Effects of Surface Chemistry on Cytotoxicity ... - ACS Publications

Sep 1, 2010 - CSIRO Materials Science and Engineering, CSIRO Future ... CSIRO Food and Nutritional Sciences, CSIRO Future Manufacturing Flagship, ...
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Effects of Surface Chemistry on Cytotoxicity, Genotoxicity, and the Generation of Reactive Oxygen Species Induced by ZnO Nanoparticles Hong Yin* and Philip S. Casey CSIRO Materials Science and Engineering, CSIRO Future Manufacturing Flagship, Clayton VIC 3168, Australia

Maxine J. McCall CSIRO Food and Nutritional Sciences, CSIRO Future Manufacturing Flagship, North Ryde NSW 2113, Australia

Michael Fenech CSIRO Food and Nutritional Sciences, CSIRO Future Manufacturing Flagship, Adelaide SA 5000, Australia Received March 15, 2010. Revised Manuscript Received August 12, 2010 The relationship between the toxicity of zinc oxide (ZnO) nanoparticles (NPs) and their surface chemistry was investigated. Cytotoxicity, genotoxicity, and the ability to generate reactive oxygen species (ROS) were assessed for wellcharacterized ZnO NPs whose surface chemistry was varied from its pristine state by coating with oleic acid (OA), poly(methacrylic acid) (PMAA), or components adsorbed from cell culture medium (medium-soaked). It was found that uncoated NPs showed ROS accumulation and diminished cell viability whereas all tested surface coatings assisted in reducing ROS production and cytotoxicity. The ability of coatings to reduce the cytotoxicity of ZnO NPs was ranked in the following order: medium-soaked ≈ PMAA > OA. However, PMAA-coated ZnO had significant genotoxicity compared to uncoated ZnO and the other coated NPs, highlighting the need to investigate thoroughly the effects of NP surface modification on both cytotoxicity and genotoxicity assays. The lowest toxicity was achieved with a surface coating of components from a cell culture medium.

1. Introduction Engineered nanoparticles (NPs) are widely used in commercial products such as catalysts, cosmetics, microelectronic devices, semiconductors, sporting goods, and textiles. Owing to their small size and large specific surface area, NPs exhibit unique physicochemical properties that may differ dramatically from their bulk counterparts. For example, zinc oxide (ZnO) NPs efficiently absorb ultraviolet light and are also highly transparent to visible light, but larger submicrometer- and micrometer-sized ZnO particles do not have this combination of properties. Thus, ZnO NPs have been widely used in products where UV protection from a transparent coating is required, such as in varnishes to protect wood and sunscreens to protect skin. The extremely small size of NPs increases the possibility of their uptake by cells and interactions with biological molecules and tissues, thereby providing advantageous opportunities but also potential risks for their application. Some recent studies have shown that ZnO NPs can be toxic to a wide variety of biological systems, including epidermal cells,1 bacteria (Streptococcus agalactiae and Staphylococcus aureus),2 zebra fish (Danio rerio),3 and *Corresponding author. E-mail: [email protected]. (1) Sharma, V.; Shukla, R. K.; Saxena, N.; Parmar, D.; Das, M.; Dhawan, A. Toxicol. Lett. 2009, 185, 211. (2) Huang, Z. B.; Zheng, X.; Yan, D. H.; Yin, G. F.; Liao, X. M.; Kang, Y. Q.; Yao, Y. D.; Huang, D.; Hao, B. Q. Langmuir 2008, 24, 4140. (3) Zhu, X. S.; Zhu, L.; Duan, Z. H.; Qi, R. Q.; Li, Y.; Lang, Y. P. J. Environ. Sci. Health, Part A: Environ. Sci. Eng. 2008, 43, 278. (4) Wang, B.; Feng, W. Y.; Wang, M.; Wang, T. C.; Gu, Y. Q.; Zhu, M. T.; Ouyang, H.; Shi, J. W.; Zhang, F.; Zhao, Y. L.; Zhai, Z. F.; Wang, H. F.; Wang, J. J. Nanopart. Res. 2008, 10, 263.

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mice.4 Moreover, ZnO was more toxic to Escherichia coli than other metal oxide NPs such as Fe2O3, Y2O3, TiO2, and CuO.5 One possible reason for the observed toxicity is that nanosized ZnO is an excellent photocatalyst in which free electrons and holes could be generated by light stronger than its band gap energy. These electron-hole pairs diffuse out to the surface and transform the surrounding oxygen or water molecules into hydroxyl radicals via strong oxidization.6 The surface chemistry of NPs is one of the critical factors determining cellular responses in vitro.7 Hoshino et al.8 investigated a series of five different surface-modified CdSe/ZnS quantum dots (QDs) in EL-4 murine cancer cells and reported that surface chemistry was an indicator of toxicity rather than the core material. It was observed that QDs coated with carboxyl groups were less toxic than QDs with an amine surface coating. Yin et al.9 reported that the cytotoxicity of nickel ferrite NPs could be reduced by an oleic acid surface layer. The NPs with one layer of oleic acid (hydrophobic surface, with -CH3 as the functional group exposed to cells) were more cytotoxic to Nero2A cells than particles with two layers of oleic acid (hydrophilic surface, with -COOH as the functional group exposed to cells). A similar impact of surface chemistry was also found for colloidal fullerene NPs that induced high levels of cytotoxicity because of the (5) Hu, X. K.; Cook, S.; Wang, P.; Hwang, H. M. Sci. Total Environ. 2009, 407, 3070. (6) Jang, Y. J.; Simer, C.; Ohm, T. Mater. Res. Bull. 2006, 41, 67. (7) Colvin, V. L. Nat. Biotechnol. 2003, 21, 1166. (8) Hoshino, A.; Fujioka, K.; Oku, T.; Suga, M.; Sasaki, Y. F.; Ohta, T.; Yasuhara, M.; Suzuki, K.; Yamamoto, K. Nano Lett. 2004, 4, 2163. (9) Yin, H.; Too, H. P.; Chow, G. M. Biomaterials 2005, 26, 5818.

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formation of O2- anions and lipid peroxidation; modification of the fullerene surface by the attachment of hydroxyl groups resulted in reduced toxicity to mouse L929 fibrosarcoma, rat C6 glioma, and U251 human glioma cell lines.10 These observations indicate that the toxicity of ZnO NPs may be controlled by changing their surface chemistry. However, few studies on coated ZnO NPs have been reported.11,12 In this article, we have investigated four variations of the surface chemistry on ZnO NPs, which included ZnO with no coating (uncoated) or ZnO with coatings of oleic acid (OA), poly(methacrylic acid) (PMAA), or components from the supplemented RPMI 1640 medium (medium-coated). It is generally accepted that a comprehensive, accurate characterization of particle size, morphology, composition, and surface chemistry is essential for developing our understanding of the parameters that contribute to nanotoxicity. Recently, Murdock et al.13 addressed this issue, and Jiang et al.14 have also emphasized the significance of understanding the state of NPs for toxicity investigations. Here, we have extensively characterized the four types of NPs studied for insight into the nature of the coatings and for their physical and chemical properties. Their effects on cells were assessed by determining the cytotoxicity, genotoxicity, and ability to generate ROS in WIL2-NS human lymphoblastoid cells. The influence of surface chemistry on the physical and chemical properties of the ZnO NPs and the associated biological effects are discussed.

2. Materials and Methods 2.1. Materials. ZnO NPs were prepared using a proprietary process developed by CSIRO Materials Science and Engineering (Melbourne, Australia) with precursors purchased from SigmaAldrich. Interested readers may contact the corresponding author to discuss the supply of these ZnO NPs for academic research. The chemicals used for surface coating, such as oleic acid (cell culture tested) and poly(methacrylic sodium salt) solution (average Mw = 4000-6000, 40 wt % in H2O), were also purchased from Sigma-Aldrich. Biological reagents used for experiments with cells, such as RPMI 1640 medium, fetal bovine serum (FBS), L-glutamine (200 mM), penicillin/streptomycin (with 10 000 units penicillin and 10 mg of streptomycin/mL), 2,7-dichlorofluoroscein diacetate (DCFH-DA, g97%), 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (methyl tetrazolium, MTT, g97.5%), cytochalasin B (cyt-B, g98%), sodium dodecyl sulfate (SDS, g98.5%), phosphate-buffered saline (PBS, pH 7.4 biotechnology performance certified), and dimethyl sulfoxide (DMSO, g99.9%) were purchased from Sigma-Aldrich. Diff Quick fixative, stain 1, and stain 2 were purchased from Lab Aids (Sydney, Australia). 2.2. Methods. 2.2.1. NPs Coated with OA. Three grams of ZnO NPs were mixed with 50 mL of a 0.1 M OA solution. The mixture was placed in an ultrasonic bath and sonicated for 30 min. The mixture was centrifuged at 10 000 rpm for 10 min, and the collected particles were washed thoroughly with deionized water. This centrifugation and washing cycle was repeated twice, and the obtained particles were dried overnight under vacuum at 50 °C.15 2.2.2. NPs Coated with PMAA. Three grams of ZnO NPs were mixed with 20 mL of 40% poly(methacrylic sodium salt) (10) Isakovic, A.; Markovic, Z.; Todorovic-Markovic, B.; Nikolic, N.; VranjesDjuric, S.; Mirkovic, M.; Dramicanin, M.; Harhaji, L.; Raicevic, N.; Nikolic, Z.; Trajkovic, V. Toxicol. Sci. 2006, 91, 173. (11) Scheckel, K. G.; Luxton, T. P.; El Badawy, A. M.; Impellitteri, C. A.; Tolaymat, T. M. Environ. Sci. Technol. 2010, 44, 1307. (12) Osmond, M. J.; McCall, M. J. Nanotoxicology 2010, 4, 15. (13) Murdock, R. C.; Braydich-Stolle, L.; Schrand, A. M.; Schlager, J. J.; Hussain, S. M. Toxicol. Sci. 2008, 101, 239. (14) Jiang, J. K.; Oberdorster, G.; Biswas, P. J. Nanopart. Res. 2009, 11, 77. (15) Tang, E. J.; Cheng, G. X.; Ma, X. L.; Pang, X. S.; Zhao, Q. Appl. Surf. Sci. 2006, 252, 5227.

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solution. The mixture was magnetically stirred at 80 °C for 30 min in a water bath. The obtained slurry was then ultrasonicated for 30 min, followed by centrifugation, washing, and drying as described above.16 2.2.3. NPs Coated with Cell Culture Medium. Three grams of ZnO NPs were soaked in supplemented cell culture medium consisting of RPMI 1640 medium, 5% FBS, 1% L-glutamine, and 1% penicillin/streptomycin for 168 h. This medium, together with the soaked NPs, was used directly for cell culturing. For the physicochemical characterization of NPs following soaking, the NPs were centrifuged and dried under vacuum overnight at room temperature. 2.2.4. Physicochemical Characterization. All physicochemical characterizations listed below were carried out at room temperature. The surfaces of NPs were analyzed by X-ray photoelectron spectroscopy (XPS; ESCA LAB 220i-XL Thermo VG Scientific, U.K.). Eight wells of a powder sample holder (two wells per sample) were filled with as-prepared coated and uncoated ZnO NPs. The sampling depth was less than 10 nm, and the analysis area was approximately 0.5 mm  0.7 mm. XPS data files were processed using the application CasaXPS software (version 2.3.13). Crystalline phases were identified by X-ray diffraction (XRD) using a Bruker D8 Advance diffractometer. Interactions between the coatings and surfaces of ZnO NPs were investigated by Fourier transform infrared (FTIR) spectroscopy using a Perkin-Elmer FTIR 2000. As-prepared coated and uncoated ZnO NPs were mixed with potassium bromide (KBr, FTIR grade) and milled into fine powder. This powder was then compressed into a thin pellet used for analysis. A pure KBr pellet was used as background. The morphologies of the NPs were studied using images taken on a transmission electron microscope (TEM, JEOL, 100CX-II, Japan); the particle size and distribution were assessed by measuring the dimensions of 100 particles of each type. The surface charges of NPs suspended in different media were measured with a Malvern Zeta Sizer 2000.

2.2.5. Dissolution of ZnO NPs. 2.2.5.1. Dialysis Method. The dissolution of NPs was investigated using Cole Parmer (Vernon Hill, IL) Spectra/Por 7 dialysis membranes with a 1000 Da molecular-weight cutoff. The 11.5-mm-diameter tubing was cut into 7 cm lengths and immersed in deionized water for 30 min and then rinsed thoroughly prior to use. Solutions placed inside the dialysis cells were prepared by adding 0.5 mg of ZnO NPs to 5 mL of supplemented RPMI 1640 medium (concentration of ZnO NPs was 100 mg/L). The dialysis cell was put into 500 mL of a receiving solution consisting of a supplemented RPMI medium that was continuously stirred at 4 °C. For each sampling point, 20 mL of receiving solution was extracted for analysis by inductively coupled plasma-atomic emission spectroscopy (ICP-AES, Varian Vista AX Simultaneous Axial) to determine the Zn2þ concentration. 2.2.5.2. Centrifugation Method. ZnO NPs (50 mg) were put into 50 mL of supplemented RPMI 1640 cell culture medium. The suspension was magnetically stirred at 4 °C. At each sampling point, 1.5 mL aliquots were taken out and centrifuged at 10 000 rpm for 30 min with cooling, 0.1 mL of supernatant was added to 0.9 mL of water, and the resulting zinc solution was measured by ICP-AES as described above. 2.2.6. MTT Assay. WIL2-NS human lymphoblastoid cells were cultured and maintained in RPMI 1640 medium supplemented with 5% FBS, 1% L-glutamine, and 1% penicillin/ streptomycin. Cells were seeded into 96-well plates with each well receiving a volume of 100 μL at a density of 1  105 cells/mL. ZnO NPs were suspended in supplemented RPMI 1640 medium at twice the desired final concentrations of 50, 35, 20, 10, and 2 mg/L and ultrasonicated for 30 min to minimize agglomeration. Then 100 μL of each particle suspension was added to the test cells, and 100 μL of supplemented medium with no ZnO NPs was added to the control cells. Cells in plates were cultured at 37 °C under a (16) Hong, R. Y.; Pan, T. T.; Qian, J. Z.; Li, H. Z. Chem. Eng. J. 2006, 119, 71.

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Figure 1. TEM images of ZnO NPs with various surface coatings. (a) Uncoated NPs dispersed in water, with particle size 29 ( 10 nm. (b) OAcoated NPs dispersed in hexane, with an inset of the same sample at higher magnification showing the uniform OA surface layer. (c) PMAAcoated NPs dispersed in water and then stained with 2% uranyl acetate. (d) Medium-coated NPs, obtained by soaking in supplemented medium for 168 h, dispersed in water. humidified atmosphere with 5% CO2 for 4, 24, or 48 h. MTT was dissolved in PBS at 5 mg/mL, and then 20 μL of this solution was added to each well to give a concentration of 0.5 mg/mL. The cells were incubated at 37 °C for another 3 h, after which 80 μL of 20% SDS in 0.02 M HCl was added to each well and mixed thoroughly, and the cells were incubated for another 4 h. The optical density (OD) at 570 nm was determined using an ELISA microplate reader (MPR-30-6VP) with 630 nm as a reference wavelength. The OD of the background was determined using supplemented medium containing NPs but no cells. The results are presented as the percentage viability of cells exposed to ZnO NPs relative to cells not exposed. All experiments were repeated between 5 and 15 times. Statistical analyses were performed using multiple t tests (Bonferroni) for different treatment groups. 2.2.7. Measurement of ROS. Levels of ROS generated by ZnO NPs in the presence of cells were determined by a fluorometric assay using the intracellular oxidation of DCFH-DA. Cells grown to confluence 24 h after seeding were treated with NPs at a concentration of 10 mg/L for 24 h, washed with PBS, and then incubated with 40 μM DCFH-DA for 30 min. At the end of the incubation, cells were washed with PBS again. The fluorescence of dichlorofluoroscein (DCF), which is the oxidized product of dichlorofluoroscein (DCFH, hydrolyzed from DCFH-DA by intracellular esterases), was measured with a Varian fluorescence spectrophotometer (Cary Eclipse) using an excitation of 485 nm and an emission of 530 nm. The DCF concentration in WIL2-NS cells not exposed to NPs was used as a control. To verify the photocatalytic activity of ZnO NPs and their effects on the formation of DCF, control experiments were carried out using NP concentrations of 10, 20, and 50 mg/L under cell-free conditions. 2.2.8. CBMN Cytome Assay. Cyt-B was dissolved in DMSO at 1.8 mg/mL and sterilized by passage through a 0.2-μm-poresize filter and stored at -20 °C. After WIL2-NS cells were cultured with 10 mg/L NPs for 24 h, Cyt-B was added to reach a final Langmuir 2010, 26(19), 15399–15408

concentration of 4.5 mg/L and the cultures were incubated at 37 °C for another 24 h. Cells were collected onto slides by centrifuging at 600 rpm for 5 min with a Cytospin (Shandon Scientific). Slides were dried in air, fixed for 10 min in Diff Quick fixative, and stained in Diff Quick stain 1 for 10 long dips then stain 2 for 6 long dips (5 s for each dip). For scoring, cells on slides were visualized under oil at an objective magnification of 100 using a Leica DM LB2 microscope. Four hundred cells from each experimental condition were scored and classified to determine the number of mononucleate, binucleate, multinucleate, apoptotic, and necrotic cells. These numbers were used to calculate the percentages of necrotic and apoptotic cells as well as the nuclear division index (NDI), which is a measure of cytostatic effects and the rate of mitotic division. NDI was calculated as follows NDI ¼ ½M1 þ 2ðM2Þ þ 3ðM3Þ þ 4ðM4Þ=N where M1, M2, M3, and M4 are the numbers of viable cells with one, two, three, or four nuclei, respectively, and N is the total number of cells scored (viable and nonviable). Biomarkers for DNA damage were scored in 1000 once-divided binucleated (BN) cells and included micronuclei (MNi), nucleoplasmic bridges (NPB) and nuclear buds (NBud). Criteria for scoring apoptotic cells, necrotic cells, and MNi, NPB, and NBud were as described by Fenech.17 Tests for comparing means of treatment groups were performed using Prism 4.0 (GraphPad, Inc., San Diego, CA) software in which the one-way analysis of variance was selected, followed by Tukey’s post hoc tests. A value of P < 0.05 was considered to be statistically significant.

3. Results and Discussion 3.1. Characterization of ZnO NPs. Analyses of ZnO NPs in TEM images such as shown in Figure 1 indicated that the size (17) Fenech, M. Nat. Protoc. 2007, 2, 1084.

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and shape of particles, the size distribution, and the state of agglomeration were largely unaffected by introducing surface coatings, although the appearance of coated NPs differed slightly compared with that of uncoated NPs. Uncoated ZnO particles (dried from a dispersion in water and shown in Figure 1a) were roughly spherical, although some of the larger particles could be described as thick plates. The size of individual particles was 29 ( 10 nm. OA-coated ZnO particles (dried from a dispersion in hexane, Figure 1b) had a uniform layer clearly visible on the surface of almost every NP. The thickness of the covering layer was measured to be 2 to 3 nm, which is consistent with the fully extended length of one OA molecule (2.5 nm). The PMAA-coated ZnO NPs, dispersed in water and then stained with 2% uranyl acetate to increase the contrast, are shown in Figure 1c. A close inspection reveals a thin, clear (unstained) coating surrounding most of the NPs against a darker (stained) background. In contrast to the clear surfaces and sharp edges of uncoated ZnO NPs, the surfaces of medium-coated NPs were mottled and the edges were less well defined (Figure 1d), which is most likely due to components of the supplemented medium randomly adsorbed on the surfaces and possibly some pitting caused by limited ZnO dissolution. X-ray diffraction patterns obtained from the coated and uncoated ZnO NPs were similar (data not shown), revealing that the internal structure was the same for all four samples with hexagonal wurtzite being the only phase identified. The composition of the surface coatings on the NPs was investigated using XPS. Figure 2a shows the XPS spectra of the C 1s core level for coated and uncoated ZnO NPs. For both medium-coated and PMAA-coated ZnO NPs, a peak was observed at a binding energy of between 288.3 and 288.8 eV that represents carboxylic carbon (O-CdO). For medium-coated ZnO NPs, the component corresponding to C-N, N-CdO, or O-C-O was also observed, indicating the presence of amino acids and/or proteins on the surface. Also on the surfaces of medium-coated ZnO NPs are calcium, phosphorus, nitrogen, and increased amounts of carbon compared with the amount on uncoated NPs, as shown in the survey spectra of medium-coated and uncoated ZnO NPs (Figure 2b). These XPS data indicate that a variety of components, including Ca2þ, PO43-, and amino acids and/or proteins, can adsorb onto the surfaces of uncoated ZnO NPs following soaking in supplemented RPMI medium. The nature of the interactions between the molecular coatings and the surfaces of the ZnO NPs was explored using FTIR spectroscopy. FTIR spectra are shown in Figure 3. For uncoated ZnO NPs, only one specific absorbance was observed at 3430 cm-1 and this was attributed to surface hydroxyl groups (-OH). For OA-coated and PMAA-coated NPs, the peaks at 2924 and 2854 cm-1 were assigned to asymmetric methylene stretches γas(CH2) and symmetric methylene stretches γs(CH2), respectively, in the OA and PMAA molecules. Both OA and PMAA contain carboxylic acid groups (-COOH) that may bind to the NP surfaces through two possible binding modes.18 In one mode, the carboxylate is bound symmetrically to the surface through both oxygen atoms and only a symmetrical C-O stretch at around 1400 cm-1 should appear in the FTIR spectrum. In the other mode, the carboxylate is connected to the surface through one oxygen atom, and both a symmetric C-O stretch (∼1420 cm-1) and an asymmetric stretch (∼1580 cm-1) should be observed. The spectrum for OA-coated ZnO NPs in Figure 3, showing all three of the above-mentioned peaks, revealed that OA molecules are chemically bound to the ZnO surface through carboxyl groups. In contrast, the spectrum for PMAA-coated (18) Yu, S.; Chow, G. M. J. Mater. Chem. 2004, 14, 2781.

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Figure 2. XPS spectra of uncoated and coated ZnO NPs. (a) C 1s core level. (b) Survey spectra of uncoated and medium-coated ZnO NPs.

Figure 3. FTIR spectra of uncoated and coated ZnO NPs.

ZnO NPs shows no peaks around 1400-1600 cm-1, indicating no chemical bonds between PMAA and ZnO or a very low concentration of PMAA on the surface. The spectrum of medium-coated Langmuir 2010, 26(19), 15399–15408

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Article Table 1. Surface Charges of ZnO NPs in Aqueous Solutions (pH 4.0 and 7.5) and RPMI1640 Cell Culture Medium

water pH 4.0 water pH 7.5 RPMI1640 pH 7.5

uncoated ZnO (mV)

OA-coated ZnO (mV)

PMAA-coated ZnO (mV)

medium-coated ZnO (mV)

17.34 4.49 -11.86

-10.98 -10.15 -11.92

-39 -40.21 -15.1

-2.56 -21.96 -11.16

ZnO NPs showed peaks at 1653 and 1538 cm-1 that are distinct vibrational modes of amide I (CdO) and amide II (asymmetric stretch of COO-), most likely from surface-adsorbed amino acids or proteins. On the basis of the structure of -CO-NH- and the properties of hydroxyl groups on the ZnO surface, the interactions between amino acids and ZnO NPs are most likely through hydrogen bonding and electrostatic forces. As reported by Yu et al,19 such interactions can result in a shift of the hydroxyl absorbance to a lower wavenumber in the FTIR spectrum. As shown in Figure 3, such a shift was observed from 3430 cm-1 in the uncoated NPs to 3350 cm-1 in the medium-coated NPs. 3.2. Surface Charges. Patil et al. reported that electrostatic interactions could play an important role in protein adsorption and the cellular uptake of cerium oxide NPs.20 Parab et al. demonstrated that a coating of poly(styrenesulfonate) on the surface of gold nanorods caused a charge reversal and was the main factor in a significant decrease in the cytotoxicity of the nanorods.21 Therefore, in this study, zeta potentials were measured in different media for uncoated and coated ZnO NPs to characterize their surface charges. As shown in Table 1, in aqueous solutions at pH 4.0 and 7.5, uncoated ZnO NPs exhibited zeta potentials of 17.34 and 4.49 mV, respectively, consistent with the reported isoelectric point of ZnO being between 8 and 9.22 In aqueous solutions at pH 4.0 and 7.5, both OA-coated and PMAA-coated ZnO NPs were negatively charged, confirming the presence of OA and PMAA molecules on the surface. The absolute zeta potentials of OAcoated NPs were much smaller than those of PMAA-coated NPs at both pH values, suggesting that the negatively charged carboxylic acid groups in OA were mainly directed toward the surface of the NPs with the hydrophobic ends exposed to solution (confirming the nature of OA binding as determined by FTIR) and the carboxylic acid groups in PMAA were more likely to be randomly arranged on the surface, as might be expected from physical adsorption. The effect of soaking uncoated NPs in supplemented medium for 168 h to produce medium-coated NPs resulted in the zeta potential changing from positive to negative in water (e.g., from 4.49 mV for uncoated NPs to -21.96 mV for medium-coated NPs at pH 7.5), confirming the adsorption of negatively charged species (such as amino acids or protein) on medium-coated ZnO.23 When the zeta potentials were measured in RPMI1640 medium rather than in water, the significant differences between the various ZnO NPs were largely eliminated (Table 1), although PMAA-coated NPs were more negatively charged (with a zeta potential of -15.1 mV) than other NPs (with zeta potentials ranging from -11.16 to -11.92 mV). The leveling out of surface charge could be explained by the very high ionic strength of the cell culture medium.24 The fact that both uncoated (19) Yu, C. H.; Al-Saadi, A.; Shih, S. J.; Qiu, L.; Tam, K. Y.; Tsang, S. C. J. Phys. Chem. C 2009, 113, 537. (20) Patil, S.; Sandberg, A.; Heckert, E.; Self, W.; Seal, S. Biomaterials 2007, 28, 4600. (21) Parab, H. J.; Chen, H. M.; Lai, T. C.; Huang, J. H.; Chen, P. H.; Liu, R. S.; Hsiao, M.; Chen, C. H.; Tsai, D. P.; Hwu, Y. K. J. Phys. Chem. C 2009, 113, 7574. (22) Chang, H.; Tsai, M. H. Rev. Adv. Mater. Sci. 2008, 18, 734. (23) Deng, Z. J.; Mortimer, G.; Schiller, T.; Musumeci, A.; Martin, D.; Minchin, R. F. Nanotechnology 2009, 20, 455101. (24) Bihari, P.; Vippola, M.; Schultes, S.; Praetner, M.; Khandoga, A. G.; Reichel, C. A.; Coester, C.; Tuomi, T.; Rehberg, M.; Krombach, F. Part. Fibre Toxicol. 2008, 5, 5.

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and coated ZnO NPs shared similar zeta potentials in RPMI1640 medium suggests that the surface charge should not be a prime contributing influence to any observed toxic effects, with the possible exception of PMAA-coated NPs. 3.3. Cytotoxicity. Cell viability was assessed by the MTT assay, which measures mitochondrial reductase activity. The effects on WIL2-NS cells caused by exposures of 4, 24, and 48 h to uncoated and coated ZnO NPs at concentrations between 2 and 50 mg/L are shown in Figure 4a-d, where data are expressed as the viability of cells exposed to NPs compared to that of cells not exposed. Statistical analyses using Bonferroni tests indicate that the profile of cell viability in response to treatment with uncoated ZnO NPs differs significantly from those observed in response to the three types of coated NPs. Cellular activity was stimulated (mitochondrial reductase activity is higher than 100% compared with untreated control) in the presence of uncoated ZnO NPs at low concentrations for short exposure times (Figure 4a, for conditions of 2 mg/L at 4 and 24 h and 10 mg/L at 4 h) but was inhibited at higher concentrations. A dose-response phenomenon characterized by low-dose stimulation and high-dose inhibition, called hormesis, has been reported for a limited number of toxicological studies.25 At higher concentrations of uncoated ZnO NPs and for longer exposure times, cell viability decreased markedly such that at the highest concentration tested (50 mg/L) only ∼55% of cells survived after 4 h and this reduced to ∼10% after 48 h. Hormesis was not observed in cells exposed to any of the coated ZnO NPs for the doses or exposure times examined. Cell viabilities in the presence of coated NPs decreased with increasing particle concentration and exposure time. However, the decreases for the coated NPs were less pronounced than for the uncoated NPs until conditions of 50 mg/L and 24 or 48 h were realized. At these points, cell viabilities of 10-30% were observed (Figure 4b-d), comparable to those displayed by the uncoated ZnO (10-20%) (Figure 4a). In general, medium-coated ZnO NPs (presoaked in supplemented medium for 168 h, Figure 4d) were much less toxic to WIL2-NS cells than uncoated ZnO (freshly prepared without presoaking, Figure 4a). PMAA-coated NPs had cytotoxicity profiles similar to medium-coated ZnO NPs (comparing Figure 4c,d), whereas cells were more sensitive to exposure times with OAcoated NPs (Figure 4b) especially at higher concentrations. It is noteworthy that cell viabilities were high in the presence of all coated NPs for short exposure times (4 h) at all tested concentrations and were also high for PMMA-coated and medium-coated NPs for long exposure times (48 h) even at 35 mg/L, relative to those for uncoated NPs. These observations imply that the coatings were stable on the surface during these times. The relationship between cell viability and concentration of ZnO NPs for cells cultured for 24 h in the presence of uncoated and coated ZnO NPs is shown in Figure 4e. For uncoated ZnO NPs, cell viability decreased sharply between 10 to 20 mg/L, whereas for all types of coated NPs the dependence was closer to a linear relationship. Quantitatively, EC50 (the effective concentration corresponding to 50% cell viability) was determined to be (25) Calabrese, E. J.; Baldwin, L. A. Nature 2003, 421, 691.

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Figure 4. Effects on WIL2-NS human lymphoblastoid cells of uncoated and coated ZnO NPs. (a-d) Dependence of cell viability on particle concentration between 2 and 50 mg/L and for cell culture times of 4, 24, and 48 h. Data are expressed as the percentage viability of cells exposed to ZnO NPs relative to cells not exposed. (e) Relation between cell viability and the concentration of ZnO NPs for cells cultured for 24 h in the presence of uncoated and coated ZnO NPs.

15.9, 28.2, 41.4, and 41.8 mg/L for uncoated, OA-coated, PMAAcoated, and medium-coated NPs, respectively. On the basis of 15404 DOI: 10.1021/la101033n

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ZnO NPs can be ranked in the order of medium-soaked ≈ PMAA > OA. Medium-coated NPs, with components from cell culture medium on the surface, were more compatible with cells and thus caused little cytotoxicity. PMAA was physically adsorbed onto the surfaces of ZnO NPs, producing a hydrophilic surface as indicated by a higher absolute value of the zeta potential in PMAAcoated NPs relative to that in the other NPs. In contrast, OA was chemically adsorbed onto the surface of ZnO NPs with the formation of new chemical bonds through carboxyl groups, thus orienting the aliphatic ends of OA away from the NP to create a hydrophobic surface. It has been reported that hydrophilic surfaces are less cytotoxic than hydrophobic surfaces. This is supported by our study with ZnO NPs, where the hydrophilic PMAA coating was less cytotoxic than the hydrohobic surface created by the oriented OA molecules.26,27 On the basis of the above observations, the cytotoxicity of coated NPs is dominated by the properties of surface coatings instead of ZnO itself. In several publications, the observed cytotoxicity of ZnO NPs has been attributed to their partial dissolution.28-30 In our study, one possibility for the decreased cytotoxicity of coated ZnO NPs relative to that of the uncoated NPs could be that shielding by the surface coatings reduced the capacity for ZnO to dissolve, resulting in lower concentrations of Zn2þ in the medium. This explanation may apply to OA-coated and PMAA-coated NPs but not the medium-coated NPs. The medium-coated NPs were prepared by first soaking uncoated NPs for 168 h in a medium that was then added directly to cells, and this lengthy soaking process should have resulted in maximum (if not equilibrium) Zn2þ concentrations being reached. However, of all the NPs tested, the presoaked medium-coated NPs were observed to be the least toxic to the cells and were far less toxic than freshly added uncoated ZnO NPs, which would be expected to have a lower total Zn2þ concentration in the medium. Understanding the differerent responses for medium-coated and uncoated ZnO NPs is complicated by not knowing if potentially dissolved Zn2þ ions are strongly complexed to biomolecules (such as albumin) in the supplemented medium or are available freely or from an exchangeable pool to assist in inducing a toxic response. In this light, it is tempting to query if the hormesis observed at low dose and short exposure times to uncoated NPs may be due to a cellular response to free Zn2þ ions before the ions are complexed by molecules in the culture medium. To clarify this, a kinetic study of the dissolution behavior of ZnO NPs in supplemented RPMI1640 medium is required. Here, we attempted two methods (dialysis and centrifugation) to separate ZnO NPs from dissolved Zn2þ in the medium, with the results being at variance with each other. Limited amounts of Zn were detected in the receiving solution by the dialysis method, and larger amounts of Zn were observed in the supernatant using the centrifuge method. This variance is most likely due to the preference for Zn2þ to bind to proteins present in the medium,31 making their complete separation from ZnO NPs difficult to achieve by either method. Work is ongoing to clarify the functions of free Zn2þ, bound Zn2þ, and undissolved ZnO NPs in cytotoxicity studies. Most recently, Bai et al. reported (26) Araki, Y.; Andoh, A.; Bamba, H.; Yoshikawa, K.; Dio, H.; Komai, Y.; Higuchi, A.; Fujiyama, Y. Oncol. Rep. 2003, 10, 1931. (27) Muller, R. H.; Maassen, S.; Weyhers, H.; Mehnert, W. J. Drug Target. 1996, 4, 161. (28) Deng, X. Y.; Luan, Q. X.; Chen, W. T.; Wang, Y. L.; Wu, M. H.; Zhang, H. J.; Jiao, Z. Nanotechnology 2009, 20, 115101. (29) Franklin, N. M.; Rogers, N. J.; Apte, S. C.; Batley, G. E.; Gadd, G. E.; Casey, P. S. Environ. Sci. Technol. 2007, 41, 8484. (30) Lin, W. S.; Xu, Y.; Huang, C. C.; Ma, Y. F.; Shannon, K. B.; Chen, D. R.; Huang, Y. W. J. Mater. Res. 2009, 11, 25. (31) Lu, J.; Stewart, A. J.; Sadler, P. J.; Pinheiro, T. J.; Blindauer, C. A. Biochem. Soc. Trans. 2008, 36, 1317.

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Figure 5. Influence of surface coatings on ZnO NPs on the generation of ROS, as determined by a fluorescence assay that detects hydroxyl radicals. (a) Levels of ROS (in the form of percentage of fluorescence relative to an untreated control) when cells were exposed to uncoated and coated ZnO NPs at 10 mg/L NPs for 24 h. (b) Relationship between EC50 and levels of ROS for the various ZnO NPs.

that three components (i.e., small nano-ZnO aggregates suspended in the medium and dissolved Zn ions and large nanoZnO aggregates deposited on the container bottom) jointly exert an influence on the tested system,32 highlighting the need for the aggregation/agglomeration behavior of ZnO NPs in cell culture medium also to be included in toxicity studies. 3.4. ROS Generation. Reactive oxygen species (ROS) are highly reactive ions or very small molecules and include oxygen ions, free radicals, and peroxides. In cellular mitochondria under normal conditions, ROS are generated at low levels and are neutralized by antioxidant enzymes such as glutathione (GSH). If exposed to excessive levels of ROS, resulting in the depletion of GSH and the accumulation of oxidized glutathione (GSSG) (i.e., in a condition of oxidative stress), cells react by mounting further protective or injurious responses.33 High ROS levels and oxidative stress have been cited as common reasons for cellular damage induced by NPs,34 including ZnO NPs.35 (32) Bai, W.; Zhang, Z. Y.; Tian, W. J.; He, X.; Ma, Y. H.; Zhao, Y. L.; Chai, Z. F. J. Nanopart. Res. 2010, 12, 1645. (33) Xiao, G. G.; Wang, M.; Li, N.; Loo, J. A.; Nel, A. E. J. Biol. Chem. 2003, 278, 50781. (34) Xia, T.; Kovochich, M.; Nel, A. Clin. Occup. Environ. Med. 2006, 5, 817. (35) Karlsson, H. L.; Cronholm, P.; Gustafsson, J.; Moller, L. Chem. Res. Toxicol. 2008, 21, 1726.

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Figure 6. Images of WIL2-NS cells exposed to PMAA-coated ZnO NPs. (a) An apoptotic cell in which the staining intensity of the nucleus, nuclear fragments, and cytoplasm is greater than that of viable cells. (b) A necrotic cell in which a pale cytoplasm and damaged/irregular cytoplasmic and nuclear membranes can be seen. (c) A binucleated (BN) cell with micronuclei (MNi), a biomarker of chromosome breakage, and/or whole chromosome loss. (d) A BN cell with a nucleoplasmic bridge (NPB), a biomarker of DNA misrepair and/or telomere end fusions. (e) A BN cell with nuclear buds (NBud), a biomarker of the elimination of amplified DNA and/or DNA repair complexes.

The influence of surface coatings on the ability of ZnO NPs to generate ROS in the presence of WIL2-NS cells was investigated using a fluorescence assay that specifically detects hydroxyl radicals.36 In this assay, hydrophobic DCFH-DA molecules, which readily penetrate cellular membranes, are hydrolyzed by intracellular esterases to yield dichlorofluoroscein (DCFH), a nonfluorescent compound; DCFH can then be oxidized by ROS to the fluorescent compound dichlorofluorescein, DCF. An important concern here is whether the photocatalytic activity of ZnO NPs could generate ROS in the absence of cells and produce a false positive in the test. ZnO NPs, cells, and DCFH-DA were co-cultured in a dark CO2 incubator for the duration of the exposure period and were exposed to light only briefly (1 to 2 min) while being processed in a biosafety hood. To verify whether ZnO NPs alone had any effect on the formation of DCF, control experiments were carried out using NP concentrations of 10, 20, and 50 mg/L under cell-free conditions. It was found that the fluorometric intensities generated in these controls were around 4000 units and were unrelated to ZnO concentrations. In comparison, the intensity increased to around 360 000 units when the cells were present. Therefore, it is reasonable to ignore the photocatalysis of ZnO NPs in our ROS testing in the dark. However, we note that this issue must be addressed in any experiments assessing the toxicity of ZnO NPs with sunlight exposure. Figure 5a shows the ROS levels in WIL2-NS cells following exposure to 10 mg/L ZnO NPs for 24 h relative to the untreated control. Decreasing levels of ROS were generated in the order of uncoated >OA-coated > PMAA-coated> medium-coated ZnO NPs. In evaluating injury from oxidative stress caused by NPs, the most appropriate dose metrics are the particle size and surface area, which influence the number of active sites at which ROS can be generated. In our study, the effect of size can largely be eliminated because a common stock of ZnO NPs with a discrete size range was used and surface coating did not significantly alter their size. Another consideration for the generation of ROS is the nature of metal ions on the NP surface. Metal ions with variable valence, such as Fe2þ, Cuþ, Mn2þ, Cr5þ, and Ni2þ, could contribute to the generation of free radicals via a Fenton-type reaction.37 However, zinc is not (36) Choi, O.; Hu, Z. Q. Environ. Sci. Technol. 2008, 42, 4583. (37) Sandstrom, B. E.; Granstrom, M.; Marklund, S. L. Free Radical Biol. Med. 1994, 16, 177. (38) Nakamura, M.; Shishido, N.; Nunomura, A.; Smith, M. A.; Perry, G.; Hayashi, Y.; Nakayama, K.; Hayashi, T. Biochemistry 2007, 46, 12737.

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considered to be a Fenton reactant because of its invariable valency (þ2).38 Figure 5b shows the observed cytotoxicity (as EC50) plotted as a function of measured ROS levels for the various ZnO NPs studied here. Except for PMAA-coated NPs, EC50 exhibited an inverse relationship with ROS levels (i.e., lower EC50 correlated with higher ROS levels). This indicates that ROS could be a main contributing factor to the observed cytotoxicity, as measured by mitochondrial reductase activity (MTT assay). In the case of uncoated ZnO NPs with the highest ROS levels and lowest EC50, the possibility for electron capture on the uncoated surface is likely to be less hindered than for coated NPs, resulting in the generation of more hydroxyl radicals and consequent oxidative stress. For coated ZnO NPs, the active electron donor and acceptor sites at the surface are either complexed or shielded by stable coatings, and thus the possibility of electron capture and ROS generation may be effectively reduced. However, for the inverse relationship to hold for PMAA-coated NPs, the measured ROS levels should be associated with more severe cytotoxicity (EC50 of around 33.2 mg/L) than what was observed (EC50 of around 41.4 mg/L), indicating that other factors may be involved in the cellular response to PMAA-coated ZnO NPs. 3.5. Genotoxicity. In biological systems, DNA in nuclei of bystander cells may be damaged by ROS generation as part of an inflammatory response when cells of the immune system are activated, by free radicals produced by chemicals that enter or associate with cells, or even indirectly as a result of intercellular signaling across cellular barriers.39 DNA damage via oxidative stress has been reported in vitro for various NPs such as TiO240,41 and ZnO.42 The CBMN (cytokinesis-block micronucleus) cytome assay is very sensitive to the DNA-damaging effects of oxidants. With good reliability and reproducibility, this assay is widely used in the pharmaceutical industry as one of the gold standards of genotoxicity testing.43 Here we have adapted the assay to investigate (39) Bhabra, G.; Sood, A.; Fisher, B.; Cartwright, L.; Saunders., M.; Evans, W. H.; Surprenant, A.; Lopez-Casrejon, G.; Mann, S.; Davis, S. A.; Halis, L. A.; Ingham, E.; Verkade, P.; Lane, J.; Heesom, K.; Newson, R.; Case, C. P. Nat. Nanotechnol. 2009, 4, 876. (40) Wang, J. J.; Sanderson, B. J. S.; Wang, H. Mutat. Res. 2007, 628, 99. (41) Rahman, Q.; Lohani, M.; Dopp, E.; Pemsel, H.; Jonas, L.; Weiss, D. G.; Schiffmann, D. Environ. Health Persp. 2002, 110, 797. (42) Yang, H.; Liu, C.; Yang, D. F.; Zhang, H. S.; Xi, Z. G. J. Appl. Toxicol. 2009, 29, 69. (43) Wu, J.; Lyons, G. H.; Graham, R. D.; Fenech, M. F. Mutagenesis 2009, 24, 225.

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Table 2. Influence of the Surface Chemistry of ZnO NPs on the Biomarkers of Apoptosis, Necrosis, and Cytostasis (NDI) and on the Frequency of Micronuclei (MNi), Nucleoplasmic Bridges (NPB), and Nuclear Buds (NBud) in Once-Divided Binucleated (BN) Cells in the CBMN Cytome Assaya,b control (cells without NP exposure)

uncoated ZnO

OA-coated ZnO

PMAA-coated ZnO

medium-coated ZnO

ANOVA p

% apoptotic cells 2.90 ( 0.86 5.75 ( 1.04 2.50 ( 0.65 2.88 ( 0.48 4.33 ( 1.26 0.0689 18.8 ( 3.2b 11.5 ( 1.5a 12.5 ( 1.9a 11.6 ( 0.7a 0.0106 % necrotic cells 10.5 ( 2.5a NDI 1.60 ( 0.01 1.51 ( 0.05 1.55 ( 0.03 1.49 ( 0.05 1.48 ( 0.04 0.0516 35.5 ( 3.4a,b 39.9 ( 6.6b 57.5 ( 3.0c 27.0 ( 6.0a 0.0009 MNi/1000BN 21.0 ( 5.3a 11.0 ( 4.7a 15.0 ( 6.2a 21.2 ( 6.2b 12.5 ( 2.5a 0.007 NPB/1000BN 8.5 ( 1.9a a a a b a 11.5 ( 3.4 11.5 ( 3.0 22.5 ( 1.0 8.0 ( 2.8 0.0006 NBud/1000BN 5.5 ( 1.0 a Groups not sharing the same superscript letter are significantly different from each other (P < 0.05). b ANOVA p values in bold are statistically significant.

potential genotoxicity induced in cells exposed to ZnO NPs with different surface chemistry. The experiments were conducted with an NP concentration of 10 mg/L and an exposure time of 24 h; under these conditions, cell viability was approximately the same for all NPs tested (Figure 4) and there were sufficient numbers of viable cells for the scoring process. Cytochalasin-B (Cyt-B), an inhibitor of microfilament ring assembly, was then added to each treatment to block cytokinesis at the once-divided stage. Cells from each experimental condition were scored and classified to determine the percentages of apoptotic and necrotic cells, the nuclear division index (NDI), and the numbers of mononucleate, binucleate, and multinucleate cells. Damage to DNA was scored in once-divided binucleated (BN) cells and included micronuclei (MNi, a biomarker of chromosome breakage and/or whole chromosome loss), nucleoplasmic bridges (NPB, a biomarker of DNA misrepair and/or telomere end fusions), and nuclear buds (NBud, a biomarker of the elimination of amplified DNA and/or DNA repair complexes). Figure 6 shows typical images of cells treated with PMAA-coated ZnO NPs, including an apoptotic cell, a necrotic cell, and binucleated cells found with MNi, NPB, and NBud. Table 2 summarizes the results obtained from the CBMN cytome assay. A statistical analysis of the data indicates that the percentages of apoptotic cells appearing in the presence of the four types of ZnO NPs were not significantly different and neither was the NDI. However, the percentage of necrotic cells associated with exposure to uncoated NPs was significantly higher when compared with the three types of coated NPs. The higher levels of necrosis (unprogrammed cell death) following exposure at 10 mg/ L for 24 h are consistent with the rapid decrease in cell viability observed for higher doses and longer exposure times to the uncoated NPs than to the coated NPs. The assessment of DNA damage indicated significant increases of 90 and 174% in the frequency of MNi in cells exposed to OA-coated and PMAA-coated NPs, respectively, whereas there was no significant elevation of MNi found in cells exposed to uncoated or medium-coated NPs. The increases in NPB and NBud in cells exposed to PMAA-coated ZnO NPs were significant, but not in cells exposed to the other types of ZnO NPs. The high frequency of MNi induced by OA-coated and PMAA-coated NPs may be associated with the medium levels of ROS generated by these NPs. The additional significant DNA damage in cells exposed to PMAA-coated NPs, in terms of high frequencies of NPB and NBud, may indeed be due to the properties of the poly(methylacrylic acid) coating, where recently it was reported that 5 mM glycidyl methacrylate caused 40% DNA damage in human lymphocytes (assessed by an alkaline version of

the comet assay) even though cell viability was high at around 80%.44 However, alternative mechanisms leading to genotoxicity cannot be precluded, such as the disruption of the mitotic spindle or kinetochore proteins resulting in whole chromosome lagging during anaphase in mitosis (which would cause MNi to form from chromosomes that failed to be included within daughter nuclei) or the disruption of telomerase activity and TRF1 protein binding to telomeres causing telomere dysfunction and the generation of telomere end fusions (which would cause NPB and NBud in BN cells).14 We have already noted that the surface charge on the PMAA-coated NPs in the cell culture medium is slightly more negative than that on the other NPs, with the potential for differing charge-related interactions of these NPs with cellular components. A hypothesis for hierarchical oxidative stress has been proposed for assessing the toxicity of NPs.45 Our study on ZnO NPs with different surface coatings to some extent agrees with this hypothesis. As shown in Figure 5b, ROS levels generated from various ZnO NPs had an inverse relationship with EC50, with the exception of PMAA-coated ZnO where the measured ROS levels were associated with a lower-than-expected EC50. Although the polymethylacrylic coating itself may be genotoxic, it is tempting to conclude that the “additional” ROS also may have induced substantial DNA damage in PMAA-coated NPs represented by the higher incidences of MNi, NPB, and NBud.

(44) Poplawski, T.; Pawlowska, E.; Wisniewska-Jarosinska, M.; Ksiazek, D.; Wozniak, K.; Szczepanska, J.; Blasiak, J. Chem.-Biol. Interact. 2009, 180, 69.

(45) Xia, T.; Kovochich, M.; Brant, J.; Hotze, M.; Sempf, J.; Oberley, T.; Sioutas, C.; Yeh, J. I.; Wiesner, M. R.; Nel, A. E. Nano Lett. 2006, 6, 1794.

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4. Conclusions To our knowledge, this is the first systematic report on the cellular response to surface-modified ZnO NPs. The surface chemistry was found to directly influence the levels of ROS generated and was identified as a major factor influencing both cytotoxicity and genotoxicity. The cytotoxicity of ZnO NPs may be controlled to a certain extent by surface chemistry. However, the significant genotoxicity observed in viable cells exposed to PMAA-coated NPs highlights the need to investigate the effects of novel NP modifications thoroughly. The treatment to which NPs were subjected prior to adding them to cells also significantly influenced ROS levels and cell viability, as evidenced by the very different results observed for medium-coated and uncoated ZnO NPs. This indicates a need to explore the effects of differing preparation procedures further, before deciding on a standard method of sample preparation for nanotoxicity testing. Our work verified that the CBMN cytome assay is very effective in assessing the genotoxicity induced by NPs. Results from the assay also highlight the need for ROS production to be monitored in toxicity studies of NPs because this metric may

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reflect the total oxidant damage to both mitochondria and DNA. Only when ROS levels in cells exposed to NPs are as low as those in untreated cells is there likely to be both low cytotoxicity and low genotoxicity. Acknowledgment. We thank Professor Ian Harper and Dr. Judy Callaghan (Monash University) and Ms. Carolyn Salisbury

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(CSIRO) for their assistance with cell culturing activities. We also thank Ms. Lynne Waddington, Dr. Thomas Gengenbach, and Ms. Yes-im G€oz€ukara, all from CSIRO, for their characterization work using TEM, XPS, and ICP-AES, respectively, Dr. Yalchin Oytam (CSIRO) for the statistical analysis of cell viability data, and Dr. Nicola Rogers (CSIRO) for informative discussions.

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