Stabilization of C60 Nanoparticles by Protein Adsorption and Its

Thus, it seems likely that C60 nanoparticles would have coagulated when their toxicities were studied. This makes it difficult to deduce the interacti...
0 downloads 0 Views 101KB Size
Download PDF
854

Chem. Res. Toxicol. 2007, 20, 854-858

Communications Stabilization of C60 Nanoparticles by Protein Adsorption and Its Implications for Toxicity Studies Shigeru Deguchi,*,† Tomoko Yamazaki,†,‡ Sada-atsu Mukai,†,§ Ron Usami,‡ and Koki Horikoshi† Extremobiosphere Research Center, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237-0061, Japan, and Department of Biological Applied Chemistry, Graduate School of Engineering, Toyo UniVersity, 2100 Kujirai, Kawagoe, Saitama 350-8585, Japan ReceiVed NoVember 13, 2006

Dispersion stability of nanoparticles of C60 under a model condition simulating a physiological environment was studied by dynamic light scattering. Although the C60 nanoparticles at a concentration of 9.3 × 10-6 M (6.7 µg/mL) coagulated and precipitated out rapidly in phosphate buffered saline, coagulation was suppressed completely when HSA was present at concentrations above 1 mg/mL. DLS results show that the HSA molecules adsorb onto the surfaces of the C60 nanoparticles, thereby forming a protective layer, and prevent salt-induced coagulation. DLS results also indicate that the HSA molecules take an expanded conformation on the surface. Our findings suggest that C60 nanoparticles can be stabilized in the physiological environment even if they are not deliberately stabilized by using stabilizers and are of significant implications for the on-going efforts to evaluate the cytotoxicity of C60 nanoparticles in which no such effect has been considered. Introduction Concern has been raised for the potential environmental impact of nanostructured materials (1), and nanoparticles of C60 have attracted special attention in this regard. The interest arises from the unique property of the C60 nanoparticles to form stable colloidal dispersions in various solvents including water, in which molecular solubility of C60 is negligibly small (2-8). Such dispersions were expected to be promising as the basis for developing new biotechnological applications of C60 without performing chemical derivatization to endow water solubility (9, 10). However, ever since it was reported that C60 nanoparticles induced oxidative stress on the brain of fish (11), the cytotoxicity of some C60 nanoparticles dispersed in water has attracted strong public attention and has been discussed intensively in the recent years (7, 12-20). Toxicological studies of the nanoparticles in general are different from those of conventional molecules in several aspects, and dispersion stability is one of them (21). In the case of C60 nanoparticles, they remain dispersed for a long period of time in pure water but coagulate rapidly upon the addition of electrolytes. Because dispersion stability is one of the key factors determining the transport and deposition of C60 nanoparticles in actual aquatic systems, it has been studied extensively with respect to electrolyte concentration, pH, and preparation procedure (5, 13, 19, 22-25). Coagulation in the * Corresponding author. Phone: +81 46 867 9679. Fax: +81 46 867 9715. E-mail: [email protected]. † Japan Agency for Marine-Earth Science and Technology (JAMSTEC). ‡ Toyo University. § Present address: Department of Research Superstar Program, Organization for the Promotion of Advanced Research, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.

presence of electrolytes can be suppressed by using appropriate stabilizers, such as surfactants or water-soluble polymers (26), but except for a few, most toxicological studies of C60 nanoparticles have been done primarily without using the stabilizer (16, 27). Thus, it seems likely that C60 nanoparticles would have coagulated when their toxicities were studied. This makes it difficult to deduce the interaction between C60 nanoparticles and living cells because change in size associated with coagulation is known to affect transport and cellular uptake kinetics of the nanoparticle (28). However, the physiological environments including body fluids or media for cell culture are far more complicated in that they contain various biological molecules such as proteins as well as various electrolytes. Because of the interaction between C60 nanoparticles and biological molecules, dispersion stability in physiological environments could be different from the results in model systems that typically contain C60 nanoparticles, water, and simple electrolytes (5, 13, 19, 22-25). For example, adsorption of proteins on surfaces (29) may prevent the coagulation induced by electrolytes, but such an effect has not been considered so far. In this communication, we report the dispersion stability of C60 nanoparticles under a model condition simulating the physiological environment. Special attention is paid to the stabilizing effect of human serum albumin (HSA), which is the major component of blood plasma and serves as a carrier of hydrophobic compounds, such as fatty acids, bilirubin, and hormones (30).

Materials and Methods Materials. C60 (>99.9% pure) was purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). Polyoxyethylene sorbitane monooleaate (Tween 80) was purchased from ICN Pharmaceuticals,

10.1021/tx6003198 CCC: $37.00 © 2007 American Chemical Society Published on Web 05/16/2007

Communications

Chem. Res. Toxicol., Vol. 20, No. 6, 2007 855

Inc. (Costa Mesa, California). Human serum albumin (HSA) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) as lyophilized powder. All of the chemicals were used as received. Preparation of Aqueous Dispersion of C60 Nanoparticles. C60 nanoparticles were prepared by the method that we call MARS (mechano assisted reduction of size) (7, 8). In this facile method, the nanoparticles were obtained by simple hand-grinding of bulk C60 solids in an agate mortar for several minutes. Ground C60 (10.4 mg) containing the nanoparticles was then mixed with 5 mL of water, and the mixture was subjected to ultrasonic treatment for 30 min at 9-11 °C. After sonication, the mixture was spun at 1000g for 30 min, and the supernatant was filtered with a membrane filter (0.45 µm nominal pore size, Millex-HV, Millipore) to obtain a stock dispersion of the C60 nanoparticles. The dispersion was found to contain 1.86 × 10-4 M (133.9 µg/mL) of C60; 6% (w/w) of C60 initially added remained dispersed in water. The mean hydrodynamic diameter of the C60 nanoparticles was 152.0 nm by dynamic light scattering (DLS) (FDLS-1200, Otsuka Electronics, Co. Ltd, Osaka, Japan). Dispersion Stability Measurements. The stock dispersion of the C60 nanoparticles (0.1 mL) was first mixed with water (1.5 mL) containing a desired amount of Tween 80 or HSA. To the mixture, 0.2 mL of a 10-strength phosphate buffered saline (PBS(-)) (NaCl (1.37 M), KCl (26.8 mM), Na2HPO4‚12H2O (81.0 mM), KH2PO4 (14.7 mM)) and 0.2 mL of an aqueous solution of CaCl2 (9 mM) and MgCl2 (3.3 mM) were added. The mixture was immediately filtered with a membrane filter (nominal pore size, 5 µm) into a cylindrical measuring cell, and the change of the hydrodynamic diameter of the C60 nanoparticles was followed by DLS at 25 °C. The measurements were done by using a He-Ne laser (λ ) 628 nm, 100 mW). The pH of the mixture was 7.4. The concentration of C60 in the final mixtures was 9.3 × 10-6 M (6.7 µg/mL) and was fixed in all of the experiments reported.

Figure 1. Size distribution of the C60 nanoparticles used in this work (mean hydrodynamic diameter, 152.0 nm). The distribution was calculated by analyzing the autocorrelation function from a DLS measurement by CONTIN.

Results Preparation of C60 Nanoparticles by MARS. Preparation of C60 nanoparticles by MARS relies on an unusual solid property of C60. A top-down approach such as grinding, in which bulk solids are reduced to small particles by applying mechanical forces, is an obvious approach to prepare small particles. However, the approach produces particles on the order of micrometers in size, and nanoparticles are not obtained unless very high energy is applied using a special device such as a high-energy ball mill (31, 32). In the case of C60, the nanoparticles including the ones as small as 20 nm can be produced by simple hand grinding of a bulk solid in an agate mortar. The nanoparticles thus obtained can be readily dispersed in water (7). As is often the case with particles prepared by a top-down approach (33), the nanoparticles obtained by MARS have a rather broad size distribution (7, 8), but centrifugation of the dispersion followed by filtration effectively narrows down the size distribution (Figure 1). Dispersion Stability in Simulated Physiological Conditions. C60 nanoparticles remained stably dispersed in pure water (Figure 2A). However, upon addition of phosphate buffered saline (PBS(+)), they coagulated rapidly and eventually precipitated at the bottom of the container (Figure 2B). The addition of a nonionic surfactant, Tween 80, at the concentration of 1 mg/mL suppressed coagulation (Figure 2C). Interestingly, coagulation was also suppressed when HSA was added at the concentration of 1 mg/mL (Figure 2E). No such stabilizing effect was seen when HSA concentration was decreased to 0.1 mg/ mL (Figure 2D). The coagulation process was studied in detail by DLS. Figure 3 shows the time-dependent change of the hydrodynamic diameter of the C60 nanoparticles measured by DLS for 3 h

Figure 2. Photograph showing dispersions of C60 nanoparticles. C60 nanoparticles stably dispersed in pure water (A) but coagulated upon addition of PBS(+) and precipitated at the bottom of the container (B). A nonionic surfactant, Tween 80, at a concentration of 1 mg/mL prevented coagulation (C). Coagulation was also suppressed by human serum albumin at a concentration of 1 mg/mL (E), but no such stabilizing effect was seen when HSA concentration was decreased to 0.1 mg/mL (D). The photograph was taken 24 h after the addition of PBS(+). [C60] ) 9.3 × 10-6 M.

after the addition of PBS(+). Without any stabilizing agents, the coagulation of C60 nanoparticles started immediately after the addition of electrolytes. The size increased rapidly with time and reached 1 µm in 3 h. However, such a steep increase in size was not seen when 1 mg/mL of Tween 80 or HSA was present, confirming the previous observation (Figure 2). The dispersion stability of the C60 nanoparticles in the simulated physiological condition depended on the concentration of HSA (Figure 4). At the concentration of 0.1 and 0.5 mg/mL, the hydrodynamic diameter increased with time after the addition of PBS(+), although the increase was much less steep compared with the case without HSA (Figure 3). Coagulation was completely suppressed when HSA concentration was increased to 1 mg/mL. Further increase of HSA concentration up to 10 mg/mL did not change the result. Preliminary experiments showed that in the presence of 5 mg/mL of HSA, the hydrodynamic diameter of the C60 nanoparticles in PBS(+)

856 Chem. Res. Toxicol., Vol. 20, No. 6, 2007

Figure 3. Change of the hydrodynamic diameter of C60 nanoparticles as a function of time after the addition of PBS(+). [C60] ) 9.3 × 10-6 M (6.7 µg/mL). [Tween80], [HSA] ) 1 mg/mL.

Figure 4. Change of the hydrodynamic diameter of C60 nanoparticles as a function of time after the addition of PBS(+). The measurements were taken in the presence of HSA at different concentrations. [C60] ) 9.3 × 10-6 M (6.7 µg/mL).

increased slightly after storage for 1 week at 4 °C, but the increase was not more than 5% of the initial size. Stabilization Mechanism. HSA is a heart-shaped molecule with approximate dimensions of 8 × 8 × 3 nm (34). It was reported that human and bovine serum albumins form complexes with water soluble derivatives of C60 by incorporating the C60 molecule into the binding pocket (35, 36). It was also predicted by computer simulation that human and bovine serum albumins could form similar complexes even with underivatized C60 molecules (36). However, such complexations cannot be expected between the HSA molecules and the nanoparticles of C60, simply because the size of the nanoparticles is significantly larger than the HSA molecule. However, serum albumin is known to nonspecifically adsorb on surfaces of various carbon materials, such as graphite and carbon nanotubes (29). The spontaneous adsorption is attributed to hydrophobic interactions (29). Although no such interaction is known for the solid surface of C60, the DLS results show that similar adsorption takes place for HSA and the C60 nanoparticles.

Communications

Figure 5. Change of the hydrodynamic diameter of C60 nanoparticles as a function of added HSA concentration. The hydrodynamic diameter in the presence of HSA was obtained by averaging the values plotted in Figure 4.

By averaging out the hydrodynamic diameters measured at different time intervals in the presence of 1, 5, and 10 mg/mL of HSA (Figure 4), the hydrodynamic diameters of the C60 nanoparticles in the presence of HSA were obtained. The values were 154.5, 155.0, and 153.6 nm in the presence of 1, 5, and 10 mg/mL of HSA, respectively, and all of the values were slightly larger than the size before the addition of HSA (152.0 nm) (Figure 5). The slight increase in size could be ascribed to the adsorption of the HSA molecules on the surfaces of C60 nanoparticles. The adsorbed HSA molecules form a protective layer on the surfaces, and the C60 nanoparticles are sterically stabilized even in PBS(+) (26). In this case, the increase in size (2.5, 3.0, and 1.6 nm in the presence of 1, 5, and 10 mg/ mL of HSA, respectively) corresponds to the thickness of the adsorbed HSA layer. The thickness does not appear to depend very much on HSA concentration, indicating that the surfaces of the C60 nanoparticles are already fully covered when HSA is added at the concentration of 1 mg/mL, and further addition of HSA does not lead to adsorption. It is interesting to note that the calculated layer thickness (1.2 nm) is significantly smaller than the size of the HSA molecule, suggesting that HSA molecules would take expanded conformations on the hydrophobic surface of the C60 nanoparticles (37).

Discussion Our results clearly demonstrate that C60 nanoparticles can be stabilized by nonspecific adsorption of HSA and remain well dispersed even in the physiological environment. Our findings are of significance in evaluating the in Vitro cytotoxicity of C60 nanoparticles using microorganisms or animal cells. For the experiments using microorganisms, coagulation of C60 nanoparticles in various culture media was studied in detail (19). It was found that coagulation could be suppressed by decreasing the concentrations of electrolytes (19), and cytotoxicity has been studied in such modified media (7, 13, 14, 19). However, nothing is known about the dispersion stability in culture media for animal cells, although the results are more relevant when considering the potential risk of C60 nanoparticles with respect to human health. However, such media typically contain serum and thus serum albumin, and our results suggest that a similar

Communications

Chem. Res. Toxicol., Vol. 20, No. 6, 2007 857

stabilization mechanism by serum albumin comes into effect in these media. The stabilizing effect of HSA was observed when its concentration was higher than 1 mg/mL under the present experimental condition (fixed C60 concentration of 9.3 × 10-6 M (6.7 µg/mL)). Serum albumin exists in the blood of most living organisms at a concentration of 40 mg/mL (38). Thus, our results suggest that stabilization by serum albumin would also work in the blood stream. However, such a stabilizing effect depends on the molar ratio between serum albumin and C60, and further studies are necessary to clarify the significance of the stabilizing effect by protein adsorption in ViVo, where substantially higher concentrations of C60 (3 orders of magnitude higher than the concentration used in this work) are sometimes administered (16). Besides dispersion stability, our findings highlight the importance of protein/C60 interactions in considering the biological activities of C60 nanoparticles. For example, it has been recently reported that cellular uptake of gold nanoparticles is mediated by nonspecific adsorption of serum proteins onto the gold surface (39). The gold nanoparticles enter into the cells via the receptor-mediated endocytosis pathway, in which the protein on the surface binds to a receptor on the cell’s surface, and the particles then enter the cell when the membrane invaginates. It is probable that HSA molecules on the surface of C60 nanoparticles affect cellular uptake (40, 41) in the same way. In summary, the dispersion stability of C60 nanoparticles in a model condition simulating a physiological environment was studied. We found that the salt-induced coagulation of C60 nanoparticles at a concentration of 9.3 × 10-6 M (6.7 µg/mL) was completely suppressed when HSA was present at concentrations above 1 mg/mL. DLS results show that the HSA molecules adsorb on the surfaces of C60 nanoparticles, thereby forming a protective layer, and prevent coagulation. Our findings suggest that C60 nanoparticles can be stabilized in the physiological environment even if they are not deliberately stabilized by using stabilizers. Such a stabilization mechanism is important in considering the interaction between living cells and C60 nanoparticles. Our findings also indicate that understanding the interactions between proteins and the solid C60 surface is crucial for toxicological studies of C60 nanoparticles.

References (1) Colvin, V. L. (2003) The potential environmental impact of engineered nanomaterials. Nat. Biotechnol. 21, 1166-1170. (2) Scrivens, W. A., Tour, J. M., Creek, K. E., and Pirisi, L. (1994) Synthesis of 14C-labeled C60, its suspension in water, and its uptake by human keratinocytes. J. Am. Chem. Soc. 116, 4517-4518. (3) Andrievsky, G. V., Kosevich, M. V., Vovk, O. M., Shelkovsky, V. S., and Vashchenko, L. A. (1995) On the production of an aqueous colloidal solution of fullerenes. J. Chem. Soc., Chem. Commun. 12811282. (4) Wei, X., Wu, M., Qi, L., and Xu, Z. (1997) Selective solution-phase generation and oxidation reaction of C60n- (n ) 1,2) and formation of an aqueous colloidal solution of C60. J. Chem. Soc., Perkin Trans. 1997, 1389-1394. (5) Deguchi, S., Alargova, R. G., and Tsujii, K. (2001) Stable dispersions of fullerenes, C60 and C70, in water. Preparation and characterization. Langmuir 17, 6013-6017. (6) Alargova, R. G., Deguchi, S., and Tsujii, K. (2001) Stable colloidal dispersions of fullerenes in polar organic solvents. J. Am. Chem. Soc. 123, 10460-10467. (7) Deguchi, S., Mukai, S., Tsudome, M., and Horikoshi, K. (2006) Facile generation of fullerene nanoparticles by hand grinding. AdV. Mater. 18, 729-732. (8) Deguchi, S., and Mukai, S. (2006) Top-down preparation of dispersions of C60 nanoparticles in organic solvents. Chem. Lett. 35, 396-397. (9) Jensen, A. W., Wilson, S. R., and Schuster, D. I. (1996) Biological applications of fullerenes. Bioorg. Med. Chem. 4, 767-779.

(10) Nakamura, E., and Isobe, H. (2003) Functionalized fullerenes in water. The first 10 years of their chemistry, biology, and nanoscience. Acc. Chem. Res. 36, 807-815. (11) Oberdo¨rster, E. (2004) Manufactured nanomaterials (fullerenes, C60) induce oxidative stress in the brain of juvenile largemouth bass. EnViron. Health Perspect. 112, 1058-1062. (12) Sayes, C. M., Fortner, J. D., Guo, W., Lyon, D., Boyd, A. M., Ausman, K. D., Tao, Y. J., Sitharaman, B., Wilson, L. J., Hughes, J. B., West, J. L., and Colvin, V. L. (2004) The differential cytotoxicity of watersoluble fullerenes. Nano Lett. 4, 1881-1887. (13) Fortner, J. D., Lyon, D. Y., Sayes, C. M., Boyd, A. M., Falkner, J. C., Hotze, E. M., Alemany, L. B., Tao, Y. J., Guo, W., Ausman, K. D., Colvin, V. L., and Hughes, J. B. (2005) C60 in water: Nanocrystal formation and microbial response. EnViron. Sci. Technol. 39, 43074316. (14) Lyon, D. Y., Fortner, J. D., Sayes, C. M., Colvin, V. L. and Hughes, J. B. (2005) Bacterial cell association and antimicrobial activity of a C60 water suspension. EnViron. Toxicol. Chem. 24, 2757-2762. (15) Andrievsky, G., Klochkov, V., and Derevyanchenko, L. (2005) Is the C60 fullerene molecule toxic? Fullerenes, Nanotubes, Carbon Nanostruct. 13, 363-376. (16) Gharbi, N., Pressac, M., Hadchouel, M., Szwarc, H., Wilson, S. R., and Moussa, F. (2005) [60]Fullerene is a powerful antioxidant in vivo with no acute or subacute toxicity. Nano Lett. 5, 2578-2585. (17) Isakovic, A., Markovic, Z., Todorovic-Markovic, B., Nikolic, N., Vranjes-Djuric, S., Mirkovic, M., Dramicanin, M., Harhaji, L., Raicevic, N., Nikolic, Z., and Trajkovic, V. (2006) Distinct cytotoxic mechanisms of pristine versus hydroxylated fullerene. Toxicol. Sci. 91, 173-183. (18) Lovern, S. B., and Klaper, R. (2006) Daphnia magna mortality when exposed to titanium dioxide and fullerene (C60) nanoparticles. EnViron. Toxicol. Chem. 25, 1132-1137. (19) Lyon, D. Y., Adams, L. K., Falkner, J. C., and Alvarez, P. J. J. (2006) Antibacterial activity of fullerene water suspensions: Effects of preparation method and particle size. EnViron. Sci. Technol. 40, 43604366. (20) Oberdo¨rster, E., Zhu, S., Blickley, T. M., McClellan-Green, P., and Haasch, M. L. (2006) Ecotoxicology of carbon-based engineered nanoparticles: effects of fullerene (C60) on aquatic organisms. Carbon 44, 1112-1120. (21) Wiesner, M. R., Lowry, G. V., Alvarez, P., Dionysiou, D., and Biswas, P. (2006) Assessing the risks of manufactured nanomaterials. EnViron. Sci. Technol. 40, 4336-4345. (22) Mchedlov-Petrossyan, N. O., Klochkov, V. K., and Andrievsky, G. V. (1997) Colloidal dispersions of fullerene C60 in water: Some properties and regularities of coagulation by electrolytes. J. Chem. Soc., Faraday Trans. 93, 4343-4346. (23) Brant, J., Lecoanet, M., Hotze, M., and Wiesner, M. (2005) Comparison of electrokinetic properties of colloidal fullerenes (n-C60) formed using two procedures. EnViron. Sci. Technol. 39, 6343-6351. (24) Brant, J., Lecoanet, H., and Wiesner, M. R. (2005) Aggregation and deposition characteristics of fullerene nanoparticles in aqueous systems. J. Nanopart. Res. 7, 545-553. (25) Brant, J. A., Labille, J., Bottero, J.-Y., and Wiesner, M. R. (2006) Characterizing the impact of preparation method on fullerene cluster structure and chemistry. Langmuir 22, 3878-3885. (26) Israelachvili, J. (1992) Intermolecular and Surface Forces, Academic Press, London. (27) Moussa, F., Trivin, F., Ceolin, R., Hadchouel, M., Sizaret, P. Y., Greugny, V., Fabre, C., Rassat, A., and Szwarc, H. (1996) Early effects of C60 administration in swiss mice: a preliminary account for in vivo C60 toxicity. Fullerene Sci. Technol. 4, 21-29. (28) Limbach, L. K., Li, Y., Grass, R. N., Brunner, T. J., Hintermann, M. A., Muller, M., Gunther, D., and Stark, W. J. (2005) Oxide nanoparticle uptake in human lung fibroblasts: effects of particle size, agglomeration, and diffusion at low concentrations. EnViron. Sci. Technol. 39, 9370-9376. (29) Chen, R. J., Bangsaruntip, S., Drouvalakis, K. A., Kam, N. W. S., Shim, M., Li, Y., Kim, W., Utz, P. J., and Dai, H. (2003) Noncovalent functionalization of carbon nanotubes for highly specific electronic biosensors. Proc. Natl. Acad. Sci. U.S.A. 100, 4984-4989. (30) Voet, D., and Voet, J. G. (2004) Biochemistry, 3rd ed., John Wiley & Sons, Hoboken, NJ. (31) Koch, C. C. (1989) Materials synthesis by mechanical alloying. Annu. ReV. Mater. Sci. 19, 121-143. (32) Basset, D., Matteazzi, P., and Miani, F. (1993) Designing a high energy ball-mill for synthesis of nanophase materials in large quantities. Mater. Sci. Eng., A 168, 149-152. (33) Maa, Y.-F., and Prestrelski, S. J. (2000) Biopharmaceutical powders: particle formation and formulation considerations. Curr. Pharm. Biotechnol. 1, 283-302.

858 Chem. Res. Toxicol., Vol. 20, No. 6, 2007 (34) Sugio, S., Kashima, A., Mochizuki, S., Noda, M., and Kobayashi, K. (1999) Crystal structure of human serum albumin at 2.5 Å resolution. Protein Eng. 12, 439-446. (35) Belgorodsky, B., Fadeev, L., Ittah, V., Benyamini, H., Zelner, S., Huppert, D., Kotlyar, A. B., and Gozin, M. (2005) Formation and characterization of stable human serum albumin-tris-malonic acid [C60]fullerene complex. Bioconjugate Chem. 16, 1058-1062. (36) Benyamini, H., Shulman-Peleg, A., Wolfson, H. J., Belgorodsky, B., Fadeev, L., and Gozin, M. (2006) Interaction of C60-fullerene and carboxyfullerene with proteins: docking and binding site alignment. Bioconjugate Chem. 17, 378-386. (37) Raffaini, G., and Ganazzoli, F. (2003) Simulation study of the interaction of some albumin subdomains with a flat graphite surface. Langmuir 19, 3403-3412. (38) Li, Y., He, W., Liu, J., Sheng, F., Hu, Z., and Chen, X. (2005) Binding of the bioactive component jatrorrhizine to human serum albumin. Biochim. Biophys. Acta 1722, 15-21.

Communications (39) Chithrani, B. D., Ghazani, A. A., and Chan, W. C. W. (2006) Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. Nano Lett. 6, 662-668. (40) Moussa, F., Chretien, P., Dubois, P., Chuniaud, L., Dessante, M., Trivin, F., Sizaret, P. Y., Agafonov, V., Ceolin, R., Szwarc, H., Greugny, V., Fabre, C., and Rassat, A. (1995) The influence of C60 powders on cultured human-leukocytes. Fullerene Sci. Technol. 3, 333-342. (41) Porter, A. E., Muller, K., Skepper, J., Midgley, P., and Welland, M. (2006) Uptake of C60 by human monocyte macrophages, its localization and implications for toxicity: studied by high resolution electron microscopy and electron tomography. Acta Biomaterialia 2, 409-419.

TX6003198