si(3)N(4), naturally occurring mineral chrysotile asbestos and carbonaceous nanoparticulate materials such as multiwall carbon nanotube aggregates and black carbon aggregates. The nanomaterials were characterized by TEM, as the primary particles, aggregates or long fiber dimensions ranged from 2nm to 20mum. Cytotoxicological assays of these nanomaterials were performed utilizing a murine alveolar macrophage cell line and human macrophage and epithelial lung cell lines as comparators. The nanoparticulate materials exhibited varying degrees of cytoxicity for all cell lines and the general trends were similar for both the murine and human macrophage cell lines. These findings suggest that representative cytotoxic responses for humans might be obtained by nanoparticulate exposures to simple murine macrophage cell line assays. Moreover, these results illustrate the utility in performing rapid in vitro assays for cytotoxicity assessments of nanoparticulate materials as a general inquiry of potential respiratory health risks in humans.

The degree and kind of agglomeration affect carbon nanotube cytotoxicity.
Wick P, Manser P, Limbach LK, Dettlaff-Weglikowska U, Krumeich F, Roth S, Stark WJ, Bruinink A.
Toxicol Lett. 2007 Jan 30;168(2):121-31.
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The urgent need for toxicological studies on carbon nanotubes (CNTs) has arisen from the rapidly emerging applications of CNTs well beyond material science and engineering. In order to provide a basis for comparison to existing epidemiological data, we have investigated CNTs at various degrees of agglomeration using an in vitro cytotoxicity study with human MSTO-211H cells. Non-cytotoxic polyoxyethylene sorbitan monooleate was found to well-disperse CNT. In the present study, the cytotoxic effects of well-dispersed CNT were compared with that of conventionally purified rope-like agglomerated CNTs and asbestos as a reference. While suspended CNT-bundles were less cytotoxic than asbestos, rope-like agglomerates induced more pronounced cytotoxic effects than asbestos fibres at the same concentrations. The study underlines the need for thorough materials characterization prior to toxicological studies and corroborates the role of agglomeration in the cytotoxic effect of nanomaterials.

Nanoparticles: pharmacological and toxicological significance.
Medina C, Santos-Martinez MJ, Radomski A, Corrigan OI, Radomski MW.
Br J Pharmacol. 2007 Jan 22; [Epub ahead of print].
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Nanoparticles are tiny materials (<1000 nm in size) that have specific physicochemical properties different to bulk materials of the same composition and such properties make them very attractive for commercial and medical development. However, nanoparticles can act on living cells at the nanolevel resulting not only in biologically desirable, but also in undesirable effects. In contrast to many efforts aimed at exploiting desirable properties of nanoparticles for medicine, there are limited attempts to evaluate potentially undesirable effects of these particles when administered intentionally for medical purposes. Therefore, there is a pressing need for careful consideration of benefits and side effects of the use of nanoparticles in medicine. This review article aims at providing a balanced update of these exciting pharmacological and potentially toxicological developments. The classes of nanoparticles, the current status of nanoparticle use in pharmacology and therapeutics, the demonstrated and potential toxicity of nanoparticles will be discussed.

Are Diamond Nanoparticles Cytotoxic?
Schrand AM, Huang H, Carlson C, Schlager JJ, Omacr Sawa E, Hussain SM, Dai L.
J Phys Chem B Condens Matter Mater Surf Interfaces Biophys. 2007 Jan 11;111(1):2-7.[ expand abstract ]

Finely divided carbon particles, including charcoal, lampblack, and diamond particles, have been used for ornamental and official tattoos since ancient times. With the recent development in nanoscience and nanotechnology, carbon-based nanomaterials (e.g., fullerenes, nanotubes, nanodiamonds) attract a great deal of interest. Owing to their low chemical reactivity and unique physical properties, nanodiamonds could be useful in a variety of biological applications such as carriers for drugs, genes, or proteins; novel imaging techniques; coatings for implantable materials; and biosensors and biomedical nanorobots. Therefore, it is essential to ascertain the possible hazards of nanodiamonds to humans and other biological systems. We have, for the first time, assessed the cytotoxicity of nanodiamonds ranging in size from 2 to 10 nm. Assays of cell viability such as mitochondrial function (MTT) and luminescent ATP production showed that nanodiamonds were not toxic to a variety of cell types. Furthermore, nanodiamonds did not produce significant reactive oxygen species. Cells can grow on nanodiamond-coated substrates without morphological changes compared to controls. These results suggest that nanodiamonds could be ideal for many biological applications in a diverse range of cell types.

Carbon nanotubes show no sign of acute toxicity but induce intracellular reactive oxygen species in dependence on contaminants.
Pulskamp K, Diabate S, Krug HF.
Toxicol Lett. 2007 Jan 10;168(1):58-74.
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Today nanosciences are experiencing massive investment worldwide although research on toxicological aspects of these nano-sized particles has just begun and to date, no clear guidelines exist to quantify the effects. In the present study, we focus on carbon nanotubes (CNTs), which represent one of the most widely investigated carbon nanoparticles. The present data indicate that CNTs are able to cross the cell membrane of rat macrophages (NR8383) and, therefore, might have an influence on cell physiology and function. NR8383 and human A549 lung cells were incubated with commercial single-walled (NT-1) and multi-walled (NT-2, NT-3) CNTs, carbon black and quartz as reference particles as well as an acid-treated single-walled CNT preparation (SWCNT a.t.) with reduced metal catalyst content. We did not observe any acute toxicity on cell viability (WST-1, PI-staining) upon incubation with all CNT products. None of the CNTs induced the inflammatory mediators NO, TNF-alpha and IL-8. A rising tendency of TNF-alpha release from LPS-primed cells due to CNT treatment could be observed. We detected however, a dose- and time-dependent increase of intracellular reactive oxygen species and a decrease of the mitochondrial membrane potential with the commercial CNTs in both cell types after particle treatment whereas incubation with the purified CNTs (SWCNT a.t.) had no effect. This leads us to the conclusion that metal traces associated with the commercial nanotubes are responsible for the biological effects.

Effects of Mechanical Flexion on the Penetration of Fullerene Amino Acid-Derivatized Peptide Nanoparticles through Skin.
Rouse JG, Yang J, Ryman-Rasmussen JP, Barron AR, Monteiro-Riviere NA.
Nano Lett. 2007 Jan 10;7(1):155-160.
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Dermatomed porcine skin was fixed to a flexing device and topically dosed with 33.5 mg.mL-1 of an aqueous solution of a fullerene-substituted phenylalanine (Baa) derivative of a nuclear localization peptide sequence (Baa-Lys(FITC)-NLS). Skin was flexed for 60 or 90 min or left unflexed (control). Confocal microscopy depicted dermal penetration of the nanoparticles at 8 h in skin flexed for 60 and 90 min, whereas Baa-Lys(FITC)-NLS did not penetrate into the dermis of unflexed skin until 24 h. TEM analysis revealed fullerene-peptide localization within the intercellular spaces of the stratum granulosum.

Nanotechnology: The Next Big Thing, or Much Ado about Nothing?
Maynard AD
Ann Occup Hyg. 2007 Jan;51(1):1-12. 2006 Oct 14.
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Nanotechnology encompasses an increasingly sophisticated ability to manipulate matter at the nanoscale, resulting in new materials, products and devices that demonstrate new and unusual behaviour. While emerging nanotechnologies have great potential for good, there are increasing concerns that the selfsame attributes that make them attractive will also lead to new risks to human health. Research to date suggests that some purposely made nanomaterials will present hazards based on their structure-as well as their chemistry-thus challenging many conventional approaches to risk assessment and management. People involved in making and using these materials need to know what the risks are and how to manage them, if safe nanotechnology-based businesses are to emerge. Yet the challenges faced by the occupational hygiene community in ensuring safe nano-workplaces are substantial. We currently know enough to suggest that some engineered nanomaterials will present new and unusual risks, but there is very little information on how these risks can be identified, assessed and controlled. And many nanomaterials are in production and use now. Good occupational hygiene practices and existing knowledge on working with hazardous substances provide a useful basis for working safely with nanomaterials. But where existing knowledge fails, new research is needed to fill the gaps: this must be strategically administered and targeted to addressing specific issues in a timely manner. Failing to take these steps will ultimately lead to people's health being endangered and emerging nanotechnologies floundering. However, with foresight, sound science and strategic research, we have the opportunity to ensure that emerging nanotechnologies are as safe as possible, while reaching their full potential.

Surface coatings determine cytotoxicity and irritation potential of quantum dot nanoparticles in epidermal keratinocytes.
Ryman-Rasmussen JP, Riviere JE, Monteiro-Riviere NA.
J Invest Dermatol. 2007 Jan;127(1):143-53.
[ expand abstract ]

Quantum dot (QD) nanoparticles have potential applications in nanomedicine as drug delivery vectors and diagnostic agents, but the skin toxicity and irritation potential of QDs are unknown. Human epidermal keratinocytes (HEKs) were used to assess if QDs with different surface coatings would cause differential effects on HEK cytotoxicity, proinflammatory cytokine release, and cellular uptake. Commercially available QDs of two different sizes, QD 565 and QD 655, with neutral (polyethylene glycol (PEG)), cationic (PEG-amine), or anionic (carboxylic acid) coatings were utilized. Live cell imaging and transmission electron microscopy were used to determine that all QDs localized intracellularly by 24 hours, with evidence of QD localization in the nucleus. Cytotoxicity and release of the proinflammatory cytokines IL-1beta, IL-6, IL-8, IL-10, and tumor necrosis factor-alpha were assessed at 24 and 48 hours. Cytotoxicity was observed for QD 565 and QD 655 coated with carboxylic acids or PEG-amine by 48 hours, with little cytotoxicity observed for PEG-coated QDs. Only carboxylic acid-coated QDs significantly increased release of IL-1beta, IL-6, and IL-8. These data indicate that QD surface coating is a primary determinant of cytotoxicity and immunotoxicity in HEKs, which is consistent across size. However, uptake of QDs by HEKs is independent of surface coating.

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2006

Biological tolerance of different materials in bulk and nanoparticulate form in a rat model: sarcoma development by nanoparticles.
Hansen T, Clermont G, Alves A, Eloy R, Brochhausen C, Boutrand JP, Gatti AM, Kirkpatrick CJ.
J R Soc Interface. 2006 Dec 22;3(11):767-75.
[ expand abstract ]

In order to study the pathobiological impact of the nanometre-scale of materials, we evaluated the effects of five different materials as nanoparticulate biomaterials in comparison with bulk samples in contact with living tissues. Five groups out of 10 rats were implanted bilaterally for up to 12 months with materials of the same type, namely TiO2, SiO2, Ni, Co and polyvinyl chloride (PVC), subcutaneously with bulk material on one side of the vertebral column and intramuscularly with nanoparticulate material on the contralateral side. At the end of each implantation time, the site was macroscopically examined, followed by histological processing according to standard techniques. Malignant mesenchymal tumours (pleomorphic sarcomas) were obtained in five out of six cases of implanted Co nanoparticle sites, while a preneoplastic lesion was observed in an animal implanted with Co in bulk form. In the Ni group, all animals rapidly developed visible nodules at the implanted sites between 4 and 6 months, which were diagnosed as rhabdomyosarcomas. Since the ratio of surface area to volume did not show significant differences between the Ni/Co group and the TiO2/SiO2/PVC group, we suggested that the induction of neoplasia was not mediated by physical effects, but was mediated by the well-known carcinogenic impact of Ni and Co. The data from the Co group show that the physical properties (particulate versus bulk form) could have a significant influence on the acceleration of the neoplastic process.

CdSe quantum dots induce apoptosis in human neuroblastoma cells via mitochondrial-dependent pathways and inhibition of survival signals.
Chan WH, Shiao NH, Lu PZ.
Toxicol Lett. 2006 Dec 15;167(3):191-200.
[ expand abstract ]

Quantum dots (QDs) may be useful as novel luminescent markers, but their cytotoxicity has not been fully investigated. In this report, we demonstrate that CdSe-core QDs can induce apoptotic biochemical changes, including JNK activation, loss of mitochondrial membrane potential, mitochondrial release of cytochrome c and activation of caspase-9 and caspase-3 in the IMR-32 human neuroblastoma cell line. Importantly, treatment of IMR-32 cells with CdSe-core QD triggered an increase in reactive oxygen species (ROS) and inhibited survival-related signaling events, such as decreased Ras and Raf-1 protein expression and decreased ERK activation. These apoptotic biochemical changes were not detected in cells treated with ZnS-coated CdSe QDs. Collectively, these results demonstrate that CdSe-core QD treatment of IMR-32 cells induced JNK activation and mitochondrial-dependent apoptotic processes while inhibiting Ras-->ERK survival signaling and that a ZnS coating could effectively reduce QD cytotoxicity.

In vitro toxicity of silica nanoparticles in human lung cancer cells.
Lin W, Huang YW, Zhou XD, Ma Y.
Toxicol Appl Pharmacol. 2006 Dec 15;217(3):252-9.
[ expand abstract ]

The cytotoxicity of 15-nm and 46-nm silica nanoparticles was investigated by using crystalline silica (Min-U-Sil 5) as a positive control in cultured human bronchoalveolar carcinoma-derived cells. Exposure to 15-nm or 46-nm SiO(2) nanoparticles for 48 h at dosage levels between 10 and 100 mug/ml decreased cell viability in a dose-dependent manner. Both SiO(2) nanoparticles were more cytotoxic than Min-U-Sil 5; however, the cytotoxicities of 15-nm and 46-nm silica nanoparticles were not significantly different. The 15-nm SiO(2) nanoparticles were used to determine time-dependent cytotoxicity and oxidative stress responses. Cell viability decreased significantly as a function of both nanoparticle dosage (10-100 mug/ml) and exposure time (24 h, 48 h, and 72 h). Indicators of oxidative stress and cytotoxicity, including total reactive oxygen species (ROS), glutathione, malondialdehyde, and lactate dehydrogenase, were quantitatively assessed. Exposure to SiO(2) nanoparticles increased ROS levels and reduced glutathione levels. The increased production of malondialdehyde and lactate dehydrogenase release from the cells indicated lipid peroxidation and membrane damage. In summary, exposure to SiO(2) nanoparticles results in a dose-dependent cytotoxicity in cultural human bronchoalveolar carcinoma-derived cells that is closely correlated to increased oxidative stress.

C60-Fullerenes: detection of intracellular photoluminescence and lack of cytotoxic effects.
Levi N, Hantgan RR, Lively MO, Carroll DL, Prasad GL.
J Nanobiotechnology. 2006 Dec 14;4:14.
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We have developed a new method of application of C60 to cultured cells that does not require water-solubilization techniques. Normal and malignant cells take-up C60 and the inherent photoluminescence of C60 is detected within multiple cell lines. Treatment of cells with up to 200 mug/ml (200 ppm) of C60 does not alter morphology, cytoskeletal organization, cell cycle dynamics nor does it inhibit cell proliferation. Our work shows that pristine C60 is non-toxic to the cells, and suggests that fullerene-based nanocarriers may be used for biomedical applications.

Carbon nanotubes as nanomedicines: From toxicology to pharmacology.
Lacerda L, Bianco A, Prato M, Kostarelos K.
Adv Drug Deliv Rev. 2006 Dec 1;58(14):1460-70.
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Various biomedical applications of carbon nanotubes have been proposed in the last few years leading to the emergence of a new field in diagnostics and therapeutics. Most of these applications will involve the administration or implantation of carbon nanotubes and their matrices into patients. The toxicological and pharmacological profile of such carbon nanotube systems developed as nanomedicines will have to be determined prior to any clinical studies undertaken. This review brings together all the toxicological and pharmacological in vivo studies that have been carried out using carbon nanotubes, to offer the first summary of the state-of-the-art in the pharmaceutical development of carbon nanotubes on the road to becoming viable and effective nanomedicines.

Nanomedicine: An unresolved regulatory issue.
Chan VS.
Regul Toxicol Pharmacol. 2006 Dec;46(3):218-24.
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Nanomedicine is a science that uses nanotechnology to maintain and improve human health at the molecular scale. Current and potential applications of nanotechnology in medicine range from research involving diagnostic devices, drug delivery vehicles to enhanced gene therapy and tissue engineering procedures. Its advantage over conventional medicine lies on its size. Particle size has effect on serum lifetime and pattern of deposition. This allows drugs of nanosize be used in lower concentration and has an earlier onset of therapeutic action. It also provides materials for controlled drug delivery by directing carriers to a specific location. Major efforts are underway, however, very little attention is devoted to assessment of health risks to human or to the ecosystem. Inhaled nanoparticles have already been related to lung injury. It is recognized that physico-chemical properties in conjunction with environmental factors and stability of the nanomaterial all contribute to the overall toxicological responses. Nanotoxicological information, currently insufficient, will be vital in aiding academia, industry and regulatory bodies in elucidating the mechanisms of action, balancing its risk and benefit, thus maximizing the utility of these materials in medicine without compromising public health and environmental integrity.

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Do nanoparticles present ecotoxicological risks for the health of the aquatic environment?
Moore MN.
Environ Int. 2006 Dec;32(8):967-76.
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Nanotechnology is a major innovative scientific and economic growth area, which may present a variety of hazards for environmental and human health. The surface properties and very small size of nanoparticles and nanotubes provide surfaces that may bind and transport toxic chemical pollutants, as well as possibly being toxic in their own right by generating reactive radicals. There is a wealth of evidence for the harmful effects of nanoscale combustion-derived particulates (ultrafines), which when inhaled can cause a number of pulmonary pathologies in mammals and humans. However, release of manufactured nanoparticles into the aquatic environment is largely an unknown. This review addresses the possible hazards associated with nanomaterials and harmful effects that may result from exposure of aquatic animals to nanoparticles. Possible nanoparticle association with naturally occurring colloids and particles is considered together with how this could affect their bioavailability and uptake into cells and organisms. Uptake by endocytotic routes are identified as probable major mechanisms of entry into cells; potentially leading to various types of toxic cell injury. The higher level consequences for damage to animal health, ecological risk and possible food chain risks for humans are also considered based on known behaviours and toxicities for inhaled and ingested nanoparticles in the terrestrial environment. It is concluded that a precautionary approach is required with individual evaluation of new nanomaterials for risk to the health of the environment. Although current toxicity testing protocols should be generally applicable to identify harmful effects associated with nanoparticles, research into new methods is required to address the special properties of nanomaterials.

Fullerene-based amino acid nanoparticle interactions with human epidermal keratinocytes.
Rouse JG, Yang J, Barron AR, Monteiro-Riviere NA.
Toxicol In Vitro. 2006 Dec;20(8):1313-20.
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The functionalization of C(60) with such complexes as amino acids has the potential to provide greater interaction between the fullerene and the biological environment yielding potential new medical and pharmacological applications. Although scientific research in the past decade has revealed much about the chemical and physical properties of C(60), the biological activities of this compound and its derivatives are still relatively unclear. In an attempt to understand the biological activity of functionalized C(60), human epidermal keratinocytes (HEK) were exposed to fullerene-based amino acid (Baa) solutions ranging in concentrations of 0.4-0.00004 mg/mL in a humidified 5% CO(2) atmosphere at 37 degrees C. MTT cell viability after 48 h significantly decreased (p<0.05) for concentrations of 0.4 and 0.04 mg/mL. In an additional study, human cytokines IL-6, IL-8, TNF-alpha, IL-1beta, and IL-10 were assessed for concentrations ranging from 0.4-0.004 mg/mL. Media was harvested at 1, 4, 8, 12, 24 and 48 h for cytokine analysis. IL-8 concentrations for the 0.04 mg/mL treatment were significantly greater (p<0.05) than all other concentrations at 8, 12, 24, and 48 h. IL-6 and IL-1beta activities were greater at the 24h and 48 h for 0.4 and 0.04 mg/mL. No significant TNF-alpha or IL-10 activity existed at any time points for any of the concentrations. These results indicate that concentrations lower than 0.04 mg/mL initiate less cytokine activity and maintain cell viability. In HEK, Baa concentrations of 0.4 and 0.04 mg/mL decrease cell viability and initiate a pro-inflammatory response.

Mammalian pharmacokinetics of carbon nanotubes using intrinsic near-infrared fluorescence.
Cherukuri P, Gannon CJ, Leeuw TK, Schmidt HK, Smalley RE, Curley SA, Weisman. Proc Natl Acad Sci USA. 2006 Nov 29; [Epub ahead of print] RB.
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Individualized, chemically pristine single-walled carbon nanotubes have been intravenously administered to rabbits and monitored through their characteristic near-infrared fluorescence. Spectra indicated that blood proteins displaced the nanotube coating of synthetic surfactant molecules within seconds. The nanotube concentration in the blood serum decreased exponentially with a half-life of 1.0 +/- 0.1 h. No adverse effects from low-level nanotube exposure could be detected from behavior or pathological examination. At 24 h after i.v. administration, significant concentrations of nanotubes were found only in the liver. These results demonstrate that debundled single-walled carbon nanotubes are high-contrast near-infrared fluorophores that can be sensitively and selectively tracked in mammalian tissues using optical methods. In addition, the absence of acute toxicity and promising circulation persistence suggest the potential of carbon nanotubes in future pharmaceutical applications.

Single-Walled Carbon Nanotube (SWCNT)-induced interstitial fibrosis in the lungs of rats is associated with increased levels of PDGF mRNA and the formation of unique intercellular carbon structures that bridge alveolar macrophages In Situ.
Mangum JB, Turpin EA, Antao-Menezes A, Cesta MF, Bermudez E, Bonner JC.
Part Fibre Toxicol. 2006 Nov 29;3:15.
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ABSTRACT: BACKGROUND: Nanotechnology is a rapidly advancing industry with many new products already available to the public. Therefore, it is essential to gain an understanding of the possible health risks associated with exposure to nanomaterials and to identify biomarkers of exposure. In this study, we investigated the fibrogenic potential of SWCNT synthesized by chemical vapor deposition using cobalt (Co) and molybdenum (Mo) as catalysts. Following a single oropharyngeal aspiration of SWCNT in rats, we evaluated lung histopathology, cell proliferation, and growth factor mRNAs at 1 and 21 days post-exposure. Comparisons were made to vehicle alone (saline containing a biocompatible nonionic surfactant), inert carbon black (CB) nanoparticles, or vanadium pentoxide (V2O5) as a known inducer of fibrosis. RESULTS: SWCNT or CB caused no overt inflammatory response at 1 or 21 days post-exposure as determined by histopathology and evaluation of cells (>95% macrophages) in bronchoalveolar lavage (BAL) fluid. However, SWCNT induced the formation of small, focal interstitial fibrotic lesions within the alveolar region of the lung at 21 days. A small fraction of alveolar macrophages harvested by BAL from the lungs of SWCNT-exposed rats at 21 days were bridged by unique intercellular carbon structures that extended into the cytoplasm of each macrophage. These "carbon bridge" structures between macrophages were also observed in situ in the lungs of SWCNT-exposed rats. No carbon bridges were observed in CB-exposed rats. SWCNT caused cell proliferation only at sites of fibrotic lesion formation as measured by bromodeoxyuridine uptake into alveolar cells. SWCNT increased platelet-derived growth factor (PDGF)-A, PDGF-B, and PDGF-C mRNA levels significantly at 1 day as measured by Taqman quantitative real-time RT-PCR. At 21 days, SWCNT did not increase any mRNAs evaluated, while V2O5 significantly increased mRNAs encoding PDGF-A, -B, and -C chains, PDGF-Ralpha, osteopontin (OPN), connective tissue growth factor (CTGF), and transforming growth factor (TGF)-beta1. CONCLUSION: Our findings indicate that SWCNT do not cause lung inflammation and yet induce the formation of small, focal interstital fibrotic lesioins in the alveolar region of the lungs of rats. Of greatest interest was the discovery of unique intercellular carbon structures composed of SWCNT that bridged lung macrophages. These "carbon bridges" offer a novel and easily identifiable biomarker of exposure.

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Safe handling of nanotechnology.
Maynard AD, Aitken RJ, Butz T, Colvin V, Donaldson K, Oberdorster G, Philbert MA, Ryan J, Seaton A, Stone V, Tinkle SS, Tran L, Walker NJ, Warheit DB.
Nature. 2006 Nov 16;444(7117):267-9.
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No abstract available

Carbon nanotubes show no sign of acute toxicity but induce intracellular reactive oxygen species in dependence on contaminants.
Pulskamp K, Diabate S, Krug HF.
Toxicol Lett. 2006 Nov 15; [Epub ahead of print].
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Today nanosciences are experiencing massive investment worldwide although research on toxicological aspects of these nano-sized particles has just begun and to date, no clear guidelines exist to quantify the effects. In the present study, we focus on carbon nanotubes (CNTs), which represent one of the most widely investigated carbon nanoparticles. The present data indicate that CNTs are able to cross the cell membrane of rat macrophages (NR8383) and, therefore, might have an influence on cell physiology and function. NR8383 and human A549 lung cells were incubated with commercial single-walled (NT-1) and multi-walled (NT-2, NT-3) CNTs, carbon black and quartz as reference particles as well as an acid-treated single-walled CNT preparation (SWCNT a.t.) with reduced metal catalyst content. We did not observe any acute toxicity on cell viability (WST-1, PI-staining) upon incubation with all CNT products. None of the CNTs induced the inflammatory mediators NO, TNF-alpha and IL-8. A rising tendency of TNF-alpha release from LPS-primed cells due to CNT treatment could be observed. We detected however, a dose- and time-dependent increase of intracellular reactive oxygen species and a decrease of the mitochondrial membrane potential with the commercial CNTs in both cell types after particle treatment whereas incubation with the purified CNTs (SWCNT a.t.) had no effect. This leads us to the conclusion that metal traces associated with the commercial nanotubes are responsible for the biological effects.

FDA holds public meeting on nanotechnology.
Traynor K.
Am J Health Syst Pharm. 2006 Nov 15;63(22):2175-7.
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No abstract available.

Particokinetics In Vitro: Dosimetry Considerations for In Vitro Nanoparticle Toxicity Assessments.
Teeguarden JG, Hinderliter PM, Orr G, Thrall BD, Pounds JG.
Toxicol Sci. 2006 Nov 10; [Epub ahead of print].
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The rapid growth in the use of in vitro methods for nanoparticle toxicity assessment has proceeded with limited consideration of the unique kinetics of these materials in solution. Particles in general and nanoparticles specifically, diffuse, settle and agglomerate in cell culture media as a function of systemic and particle properties: media density and viscosity, particle size, shape and density, for example. Cellular dose then is also a function of these factors as they determine the rate of transport of nanoparticles to cells in culture. Here we develop and apply the principles of dosimetry in vitro and outline an approach for simulation of nanoparticle particokinetics in cell culture systems. We illustrate that where equal mass concentrations (microg/ml) imply equal doses for dissimilar materials, the corresponding particle number or surface area concentration doses differ by orders of magnitude. More importantly, when rates of diffusional and gravitational particle delivery are accounted for, the trends and magnitude of cellular dose as a function of particle size and density differ significantly from those implied by "concentration" doses. For example, 15 nm silver nanoparticles appear approximately 4000 times more potent than micron sized cadmium oxide particles on a cm(2)/ml media basis, but are only approximately 50 times more potent when differences in delivery to adherent cells are considered. We conclude that simple surrogates of dose can cause significant misinterpretation of response and uptake data for nanoparticles in vitro. Incorporating particokinetics and principles of dosimetry would significantly improve the basis for nanoparticle toxicity assessment, increasing the predictive power and scalability of such assays.

Loading Magnetic Nanoparticles into Sperm Cells Does Not Affect Their Functionality.
Ben-David Makhluf S, Qasem R, Rubinstein S, Gedanken A, Breitbart H.
Langmuir. 2006 Nov 7;22(23):9480-9482.
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The spontaneous loading of magnetite nanoparticles into sperm cell was carried out by mixing an aqueous colloidal solution of Fe(3)O(4)-PVA with sperm cells (10(8) cells/ml) for 2 h at 37 degrees C suspended in glucose-free modified Tyrode solution. The penetration of the magnetite nanoparticles into the sperm cells was monitored by conventional analytical chemistry. We have demonstrated that the motility and the ability to undergo the acrosome reaction (i.e., the ability to fertilize the egg) were not affected by the presence of the magnetite nanoparticles.

An attempt to directly trace polymeric nanoparticles in vivo with electron microscopy.
Sun W, Wang H, Xie C, Hu Y, Yang X, Xu H.
J Control Release. 2006 Oct 27;115(3):259-265.
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This work attempted to directly observe polymeric nanoparticles in vivo by analytical electron microscopy (AEM) using copper chlorophyll as the contrast agent, based on the experiments concerned with the copper chlorophyll labeled poly-dl-lactide nanoparticles and the in vivo distribution of the polysorbate 80 (T-80)-coated nanoparticles in brain tissues. With the nanoprecipitation method without surfactants, copper chlorophyll is aggregated in the coordinately saturated form and encapsulated by the matrices of nanoparticles, which may ensure the stability of copper chlorophyll during the in vivo experiments. From both morphological information and chemical information, only the labeled nanoparticles with the T-80 coating were directly traced in the brain by AEM. The results not only support the mechanism of endocytosis and/or transcytosis of T-80-coated nanoparticles targeted to the brain but also verify that it is practical to probe polymeric nanoparticles in vivo using AEM together with copper chlorophyll as the contrast agent.

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Pulmonary Bioassay Studies with Nanoscale and Fine Quartz Particles in Rats: Toxicity is not dependent upon Particle Size but on Surface Characteristics.
Warheit DB, Webb TR, Colvin VL, Reed KL, Sayes CM.
Toxicol Sci. 2006 Oct 9; [Epub ahead of print] .
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Pulmonary toxicology studies in rats demonstrate that nanoparticles are more toxic than fine-sized particles of similar chemistry. This study, however, provides evidence to contradict this theory. The aims of the study were 1) to compare the toxicity of synthetic 50 nm nanoquartz-I particles vs. (mined) Min-U-Sil quartz ( approximately 500 nm); the toxicity of synthetic 12 nm nanoquartz-II particles vs. (mined) Min-U-Sil ( approximately 500 nm) vs. (synthetic) fine quartz particles (300 nm); and 2) to evaluate the surface activities among the samples as they relate to toxicity. Well characterized samples were tested for surface activity and hemolytic potential. In addition, groups of rats were instilled either with doses of 1 or 5 mg/kg of carbonyl iron or various alpha-quartz particle-types in phosphate buffered saline solution and subsequently assessed using bronchoalveolar lavage fluid biomarkers, cell proliferation, and histopathological evaluation of lung tissue at 24 hrs, 1 week, 1 month and 3 months postexposure. Exposures to the various alpha-quartz particles produced differential degrees of pulmonary inflammation and cytotoxicity, which were not always consistent with particle size but correlated with surface activity, particularly hemolytic potential. Lung tissue evaluations of three of the quartz samples demonstrated "typical" quartz-related effects - dose-dependent lung inflammatory macrophage accumulation responses concomitant with early development of pulmonary fibrosis. The various alpha-quartz related effects were similar qualitatively but with different potencies. The range of particle-related toxicities and histopathological effects in descending order were nanoscale quartz II = Min-U-Sil quartz > fine quartz > nanoscale quartz I > carbonyl iron particles. The results demonstrate that the pulmonary toxicities of alpha-quartz particles appear to correlate better with surface activity than particle size and surface area.

Proteomic identification of macrophage migration inhibitory factor upon exposure to TiO2 particles.
Cha MH, Rhim T, Kim KH, Jang AS, Paik YK, Park CS.
Mol Cell Proteomics. 2006 Oct 6; [Epub ahead of print] .
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Inhalation of particulate matter aggravates respiratory symptoms in patients with chronic airway diseases, but the mechanisms underlying this response remain poorly understood. We employed a proteomics approach to examine this phenomenon. Treatment of epithelial cells with BSA-coated titanium dioxide(TiO2) particles altered twenty protein spots on the 2-dimensional gel and these were then analyzed by 2D-nano liquid chromatography tandem mass spectrometry (2D Nano-LC-MS/MS). These proteins included defense-related, cell-activating, and cytoskeletal proteins implicated in the response to oxidative stress. The proteins were classified into four groups according to the time-course of their expression patterns. For validation, RT-PCR was performed on extracts of in vitro TiO2-treated cells, and lung issues from TiO2-treated rats were analyzed by immunohistochemical staining and enzyme immunoassay. TiO2 treatment was found to increase the amount of mRNA for macrophage migration inhibitory factor (MIF). MIF was expressed primarily in epithelium and was elevated in lung tissues and bronchoalveolar lavage fluids of TiO2-treated rats, as compared to sham-treated rats. Carbon black and diesel exhaust particles also induced expression of MIF protein in the epithelial cells.

Trace analysis of fullerenes in biological samples by simplified liquid-liquid extraction and high-performance liquid chromatography.
Xia XR, Monteiro-Riviere NA, Riviere JE.
J Chromatogr A. 2006 Oct 6;1129(2):216-22.
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Fullerene (C(60)) has several potential biomedical and industrial applications. While pure fullerene is not soluble in water, nanoparticles of the fullerene aggregates (nano-C(60)) can be prepared in water solutions. The concentration of nano-C(60) in biological media after systemic exposure could be very low and requires trace analytical methods to be developed for the toxicological and pharmacokinetic studies of the nanomaterial. A serious drop in extraction efficiency was observed when the concentration was under 0.5mug/mL using traditional liquid-liquid extraction (LLE) protocols. The evaporation of the solvent extract to dryness was found to be the main reason for the efficiency drop and an improved evaporation method was proposed to overcome this problem. Optimal proportion of glacial acetic acid (GAA) was used to solublize the proteins and surfactants in the biological samples, so that the emulsion problem was eliminated during LLE. Magnesium perchlorate was used to destabilize the nano-C(60) particles in the water solution and promoted the solvent extraction. A simplified LLE method was developed for high throughput while preserved the advantages of the traditional LLE. The developed method was used for trace analysis of fullerenes in protein containing media and tape-stripped skin samples. Under optimal experimental conditions, the detection limit was 0.34ng/mL and the recovery was in the range of 94-100% (n=5) at a concentration of 10ng/mL nano-C(60) in the biological media.

The 3p21.3 Tumor Suppressor NPRL2 Plays an Important Role in Cisplatin-Induced Resistance in Human Non-Small-Cell Lung Cancer Cells.
Ueda K, Kawashima H, Ohtani S, Deng WG, Ravoori M, Bankson J, Gao B, Girard L, Minna JD, Roth JA, Kundra V, Ji L.
Cancer Res. 2006 Oct 1;66(19):9682-90.
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NPRL2 is one of the novel candidate tumor suppressor genes identified in the human chromosome 3p21.3 region. The NPRL2 has shown potent tumor suppression activity in vitro and in vivo and has been suggested to be involved in DNA mismatch repair, cell cycle checkpoint signaling, and regulation of the apoptotic pathway. In this study, we analyzed the endogenous expression of the NPRL2 protein and the cellular response to cisplatin in 40 non-small-cell lung cancer cell lines and found that expression of NPRL2 was significantly and reciprocally correlated to cisplatin sensitivity, with a Spearman correlation coefficient of -0.677 (P < 0.00001). Exogenously introduced expression of NPRL2 by N-[1-(2,3-dioleoyloxyl)propyl]-NNN-trimethylammoniummethyl sulfate:cholesterol nanoparticle-mediated gene transfer significantly resensitized the response to cisplatin, yielding a 40% greater inhibition of tumor cell viability and resulting in a 2- to 3-fold increase in induction of apoptosis by activation of multiple caspases in NPRL2-transfected cells compared with untransfected cells at an equal dose of cisplatin. Furthermore, a systemic treatment with a combination of NPRL2 nanoparticles and cisplatin in a human H322 lung cancer orthotopic mouse model significantly enhanced the therapeutic efficacy of cisplatin and overcame cisplatin-induced resistance (P < 0.005). These findings implicate the potential of NPRL2 as a biomarker for predicting cisplatin response in lung cancer patients and as a molecular therapeutic agent for enhancing response and resensitizing nonresponders to cisplatin treatment.

Stable isotopic tracing-a way forward for nanotechnology.
Gulson B, Wong H.
Environ Health Perspect. 2006 Oct;114(10):1486-8.
[ expand abstract ]

Numerous publications and reports have expressed health and safety concerns about the production and use of nanoparticles, especially in areas of exposure monitoring, personal use, and environmental fate and transport. We suggest that stable isotopic tracers, which have been used widely in the earth sciences and in metabolic and other health-related studies for several decades, could be used to address many of these issues. One such example we are pursuing is the use of stable isotopes to monitor dermal absorption of zinc and titanium oxides in sunscreen preparations and other personal care products. Other potential applications of this tracing approach are discussed.

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Multiphoton microscopy for the investigation of dermal penetration of nanoparticle-borne drugs.
Stracke F, Weiss B, Lehr CM, Konig K, Schaefer UF, Schneider M.
J Invest Dermatol. 2006 Oct;126(10):2224-33.
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Multiphoton microscopy (MPM) of a dually fluorescence-labeled model system in excised human skin is employed for high-resolution three-dimensional (3D) visualization in order to study the release, accumulation, and penetration properties of drugs released from nanoscale carrier particles in dermal administration. Polymer particles were covalently labeled with fluorescein, whereas Texas Red as a drug-model was dissolved in the particles to be released to the formulation matrix. Single nanoparticles on skin could easily be localized and imaged with diffraction-limited resolution. The temporal evolution of the fluorescent drug-model concentration in various skin compartments over more than 5 hours was investigated by multiphoton spectral imaging of the same area of the specimen. The 3D penetration profile of the drug model in correlation with skin morphology and particle localization information is obtained by multiple laser line excitation experiments. MPM combined with spectral imaging was found to allow noninvasive long-term studies of particle-borne drug-model penetration into skin with subcellular resolution. By dual color labeling, a clear discrimination between particle-bound and released drug model was possible. The introduced technique was shown to be a powerful tool in revealing the dermal penetration properties and pathways of drugs and nanoscale drug vehicles on microscopic level.

Cytotoxicity of single-wall carbon nanotubes on human fibroblasts.
Tian F, Cui D, Schwarz H, Estrada GG, Kobayashi H.
Toxicol In Vitro. 2006 Oct;20(7):1202-12.
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We present a toxicological assessment of five carbon nanomaterials on human fibroblast cells in vitro. We correlate the physico-chemical characteristics of these nanomaterials to their toxic effect per se, i.e. excluding catalytic transition metals. Cell survival and attachment assays were evaluated with different concentrations of refined: (i) single-wall carbon nanotubes (SWCNTs), (ii) active carbon, (iii) carbon black, (iv) multi-wall carbon nanotubes, and (v) carbon graphite. The refined nanomaterial that introduced the strongest toxic effect was subsequently compared to its unrefined version. We therefore covered a wide range of variables, such as: physical dimensions, surface areas, dosages, aspect ratios and surface chemistry. Our results are twofold. Firstly, we found that surface area is the variable that best predicts the potential toxicity of these refined carbon nanomaterials, in which SWCNTs induced the strongest cellular apoptosis/necrosis. Secondly, we found that refined SWCNTs are more toxic than its unrefined counterpart. For comparable small surface areas, dispersed carbon nanomaterials due to a change in surface chemistry, are seen to pose morphological changes and cell detachment, and thereupon apoptosis/necrosis. Finally, we propose a mechanism of action that elucidates the higher toxicity of dispersed, hydrophobic nanomaterials of small surface area.

Lung dosimetry and risk assessment of nanoparticles: evaluating and extending current models in rats and humans.
Kuempel ED, Tran CL, Castranova V, Bailer AJ.
Inhal Toxicol. 2006 Sep;18(10):717-24.
[ expand abstract ]

Risk assessment of occupational exposure to nanomaterials is needed. Human data are limited, but quantitative data are available from rodent studies. To use these data in risk assessment, a scientifically reasonable approach for extrapolating the rodent data to humans is required. One approach is allometric adjustment for species differences in the relationship between airborne exposure and internal dose. Another approach is lung dosimetry modeling, which provides a biologically-based, mechanistic method to extrapolate doses from animals to humans. However, current mass-based lung dosimetry models may not fully account for differences in the clearance and translocation of nanoparticles. In this article, key steps in quantitative risk assessment are illustrated, using dose-response data in rats chronically exposed to either fine or ultrafine titanium dioxide (TiO2), carbon black (CB), or diesel exhaust particulate (DEP). The rat-based estimates of the working lifetime airborne concentrations associated with 0.1% excess risk of lung cancer are approximately 0.07 to 0.3 mg/m3 for ultrafine TiO2, CB, or DEP, and 0.7 to 1.3 mg/m3 for fine TiO2. Comparison of observed versus model-predicted lung burdens in rats shows that the dosimetry models predict reasonably well the retained mass lung burdens of fine or ultrafine poorly soluble particles in rats exposed by chronic inhalation. Additional model validation is needed for nanoparticles of varying characteristics, as well as extension of these models to include particle translocation to organs beyond the lungs. Such analyses would provide improved prediction of nanoparticle dose for risk assessment.

When nanoparticles get in the way: impact of projected area on in vivo and in vitro macrophage function.
Moss OR, Wong VA.
Inhal Toxicol. 2006 Sep;18(10):711-6.
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Previous reports by others establish that particle surface area is related to a change in macrophage function as measured by the ability to clear particles from the alveolar spaces. However, for nanoparticles the relation may not be strictly due to surface chemistry: The cumulative projected area of the particles may reflect the degree to which the inner or outer surface of the macrophage is shielded from other objects or molecules. We apply this alternative interpretation to in vitro measurements of macrophage uptake of 26-nm-diameter fluorescent beads and to in vivo data presented in a classic inhalation toxicology paper on nano-sized TiO2 particles. In their paper, Oberdorster et al. (Environ. Health Perspect. 102[suppl. 5]:173-179, 1994) reported that following inhalation exposure to 20-nm or 250-nm TiO2 particles, the half-times for alveolar clearance of polystyrene test particles were proportional to square centimeters of TiO2 particle surface per million macrophages; macrophage toxicity from TiO2 particle surface was assumed to be the cause of the decrease in the clearance rate of polystyrene test particles. When TiO2 particle projected area was incorporated into the in vivo macrophage dosimetry calculations, particle projected areas ranged in value from covering only a fraction (0.1) of the macrophage surface to covering the cell surface 4 times over. The observed decrease in macrophage mediated alveolar clearance of polystyrene test particles was directly related to the potential for TiO2 particles to mask the surface of the macrophage-a possibility that was visualized in vitro with confocal laser scanning microscopy.

Nanomedicine-emerging or re-emerging ethical issues? A discussion of four ethical themes.
Lenk C, Biller-Andorno N.
Med Health Care Philos. 2006 Aug 30; [Epub ahead of print] .
[ expand abstract ]

Nanomedicine plays a prominent role among emerging technologies. The spectrum of potential applications is as broad as it is promising. It includes the use of nanoparticles and nanodevices for diagnostics, targeted drug delivery in the human body, the production of new therapeutic materials as well as nanorobots or nanoprotheses. Funding agencies are investing large sums in the development of this area, among them the European Commission, which has launched a large network for life-sciences related nanotechnology. At the same time government agencies as well as the private sector are putting forward reports of working groups that have looked into the promises and risks of these developments. This paper will begin with an introduction to the central ethical themes as identified by selected reports from Europe and beyond. In a next step, it will analyse the most frequently invoked ethical concerns-risk assessment and management, the issues of human identity and enhancement, possible implications for civil liberties (e.g. nanodevices that might be used for covert surveillance), and concerns about equity and fair access. Although it seems that the main ethical issues are not unique to nanotechnologies, the conclusion will argue against shrugging them off as non-specific items that have been considered before in the context of other biomedical technologies, such as gene therapy or xenotransplantation. Rather, the paper will call on ethicists to help foster a rational, fair and participatory discourse on the different potential applications of nanotechnologies in medicine, which can form the basis for informed and responsible societal and political decisions.

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Investigation of the proinflammatory potential of biodegradable nanoparticle drug delivery systems in the lung.
Dailey LA, Jekel N, Fink L, Gessler T, Schmehl T, Wittmar M, Kissel T, Seeger W.
Toxicol Appl Pharmacol. 2006 Aug 15;215(1):100-8.
[ expand abstract ]

Particulate nanocarriers have been praised for their advantageous drug delivery properties in the lung, such as avoidance of macrophage clearance mechanisms and long residence times. However, instilled non-biodegradable polystyrene nanospheres with small diameters and thus large surface areas have been shown to induce pulmonary inflammation. This study examines the potential of biodegradable polymeric nanoparticles composed of poly(lactic-co-glycolic acid) (PLGA) and the novel PLGA derivative, diethylaminopropylamine polyvinyl alcohol-grafted-poly(lactic-co-glycolic acid) (DEAPA-PVAL-g-PLGA), to provoke inflammatory responses in the murine lung after intratracheal instillation. Lactate dehydrogenase (LDH) release, protein concentration, MIP-2 mRNA induction, and polymorphonucleocyte (PMN) recruitment in the bronchial alveolar lavage fluid (BALF) were used to evaluate an inflammatory response in Balb-C mice. Two sizes of polystyrene (PS) nanospheres (diameters: 75 nm and 220 nm) were included in the study for comparison. All nanoparticle suspensions were instilled at concentrations of 1 mug/mul and 2.5 mug/mul, representative of an estimated "therapeutic dose" and a concentrated "dose" of particles. In all experiments, the 75 nm PS particles exhibited elevated responses for the inflammatory markers investigated. In contrast, biodegradable particles of comparable hydrodynamic diameter showed a significantly lower inflammatory response. The most marked differences were observed in the extent of PMN recruitment. While the 75 nm and 220 nm PS nanospheres exhibited 41 and 74% PMN within the total BALF cell population after 24 h, respectively, PMN recruiting in lungs instilled with both types of biodegradable particles did not exceed values of the negative isotonic glucose control. In conclusion, evidence suggests that biodegradable polymeric nanoparticles designed for pulmonary drug delivery may not induce the same inflammatory response as non-biodegradable polystyrene particles of comparable size.

Surface Coatings Determine Cytotoxicity and Irritation Potential of Quantum Dot Nanoparticles in Epidermal Keratinocytes.
Ryman-Rasmussen JP, Riviere JE, Monteiro-Riviere NA.
J Invest Dermatol. 2006 Aug 10; [Epub ahead of print].
[ expand abstract ]

Quantum dot (QD) nanoparticles have potential applications in nanomedicine as drug delivery vectors and diagnostic agents, but the skin toxicity and irritation potential of QDs are unknown. Human epidermal keratinocytes (HEKs) were used to assess if QDs with different surface coatings would cause differential effects on HEK cytotoxicity, proinflammatory cytokine release, and cellular uptake. Commercially available QDs of two different sizes, QD 565 and QD 655, with neutral (polyethylene glycol (PEG)), cationic (PEG-amine), or anionic (carboxylic acid) coatings were utilized. Live cell imaging and transmission electron microscopy were used to determine that all QDs localized intracellularly by 24 hours, with evidence of QD localization in the nucleus. Cytotoxicity and release of the proinflammatory cytokines IL-1beta, IL-6, IL-8, IL-10, and tumor necrosis factor-alpha were assessed at 24 and 48 hours. Cytotoxicity was observed for QD 565 and QD 655 coated with carboxylic acids or PEG-amine by 48 hours, with little cytotoxicity observed for PEG-coated QDs. Only carboxylic acid-coated QDs significantly increased release of IL-1beta, IL-6, and IL-8. These data indicate that QD surface coating is a primary determinant of cytotoxicity and immunotoxicity in HEKs, which is consistent across size. However, uptake of QDs by HEKs is independent of surface coating.

Comparison of the Abilities of Ambient and Manufactured Nanoparticles To Induce Cellular Toxicity According to an Oxidative Stress Paradigm.
Xia T, Kovochich M, Brant J, Hotze M, Sempf J, Oberley T, Sioutas C, Yeh JI, Wiesner MR, Nel AE.
Nano Lett. 2006 Aug 9;6(8):1794-1807.
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Nanomaterial properties differ from those bulk materials of the same composition, allowing them to execute novel activities. A possible downside of these capabilities is harmful interactions with biological systems, with the potential to generate toxicity. An approach to assess the safety of nanomaterials is urgently required. We compared the cellular effects of ambient ultrafine particles with manufactured titanium dioxide (TiO(2)), carbon black, fullerol, and polystyrene (PS) nanoparticles (NPs). The study was conducted in a phagocytic cell line (RAW 264.7) that is representative of a lung target for NPs. Physicochemical characterization of the NPs showed a dramatic change in their state of aggregation, dispersibility, and charge during transfer from a buffered aqueous solution to cell culture medium. Particles differed with respect to cellular uptake, subcellular localization, and ability to catalyze the production of reactive oxygen species (ROS) under biotic and abiotic conditions. Spontaneous ROS production was compared by using an ROS quencher (furfuryl alcohol) as well as an NADPH peroxidase bioelectrode platform. Among the particles tested, ambient ultrafine particles (UFPs) and cationic PS nanospheres were capable of inducing cellular ROS production, GSH depletion, and toxic oxidative stress. This toxicity involves mitochondrial injury through increased calcium uptake and structural organellar damage. Although active under abiotic conditions, TiO(2) and fullerol did not induce toxic oxidative stress. While increased TNF-alpha production could be seen to accompany UFP-induced oxidant injury, cationic PS nanospheres induced mitochondrial damage and cell death without inflammation. In summary, we demonstrate that ROS generation and oxidative stress are a valid test paradigm to compare NP toxicity. Although not all materials have electronic configurations or surface properties to allow spontaneous ROS generation, particle interactions with cellular components are capable of generating oxidative stress.

Direct and indirect effects of single walled carbon nanotubes on RAW 264. 7 macrophages: role of iron.
Kagan VE, Tyurina YY, Tyurin VA, Konduru NV, Potapovich AI, Osipov AN, Kisin ER, Schwegler-Berry D, Mercer R, Castranova V, Shvedova AA.
Toxicol Lett. 2006 Aug 1;165(1):88-100. Epub 2006 Mar 9.
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Single-walled carbon nanotubes (SWCNT), nano-cylinders with an extremely small diameter (1-2 nm) and high aspect ratio, have unique physico-chemical, electronic and mechanical properties and may exhibit unusual interactions with cells and tissues, thus necessitating studies of their toxicity and health effects. Manufactured SWCNT usually contain significant amounts of iron that may act as a catalyst of oxidative stress. Because macrophages are the primary responders to different particles that initiate and propagate inflammatory reactions and oxidative stress, we utilized two types of SWCNT: (1) iron-rich (non-purified) SWCNT (26 wt.% of iron) and (2) iron-stripped (purified) SWCNT (0.23 wt.% of iron) to study their interactions with RAW 264.7 macrophages. Ultrasonication resulted in predominantly well-dispersed and separated SWCNT strands as evidenced by scanning electron microscopy. Neither purified nor non-purified SWCNT were able to generate intracellular production of superoxide radicals or nitric oxide in RAW 264.7 macrophages as documented by flow-cytometry and fluorescence microscopy. SWCNT with different iron content displayed different redox activity in a cell-free model system as revealed by EPR-detectable formation of ascorbate radicals resulting from ascorbate oxidation. In the presence of zymosan-stimulated RAW 264.7 macrophages, non-purified iron-rich SWCNT were more effective in generating hydroxyl radicals (documented by EPR spin-trapping with 5,5-dimethyl-1-pyrroline-N-oxide, DMPO) than purified SWCNT. Similarly, EPR spin-trapping experiments in the presence of zymosan-stimulated RAW 264.7 macrophages showed that non-purified SWCNT more effectively converted superoxide radicals generated by xanthine oxidase/xanthine into hydroxyl radicals as compared to purified SWCNT. Iron-rich SWCNT caused significant loss of intracellular low molecular weight thiols (GSH) and accumulation of lipid hydroperoxides in both zymosan-and PMA-stimulated RAW 264.7 macrophages. Catalase was able to partially protect macrophages against SWCNT induced elevation of biomarkers of oxidative stress (enhancement of lipid peroxidation and GSH depletion). Thus, the presence of iron in SWCNT may be important in determining redox-dependent responses of macrophages.

Nanomedicines and nanotoxicology: some physiological principles.
Garnett MC, Kallinteri P.
Occup Med (Lond). 2006 Aug;56(5):307-11.
[ expand abstract ]

Nanosized materials have been investigated as potential medicines for several decades. Consequently, a great deal of work has been conducted on how to exploit constructs of this size range in a beneficial way. Similarly, a number of the consequences from the use of these materials have already been considered. Nanosized materials do behave differently to low-molecular-weight drugs, the biological properties of nanomaterials being mainly dependent on relevant physiology and anatomy, which are reviewed in this article. Biodistribution, movement of materials through tissues, phagocytosis, opsonization and endocytosis of nanosized materials are all likely to have an impact on potential toxicity. In turn these processes are most likely to depend on the nanoparticle surface. Evidence from the literature is considered which suggests that our understanding of these areas is incomplete, and that biodistribution to specific sites can occur for nanoparticles with particular characteristics. However, our current knowledge does indicate which areas are of concern and deserve further investigation to understand how individual nanoparticles behave and what toxicity may be expected from them.

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Nanotechnology--what is it? Should we be worried?
Whatmore RW.
Occup Med (Lond). 2006 Aug;56(5):295-9.
[ expand abstract ]

This paper describes the origins of nanoscience from theoretical reasoning to its realization in terms of mechanical manipulation of atoms. The ability to visualize and manipulate matter at the nanoscale has led to a diverse technology that ranges from better and faster electronics and more efficient fuel usage to sensing, drug discovery and stronger, more resistant materials. It has the prospect of affecting the lives of all of us and already a number of applications are in the market-place. But in our development of these technologies, we need to take care to reduce the risks of the adverse consequences that usually attend new applications of science.

Trace analysis of fullerenes in biological samples by simplified liquid-liquid extraction and high-performance liquid chromatography.
Xia XR, Monteiro-Riviere NA, Riviere JE.
J Chromatogr A. 2006 Jul 29; [Epub ahead of print].
[ expand abstract ]

Fullerene (C(60)) has several potential biomedical and industrial applications. While pure fullerene is not soluble in water, nanoparticles of the fullerene aggregates (nano-C(60)) can be prepared in water solutions. The concentration of nano-C(60) in biological media after systemic exposure could be very low and requires trace analytical methods to be developed for the toxicological and pharmacokinetic studies of the nanomaterial. A serious drop in extraction efficiency was observed when the concentration was under 0.5mug/mL using traditional liquid-liquid extraction (LLE) protocols. The evaporation of the solvent extract to dryness was found to be the main reason for the efficiency drop and an improved evaporation method was proposed to overcome this problem. Optimal proportion of glacial acetic acid (GAA) was used to solublize the proteins and surfactants in the biological samples, so that the emulsion problem was eliminated during LLE. Magnesium perchlorate was used to destabilize the nano-C(60) particles in the water solution and promoted the solvent extraction. A simplified LLE method was developed for high throughput while preserved the advantages of the traditional LLE. The developed method was used for trace analysis of fullerenes in protein containing media and tape-stripped skin samples. Under optimal experimental conditions, the detection limit was 0.34ng/mL and the recovery was in the range of 94-100% (n=5) at a concentration of 10ng/mL nano-C(60) in the biological media.

Do nanoparticles present ecotoxicological risks for the health of the aquatic environment?
Moore MN.
Environ Int. 2006 Jul 19; [Epub ahead of print].
[ expand abstract ]

Nanotechnology is a major innovative scientific and economic growth area, which may present a variety of hazards for environmental and human health. The surface properties and very small size of nanoparticles and nanotubes provide surfaces that may bind and transport toxic chemical pollutants, as well as possibly being toxic in their own right by generating reactive radicals. There is a wealth of evidence for the harmful effects of nanoscale combustion-derived particulates (ultrafines), which when inhaled can cause a number of pulmonary pathologies in mammals and humans. However, release of manufactured nanoparticles into the aquatic environment is largely an unknown. This review addresses the possible hazards associated with nanomaterials and harmful effects that may result from exposure of aquatic animals to nanoparticles. Possible nanoparticle association with naturally occurring colloids and particles is considered together with how this could affect their bioavailability and uptake into cells and organisms. Uptake by endocytotic routes are identified as probable major mechanisms of entry into cells; potentially leading to various types of toxic cell injury. The higher level consequences for damage to animal health, ecological risk and possible food chain risks for humans are also considered based on known behaviours and toxicities for inhaled and ingested nanoparticles in the terrestrial environment. It is concluded that a precautionary approach is required with individual evaluation of new nanomaterials for risk to the health of the environment. Although current toxicity testing protocols should be generally applicable to identify harmful effects associated with nanoparticles, research into new methods is required to address the special properties of nanomaterials.

Correlating nanoscale titania structure with toxicity: a cytotoxicity and inflammatory response study with human dermal fibroblasts and human lung epithelial cells.
Sayes CM, Wahi R, Kurian PA, Liu Y, West JL, Ausman KD, Warheit DB, Colvin VL.
Toxicol Sci. 2006 Jul;92(1):174-85; Epub 2006 Apr 12.
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Nanocrystalline titanium dioxide (nano-TiO(2)) is an important material used in commerce today. When designed appropriately it can generate reactive species (RS) quite efficiently, particularly under ultraviolet (UV) illumination; this feature is exploited in applications ranging from self-cleaning glass to low-cost solar cells. In this study, we characterize the toxicity of this important class of nanomaterials under ambient (e.g., no significant light illumination) conditions in cell culture. Only at relatively high concentrations (100 mug/ml) of nanoscale titania did we observe cytotoxicity and inflammation; these cellular responses exhibited classic dose-response behavior, and the effects increased with time of exposure. The extent to which nanoscale titania affected cellular behavior was not dependent on sample surface area in this study; smaller nanoparticlulate materials had effects comparable to larger nanoparticle materials. What did correlate strongly to cytotoxicity, however, was the phase composition of the nanoscale titania. Anatase TiO(2), for example, was 100 times more toxic than an equivalent sample of rutile TiO(2). The most cytotoxic nanoparticle samples were also the most effective at generating reactive oxygen species; ex vivo RS species generation under UV illumination correlated well with the observed biological response. These data suggest that nano-TiO(2) samples optimized for RS production in photocatalysis are also more likely to generate damaging RS species in cell culture. The result highlights the important role that ex vivo measures of RS production can play in developing screens for cytotoxicity.

Research Strategies for Safety Evaluation of Nanomaterials, Part VIII: International Efforts to Develop Risk-Based Safety Evaluations for Nanomaterials.
Thomas K, Aguar P, Kawasaki H, Morris J, Nakanishi J, Savage N.
Toxicol Sci. 2006 Jul;92(1):23-32; Epub 2006 May 9.
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The use of nanotechnology in consumer and industrial applications will likely have a profound impact on a number of products from a variety of industrial sectors. Nanomaterials exhibit unique physical/chemical properties and impart enhancements to engineered materials, including better magnetic properties, improved electrical activity, and increased optical properties. The United States, Europe, and Japan have each initiated comprehensive programs to promote and expand the utility of nanotechnology for commercial applications. An important component of these programs is the development of reliable risk and safety evaluations for these materials to ensure their safety for human health and the environment. The scope of each of these programs includes efforts to assess the hazards posed by nanomaterials in realistic exposure conditions.

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Biocompatibility of cluster-assembled nanostructured TiO2 with primary and cancer cells.
Carbone R, Marangi I, Zanardi A, Giorgetti L, Chierici E, Berlanda G, Podesta A, Fiorentini F, Bongiorno G, Piseri P, Pelicci PG, Milani P.
Biomaterials. 2006 Jun;27(17):3221-9; Epub 2006 Feb 28.
[ expand abstract ]

We have characterized the biocompatibility of nanostructured TiO2 films produced by the deposition of a supersonic beam of TiOx clusters. Physical analysis shows that these films possess, at the nanoscale, a granularity and porosity mimicking those of typical extracellular matrix structures and adsorption properties that could allow surface functionalization with different macromolecules such as DNA, proteins, and peptides. To explore the biocompatibility of this novel nanostructured surface, different cancer and primary cells were analyzed in terms of morphological appearance (by bright field microscopy and immunofluorescence) and growth properties, with the aim to evaluate cluster-assembled TiO2 films as substrates for cell-based and tissue-based applications. Our results strongly suggest that this new biomaterial supports normal growth and adhesion of primary and cancer cells with no need for coating with ECM proteins; we thus propose this new material as an optimal substrate for different applications in cell-based assays, biosensors or microfabricated medical devices.

Cytotoxicity of water-soluble fullerene in vascular endothelial cells.
Yamawaki H, Iwai N.
Am J Physiol Cell Physiol. 2006 Jun;290(6):C1495-502; Epub 2006 Jan 11.
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Nanoscale materials are presently under development for diagnostic (nanomedicine) and electronic purposes. In contrast to the potential benefits of nanotechnology, the effects of nanomaterials on human health are poorly understood. Nanomaterials are known to translocate into the circulation and could thus directly affect vascular endothelial cells (ECs), causing vascular injury that might be responsible for the development of atherosclerosis. To explore the direct effects of nanomaterials on endothelial toxicity, human umbilical vein ECs were treated with 1-100 microg/ml hydroxyl fullerene [C60(OH)24; mean diameter, 7.1 +/- 2.4 nm] for 24 h. C60(OH)24 induced cytotoxic morphological changes such as cytosolic vacuole formation and decreased cell density in a dose-dependent manner. Lactate dehydrogenase assay revealed that a maximal dose of C60(OH)24 (100 microg/ml) induced cytotoxic injury. Proliferation assay also showed that a maximal dose of C60(OH)24 inhibited EC growth. C60(OH)24 did not seem to induce apoptosis but caused the accumulation of polyubiquitinated proteins and facilitated autophagic cell death. Formation of autophagosomes was confirmed on the basis of Western blot analysis using a specific marker, light chain 3 antibody, and electron microscopy. Chronic treatment with low-dose C60(OH)24 (10 microg/ml for 8 days) inhibited cell attachment and delayed EC growth. In the present study, we have examined, for the first time, the toxicity of water-soluble fullerenes to ECs. Although fullerenes changed morphology in a dose-dependent manner, only maximal doses of fullerenes caused cytotoxic injury and/or death and inhibited cell growth. EC death seemed to be caused by activation of ubiquitin-autophagy cell death pathways. Although exposure to nanomaterials appears to represent a risk for cardiovascular disorders, further in vivo validations are necessary.

Research Strategies for Safety Evaluation of Nanomaterials, Part VIII: International Efforts to Develop Risk-Based Safety Evaluations for Nanomaterials.
Karluss T, Pilar A, Hajime K, Jeff M, Junko N, Nora S.
Toxicol Sci. 2006 May 9; [Epub ahead of print] .
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No abstract available

Cytotoxic evaluation of injectable cyclodextrin nanoparticles.
Memisoglu-Bilensoy E, Dogan AL, Hincal AA.
J Pharm Pharmacol. 2006 May;58(5):585-9.
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Nanoparticles were prepared using beta-CDC6, which is an amphiphilic beta-cyclodextrin derivative modified on the secondary face with 6C aliphatic esters. A nanoprecipitation technique was used to prepare the blank nanoparticles without any surfactant and nanoparticles containing Pluronic F68 as surfactant in a concentration range of 0.1 to 1%. Nanoparticle formulations were characterized by particle size distribution and zeta potential measurements. Entrapment efficiency and in-vitro release profiles were determined and the cytotoxicity of these injectable nanospheres was evaluated against mouse fibroblast L929 cell line and human polymorphonuclear cells by methlythiazolyltetrazolium assay. As far as particle size and zeta potential are concerned, there is a relationship between surfactant presence and nanoparticle characteristics. However, these effects are not significant. It was also found that surfactant presence has no effect on model drug nimodipine encapsulation but accelerates the in-vitro release of the drug. Cell culture studies on mouse fibroblasts and human polymorphonuclear cells revealed a concentration-dependent cytotoxicity more pronounced in fibroblast cells. This led to the conclusion that the use of surfactants in injectable nanoparticles prepared from amphiphilic beta-cyclodextrins may lead to altered in-vitro properties and impaired safety for the drug delivery system.

Penetration of intact skin by quantum dots with diverse physicochemical properties.
Ryman-Rasmussen JP, Riviere JE, Monteiro-Riviere NA.
Toxicol Sci. 2006 May;91(1):159-65; Epub 2006 Jan 27.
[ expand abstract ]

Skin is the largest organ of the body and is a potential route of exposure to engineered nanomaterials, but the permeability of the skin to these nanomaterials is unknown. We selected commercially available quantum dots (QD) of two core/shell sizes and shapes and three different surface coatings to determine if QD could penetrate intact skin in a size- or coating-dependent manner. Spherical 4.6 nm core/shell diameter QD 565 and ellipsoid 12 nm (major axis) by 6 nm (minor axis) core/shell diameter QD 655 with neutral (polyethylene glycol), anionic (carboxylic acids) or cationic (polyethylene glycol-amine) coatings were topically applied to porcine skin in flow-through diffusion cells at an occupationally relevant dose for 8 h and 24 h. Confocal microscopy revealed that spherical QD 565 of each surface coating penetrated the stratum corneum and localized within the epidermal and dermal layers by 8 h. Similarly, polyethylene glycol- and polyethylene glycol-amine-coated ellipsoid QD 655 localized within the epidermal layers by 8 h. No penetration of carboxylic acid-coated QD 655 was evident until 24 h, at which time localization in the epidermal layers was observed. This study showed that quantum dots of different sizes, shapes, and surface coatings can penetrate intact skin at an occupationally relevant dose within the span of an average-length work day. These results suggest that skin is surprisingly permeable to nanomaterials with diverse physicochemical properties and may serve as a portal of entry for localized, and possibly systemic, exposure of humans to QD and other engineered nanoscale materials.

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Research strategies for safety evaluation of nanomaterials, part VII: evaluating consumer exposure to nanoscale materials.
Thomas T, Thomas K, Sadrieh N, Savage N, Adair P, Bronaugh R.
Toxicol Sci. 2006 May;91(1):14-9; Epub 2006 Feb 13.
[ expand abstract ]

Considerable media attention has recently been given to novel applications for products that contain nanoscale materials. These products could have utility in several industries that market consumer products, including textiles, sporting equipment, cosmetics, consumer electronics, and household cleaners. Some of the purported benefits of these products include improved performance, convenience, lower cost, as well as other desirable features, when compared to the conventional products that do not contain nanoscale materials. Although there are numerous likely consumer advantages from products containing nanoscale materials, there is very little information available regarding consumer exposure to the nanoscale materials in these products or any associated risks from these exposures. This paper seeks to review a limited subset of products that contain nanoscale materials, assess the available data for evaluating the consumer exposures and potential hazards associated with these products, and discuss the capacity of U.S. regulatory agencies to address the potential risks associated with these products.

Characterization of quantum dot bioconjugates by capillary electrophoresis with laser-induced fluorescent detection.
Huang X, Weng J, Sang F, Song X, Cao C, Ren J.
J Chromatogr A. 2006 Apr 28;1113(1-2):251-4; Epub 2006 Mar 24.
[ expand abstract ]

In this paper, we present a universal, highly efficient and sensitive method for the characterization of quantum dot (QD) bioconjugates based on capillary electrophoresis with laser-induced fluorescent (LIF) detection. We first prepared CdTe QDs in aqueous phase by a chemical route with mercaptopropionic acid as a ligand, and then were coupled to certain proteins using bifunctional linkage reagent or electrostatic attraction. The QD bioconjugates were characterized by capillary electrophoresis with LIF detection. We found that QD bioconjugates were efficiently separated with free QDs by the optimization of buffer pH. Furthermore, we found that ultrafiltration was an effective and simple approach to purify QD conjugates with bovine serum albumin (BSA). Due to their broad absorption spectra and size dependent emission wavelength tunability, QDs can be excited to emit different colour fluorescence using a single wavelength laser source, and therefore, we believe that CE with LIF detection will become a universal and efficient tool for the characterization of QD bioconjugates.

Cellular Effect of High Doses of Silica-Coated Quantum Dot Profiled with High Throughput Gene Expression Analysis and High Content Cellomics Measurements.
Zhang T, Stilwell JL, Gerion D, Ding L, Elboudwarej O, Cooke PA, Gray JW, Alivisatos AP, Chen FF.
Nano Lett. 2006 Apr 12;6(4):800-808.
[ expand abstract ]

Quantum dots (Qdots) are now used extensively for labeling in biomedical research, and this use is predicted to grow because of their many advantages over alternative labeling methods. Uncoated Qdots made of core/shell CdSe/ZnS are toxic to cells because of the release of Cd(2+) ions into the cellular environment. This problem has been partially overcome by coating Qdots with polymers, poly(ethylene glycol) (PEG), or other inert molecules. The most promising coating to date, for reducing toxicity, appears to be PEG. When PEG-coated silanized Qdots (PEG-silane-Qdots) are used to treat cells, toxicity is not observed, even at dosages above 10-20 nM, a concentration inducing death when cells are treated with polymer or mercaptoacid coated Qdots. Because of the importance of Qdots in current and future biomedical and clinical applications, we believe it is essential to more completely understand and verify this negative global response from cells treated with PEG-silane-Qdots. Consequently, we examined the molecular and cellular response of cells treated with two different dosages of PEG-silane-Qdots. Human fibroblasts were exposed to 8 and 80 nM of these Qdots, and both phenotypic as well as whole genome expression measurements were made. PEG-silane-Qdots did not induce any statistically significant cell cycle changes and minimal apoptosis/necrosis in lung fibroblasts (IMR-90) as measured by high content image analysis, regardless of the treatment dosage. A slight increase in apoptosis/necrosis was observed in treated human skin fibroblasts (HSF-42) at both the low and the high dosages. We performed genome-wide expression array analysis of HSF-42 exposed to doses 8 and 80 nM to link the global cell response to a molecular and genetic phenotype. We used a gene array containing approximately 22,000 total probe sets, containing 18,400 probe sets from known genes. Only approximately 50 genes ( approximately 0.2% of all the genes tested) exhibited a statistically significant change in expression level of greater than 2-fold. Genes activated in treated cells included those involved in carbohydrate binding, intracellular vesicle formation, and cellular response to stress. Conversely, PEG-silane-Qdots induce a down-regulation of genes involved in controlling the M-phase progression of mitosis, spindle formation, and cytokinesis. Promoter analysis of these results reveals that expression changes may be attributed to the down-regulation of FOXM and BHLB2 transcription factors. Remarkably, PEG-silane-Qdots, unlike carbon nanotubes, do not activate genes indicative of a strong immune and inflammatory response or heavy-metal-related toxicity. The experimental evidence shows that CdSe/ZnS Qdots, if appropriately protected, induce negligible toxicity to the model cell system studied here, even when exposed to high dosages. This study indicates that PEG-coated silanized Qdots pose minimal impact to cells and are a very promising alternative to uncoated Qdots.

Research Strategies for Safety Evaluation of Nanomaterials. Part VI. Characterization of Nanoscale Particles for Toxicological Evaluation.
Powers KW, Brown SC, Krishna VB, Wasdo SC, Moudgil BM, Roberts SM.
Toxicol Sci. 2006 Apr;90(2):296-303. Epub 2006 Jan 11.
[ expand abstract ]

To properly assign mechanisms or causes for toxic effects of nanoscale materials, their properties and characteristics both outside and within the biological environment must be well understood. Scientists have many tools for studying the size, shape, and surface properties of particulates outside of the physiological environment; however, it is difficult to measure many of these same properties in situ without perturbing the environment, leading to spurious findings. Characterizing nanoparticle systems in situ can be further complicated by an organism's active clearance, defense, and/or immune responses. As toxicologists begin to examine nanomaterials in a systematic fashion, there is consensus that a series of guidelines or recommended practices is necessary for basic characterization of nanomaterials. These recommended practices should be developed jointly by physical scientists skilled in nano characterization and biological scientists experienced in toxicology research. In this article, basic nanoparticle characterization techniques are discussed, along with the some of the issues and implications associated with measuring nanoparticle properties and their interactions with biological systems. Recommendations regarding how best to approach nanomaterial characterization include using proper sampling and measurement techniques, forming multidisciplinary teams, and making measurements as close to the biological action point as possible.

Detecting cryptic epitopes created by nanoparticles.
Lynch I, Dawson KA, Linse S.
Sci STKE. 2006 Mar 21;2006(327):pe14.
[ expand abstract ]

As potential applications of nanotechnology and nanoparticles increase, so too does the likelihood of human exposure to nanoparticles. Because of their small size, nanoparticles are easily taken up into cells (by receptor-mediated endocytosis), whereupon they have essentially free access to all cellular compartments. Similarly to macroscopic biomaterial surfaces (that is, implants), nanoparticles become coated with a layer of adsorbed proteins immediately upon contact with physiological solutions (unless special efforts are taken to prevent this). The process of adsorption often results in conformational changes of the adsorbed protein, which may be affected by the larger curvature of nanoparticles compared with implant surfaces. Protein adsorption may result in the exposure at the surface of amino acid residues that are normally buried in the core of the native protein, which are recognized by the cells as "cryptic epitopes." These cryptic epitopes may trigger inappropriate cellular signaling events (as opposed to being rejected by the cells as foreign bodies). However, identification of such surface-exposed epitopes is nontrivial, and the molecular nature of the adsorbed proteins should be investigated using biological and physical science methods in parallel with systems biology studies of the induced alterations in cell signaling.

Investigation of the proinflammatory potential of biodegradable nanoparticle drug delivery systems in the lung.
Dailey LA, Jekel N, Fink L, Gessler T, Schmehl T, Wittmar M, Kissel T, Seeger W.
Toxicol Appl Pharmacol. 2006 Mar 19; [Epub ahead of print] .
[ expand abstract ]

Particulate nanocarriers have been praised for their advantageous drug delivery properties in the lung, such as avoidance of macrophage clearance mechanisms and long residence times. However, instilled non-biodegradable polystyrene nanospheres with small diameters and thus large surface areas have been shown to induce pulmonary inflammation. This study examines the potential of biodegradable polymeric nanoparticles composed of poly(lactic-co-glycolic acid) (PLGA) and the novel PLGA derivative, diethylaminopropylamine polyvinyl alcohol-grafted-poly(lactic-co-glycolic acid) (DEAPA-PVAL-g-PLGA), to provoke inflammatory responses in the murine lung after intratracheal instillation. Lactate dehydrogenase (LDH) release, protein concentration, MIP-2 mRNA induction, and polymorphonucleocyte (PMN) recruitment in the bronchial alveolar lavage fluid (BALF) were used to evaluate an inflammatory response in Balb-C mice. Two sizes of polystyrene (PS) nanospheres (diameters: 75 nm and 220 nm) were included in the study for comparison. All nanoparticle suspensions were instilled at concentrations of 1 mug/mul and 2.5 mug/mul, representative of an estimated "therapeutic dose" and a concentrated "dose" of particles. In all experiments, the 75 nm PS particles exhibited elevated responses for the inflammatory markers investigated. In contrast, biodegradable particles of comparable hydrodynamic diameter showed a significantly lower inflammatory response. The most marked differences were observed in the extent of PMN recruitment. While the 75 nm and 220 nm PS nanospheres exhibited 41 and 74% PMN within the total BALF cell population after 24 h, respectively, PMN recruiting in lungs instilled with both types of biodegradable particles did not exceed values of the negative isotonic glucose control. In conclusion, evidence suggests that biodegradable polymeric nanoparticles designed for pulmonary drug delivery may not induce the same inflammatory response as non-biodegradable polystyrene particles of comparable size.

Injection of PEGylated liposomes in rats elicits PEG-specific IgM, which is responsible for rapid elimination of a second dose of PEGylated liposomes.
Ishida T, Ichihara M, Wang X, Yamamoto K, Kimura J, Majima E, Kiwada H.
J Control Release. 2006 Mar 1; [Epub ahead of print].
[ expand abstract ]

Steric stabilization of the surface of liposomes by a PEG conjugated lipid results in reduced recognition of the liposomes by the cells of the mononuclear phagocyte system and consequently extended their circulation times (t(1/2) approximately 20h in rat). Recently, we reported on the "accelerated blood clearance phenomenon", causing 'invisible' PEGylated liposomes to be cleared very rapidly from the circulation upon repeated injection. In addition, we reported that certain serum factor(s) secreted into the blood after the first dose of PEGylated liposomes play an essential role in the phenomenon. The aim of the present study was to identify the major serum protein(s) responsible for the phenomenon and to unravel their mode of action. The amount of protein binding to PEGylated liposomes during incubation with serum hardly correlated with the extent of the phenomenon. PEGylated liposomes incubated with serum obtained from rats pre-injected 5 days before with the same liposomes showed a much more complex pattern of bound proteins than when incubated with naive serum, as revealed by 2D-PAGE and SDS-PAGE. Subsequent analysis with LC-MS/MS and Western blot showed that the major pre-treated serum protein binding to PEGylated liposomes was IgM. Semi-quantitative analysis showed that larger amount of IgM bound to PEGylated liposomes compared to conventional liposomes. It was further demonstrated that PEGylated liposomes could activate the complement system due to IgM binding when incubated in serum from rats pre-injected with PEGylated liposomes, while conventional liposomes were not. These findings suggest that the selective binding of IgM to the second injected PEGylated liposomes and the subsequent complement activation by IgM resulted in the accelerated clearance and enhanced hepatic uptake of the second injected PEGylated liposomes. Based on the results described here, we are drawing attention to the potential occurrence of unexpected immune reactions upon intravenous administration of PEGylated liposomes or other particles and, by extension, PEGylated proteins and DNAs.

Chemical modification of SWNT alters in vitro cell-SWNT interactions.
Nimmagadda A, Thurston K, Nollert MU, McFetridge PS.
J Biomed Mater Res A. 2006 Mar 1;76(3):614-25.
[ expand abstract ]

Single-walled carbon nanotubes (SWNT) have been the focus of considerable attention as a material with extraordinary mechanical and electrical properties. SWNT have been proposed in a number of biomedical applications, including neural, bone, and dental tissue engineering. In these applications, it is clear that surrounding tissues will come into surface contact with SWNT composites, and compatibility between SWNT and host cells must be addressed. This investigation describes the gross physical and chemical effects of different SWNT preparations on in vitro cell viability and metabolic activity. Three different SWNT preparations were analyzed: as purchased (AP-NT), purified (PUR-NT), and functionalized with glucosamine (GA-NT), over concentrations of 0.001-1.0% (wt/vol). With the exception of the lowest SWNT concentrations, increasing concentrations of SWNT resulted in a decrease of cell viability, which was dependent on SWNT preparation. The metabolic activity of 3T3 cells was also dependent on SWNT preparation and concentration. These investigations have shown that these SWNT preparations have significant effects on in vitro cellular function that cannot be attributed to one factor alone, but are more likely the result of several unfavorable interactions. Effects, such as destabilizing the cell membrane, soluble toxic contaminants, and limitations in mass transfer as the SWNT coalesce into sheets, may all play a role in these interactions. Using comprehensive purification processes and modifying the NT-surface chemistry to introduce functional groups or reduce hydrophobicity or both, these interactions can be significantly improved. (c) 2005 Wiley Periodicals, Inc. J Biomed Mater Res, 2006.

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Research strategies for safety evaluation of nanomaterials, part V: role of dissolution in biological fate and effects of nanoscale particles.
Borm P, Klaessig FC, Landry TD, Moudgil B, Pauluhn J, Thomas K, Trottier R, Wood S.
Toxicol Sci. 2006 Mar;90(1):23-32.
[ expand abstract ]

Dissolution, translocation, and disposition have been shown to play a key role in the fate and effects of inhaled particles and fibers. Concepts that have been applied in the micron size range may be usefully applied to the nanoscale range, but new challenges are presented based on the small size and possible change in the dissolution:translocation relationship. The size of the component molecule itself may be on the nanoscale. Solute concentration, surface area, surface morphology, surface energy, dissolution layer properties, adsorbing species, and aggregation are relevant parameters in considering dissolution at the nanoscale. With regard to the etiopathology caused by these types of particulates, the metrics of dose (particle number, surface area, mass or shape) is not yet well defined. Analytical procedures for assessing dissolution and translocation include chemical assay and particle characterization. Leaching of substituents from particle surfaces may also be important. Compartmentalization within the respiratory tract may add another dimension of complexity. Dissolution may be a critical step for some nanoscale materials in determining fate in the environment and within the body. This review, combining aspects of particle toxicology, material science, and analytical chemistry, is intended to provide a useful basis for developing relevant dissolution assay(s) for nanoscale particles.

Cellular Response to Magnetic Nanoparticles "PEGylated" via Surface-Initiated Atom Transfer Radical Polymerization.
Hu F, Neoh KG, Cen L, Kang ET.
Biomacromolecules. 2006 Mar;7(3):809-16.
[ expand abstract ]

A new method to PEGylate magnetic nanoparticles with a dense layer of poly(poly(ethylene glycol) monomethacrylate) (P(PEGMA)) by surface-initiated atom transfer radical polymerization (ATRP) is reported. In this approach, an initiator for ATRP was first immobilized onto the magnetic nanoparticle surface, and then P(PEGMA) was grafted onto the surface of magnetic nanoparticle via copper-mediated ATRP. The modified nanoparticles were subjected to detailed characterization using FTIR, XPS, and TGA. The P(PEGMA)-immobilized nanoparticles dispersed well in aqueous media. The saturation magnetization values of the P(PEGMA)-immobilized nanoparticles were 19 emu/g and 11 emu/g after 2 and 4 h polymerization respectively, compared to 52 emu/g for the pristine magnetic nanoparticles. The response of macrophage cells to pristine and P(PEGMA)-immobilized nanoparticles was compared. The results showed that the macrophage cells are very effective in cleaning up the pristine magnetic nanoparticles. With the P(PEGMA)-immobilized nanoparticles, the amount of nanoparticles internalized into the cells is greatly reduced to <2 pg/cell over a 5 day period. With this amount of nanoparticles uptake, no significant cytotoxicity effects were observed.

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Pulmonary Instillation Studies with Nanoscale TiO2 Rods and Dots in Rats: Toxicity is not Dependent Upon Particle Size and Surface Area.
Warheit D, Webb T, Sayes C, Colvin V, Reed K.
Toxicol Sci. 2006 Feb 22; [Epub ahead of print] .
[ expand abstract ]

Pulmonary toxicology studies in rats demonstrate that nanoparticles administered to the lung are more toxic than larger, fine-sized particles of similar chemistry at identical mass concentrations. The aim of this study was to evaluate the acute lung toxicity in rats of intratracheally instilled pigment-grade TiO2 particles (rutile-type- particle size = approximately 300 nm) vs. Nanoscale TiO2 rods (anatase - 200 nm x 35 nm) or Nanoscale TiO2 dots (anatase - approximately 10 nm) compared with a positive control particle-type, quartz. Groups of rats were instilled either with doses of 1 or 5 mg/kg of the various particle- types in phosphate-buffered saline (PBS). Subsequently, the lungs of PBS and particle-exposed rats were assessed using bronchoalveolar lavage (BAL) fluid biomarkers, cell proliferation methods, and by histopathological evaluation of lung tissue at 24 hrs, 1 week, 1 month and 3 months post-instillation exposure. Exposures to Nanoscale TiO2 rods or Nanoscale TiO2 dots produced transient inflammatory and cell injury effects at 24 hours postexposure (pe) and were not different from the pulmonary effects of larger-sized TiO2 particle exposures. In contrast, pulmonary exposures to quartz particles in rats produced a dose-dependent lung inflammatory response characterized by neutrophils and foamy lipid-containing alveolar macrophage accumulation as well as evidence of early lung tissue thickening consistent with the development of pulmonary fibrosis. The results described herein provide the first example of nanoscale particle-types which are not more cytotoxic or inflammogenic to the lung compared to larger-sized particles of similar composition. Furthermore, these findings run counter to the postulation that surface area is a major factor associated with the pulmonary toxicity of nanoscale particle-types.

Tissue biodistribution and blood clearance rates of intravenously administered carbon nanotube radiotracers.
Singh R, Pantarotto D, Lacerda L, Pastorin G, Klumpp C, Prato M, Bianco A, Kostarelos K.
Proc Natl Acad Sci U S A. 2006 Feb 21; [Epub ahead of print] .
[ expand abstract ]

Carbon nanotubes (CNT) are intensively being developed for biomedical applications including drug and gene delivery. Although all possible clinical applications will require compatibility of CNT with the biological milieu, their in vivo capabilities and limitations have not yet been explored. In this work, water-soluble, single-walled CNT (SWNT) have been functionalized with the chelating molecule diethylentriaminepentaacetic (DTPA) and labeled with indium ((111)In) for imaging purposes. Intravenous (i.v.) administration of these functionalized SWNT (f-SWNT) followed by radioactivity tracing using gamma scintigraphy indicated that f-SWNT are not retained in any of the reticuloendothelial system organs (liver or spleen) and are rapidly cleared from systemic blood circulation through the renal excretion route. The observed rapid blood clearance and half-life (3 h) of f-SWNT has major implications for all potential clinical uses of CNT. Moreover, urine excretion studies using both f-SWNT and functionalized multiwalled CNT followed by electron microscopy analysis of urine samples revealed that both types of nanotubes were excreted as intact nanotubes. This work describes the pharmacokinetic parameters of i.v. administered functionalized CNT relevant for various therapeutic and diagnostic applications.

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Functionalization density dependence of single-walled carbon nanotubes cytotoxicity in vitro.
Sayes CM, Liang F, Hudson JL, Mendez J, Guo W, Beach JM, Moore VC, Doyle CD, West JL, Billups WE, Ausman KD, Colvin VL.
Toxicol Lett. 2006 Feb 20;161(2):135-42.
[ expand abstract ]

The cytotoxic response of cells in culture is dependant on the degree of functionalization of the single-walled carbon nanotube (SWNT). After characterizing a set of water-dispersible SWNTs, we performed in vitro cytotoxicity screens on cultured human dermal fibroblasts (HDF). The SWNT samples used in this exposure include SWNT-phenyl-SO(3)H and SWNT-phenyl-SO(3)Na (six samples with carbon/-phenyl-SO(3)X ratios of 18, 41, and 80), SWNT-phenyl-(COOH)(2) (one sample with carbon/-phenyl-(COOH)(2) ratio of 23), and underivatized SWNT stabilized in 1% Pluronic F108. We have found that as the degree of sidewall functionalization increases, the SWNT sample becomes less cytotoxic. Further, sidewall functionalized SWNT samples are substantially less cytotoxic than surfactant stabilized SWNTs. Even though cell death did not exceed 50% for cells dosed with sidewall functionalized SWNTs, optical and atomic force microscopies show direct contact between cellular membranes and water-dispersible SWNTs; i.e. the SWNTs in aqueous suspension precipitate out and selectively deposit on the membrane.

Carbon Nanotubes: a Review of Their Properties in Relation to Pulmonary Toxicology and Workplace Safety.
Donaldson K, Aitken R, Tran L, Stone V, Duffin R, Forrest G, Alexander A.
Toxicol Sci. 2006 Feb 16; [Epub ahead of print] .
[ expand abstract ]

Carbon nanotubes (CNT) are an important new class of technological materials that have numerous novel and useful properties. The forecast increase in manufacture makes it likely that increasing human exposure will occur and as a result, CNT are beginning to come under toxicological scrutiny. This review seeks to set out the toxicological paradigms applicable to the toxicity of inhaled CNT, building on the toxicological database on nanoparticles and fibres. Relevant workplace regulation regarding exposure is also considered in the light of our knowledge of CNT. CNT could have features of both nanoparticles and conventional fibres and so the current paradigm for fibre toxicology, which is based on mineral fibres and synthetic vitreous fibres, is discussed. The nanoparticle toxicology paradigm is also discussed in relation to CNT. The available peer-reviewed literature suggests that CNT may have unusual toxicity properties. In particular CNT seem to have a special ability to stimulate mesenchymal cell growth and to cause granuloma formation and fibrogenesis. In several studies CNT have more adverse effects than the same mass of nanoparticle carbon and quartz, the latter a commonly used benchmark of particle toxicity. There is however no definitive inhalation study available that would avoid the potential for artefactual effects due to large mats and aggregates forming during instillation exposure procedures. Studies also show that CNT may exhibit some of their effects through oxidative stress and inflammation. CNT represent a group of particles that are growing in production and use and therefore research into their toxicology and safe use is warranted.

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The internalized CdSe/ZnS quantum dots impair the chondrogenesis of bone marrow mesenchymal stem cells.
Hsieh SC, Wang FF, Hung SC, Chen YJ, Wang YJ.
J Biomed Mater Res B Appl Biomater. 2006 Feb 9; [Epub ahead of print] .
[ expand abstract ]

Mesenchymal stem cells (MSCs) are capable of differentiating into multiple cell lineages and are useful for therapeutic applications. Labeling the MSCs with fluorescent probes is beneficial in tracing the fate of MSCs after implantation. We have introduced the CdSe/ZnS quantum dots (QDs) into the human bone marrow MSCs and examined the effects of QDs on the proliferation and chondrogenesis of the cells. The internalized QDs were found localized in perinuclear regions and remained there after a number of cell passages. The presence of QDs did not affect the proliferation of cells or the size of chondrospheres formed, when subjected to chondrogenesis induction. However, the expression of mRNA and protein of type II collagen and aggrecan in the chondrospheres was significantly inhibited in cells labeled with QDs, suggesting impaired chondrogenesis. Our results that the presence of QDs interferes with the chondrogenic differentiation of MSCs raise concerns in using the QDs as fluorescence tracers for stem cells. (c) 2006 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2006.

Toxic potential of materials at the nanolevel.
Nel A, Xia T, Madler L, Li N.
Science. 2006 Feb 3;311(5761):622-7.
[ expand abstract ]

Nanomaterials are engineered structures with at least one dimension of 100 nanometers or less. These materials are increasingly being used for commercial purposes such as fillers, opacifiers, catalysts, semiconductors, cosmetics, microelectronics, and drug carriers. Materials in this size range may approach the length scale at which some specific physical or chemical interactions with their environment can occur. As a result, their properties differ substantially from those bulk materials of the same composition, allowing them to perform exceptional feats of conductivity, reactivity, and optical sensitivity. Possible undesirable results of these capabilities are harmful interactions with biological systems and the environment, with the potential to generate toxicity. The establishment of principles and test procedures to ensure safe manufacture and use of nanomaterials in the marketplace is urgently required and achievable.

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A toxicologic review of quantum dots: toxicity depends on physicochemical and environmental factors.
Hardman R.
Environ Health Perspect. 2006 Feb;114(2):165-72.
[ expand abstract ]

As a growing applied science, nanotechnology has considerable global socioeconomic value, and the benefits afforded by nanoscale materials and processes are expected to have significant impacts on almost all industries and all areas of society. A diverse array of engineered nanoscale products and processes have emerged [e.g., carbon nanotubes, fullerene derivatives, and quantum dots (QDs)], with widespread applications in fields such as medicine, plastics, energy, electronics, and aerospace. With the nanotechnology economy estimated to be valued at $1 trillion by 2012, the prevalence of these materials in society will be increasing, as will the likelihood of exposures. Importantly, the vastness and novelty of the nanotechnology frontier leave many areas unexplored, or underexplored, such as the potential adverse human health effects resulting from exposure to novel nanomaterials. It is within this context that the need for understanding the potentially harmful side effects of these materials becomes clear. The reviewed literature suggests several key points: Not all QDs are alike; engineered QDs cannot be considered a uniform group of substances. QD absorption, distribution, metabolism, excretion, and toxicity depend on multiple factors derived from both inherent physicochemical properties and environmental conditions; QD size, charge, concentration, outer coating bioactivity (capping material and functional groups), and oxidative, photolytic, and mechanical stability have each been implicated as determining factors in QD toxicity. Although they offer potentially invaluable societal benefits such as drug targeting and in vivo biomedical imaging, QDs may also pose risks to human health and the environment under certain conditions. Key words: environment, human health, nanomaterials, nanosized particles, nanotechnology, nanotoxicology, quantum dots, toxicology.

Penetration of Intact Skin by Quantum Dots with Diverse Physicochemical Properties.
Ryman-Rasmussen JP, Riviere JE, Monteiro-Riviere NA.
Toxicol Sci. 2006 Jan 27; [Epub ahead of print] .
[ expand abstract ]

Skin is the largest organ of the body and is a potential route of exposure to engineered nanomaterials, but the permeability of the skin to these nanomaterials is unknown. We selected commercially available quantum dots (QD) of two core/shell sizes and shapes and three different surface coatings to determine if QD could penetrate intact skin in a size- or coating-dependent manner. Spherical 4.6 nm core/shell diameter QD 565 and ellipsoid 12 nm (major axis) by 6 nm (minor axis) core/shell diameter QD 655 with neutral (polyethylene glycol), anionic (carboxylic acids) or cationic (polyethylene glycol-amine) coatings were topically applied to porcine skin in flow-through diffusion cells at an occupationally relevant dose for 8 h and 24 h. Confocal microscopy revealed that spherical QD 565 of each surface coating penetrated the stratum corneum and localized within the epidermal and dermal layers by 8 h. Similarly, polyethylene glycol and polyethylene glycol-amine-coated ellipsoid QD 655 localized within the epidermal layers by 8 h. No penetration of carboxylic acid-coated QD 655 was evident until 24 h, at which time localization in the epidermal layers was observed. This study showed that quantum dots of different sizes, shapes, and surface coatings can penetrate intact skin at an occupationally relevant dose within the span of an average-length work day. These results suggest that skin is surprisingly permeable to nanomaterials with diverse physicochemical properties and may serve as a portal of entry for localized, and possibly systemic, exposure of humans to QD and other engineered nanoscale materials.

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In vitro study of the pulmonary translocation of nanoparticles: a preliminary study.
Geys J, Coenegrachts L, Vercammen J, Engelborghs Y, Nemmar A, Nemery B, Hoet PH.
Toxicol Lett. 2006 Jan 25;160(3):218-26.
[ expand abstract ]

Recent studies indicate that inhaled ultrafine particles can pass into the circulation. To study this translocation in an in vitro model three types of pulmonary epithelial cells were examined. The integrity of the cell monolayer was verified by measuring the transepithelial electrical resistance (TEER) and passage of sodium fluorescein. TEER was too low in A549 cells. In these preliminary experiments, TEER values of 1007+/-300 and 348+/-62 Omega cm2 were reached for the Calu-3 cell line, using permeable membranes of 0.4 and 3 microm pore size, respectively. Growing primary rat type II pneumocytes on 0.4 microm pores, a TEER value of 241+/-90 Omega cm2 was reached on day 5; on 3 microm pores, no acceptable high TEER value was obtained. Translocation studies were done using 46 nm fluorescent polystyrene particles. When incubating polystyrene particles on membranes without a cellular monolayer, significant translocation was only observed using 3 microm pores: 67.5% and 52.7% for carboxyl- and amine-modified particles, respectively. Only the Calu-3 cell line was used in an initial experiment to investigate the translocation: on 0.4 microm pores no translocation was observed, on 3 microm pores approximately 6% translocation was observed both for carboxyl- and amine-modified particles.

Cytotoxicity of water soluble fullerene in vascular endothelial cells.
Yamawaki H, Iwai N.
Am J Physiol Cell Physiol. 2006 Jan 11; [Epub ahead of print].
[ expand abstract ]

Nano-scaled materials for diagnostic (nanomedicine) and electronics purposes are presently under development. Contrasting with the potential benefits of nanotechnology, the effects on human health are poorly understood. Nanomaterials are known to translocate into the circulation, and could thus directly affect vascular endothelial cells (ECs), causing vascular injury that might be responsible for the development of atherosclerosis. To explore the direct effects of nanomaterials on endothelial toxicity, human umbilical vein ECs were treated with 1-100 microg/ml of hydroxyl fullerene (C60(OH)24; mean diameter, 7.1 +/-2.4 nm) for 24 h. C60(OH)24 induced cytotoxic morphological changes such as cytosolic vacuole formation and decreased cell density in a dose-dependent manner. Lactate dehydrogenase assay revealed that maximal dose C60(OH)24 (100 microg/ml) induced cytotoxic injury. Proliferation assay also showed that maximal dose C60(OH)24 inhibited ECs growth. C60(OH)24 did not seem to induce apoptosis, but caused the accumulation of polyubiquitylated proteins and facilitated autophagic cell death. Formation of autophagosomes was confirmed by Western blotting using a specific marker, light chain 3 antibody and electron microscopy. Chronic treatment with low dose C60(OH)24 (10 microg/ml, 8 days) inhibited cell attachment and delayed ECs growth. The present study examined for the first time the toxicity of water-soluble fullerene to ECs. While fullerene changed morphology in a dose-dependent manner, only maximal dose fullerene caused cytotoxic injury/death and inhibited cell growth. ECs death seemed to be caused by activation of ubiquitin-autophagic death pathways. Although exposure to nanomaterials appears to represent a risk for cardiovascular disorders, further in vivo validations are necessary.

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Multi-walled carbon nanotubes induce T lymphocyte apoptosis.
Bottini M, Bruckner S, Nika K, Bottini N, Bellucci S, Magrini A, Bergamaschi A, Mustelin T.
Toxicol Lett. 2006 Jan 5;160(2):121-6.
[ expand abstract ]

Carbon nanotubes are a man-made form of carbon that did not exist in our environment until very recently. Due to their unique chemical, physical, optical, and magnetic properties, carbon nanotubes have found many uses in industrial products and in the field of nanotechnology, including in nanomedicine. However, very little is yet known about the toxicity of carbon nanotubes. Here, we compare the toxicity of pristine and oxidized multi-walled carbon nanotubes on human T cells and find that the latter are more toxic and induce massive loss of cell viability through programmed cell death at doses of 400 microg/ml, which corresponds to approximately 10 million carbon nanotubes per cell. Pristine, hydrophobic, carbon nanotubes were less toxic and a 10-fold lower concentration of either carbon nanotube type were not nearly as toxic. Our results suggest that carbon nanotubes indeed can be very toxic at sufficiently high concentrations and that careful toxicity studies need to be undertaken particularly in conjunction with nanomedical applications of carbon nanotubes.

The Role of Dissolution in Biological Fate and Effects of Nanoscale Particles.
Borm P, Klaessig FC, Landry TD, Moudgil B, Pauluhn J, Thomas K, Trottier R, Wood S.
Toxicol Sci. 2006 Jan 4; [Epub ahead of print].
[ expand abstract ]

Dissolution, translocation, and disposition have been shown to play a key role in the fate and effects of inhaled particles and fibers. Concepts that have been applied in the micron size range may be usefully applied to the nanoscale range, but new challenges are presented based on the small size and possibly change in the dissolution : translocation relationship. The size of the component molecule itself may be on the nanoscale. Solute concentration, surface area, surface morphology, surface energy, dissolution layer properties, adsorbing species and aggregation are relevant parameters in considering dissolution at the nanoscale. With regard to the etiopathology caused by these types of particulates, the metrics of dose (particle number, surface area, mass or shape) is not yet well-defined. Analytical procedures for assessing dissolution and translocation include chemical assay and particle characterization. Leaching of substituents from particle surfaces may also be important. Compartmentalization within the respiratory tract may add another dimension of complexity. Dissolution may be a critical step for some nanoscale materials in determining fate in the environment and within the body. This review, combining aspects of particle toxicology, material science, and analytical chemistry, is intended to provide a useful basis for developing relevant dissolution assay(s) for nanoscale particles.

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

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

Toxicity and tissue distribution of magnetic nanoparticles in mice.
Kim JS, Yoon TJ, Yu KN, Kim BG, Park SJ, Kim HW, Lee KH, Park SB, Lee JK, Cho MH.
Toxicol Sci. 2006 Jan;89(1):338-47.
[ expand abstract ]

The development of technology enables the reduction of material size in science. The use of particle reduction in size from micro to nanoscale not only provides benefits to diverse scientific fields but also poses potential risks to humans and the environment. For the successful application of nanomaterials in bioscience, it is essential to understand the biological fate and potential toxicity of nanoparticles. The aim of this study was to evaluate the biological distribution as well as the potential toxicity of magnetic nanoparticles to enable their diverse applications in life science, such as drug development, protein detection, and gene delivery. We recently synthesized biocompatible silica-overcoated magnetic nanoparticles containing rhodamine B isothiocyanate (RITC) within a silica shell of controllable thickness [MNPs@SiO2(RITC)]. In this study, the MNPs@SiO2(RITC) with 50-nm thickness were used as a model nanomaterial. After intraperitoneal administration of MNPs@SiO2(RITC) for 4 weeks into mice, the nanoparticles were detected in the brain, indicating that such nanosized materials can penetrate blood-brain barrier (BBB) without disturbing its function or producing apparent toxicity. After a 4-week observation, MNPs@SiO2(RITC) was still present in various organs without causing apparent toxicity. Taken together, our results demonstrated that magnetic nanoparticles of 50-nm size did not cause apparent toxicity under the experimental conditions of this study.

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Research strategies for safety evaluation of nanomaterials, part IV: risk assessment of nanoparticles.
Tsuji JS, Maynard AD, Howard PC, James JT, Lam CW, Warheit DB, Santamaria AB.
Toxicol Sci. 2006 Jan;89(1):42-50.
[ expand abstract ]

Nanoparticles are small-scale substances (<100 nm) with unique properties and, thus, complex exposure and health risk implications. This symposium review summarizes recent findings in exposure and toxicity of nanoparticles and their application for assessing human health risks. Characterization of airborne particles indicates that exposures will depend on particle behavior (e.g., disperse or aggregate) and that accurate, portable, and cost-effective measurement techniques are essential for understanding exposure. Under many conditions, dermal penetration of nanoparticles may be limited for consumer products such as sunscreens, although additional studies are needed on potential photooxidation products, experimental methods, and the effect of skin condition on penetration. Carbon nanotubes apparently have greater pulmonary toxicity (inflammation, granuloma) in mice than fine-scale carbon graphite, and their metal content may affect toxicity. Studies on TiO2 and quartz illustrate the complex relationship between toxicity and particle characteristics, including surface coatings, which make generalizations (e.g., smaller particles are always more toxic) incorrect for some substances. These recent toxicity and exposure data, combined with therapeutic and other related literature, are beginning to shape risk assessments that will be used to regulate the use of nanomaterials in consumer products.

2005

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

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

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Molecular Characterization of the Cytotoxic Mechanism of Multiwall Carbon Nanotubes and Nano-Onions on Human Skin Fibroblast.
Ding L, Stilwell J, Zhang T, Elboudwarej O, Jiang H, Selegue JP, Cooke PA, Gray JW, Chen FF.
Nano Lett. 2005 Dec 14;5(12):2448-2464.
[ expand abstract ]

The increasing use of nanotechnology in consumer products and medical applications underlies the importance of understanding its potential toxic effects to people and the environment. Although both fullerene and carbon nanotubes have been demonstrated to accumulate to cytotoxic levels within organs of various animal models and cell types and carbon nanomaterials have been exploited for cancer therapies, the molecular and cellular mechanisms for cytotoxicity of this class of nanomaterial are not yet fully apparent. To address this question, we have performed whole genome expression array analysis and high content image analysis based phenotypic measurements on human skin fibroblast cell populations exposed to multiwall carbon nano-onions (MWCNOs) and multiwall carbon nanotubes (MWCNTs). Here we demonstrate that exposing cells to MWCNOs and MWCNTs at cytotoxic doses induces cell cycle arrest and increases apoptosis/necrosis. Expression array analysis indicates that multiple cellular pathways are perturbed after exposure to these nanomaterials at these doses, with material-specific toxigenomic profiles observed. Moreover, there are also distinct qualitative and quantitative differences in gene expression profiles, with each material at different dosage levels (6 and 0.6 mug/mL for MWCNO and 0.6 and 0.06 mug/mL for MWCNT). MWCNO and MWCNT exposure activates genes involved in cellular transport, metabolism, cell cycle regulation, and stress response. MWCNTs induce genes indicative of a strong immune and inflammatory response within skin fibroblasts, while MWCNO changes are concentrated in genes induced in response to external stimuli. Promoter analysis of the microarray results demonstrate that interferon and p38/ERK-MAPK cascades are critical pathway components in the induced signal transduction contributing to the more adverse effects observed upon exposure to MWCNTs as compared to MWCNOs.

Dendrimer biocompatibility and toxicity.
Duncan R, Izzo L.
Adv Drug Deliv Rev. 2005 Dec 14;57(15):2215-37.
[ expand abstract ]

The field of biomedical dendrimers is still in its infancy, but the explosion of interest in dendrimers and dendronised polymers as inherently active therapeutic agents, as vectors for targeted delivery of drugs, peptides and oligonucleotides, and as permeability enhancers able to promote oral and transdermal drug delivery makes it timely to review current knowledge regarding the toxicology of these dendrimer chemistries (currently under development for biomedical applications). Clinical experience with polymeric excipients, plasma expanders, and most recently the development of more 'classical polymer'-derived therapeutics can be used to guide development of "safe" dendritic polymers. Moreover, in future it will only ever be possible to designate a dendrimer as "safe" when related to a specific application. The so far limited clinical experience using dendrimers make it impossible to designate any particular chemistry intrinsically "safe" or "toxic". Although there is widespread concern as to the safety of nano-sized particles, preclinical and clinical experience gained during the development of polymeric excipients, biomedical polymers and polymer therapeutics shows that judicious development of dendrimer chemistry for each specific application will ensure development of safe and important materials for biomedical and pharmaceutical use.

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Manikin-Based Performance Evaluation of N95 Filtering-Facepiece Respirators Challenged with Nanoparticles.
Balazy A, Toivola M, Reponen T, Podgorski A, Zimmer A, Grinshpun SA.
Ann Occup Hyg. 2005 Dec 12; [Epub ahead of print].
[ expand abstract ]

Protection of the human respiratory system from exposure to nanoparticles is becoming an emerging issue in occupational hygiene. The potential adverse health effects associated with particles of approximately 1-100 nm are probably greater than submicron or micron-sized particles. The performance of two models of N95 half-facepiece-filtering respirators against nano-sized particles was evaluated at two inhalation flow rates, 30 and 85 l min(-1), following a manikin-based protocol. The aerosol concentration was measured outside and inside the facepiece using the Wide-Range Particle Spectrometer. Sodium chloride particles, conventionally used to certify N-series respirators under NIOSH 42 CFR 84 regulations, were utilized as the challenge aerosol. The targeted particle sizes ranged from 10 to 600 nm, although the standard certification tests are performed with particles of approximately 300 nm, which is assumed to be the most penetrating size. The results indicate that the nanoparticle penetration through a face-sealed N95 respirator may be in excess of the 5% threshold, particularly at high respiratory flow rates. Thus, N95 respirators may not always provide the expected respiratory protection for workers. The highest penetration values representing the poorest respirator protection conditions were observed in the particle diameter range of approximately 30-70 nm. Based on the theoretical simulation, we have concluded that for respirators utilizing mechanical filters, the peak penetration indeed occurs at the particle diameter of approximately 300 nm; however, for pre-charged fiber filters, which are commonly used for N95 respirators, the peak shifts toward nano-sizes. This study has confirmed that the neutralization of particles is a crucial element in evaluating the efficiency of a respirator. The variability of the respirator's performance was determined for both models and both flow rates. The analysis revealed that the coefficient of variation of the penetration ranged from 0.10 to 0.54 for particles of 20-100 nm in diameter. The fraction of N95 respirators for which the performance test at 85 l min(-1) demonstrated excessive (>5%) penetration of nanoparticles was as high as 9/10. The test results obtained in a relatively small (0.096 m(3)) test chamber and in a large (24.3 m(3)) walk-in chamber were found essentially the same, thus, suggesting that laboratory-based evaluations have a good potential to adequately represent the respirator field performance.

Nanotechnology. Calls rise for more research on toxicology of nanomaterials.
Service RF.
Science. 2005 Dec 9;310(5754):1609.
[ expand abstract ]

No abstract available

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Carbon black nanoparticles induce type II epithelial cells to release chemotaxins for alveolar macrophages.
Barlow PG, Clouter-Baker A, Donaldson K, Maccallum J, Stone V.
Part Fibre Toxicol. 2005 Dec 6;2(1):11.
[ expand abstract ]

BACKGROUND: Alveolar macrophages are a key cell in dealing with particles deposited in the lungs and in determining the subsequent response to that particle exposure. Nanoparticles are considered a potential threat to the lungs and the mechanism of pulmonary response to nanoparticles is currently under intense scrutiny. The type II alveolar epithelial cell has previously been shown to release chemoattractants which can recruit alveolar macrophages to sites of particle deposition. The aim of this study was to assess the responses of a type II epithelial cell line (L-2) to both fine and nanoparticle exposure in terms of secretion of chemotactic substances capable of inducing macrophage migration. RESULTS: Exposure of type II cells to carbon black nanoparticles resulted in significant release of macrophage chemoattractant compared to the negative control and to other dusts tested (fine carbon black and TiO2 and nanoparticle TiO2) as measured by macrophage migration towards type II cell conditioned medium. SDS-PAGE analysis of the conditioned medium from particle treated type II cells revealed that a higher number of protein bands were present in the conditioned medium obtained from type II cells treated with nanoparticle carbon black compared to other dusts tested. Size-fractionation of the chemotaxin-rich supernatant determined that the chemoattractants released from the epithelial cells were between 5 and 30 kDa in size. CONCLUSIONS: The highly toxic nature and reactive surface chemistry of the carbon black nanoparticles has very likely induced the type II cell line to release pro-inflammatory mediators that can potentially induce migration of macrophages. This could aid in the rapid recruitment of inflammatory cells to sites of particle deposition and the subsequent removal of the particles by phagocytic cells such as macrophages and neutrophils. Future studies in this area could focus on the exact identity of the substance(s) released by the type II cells in response to particle exposure.

Research strategies for safety evaluation of nanomaterials, Part III: nanoscale technologies for assessing risk and improving public health.
Balshaw DM, Philbert M, Suk WA.
Toxicol Sci. 2005 Dec;88(2):298-306.
[ expand abstract ]

Risk assessment in the environmental health sciences focuses on understanding the nature of environmental exposures and the potential harm posed by those exposures which in turn is determined by the perturbation of biological pathways and the individual's susceptibility to damage. While there are extensive research efforts ongoing in these areas, progress in each is currently slowed by technological limitations including comprehensive assessment of multiple exposures in real time and dynamic assessment of biological response with high temporal and quantitative resolution. This Forum article discusses recent technological innovations capitalizing on the emergent properties of nanoscale materials and their potential adaptation to improving individual exposure assessment, determination of biological response, and environmental remediation. The ultimate goal is to raise the environmental health science community's awareness of these possibilities and encourage the development of improved strategies for assessing risk and improving public health.

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In vitro cytotoxicity of nanoparticles in mammalian germline stem cells.
Braydich-Stolle L, Hussain S, Schlager JJ, Hofmann MC.
Toxicol Sci. 2005 Dec;88(2):412-9.
[ expand abstract ]

Gametogenesis is a complex biological process that is particularly sensitive to environmental insults such as chemicals. Many chemicals have a negative impact on the germline, either by directly affecting the germ cells, or indirectly through their action on the somatic nursing cells. Ultimately, these effects can inhibit fertility, and they may have negative consequences for the development of the offspring. Recently, nanomaterials such as nanotubes, nanowires, fullerene derivatives (buckyballs), and quantum dots have received enormous national attention in the creation of new types of analytical tools for biotechnology and the life sciences. Despite the wide application of nanomaterials, there is a serious lack of information concerning their impact on human health and the environment. Thus, there are limited studies available on toxicity of nanoparticles for risk assessment of nanomaterials. The purpose of this study was to assess the suitability of a mouse spermatogonial stem cell line as a model to assess nanotoxicity in the male germline in vitro. The effects of different types of nanoparticles on these cells were evaluated by light microscopy, and by cell proliferation and standard cytotoxicity assays. Our results demonstrate a concentration-dependent toxicity for all types of particles tested, whereas the corresponding soluble salts had no significant effect. Silver nanoparticles were the most toxic while molybdenum trioxide (MoO(3)) nanoparticles were the least toxic. Our results suggest that this cell line provides a valuable model with which to assess the cytotoxicity of nanoparticles in the germ line in vitro.

Nano-C60 cytotoxicity is due to lipid peroxidation.
Sayes CM, Gobin AM, Ausman KD, Mendez J, West JL, Colvin VL.
Biomaterials. 2005 Dec;26(36):7587-95.
[ expand abstract ]

This study examines the biological effects of water-soluble fullerene aggregates in an effort to evaluate the fundamental mechanisms that contribute to the cytotoxicity of a classic engineered nanomaterial. For this work we used a water-soluble fullerene species, nano-C60, a fullerene aggregate that readily forms when pristine C60 is added to water. Nano-C60 was cytotoxic to human dermal fibroblasts, human liver carcinoma cells (HepG2), and neuronal human astrocytes at doses>or= 50 ppb (LC50=2-50 ppb, depending on cell type) after 48 h exposure. This water-soluble nano-C60 colloidal suspension disrupts normal cellular function through lipid peroxidation; reactive oxygen species are responsible for the membrane damage. Cellular viability was determined through live/dead staining and LDH release. DNA concentration and mitochondrial activity were not affected by the nano-C60 inoculations to cells in culture. The integrity of cellular membrane was examined by monitoring the peroxy-radicals on the lipid bilayer. Subsequently, glutathione production was measured to assess the cell's reaction to membrane oxidation. The damage to cell membranes was observed both with chemical assays, and confirmed physically by visualizing membrane permeability with high molecular weight dyes. With the addition of an antioxidant, L-ascorbic acid, the oxidative damage and resultant toxicity of nano-C60 was completely prevented.

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Chemical modification of SWNT alters in vitro cell-SWNT interactions.
Nimmagadda A, Thurston K, Nollert MU, McFetridge PS.
J Biomed Mater Res A. 2005 Nov 28; [Epub ahead of print].
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Single-walled carbon nanotubes (SWNT) have been the focus of considerable attention as a material with extraordinary mechanical and electrical properties. SWNT have been proposed in a number of biomedical applications, including neural, bone, and dental tissue engineering. In these applications, it is clear that surrounding tissues will come into surface contact with SWNT composites, and compatibility between SWNT and host cells must be addressed. This investigation describes the gross physical and chemical effects of different SWNT preparations on in vitro cell viability and metabolic activity. Three different SWNT preparations were analyzed: as purchased (AP-NT), purified (PUR-NT), and functionalized with glucosamine (GA-NT), over concentrations of 0.001-1.0% (wt/vol). With the exception of the lowest SWNT concentrations, increasing concentrations of SWNT resulted in a decrease of cell viability, which was dependent on SWNT preparation. The metabolic activity of 3T3 cells was also dependent on SWNT preparation and concentration. These investigations have shown that these SWNT preparations have significant effects on in vitro cellular function that cannot be attributed to one factor alone, but are more likely the result of several unfavorable interactions. Effects, such as destabilizing the cell membrane, soluble toxic contaminants, and limitations in mass transfer as the SWNT coalesce into sheets, may all play a role in these interactions. Using comprehensive purification processes and modifying the NT-surface chemistry to introduce functional groups or reduce hydrophobicity or both, these interactions can be significantly improved.

Relative risk analysis of several manufactured nanomaterials: an insurance industry context.
Robichaud CO, Tanzil D, Weilenmann U, Wiesner MR.
Environ Sci Technol. 2005 Nov 15;39(22):8985-94.
[ expand abstract ]

A relative risk assessment is presented for the industrial fabrication of several nanomaterials. The production processes for five nanomaterials were selected for this analysis, based on their current or near-term potential for large-scale production and commercialization: single-walled carbon nanotubes, bucky balls (C60), one variety of quantum dots, alumoxane nanoparticles, and nano-titanium dioxide. The assessment focused on the activities surrounding the fabrication of nanomaterials, exclusive of any impacts or risks with the nanomaterials themselves. A representative synthesis method was selected for each nanomaterial based on its potential for scaleup. A list of input materials, output materials, and waste streams for each step of fabrication was developed and entered into a database that included key process characteristics such as temperature and pressure. The physical-chemical properties and quantities of the inventoried materials were used to assess relative risk based on factors such as volatility, carcinogenicity, flammability, toxicity, and persistence. These factors were first used to qualitatively rank risk, then combined using an actuarial protocol developed by the insurance industry for the purpose of calculating insurance premiums for chemical manufacturers. This protocol ranks three categories of risk relative to a 100 point scale (where 100 represents maximum risk): incident risk, normal operations risk, and latent contamination risk. Results from this analysis determined that relative environmental risk from manufacturing each of these five materials was comparatively low in relation to other common industrial manufacturing processes.

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Acute toxicological effects of copper nanoparticles in vivo.
Chen Z, Meng H, Xing G, Chen C, Zhao Y, Jia G, Wang T, Yuan H, Ye C, Zhao F, Chai Z, Zhu C, Fang X, Ma B, Wan L.
Toxicol Lett. 2005 Nov 12; [Epub ahead of print].
[ expand abstract ]

To assess the toxicity of copper nanoparticles (23.5nm) in vivo, LD(50), morphological changes, pathological examinations and blood biochemical indexes of experimental mice are studied comparatively with micro-copper particles (17mum) and cupric ions (CuCl(2).2H(2)O). The LD(50) for the nano-, micro-copper particles and cupric ions exposed to mice via oral gavage are 413, >5000 and 110mg/kg body weight, respectively. The toxicity classes of nano and ionic copper particles both are class 3 (moderately toxic), and micro-copper is class 5 (practically non-toxic) of Hodge and Sterner Scale. Kidney, liver and spleen are found to be target organs of nano-copper particles. Nanoparticles induce gravely toxicological effects and heavy injuries on kidney, liver and spleen of experimental mice, but micro-copper particles do not, on mass basis. Results indicate a gender dependent feature of nanotoxicity. Several factors such as huge specific surface area, ultrahigh reactivity, exceeding consumption of H(+), etc. that likely cause the grave nanotoxicity observed in vivo are discussed

Cationic liposomes induce apoptosis through p38 MAP kinase-caspase-8-Bid pathway in macrophage-like RAW264.7 cells.
Iwaoka S, Nakamura T, Takano S, Tsuchiya S, Aramaki Y.
J Leukoc Biol. 2005 Nov 7; [Epub ahead of print].
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We have demonstrated that cationic liposomes composed of stearylamine (SA-liposomes) induce apoptosis in a variety of cells, but the mechanism responsible for the cellular death is not clear. In this paper, we investigated the signaling pathways implicated in SA-liposome-induced apoptosis in the macrophage-like cell line RAW264.7. Treatment with SA-liposomes caused the activation of mitogen-activated protein kinases (MAPKs), especially p38 and c-jun N-terminal kinase, and apoptosis was only inhibited upon the addition of a specific inhibitor for p38. N-acetylcysteine, a scavenger of reactive oxygen species (ROS), effectively inhibited the activation of p38 and cellular death, indicating that the activation induced by ROS is an initial step in the process of apoptosis triggered by SA-liposomes. Caspase-8 was activated by p38, and caspase-8-dependent cleavage of Bid was also observed. No down-regulation of bcl-2 expression, and no cleavage of Bax protein were observed. Taken together, our results suggest that apoptosis of RAW264.7 by SA-liposomes was mediated by the MAPK p38 and a caspase-8-dependent Bid-cleavage pathway. Moreover, we found that ROS can contribute intimately to the SA-liposome-induced cell death in RAW264.7.

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Hypersensitivity and Loss of Disease Site Targeting Caused by Antibody Responses to PEGylated Liposomes.
Judge A, McClintock K, Phelps JR, Maclachlan I.
Mol Ther. 2005 Nov 4; [Epub ahead of print].
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The systemic application of nucleic acid drugs requires delivery systems that overcome the poor pharmacokinetics, limited biodistribution, and inefficient uptake of nucleic acids. PEGylated liposomes show considerable promise because of their intrinsic ability to accumulate at disease sites and facilitate transfection of target cells. Unlike many viral vectors, PEGylated liposomes are generally considered to be nonimmunogenic. We have developed a PEGylated liposome for the systemic administration of plasmid DNA that achieves high levels of selective gene expression at distal tumor sites. Here we report that the in vivo efficacy and safety of these systems can be severely compromised following repeat administration. This phenomenon is characterized by a loss of disease site targeting, accelerated clearance from the blood, and acute hypersensitivity. These effects are fully attributable to a surprisingly robust, long-lived antibody response generated against polyethylene glycol (PEG) that results from the strong adjuvant effect of the plasmid payload. Importantly, immunogenicity may be substantially reduced by modifying the alkyl chain of the PEG-lipid conjugate, thereby allowing successful repeat dosing of the modified plasmid formulations without adverse side effects. Immunogenicity is a relevant concern for a number of nonviral delivery systems given the potent immunostimulatory properties of many nucleic acid drugs.

Smaller is not always better: nanotechnology yields nanotoxicology.
Kipen HM, Laskin DL.
Am J Physiol Lung Cell Mol Physiol. 2005 Nov;289(5):L696-7.
[ expand abstract ]

No abstract available

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Blood compatibility of cetyl alcohol/polysorbate-based nanoparticles.
Koziara JM, Oh JJ, Akers WS, Ferraris SP, Mumper RJ.
Pharm Res. 2005 Nov;22(11):1821-8.
[ expand abstract ]

PURPOSE: Pegylated and nonpegylated cetyl alcohol/polysorbate nanoparticles (E78 NPs) are being tested as drug carriers for specific tumor and brain targeting. Because these nanoparticle formulations are designed for systemic administration, it is important to test the compatibility of these lipid-based NPs with blood and blood cells. METHODS: The hemocompatibility of E78 NPs was evaluated with a particular focus on hemolytic activity, platelet function, and blood coagulation. Human red blood cell lysis was determined by measuring hemoglobin release. Activation and aggregation of human platelets were determined using flow cytometry and aggregometry, respectively. Finally, the whole blood clotting time was measured using human blood. RESULTS: E78 NPs did not cause in vitro red blood cell lysis at concentrations up to 1 mg/mL. In addition, under conditions tested, E78 and polyethylene glycol (PEG)-coated E78 NPs (PEG-E78 NPs) did not activate platelets. In fact, both NP formulations very rapidly inhibited agonist-induced platelet activation and aggregation in a dose-dependent manner. Additionally, E78 NPs significantly prolonged in vitro whole blood clotting time at a concentration of 500 mug/mL or greater. CONCLUSIONS: It was concluded that PEG-coated and nonpegylated E78 NPs have potential blood compatibility at clinically relevant doses. Based on the calculated nanoparticle-to-platelet ratio, the concentration at which E78 NPs could potentially affect platelet function in vivo was approximately 1 mg/mL.

Unusual inflammatory and fibrogenic pulmonary responses to single-walled carbon nanotubes in mice.
Shvedova AA, Kisin ER, Mercer R, Murray AR, Johnson VJ, Potapovich AI, Tyurina YY, Gorelik O, Arepalli S, Schwegler-Berry D, Hubbs AF, Antonini J, Evans DE, Ku BK, Ramsey D, Maynard A, Kagan VE, Castranova V, Baron P.
Am J Physiol Lung Cell Mol Physiol. 2005 Nov;289(5):L698-708.
[ expand abstract ]

Single-walled carbon nanotubes (SWCNT) are new materials of emerging technological importance. As SWCNT are introduced into the life cycle of commercial products, their effects on human health and environment should be addressed. We demonstrated that pharyngeal aspiration of SWCNT elicited unusual pulmonary effects in C57BL/6 mice that combined a robust but acute inflammation with early onset yet progressive fibrosis and granulomas. A dose-dependent increase in the protein, LDH, and gamma-glutamyl transferase activities in bronchoalveolar lavage were found along with accumulation of 4-hydroxynonenal (oxidative biomarker) and depletion of glutathione in lungs. An early neutrophils accumulation (day 1), followed by lymphocyte (day 3) and macrophage (day 7) influx, was accompanied by early elevation of proinflammatory cytokines (TNF-alpha, IL-1beta; day 1) followed by fibrogenic transforming growth factor (TGF)-beta1 (peaked on day 7). A rapid progressive fibrosis found in mice exhibited two distinct morphologies: 1) SWCNT-induced granulomas mainly associated with hypertrophied epithelial cells surrounding SWCNT aggregates and 2) diffuse interstitial fibrosis and alveolar wall thickening likely associated with dispersed SWCNT. In vitro exposure of murine RAW 264.7 macrophages to SWCNT triggered TGF-beta1 production similarly to zymosan but generated less TNF-alpha and IL-1beta. SWCNT did not cause superoxide or NO.production, active SWCNT engulfment, or apoptosis in RAW 264.7 macrophages. Functional respiratory deficiencies and decreased bacterial clearance (Listeria monocytogenes) were found in mice treated with SWCNT. Equal doses of ultrafine carbon black particles or fine crystalline silica (SiO2) did not induce granulomas or alveolar wall thickening and caused a significantly weaker pulmonary inflammation and damage.

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Researchers size up nanotechnology risks.
Hampton T.
JAMA. 2005 Oct 19;294(15):1881-3.
[ expand abstract ]

No abstract available

Toxicity and Tissue Distribution of Magnetic Nanoparticles in Mice.
Kim JS, Yoon TJ, Yu KN, Kim BG, Park SJ, Kim HW, Lee KH, Park SB, Lee JK, Cho MH.
Toxicol Sci. 2005 Oct 19; [Epub ahead of print].
[ expand abstract ]

The development of technology enables the reduction of material size in science. The use of particle reduction in size from micro to nanoscale not only provides benefits to diverse scientific fields but also poses potential risks to human and the environment. For the successful application of nanomaterials in bioscience, it is essential to understand the biological fate and potential toxicity of nanoparticles. The aim of this study was to evaluate the biological distribution as well as the potential toxicity of magnetic nanoparticles to enable their diverse applications in life science such as drug development, protein detection, and gene delivery. We recently synthesized biocompatible silica-overcoated magnetic nanoparticles containing rhodamine B isothiocyanate (RITC) within a silica shell of controllable thickness [MNPs@SiO2(RITC)]. In this study, the MNPs@SiO2(RITC) with 50 nm thickness were used as a model nanomaterial. After intraperitoneal administration of MNPs@SiO2(RITC) for 4 weeks into mice, the nanoparticles were detected in the brain, indicating that such nanosized materials can penetrate blood-brain-barrier (BBB) without disturbing its function or producing apparent toxicity. After a 4-week observation, MNPs@SiO2(RITC) was still present in various organs without causing apparent toxicity. Taken together, our results demonstrated that magnetic nanoparticles of 50-nm size did not cause apparent toxicity under the experimental conditions of this study

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Research strategies for safety evaluation of nanomaterials, Part I: evaluating the human health implications of exposure to nanoscale materials.
Thomas K, Sayre P.
Toxicol Sci. 2005 Oct;87(2):316-21.
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Nanotechnology has the potential to dramatically improve the effectiveness of a number of existing consumer and industrial products and could have a substantial impact on the development of new products ranging from disease diagnosis and treatment to environmental remediation. The broad range of possible nanotechnology applications could lead to substantive changes in industrial productivity, economic growth, and international trade. A continuing evaluation of the human health implications of exposure to nanoscale materials will be essential before the commercial benefits of these materials can be fully realized. The purpose of this article is to review the human health implications of exposure to nanoscale materials in the context of a toxicological risk evaluation, the current scope of U.S. Federal research on nanoscale materials, and selected toxicological studies associated with nanoscale materials to note emerging research in this area.

Blood Compatibility of Cetyl Alcohol/Polysorbate-Based Nanoparticles.
Koziara JM, Oh JJ, Akers WS, Ferraris SP, Mumper RJ.
Pharm Res. 2005 Aug 26; [Epub ahead of print].
[ expand abstract ]

PURPOSE: Pegylated and nonpegylated cetyl alcohol/polysorbate nanoparticles (E78 NPs) are being tested as drug carriers for specific tumor and brain targeting. Because these nanoparticle formulations are designed for systemic administration, it is important to test the compatibility of these lipid-based NPs with blood and blood cells. METHODS: The hemocompatibility of E78 NPs was evaluated with a particular focus on hemolytic activity, platelet function, and blood coagulation. Human red blood cell lysis was determined by measuring hemoglobin release. Activation and aggregation of human platelets were determined using flow cytometry and aggregometry, respectively. Finally, the whole blood clotting time was measured using human blood. RESULTS: E78 NPs did not cause in vitro red blood cell lysis at concentrations up to 1 mg/mL. In addition, under conditions tested, E78 and polyethylene glycol (PEG)-coated E78 NPs (PEG-E78 NPs) did not activate platelets. In fact, both NP formulations very rapidly inhibited agonist-induced platelet activation and aggregation in a dose-dependent manner. Additionally, E78 NPs significantly prolonged in vitro whole blood clotting time at a concentration of 500 mug/mL or greater. CONCLUSIONS: It was concluded that PEG-coated and nonpegylated E78 NPs have potential blood compatibility at clinically relevant doses. Based on the calculated nanoparticle-to-platelet ratio, the concentration at which E78 NPs could potentially affect platelet function in vivo was approximately 1 mg/mL.

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Multi-walled carbon nanotubes induce T lymphocyte apoptosis.
Bottini M, Bruckner S, Nika K, Bottini N, Bellucci S, Magrini A, Bergamaschi A, Mustelin T.
Toxicol Lett. 2005 Aug 24; [Epub ahead of print].
[ expand abstract ]

Carbon nanotubes are a man-made form of carbon that did not exist in our environment until very recently. Due to their unique chemical, physical, optical, and magnetic properties, carbon nanotubes have found many uses in industrial products and in the field of nanotechnology, including in nanomedicine. However, very little is yet known about the toxicity of carbon nanotubes. Here, we compare the toxicity of pristine and oxidized multi-walled carbon nanotubes on human T cells and find that the latter are more toxic and induce massive loss of cell viability through programmed cell death at doses of 400mug/ml, which corresponds to approximately 10 million carbon nanotubes per cell. Pristine, hydrophobic, carbon nanotubes were less toxic and a 10-fold lower concentration of either carbon nanotube type were not nearly as toxic. Our results suggest that carbon nanotubes indeed can be very toxic at sufficiently high concentrations and that careful toxicity studies need to be undertaken particularly in conjunction with nanomedical applications of carbon nanotubes.

In vitro toxicity of nanoparticles in BRL 3A rat liver cells.
Hussain SM, Hess KL, Gearhart JM, Geiss KT, Schlager JJ.
Toxicol In Vitro. 2005 Aug 24; [Epub ahead of print].
[ expand abstract ]

This study was undertaken to address the current deficient knowledge of cellular response to nanosized particle exposure. The study evaluated the acute toxic effects of metal/metal oxide nanoparticles proposed for future use in industrial production methods using the in vitro rat liver derived cell line (BRL 3A). Different sizes of nanoparticles such as silver (Ag; 15, 100nm), molybdenum (MoO(3); 30, 150nm), aluminum (Al; 30, 103nm), iron oxide (Fe(3)O(4); 30, 47nm), and titanium dioxide (TiO(2); 40nm) were evaluated for their potential toxicity. We also assessed the toxicity of relatively larger particles of cadmium oxide (CdO; 1mum), manganese oxide (MnO(2); 1-2mum), and tungsten (W; 27mum), to compare the cellular toxic responses with respect to the different sizes of nanoparticles with different core chemical compositions. For toxicity evaluations, cellular morphology, mitochondrial function (MTT assay), membrane leakage of lactate dehydrogenase (LDH assay), reduced glutathione (GSH) levels, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were assessed under control and exposed conditions (24h of exposure). Results showed that mitochondrial function decreased significantly in cells exposed to Ag nanoparticles at 5-50mug/ml. However, Fe(3)O(4), Al, MoO(3) and TiO(2) had no measurable effect at lower doses (10-50mug/ml), while there was a significant effect at higher levels (100-250mug/ml). LDH leakage significantly increased in cells exposed to Ag nanoparticles (10-50mug/ml), while the other nanoparticles tested displayed LDH leakage only at higher doses (100-250mug/ml). In summary the Ag was highly toxic whereas, MoO(3) moderately toxic and Fe(3)O(4), Al, MnO(2) and W displayed less or no toxicity at the doses tested. The microscopic studies demonstrated that nanoparticle-exposed cells at higher doses became abnormal in size, displaying cellular shrinkage, and an acquisition of an irregular shape. Due to toxicity of silver, further study conducted with reference to its oxidative stress. The results exhibited significant depletion of GSH level, reduced mitochondrial membrane potential and increase in ROS levels, which suggested that cytotoxicity of Ag (15, 100nm) in liver cells is likely to be mediated through oxidative stress

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Interactions of blood proteins with poly(isobutylcyanoacrylate) nanoparticles decorated with a polysaccharidic brush.
Labarre D, Vauthier C, Chauvierre C, Petri B, Muller R, Chehimi MM.
Biomaterials. 2005 Aug;26(24):5075-84.
[ expand abstract ]

The aim of this work was to examine the in vitro interactions of core-shell poly(isobutylcyanoacrylate)-polysaccharide nanoparticles (NP) with blood proteins. The particles were prepared by initiating the emulsion polymerization of isobutylcyanoacrylate (IBCA) in the presence of dextran 71 or 15 kDa, heparin, a blend of dextran 71 and heparin, or dextran sulphate in aqueous medium at pH 1. The mechanisms of polymerisation were redox radical (Rad) or anionic (An), resulting in differences in the spatial arrangement of the polysaccharide chains at the NP surface, i.e. "loops" and "trains" by anionic polymerization, "brush" by radical polymerization. Surface composition of NPs was determined by X-ray photo-electron spectroscopy (XPS) and surface charge by zeta potential measurements. In the presence of citrated blood plasma, efficacy of the steric repulsive effect of the NP dextran shell towards protein adsorption decreased in the order: Dex71-Rad > Dex15-Rad > Dex71-AnDex15-An. Dextran-coated NPs adsorbed ApoA-I and fibrinogen from plasma. Concerning activation of complement in serum, the effect was sharp: Dex71-Rad was a very low activator whereas Dex15-An, Dex15-Rad and Dex71-An were strong activators. In citrated plasma, the steric repulsive effects of Hep-Rad and Dex-Hep-Rad NPs were similar to Dex71-An, and Dex-Sulph-Rad NPs adsorbed twice more proteins than Hep-Rad. Hep-Rad, Dex-Hep-Rad and Dex-Sulph-Rad NPs adsorbed IgG and fibrinogen. Complement was not activated in serum in the presence of Hep-Rad and Dex-Hep-Rad and a slight adsorption of C3 was noted. C3 was completely adsorbed on Dex-Sulph-Rad. The exquisite sensitivity of blood proteins to differences in the nature and outermost structure of the polysaccharides-coated NPs is highlighted by the present results.

Research Strategies for Safety Evaluation of Nanomaterials, Part I: Evaluating the Human Health Implications of Exposure to Nanoscale Materials.
Karluss T, Philip S.
Toxicol Sci. 2005 Jul 27; [Epub ahead of print].
[ No abstract available ]

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Nano-C(60) cytotoxicity is due to lipid peroxidation.
Sayes CM, Gobin AM, Ausman KD, Mendez J, West JL, Colvin VL.
Biomaterials. 2005 Jul 7; [Epub ahead of print].
[ expand abstract ]

This study examines the biological effects of water-soluble fullerene aggregates in an effort to evaluate the fundamental mechanisms that contribute to the cytotoxicity of a classic engineered nanomaterial. For this work we used a water-soluble fullerene species, nano-C(60), a fullerene aggregate that readily forms when pristine C(60) is added to water. Nano-C(60) was cytotoxic to human dermal fibroblasts, human liver carcinoma cells (HepG2), and neuronal human astrocytes at doses 50ppb (LC(50)=2-50ppb, depending on cell type) after 48h exposure. This water-soluble nano-C(60) colloidal suspension disrupts normal cellular function through lipid peroxidation; reactive oxygen species are responsible for the membrane damage. Cellular viability was determined through live/dead staining and LDH release. DNA concentration and mitochondrial activity were not affected by the nano-C(60) inoculations to cells in culture. The integrity of cellular membrane was examined by monitoring the peroxy-radicals on the lipid bilayer. Subsequently, glutathione production was measured to assess the cell's reaction to membrane oxidation. The damage to cell membranes was observed both with chemical assays, and confirmed physically by visualizing membrane permeability with high molecular weight dyes. With the addition of an antioxidant, l-ascorbic acid, the oxidative damage and resultant toxicity of nano-C(60) was completely prevented.

Nanotechnology. EPA ponders voluntary nanotechnology regulations.
Service RF.
Science. 2005 Jul 1;309(5731):36.
[ No abstract available ]

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"Nanotechnology: Environmental Implications and Solutions " A discussion.
Oberdorster E.
Environ Health Perspect. 2005 Jul;113(7):a488.
[ expand abstract ]

This book, written mainly for engineering students, gives an excellent summary of traditional environmental pollution issues. Ten chapters cover current legislation regarding environmental pollutants; an overview of chemistry and current nanotechnology processes; air, water and solid-waste issues; multimedia analysis; both health and hazard risk assessment; ethical considerations; and concluding remarks on future trends.

Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles.
Oberdorster G, Oberdorster E, Oberdorster J.
Environ Health Perspect. 2005 Jul;113(7):823-39.
[ expand abstract ]

Although humans have been exposed to airborne nanosized particles (NSPs; < 100 nm) throughout their evolutionary stages, such exposure has increased dramatically over the last century due to anthropogenic sources. The rapidly developing field of nanotechnology is likely to become yet another source through inhalation, ingestion, skin uptake, and injection of engineered nanomaterials. Information about safety and potential hazards is urgently needed. Results of older biokinetic studies with NSPs and newer epidemiologic and toxicologic studies with airborne ultrafine particles can be viewed as the basis for the expanding field of nanotoxicology, which can be defined as safety evaluation of engineered nanostructures and nanodevices. Collectively, some emerging concepts of nanotoxicology can be identified from the results of these studies. When inhaled, specific sizes of NSPs are efficiently deposited by diffusional mechanisms in all regions of the respiratory tract. The small size facilitates uptake into cells and transcytosis across epithelial and endothelial cells into the blood and lymph circulation to reach potentially sensitive target sites such as bone marrow, lymph nodes, spleen, and heart. Access to the central nervous system and ganglia via translocation along axons and dendrites of neurons has also been observed. NSPs penetrating the skin distribute via uptake into lymphatic channels. Endocytosis and biokinetics are largely dependent on NSP surface chemistry (coating) and in vivo surface modifications. The greater surface area per mass compared with larger-sized particles of the same chemistry renders NSPs more active biologically. This activity includes a potential for inflammatory and pro-oxidant, but also antioxidant, activity, which can explain early findings showing mixed results in terms of toxicity of NSPs to environmentally relevant species. Evidence of mitochondrial distribution and oxidative stress response after NSP endocytosis points to a need for basic research on their interactions with subcellular structures. Additional considerations for assessing safety of engineered NSPs include careful selections of appropriate and relevant doses/concentrations, the likelihood of increased effects in a compromised organism, and also the benefits of possible desirable effects. An interdisciplinary team approach (e.g., toxicology, materials science, medicine, molecular biology, and bioinformatics, to name a few) is mandatory for nanotoxicology research to arrive at an appropriate risk assessment. Key words: biokinetics, central nervous system, engineered nanomaterials, environmental health, human health, nanosized particles, respiratory tract, risk assessment, skin, ultrafine particles.