Direct Evidence for Hydroxyl Radical Scavenging Activity of Cerium

Mar 1, 2011 - (17, 23) The generated hydroxyl radical can react with MV by attacking its −C═C− where it has a higher density of electron cloud i...
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Direct Evidence for Hydroxyl Radical Scavenging Activity of Cerium Oxide Nanoparticles Ying Xue,† Qingfen Luan,† Dan Yang,† Xin Yao,*,†,‡ and Kebin Zhou*,† †

College of Chemistry and Chemical Engineering, Graduate University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China ‡ State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, People’s Republic of China

bS Supporting Information ABSTRACT: Cerium oxide (CeO2) nanoparticles have been considered as excellent antioxidants and have become a focus of numerous studies. However, the mechanism behind the antioxidant role in the complex biological system has not been wellunderstood. In this work, direct evidence for the hydroxyl radical (•OH) scavenging activity of CeO2 nanoparticles was established by a simple photometric system in vitro. When methyl violet (MV) reacted with hydroxyl radical, the absorbance change indicated the hydroxyl radical level. The presence of CeO2 nanoparticles protected MV by competitively reacting with the •OH, so their hydroxyl radical scavenging activity was directly seen through the absorbance change. This activity was also proved to be size-dependent and was believed to have a close correlation with Ce3þ at the surface of the particles.

’ INTRODUCTION Recently, cerium oxide (CeO2) nanoparticles have been considered as excellent antioxidants and become a focus of numerous studies.1-9 Because of their good biocompatibility and unique redox property, CeO2 nanoparticles have been widely used to prevent retinal degeneration induced by intracellular peroxide molecules,2 protect the normal cells against radiation damage as opposed to tumor cells,3 offer neuroprotection to adult rat nervous cells,4,9 and inhibit cardiovascular myopathy and other inflammatory diseases in biological systems.5-8 These animal and cell culture studies have demonstrated the effective antioxidant role of CeO2 nanoparticles, which is speculated to be due to their ability of scavenging free radicals.10,11 Generally, the biological systems are very complex and have some uncertain factors. So far, the mechanism behind the protective effect of CeO2 nanoparticles has not been well-understood. Further investigation is required to confirm CeO2 nanoparticles’ free radical scavenging activity, which is considered to be responsible for the protection. Up to now, the only specific free radical scavenging activity of CeO2 nanoparticles studied without the relatively complex cell system is the superoxide dismutase mimetic activity, which proves the superoxide-scavenging ability of CeO2 nanoparticles.12,13 However, besides superoxide (O2-), there are many other kinds of free radicals, such as nitric oxide (NO), peroxynitrite (ONOO-), and hydroxyl radical (•OH), which are also commonly recognized to play an important role in physiological and pathological processes of the organisms.11,14 Especially, the highly active hydroxyl radical is known as one of the strongest oxidants, whose disbalance between generation and r 2011 American Chemical Society

elimination can potentially induce DNA damage, protein carbonylation, and lipid peroxidation, eventually leading to a variety of health problems, such as cancer, aging, and many chronic inflammations.14-17 Therefore, to study whether antioxidative CeO2 nanoparticles could scavenge hydroxyl radical as well as the scavenging mechanism is of great significance. To date, direct evidence for the hydroxyl radical scavenging activity of cerium oxide nanoparticles has been scarcely reported. In this work, methyl violet (MV) was used to investigate the hydroxyl radical scavenging activity of CeO2 nanoparticles. The absorbance change during the reaction of MV with hydroxyl radical can be used to research the free radical scavenging role of antioxidants.18 With this simple photometric method, direct evidence for the hydroxyl radical scavenging activity of cerium oxide nanoparticles was confirmed. The effect of particle size and the probable scavenging mechanism were also explored.

’ EXPERIMENTAL SECTION Materials and Reagents. Methyl violet (MV), iron(II) sulfate heptahydrate (FeSO4 3 7H2O), hydrogen peroxide (30%), hexamethylenetetramine (HMT), and cerium(III) nitrate hexahydrate (CeN) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). The stock solutions of MV and FeSO4 were prepared in water at concentrations of 2.0  10-4 M and 15 mM, respectively. All reagents were of analytical grade and Received: October 13, 2010 Revised: January 31, 2011 Published: March 01, 2011 4433

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The Journal of Physical Chemistry C used without further purification. Millipore Milli Q (18 MΩ cm) water was used in all experiments. Instruments. The nanoparticle morphology was studied using a FEI Tecnai G2 T20 transmission electron microscope (TEM) by depositing a drop of suspended CeO2 solution on a holey carbon-coated copper grid. X-ray diffraction (XRD) patterns were obtained by using a Bruker D8 Advance X-ray diffractometer with Cu KR radiation (λ = 1.5418 Å). The data were recorded at a scan rate of 2°/min. Dynamic light scattering (DLS) was performed by analyzing the suspended CeO2 solution using a Zatasizer Nano ZS particle analyzer. The surface chemistry of CeO2 nanoparticles was studied using X-ray photoelectron spectroscopy (XPS). The XPS data were obtained by using an ESCA Lab220i-XL spectrometer from VG Scientific with 300 W Al KR X-ray radiation. The base pressure during XPS analysis was about 3  10-9 mbar, and the binding energies were referenced to the C 1s line at 284.8 eV from adventitious carbon. The 3d peak positions of CeO2 were then fitted by using PeakFit (version 4.0) software. The UV-vis absorption spectrum was achieved with a UV-2550 spectrophotometer. Nanoparticle Synthesis. Cerium oxide nanoparticles were prepared by a hydrothermal synthesis method as described in the literature with a little modification.19 In brief, 0.125 M HMT was added to 0.025 M CeN solution dropwise under continuous stirring. The mixed aqueous solution was hydrothermally treated in a 50 mL autoclave at 100 °C for 2 h or 150 °C for 12 h and then was cooled to room temperature. The cerium oxide powder was obtained by centrifuging and washing with water and ethanol, followed by oven-drying. UV-vis Photometric Experiments. The stock suspended solution of CeO2 nanoparticles was prepared at a concentration of 10 μM by dispersing in 0.1 M Tris-HCl buffer, pH 4.7, sonicated prior to use. The reaction solution for photometric determination contained 1.2  10-5 M MV, 0.15 mM FeSO4, 1.0 M H2O2, 0.1 M Tris-HCl buffer (pH 4.7), and appropriate CeO2 in a final volume of 5 mL (denoted as MV/FeSO4/H2O2/ CeO2 solution). Solutions at these concentrations were used without special note. After incubation for 5 min or more time at room temperature, the absorbance of the reaction solution was measured.

’ RESULTS AND DISCUSSION Characterization of CeO2 Nanoparticles. The size and morphology of the synthesized nanoparticles were examined. Figure 1a indicates the XRD diffraction patterns of the nanoparticles that were, respectively, hydrothermally treated at 100 °C for 2 h (curve 1) and at 150 °C for 12 h (curve 2), showing that the particles are cerium oxide of typical fluorite cubic structures (JCPDS card: 34-0394). The broadening peaks in curve 1 indicate that the particles treated at 100 °C for 2 h are smaller than the ones treated at 150 °C for 12 h (curve 2). This is consistent with the Debye-Scherrer formula, which shows that crystallite size is inversely proportional to the full width at halfmaximum (fwhm).20 The fwhm values of CeO2 nanoparticles corresponding to each plane of them are clearly presented in Table S1 in the Supporting Information. Their TEM images in Figure 1b,c exhibit that the size of nanoparticles is, respectively, 5-10 nm and 15-20 nm. That is, the particles treated with higher temperature and a longer time are larger, which is consistent with the result of XRD patterns. Because the hydrothermal treatment is a process of Ostwald

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Figure 1. (a) XRD patterns of 5-10 nm (1) and 15-20 nm (2) CeO2 nanoparticles. (b) TEM image of 5-10 nm CeO2 nanoparticles. (c) TEM image of 15-20 nm CeO2 nanoparticles.

ripening and the solubility of the smaller particle is higher than that of the larger ones, the particles with a small size slowly dissolve and those with a larger size grow into much larger ones with time during hydrothermal treatment. The solubility of the particles increases at higher temperature. Therefore, the particles treated with higher temperature and a longer time are larger, which is also in line with ref 21. Additionally, both of them have a certain level of aggregation, but this does not affect their properties, which has been proved by the following experiments. Free Radical Scavenging Activity of CeO2. As is known, the chromogenic reagent methyl violet has a maximum absorbance at about 582 nm,22 and the typical Fenton reaction is such an oxidation system in which H2O2 can be catalyzed by Fe2þ to produce the extremely reactive hydroxyl radical. One main step of this chain reaction is shown in eq 1.17,23 The generated hydroxyl radical can react with MV by attacking its -CdCwhere it has a higher density of electron cloud in the way of electrophilic addition (shown in eq 2),24 leading purple MV into a colorless product, and correspondingly, its maximum 4434

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Figure 2. UV-vis absorption spectra of MV: (a) MV, (b) MV/H2O2/ 10 nM CeO2 (5-10 nm), (c) MV/FeSO4/H2O2, and (d) MV/FeSO4/ H2O2/10 nM CeO2 (5-10 nm) solutions at an incubation time of 5 min.

absorbance decreases. The change of absorbance (denoted as ΔA) indirectly indicates the amount of generated •OH. The bigger ΔA is, the more •OH there is. When a certain amount of CeO2 is added to the above system, if CeO2 is able to eliminate the hydroxyl radical, part of the MV will be protected from being attacked, and ΔA will decrease. Fe2þ þ H2 O2 f Fe3þ þ OH- þ •OH

ð1Þ

According to the principle presented above, the absorption spectra of MV and MV/H2O2/CeO2 (5-10 nm) solution systems were, respectively, determined (shown in Figure 2, spectra a and b). Both solutions have a maximum absorption at 582 nm, and the absorbance of the MV/H2O2/CeO2 solution has not obviously changed compared to that of MV existing alone. All the absorption of MV refers to that at 582 nm if there is no special note. To demonstrate that Fe2þ has no effect on the absorption of MV, the absorption spectrum of the MV/FeSO4/10 nM CeO2 (5-10 nm) solution was also determined (the curve not given here). All results indicate that the presence of Fe2þ, H2O2, and CeO2 almost has no effect on the UV-vis absorption of the MV complex. Moreover, Figure 2, spectrum b, shows that, without the addition of Fe2þ, the absorbance of the MV system remains, meaning that almost no free radical that can cause an obvious absorbance change is produced in the incubation time. When Fe2þ was added to the MV/H2O2 solution, as is seen in Figure 2, spectrum c, the absorbance of the MV system decreased, which indicates that the Fenton reaction occurs and the generated hydroxyl radical attacks MV, causing its fading (the reaction process shown in eqs 1 and 2). However, after the addition of 10 nM CeO2 (5-10 nm), the absorbance partially increases and ΔA decreases (Figure 2, spectrum d), which proves that CeO2 indeed scavenges part of •OH and protects MV from further fading as a result. Therefore, this photometric method is feasible to demonstrate the free radical scavenging activity of CeO2. In addition, from Figure 2, we can see that the absorbance does not

Figure 3. Change of ΔA with incubation time in the reactive solutions when the CeO2 concentration is 0 nM (a), 1 nM (5-10 nm) (b), 10 nM (5-10 nm) (c), 100 nM (5-10 nm) (d), 10 nM (15-20 nm) (e), and 100 nM (15-20 nm) (f). The reactive solutions contain 1.2  10-5 M MV, 0.15 mM FeSO4, 1.0 M H2O2, and different concentrations of CeO2 (5-10 and 15-20 nm). The relative standard deviations (percentage of RSD) are all less than 8%.

completely recover, which indicates that CeO2 can only partly, not completely, eliminate the generated hydroxyl radical at this experimental condition. Besides, it is worth noting that there are some differences with the absorbance of MV in the wavelength less than 450 nm. That is due to the absorption of FeSO4, CeO2, and H2O2 in this wavelength range besides MV as well as the possible effect of the hydroxyl radical generated by the Fenton reagent, though all of them have no absorption at 582 nm (shown in Figure S1, Supporting Information). To further investigate the scavenging activity of CeO2 nanoparticles to hydroxyl radical, ΔA of the solutions containing different concentrations of CeO2 (5-10 nm) in longer incubation times were determined, as shown in Figure 3, curves a-d. It can be seen that, without addition of CeO2 (Figure 3, curve a), the ΔA at 5 min is 0.060, and then it increases to 0.075 when incubated for 25 min. This indicates that the hydroxyl radical is continuously generated by the Fenton reaction. When the CeO2 (5-10 nm) concentration increases to 1 nM (Figure 3, curve b), the ΔA at 5 min decreases to 0.048, and at other incubation times, it is smaller than that of the solution without CeO2 (Figure 3, curve a). This demonstrates that CeO2 has scavenged the •OH and protected MV from being attacked to some extent. Similarly, when the CeO2 (5-10 nm) concentration increases from 1 to 10 nM (Figure 3, curve c) and 100 nM (Figure 3, curve d), ΔA at 5 min decreases from 0.048 to 0.028 and 0.022, respectively, and ΔA at other incubation times also becomes smaller, which proves that higher concentrations of CeO2 can eliminate more hydroxyl radical. That is, with the increase of concentration, the hydroxyl radical scavenging ability of CeO2 is enhanced. On the other hand, it can be seen that the ΔA increases with the prolonged time at different concentrations of CeO2. We believe that this may be because the concentration of H2O2 (1.0 M) is far higher than that of CeO2 nanoparticles and there is too much •OH continuously generated. However, obviously, the protective activity of CeO2 nanoparticles with each concentration exists throughout the whole incubation time. In addition, considering its biological safety of potential applications in the organism,5 higher concentrations of CeO2 is not included within the scope of this study. Effect of Particle Size of CeO2. Having demonstrated the scavenging activity of CeO2 to •OH, the 15-20 nm CeO2 nanoparticles in different concentrations (Figure 3, curves e 4435

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Figure 4. XPS analysis of 5-10 nm (a) and 15-20 nm (b) CeO2 nanoparticles.

and f) were also employed to investigate the effect of particle size on it. From Figure 3, curve e, the ΔA of the solution with 10 nM CeO2 (15-20 nm) at each incubation time is smaller than the value when there is no CeO2 added (Figure 3, curve a), indicating that the larger particles also have the ability to scavenge •OH; however, it is bigger compared with that of 5-10 nm CeO2 (Figure 3, curve c), showing that they capture less •OH and finally protect less MV from being attacked than the smaller ones. Similarly, on comparison of curves d and f in Figure 3, the same situation occurs with the 5-10 nm and 15-20 nm CeO2 when the concentration increases to 100 nM. Therefore, the free radical scavenging ability of CeO2 nanoparticles shows an increase with the decrease in particle size. To clearly explain the size-dependent free radical scavenging activity, the surface chemistry of CeO2 was studied by the XPS analysis (shown in Figure 4), which reveals the presence of a mixed valence state (Ce3þ and Ce4þ) for both of the 5-10 nm (Figure 4, spectrum a) and 15-20 nm (Figure 4, spectrum b) CeO2 nanoparticles. The binding energy peaks at 885.0 and 903.5 eV belong to Ce3þ, and peaks at 882.1, 888.1, 898.0, 900.9, 906.4, and 916.40 eV are indicative of the presence of Ce4þ. Obviously, the two peaks of Ce3þ in Figure 4, spectrum a, are bigger than those in Figure 4, spectrum b, so there is more Ce3þ at the surface of the smaller size particles. To further determine the amount of Ce3þ in both samples, the peak positions were fitted using PeakFit (version 4.0) software, through which we can get the peak area of each Ce3þ peak as well as each Ce4þ peak. The ratio of integrated peak areas of Ce3þ to the sum of Ce3þ and Ce4þ is the atomic fraction of Ce3þ.12,20 By calculating, the Ce3þ concentration at the surface of 5-10 nm particles and 1520 nm ones is, respectively, 30.4% and 20.9%. Therefore, it can be confirmed that there is more Ce3þ at the surface of the smaller size particles, which is consistent with the literature.12,25 As is reported, there are more oxygen vacancies on the surface of the smaller particles,25 whose formation may be accompanied with the generation of more Ce3þ ions. The Ce3þ ions, presumed to acted as active sites, can easily react with the oxidative free radicals.3,26-28 Therefore, the smaller particles with more Ce3þ are able to capture more •OH, protecting more MV from fading. That is to say, the Ce3þ at the surface of the particles may be mainly responsible for the size-dependent hydroxyl radical scavenging ability of CeO2 nanoparticles. Probable Mechanism. To exclude the possibility that the decrease of •OH may be due to the direct reaction of CeO2 and H2O2, the following experiments were designed. H2O2 (1 M) and 10 nM CeO2 were mixed prior for 30 min before the addition of MV and Fe2þ, then the absorbance of the MV system at an

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Figure 5. UV-vis absorption spectra of MV: (a) MV/FeSO4/H2O2/ 10 nM CeO2 (5-10 nm) solution at an incubation time of 5 min with prior mixing of H2O2 and CeO2 for 30 min, and (b) solution (a) without prior mixing. (Inset) UV-vis absorption spectra of H2O2: (a) the mixed solution of 1 mM H2O2 and 10 nM CeO2 (5-10 nm) incubated for 30 min, and (b) solution (a) without incubation.

incubation time of 5 min was determined. It is shown that its absorbance (Figure 5, spectrum a) is almost the same as that of the solution that was directly mixed (Figure 5, spectrum b), indicating that the prior 30 min of incubation of CeO2 and H2O2 cannot obviously affect the following photometric determination at an incubation time of 5 min. In addition, the mixture solution of 1 mM H2O2 (1000 times less than 1 M used in the Fenton reaction) and 10 nM CeO2 was made and incubated for 30 min, and then the absorption of H2O2 was determined (inset, spectrum a, of Figure 5). The absorbance of H2O2 does not change compared to that when H2O2 exists alone (inset, spectrum b, of Figure 5), confirming that the decrease of ΔA is not due to the consumption of H2O2 by CeO2. Therefore, there is no doubt that it is the reaction of CeO2 and •OH (not H2O2) that causes the recovery of the absorbance of MV under the present experimental conditions. How does the scavenging reaction of CeO2 and •OH take place? To describe it in detail, we should begin with the Ce3þ at the surface of CeO2 nanoparticles. As is discussed above, the Ce3þ, which is related to the formation of oxygen vacancies, is presumed to act as active sites.3,26-28 The relatively high Ce3þ concentration of 30.4% or 20.9% at the surface of CeO2 nanoparticles allows them to react with the highly oxidative •OH by reversibly switching between the Ce3þ and Ce4þ.2,4,8 One oxidation-reduction cycle detailing this unique activity of CeO2 nanoparticles is suggested in Scheme 1. Combined with the photometric results, it is believed that, during the scavenging process, Ce3þ ions are converted to Ce4þ by competing with MV to react with •OH, protecting part of MV from fading. A regenerated process of Ce3þ is then considered to take place. It may be completed via a series of surface chemical reactions between ions in solution (such as Hþ) and the Ce4þ on the nanoparticle surface.28 If Ce3þ is not able to regenerate, according to the stoichiometric shown in Scheme 1 and supposing that there is only Ce3þ in 100 nM CeO2 nanoparticles, when 100 nM CeO2 scavenges hydroxyl radical, there will be 100 nM MV protected and then MV absorption would change. However, seen from Figure S2a,b (Supporting Information), the 100 nM concentration change of MV can hardly induce any absorbance change. In fact, the absorbance change due to the protection of 100 nM CeO2 is equivalent to that of 1120 nM MV (11.2 times more than 100 nM) (Figure S2c,d, Supporting Information). Moreover, there is only 30.4% Ce3þ in CeO2, and CeO2 with a 4436

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The Journal of Physical Chemistry C Scheme 1. Schematic Diagram Detailing the OxidationReduction Cycle as the Probable Mechanism of Hydroxyl Radical Scavenging Activity of CeO2 Nanoparticles

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’ ASSOCIATED CONTENT

bS

Supporting Information. The full width at half-maximum (fwhm) values of CeO2; UV-vis absorption spectra of MV, FeSO4, CeO2, H2O2, and the Fenton reagent; UV-vis absorption spectra of different concentrations of MV; and the DLS patterns of 5-10 nm and 15-20 nm CeO2 nanoparticles. This material is available free of charge via the Internet at http:// pubs.acs.org.

’ AUTHOR INFORMATION lower concentration of 1 nM also has the persistent protection ability. Therefore, in order to induce such a big protective effect, the amount of hydroxyl radical that CeO2 nanoparticles scavenge is much larger than the concentration of its own. Therefore, it can be concluded that the reversible cycle between Ce3þ and Ce4þ imparts to CeO2 nanoparticles the protective activity that can maintain throughout the whole incubation time. After Ce4þ is converted back to Ce3þ, more hydroxyl radical will be continuously scavenged in the following cycles. The more initial Ce3þ in the cycle there is, the more hydroxyl radicals can be scavenged. CeO2 nanoparticles with a higher concentration or smaller particle size, which own more Ce3þ, have an enhanced free radical scavenging ability, which is consistent with the photometric experimental results. As is shown in Figure 3, when the CeO2 (5-10 nm) concentration increases, ΔA at 5 min decreases from 0.048 (1 nM) to 0.028 (10 nM) and 0.022 (100 nM), and when the CeO2 (10 nM) size decreases from 1520 to 5-10 nm, ΔA at 5 min decreases from 0.036 to 0.028. In addition, this regenerated property imparts to the radical scavenger CeO2 nanoparticles some advantages compared to other antioxidants so that, with a small dose, a relatively obvious effect could be achieved in potential applications.5 Besides, according to the TEM images (Figure 1b,c), it can be found that there is some agglutination with the CeO2 nanoparticles. The DLS analysis (Figures S3 and S4, Supporting Information) of a CeO2 suspended solution shows that the average size of the aggregates is, respectively, about 170 and 230 nm, also indicating the existence of agglutination. However, we believe that it would not change the scavenging activity of CeO2. That is, because it is the Ce3þ at the surface of CeO2 nanocrystals that plays the role in the process of scavenging hydroxyl radical, and the concentration of Ce3þ is determined by the size of the nanocrystal, but not the size of aggregated particles. This has been proved by the XPS analysis (Figure 4), which shows that there is still more Ce3þ at the surface of smaller-sized CeO2, though they agglutinate more. Therefore, although there is some agglutination, the performance of scavenging hydroxyl radical still conserves.

’ CONCLUSION In this work, the hydroxyl radical scavenging activity of CeO2 nanoparticles was clearly demonstrated by a simple photometric method in vitro. This activity of CeO2 nanoparticles was proved to be size-dependent and believed to have a close correlation with Ce3þ at the surface of the particles. On the basis of the study, direct evidence for the hydroxyl radical scavenging activity of CeO2 nanoparticles is definitely established. The results will give more reference for their further biomedical application in vivo for the diseases caused by free radicals.

Corresponding Author

*Phone: 86-10-8825-6414. Fax: 86-10-8825-6092. E-mail: [email protected] (X.Y.), [email protected] (K.Z.).

’ ACKNOWLEDGMENT This work was supported by grants from the National Natural Science Foundation of China (20703065, 20877097), the Ministry of Science and Technology of China (2008AA06Z324), the Ministry of Science and Technology of China (973 program 2010CB833101), and the Knowledge Innovation Program of the Chinese Academy of Sciences. ’ REFERENCES (1) Karakoti, A. S.; Monteiro-Riviere, N. A.; Aggarwal, R.; Davis, J. P.; Narayan, R. J.; Self, W. T.; McGinnis, J.; Seal, S. JOM 2008, 60, 33. (2) Chen, J. P.; Patil, S.; Seal, S.; McGinnis, J. F. Nat. Nanotechnol. 2006, 1, 142. (3) Tarnuzzer, R. W.; Colon, J.; Patil, S.; Seal, S. Nano Lett. 2005, 5, 2573. (4) Das, M.; Patil, S.; Bhargava, N.; Kang, J. F.; Riedel, L. M.; Seal, S.; Hickman, J. J. Biomaterials 2007, 28, 1918. (5) Colon, J.; Herrera, L.; Smith, J.; Patil, S.; Komanski, C.; Kupelian, P.; Seal, S.; Jenkins, D. W.; Baker, C. H. Nanomedicine 2009, 5, 225. (6) Niu, J. L.; Azfer, A.; Rogers, L. M.; Wang, X. H.; Kolattukudy, P. E. Cardiovasc. Res. 2007, 73, 549. (7) Yu, L.; Lu, Y.; Man, N.; Yu, S. H.; Wen, L. P. Small 2009, 5, 2784. (8) Hirst, S. M.; Karakoti, A. S.; Tyler, R. D.; Sriranganathan, N.; Seal, S.; Reilly, C. M. Small 2009, 5, 2848. (9) Schubert, D.; Dargusch, R.; Raitano, J.; Chan, S. W. Biochem. Biophys. Res. Commun. 2006, 342, 86. (10) Karakoti, A. S.; Singh, S.; Kumar, A.; Malinska, M.; Kuchibhatla, S.; Wozniak, K.; Self, W. T.; Seal, S. J. Am. Chem. Soc. 2009, 131, 14144. (11) Rzigalinski, B. A.; Meehan, K.; Davis, R. M.; Xu, Y.; Miles, W. C.; Cohen, C. A. Nanomedicine 2006, 1, 399. (12) Korsvik, C.; Patil, S.; Seal, S.; Self, W. T. Chem. Commun. 2007, 1056. (13) Heckert, E. G.; Karakoti, A. S.; Seal, S.; Self, W. T. Biomaterials 2008, 29, 2705. (14) Bauer, V.; Bauer, F. Gen. Physiol. Biophys. 1999, 18, 7. (15) Ames, B. N.; Shigenaga, M. K.; Hagen, T. M. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 7915. (16) Battino, M.; Bullon, P.; Wilson, M.; Newman, H. Crit. Rev. Oral Biol. Med. 1999, 10, 458. (17) Jia, S. P.; Liang, M. M.; Guo, L. H. J. Phys. Chem. B 2008, 112, 4461. (18) Yang, Q. M.; Pan, X. H.; Kong, W. B.; Yang, H.; Su, Y. D.; Zhang, L.; Zhang, Y. N.; Yang, Y. L.; Ding, L.; Liu, G. A. Food Chem. 2010, 118, 84. (19) Tok, A. I. Y.; Du, S. W.; Boey, F. Y. C.; Chong, W. K. Mater. Sci. Eng., A 2007, 466, 223. (20) Viswanathan, V.; Filmalter, R.; Patil, S.; Deshpande, S.; Seal, S. J. Am. Ceram. Soc. 2007, 90, 870. 4437

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