Insight into Several Factors that Affect the Conversion between

Aug 22, 2016 - Many conflicting results have been reported related to the antioxidant and oxidant activities of nanoceria. On the basis of this resear...
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Insight into Several Factors that Affect the Conversion between Antioxidant and Oxidant Activities of Nanoceria Mei Lu, Yan Zhang, Yiwen Wang, Miao Jiang, and Xin Yao* School of Chemistry and Chemical Engineering, University of Chinese Academy of Sciences, Beijing 100049, P.R. China S Supporting Information *

ABSTRACT: Many conflicting results have been reported related to the antioxidant and oxidant activities of nanoceria. On the basis of this research, many factors might affect the antioxidant activity of nanoceria. However, all of the factors reported only affect the antioxidant activity of nanoceria to a limited extent or cause the antioxidant activity to be lost. We found that several factors can induce conversion between the protective effect and toxicity of nanoceria. At low concentrations of hydroxyl radicals (•OH) and nanomaterials, nanoceria exhibited antioxidant activity but could produce greater amounts of •OH at higher •OH or nanomaterial concentrations and subsequently exhibit oxidant activity. Moreover, the morphology and size of nanoceria can also affect this conversion. We found that high concentrations of •OH and nanoceria could introduce a high amount of Ce3+ in the system, which might be the reason that nanoceria converted from exhibiting antioxidant to oxidant activity. Under this condition, nanoceria act as a catalyst similar to Fe2+ to promote •OH production in a Fenton system and also as a catalyst promoter to boost Fe2+ production of additional •OH during the redox reaction. These conclusions support a better understanding of conflicting reports on medicinal applications for nanoceria and promote their practical application. KEYWORDS: nanoceria, antioxidant activity, oxidant activity, conversion factors, hydroxyl radicals nanoceria is due to oxidative stress,25,27 production of ROS,28,29 DNA damage,5 cell membrane damage,30 or an increase in intracellular ROS caused by direct contact between the cells and the nanoparticles.31 These findings have raised conflicts related to the protective effect and toxicity of nanoceria in biological systems. Until now, this paradoxical phenomenon has not been well-understood, which greatly hinders the medical application of nanoceria. On the basis of related reports, we found that many factors could affect the antioxidant activity of nanoceria. Zhou et al. studied the concentration effect of nanoceria and found that ceria promoted differentiation of osteoblasts and that the promotion rate was enhanced with increasing ceria concentration.32 Through different synthesis procedures, ceria of different sizes, morphologies, and surface properties were obtained,33−37 and these differences could have an impact on antioxidant

1. INTRODUCTION Recently, highly conflicting results have been published on the antioxidant and oxidant activities of nanoceria in biomedical science.1−6 Nanoceria display the antioxidant activity of scavenging reactive oxygen species (ROS) by switching between Ce3+ and Ce4+ on their surface.7−18 Previous studies showed that nanoceria could protect photoreceptor cells,2 cardiac progenitor cells,19 gastrointestinal epithelium,20 and human endothelial cells (ECs)21 by scavenging •OH and might also offer radioprotection to normal cells but not to tumor cells.3 Because of this antiradiation property, nanoceria could prevent •OH-related diseases such as inflammation8,22 and xerostomia.23 However, nanoceria toxicity has also been reported.24−31 Park et al. found that nanoceria could induce delayed-type hypersensitivity and granuloma in the lungs of mice after intratracheal instillation.24 Furthermore, Cheng et al. showed that nanoceria decreased the cell viability of human SMMC-7721.25 Moreover, nanoceria could be one of the prooxidants that change the intracellular redox status of normal and cancer cells.26 It has been proposed that the toxicity of © XXXX American Chemical Society

Received: July 7, 2016 Accepted: August 22, 2016

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DOI: 10.1021/acsami.6b08219 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 1. XRD (A) and XPS (B) patterns of CeO2: 20−25 nm nanocubes (a), 30−40 nm nanocubes (b), 5−10 nm nanoparticles (c), and 15−20 nm nanoparticles (d).

different morphologies and sizes also displayed different antioxidant properties. Furthermore, the mechanism of the described phenomenon was explored. These conclusions support a better understanding of the conflicting reports on medicinal application of nanoceria and promote practical applications.

activity. Xue et al. proved that the antioxidant activity of 5−10 nm nanoceria is greater than that of 15−20 nm ceria particles because the smaller particles can scavenge a greater amount of •OH.38 Additionally, Ji et al. also reported that smaller ceria nanorods were nontoxic, whereas the larger counterparts obviously cause cell death.39 The Ce3+ on the surface of nanoceria are presumed to be active sites and can easily react with ROS, and a greater amount of Ce3+ can scavenge greater amounts of ROS.13,34,40−42 Previous studies showed that exposed crystal planes could affect the antioxidant property of nanoceria.43 At the same time, certain researchers focused on the effect of pH.8,44,45 Perez et al. demonstrated that Ce3+ on the surface of nanoceria were able to return to their initial amount at pH 7.4, but this process was nonreversible at lower pH.8 Water-soluble nanoceria coated with several polymers were also reported, and the coatings had no effect on antioxidant activity.8,9,46−48 These factors affect only the antioxidant activity of nanoceria to a limited extent. However, this activity is completely lost in solutions of phosphate or bicarbonate ions.45,49 Although many factors of influence have been discovered, it is still difficult to explain the controversial reports that refer to the protective effect and toxicity of nanoceria. It was found that all of the reported factors only affected the antioxidant activity of nanoceria to a limited degree or caused the antioxidant activity to be lost. No findings have been reported on the factors that could result in the toxicity of nanoceria. In analyzing the existing reports, we found that researchers use different synthetic methods and thus produce nanoceria products with various morphologies and sizes, which were applied in different types of cells during biological effect studies. This diversity of experimental conditions might be the primary reason for conflicting conclusions. In this study, we used a simple UV−vis spectroscopic method with a methyl violet (MV)-Fenton reagent system to identify the factors that could result in conversion between the protective effect and toxicity of nanoceria in biological systems. In considering the reaction and the reported influential factors, the concentration of •OH was first investigated to explore its relationship with nanoceria’s properties. In this work, different concentrations of •OH were obtained by adjusting the ratio of H2O2 and Fe2+ in the Fenton systems. The concentration effect of nanoceria was also studied, and the concentration conversion points of nanoceria between a protective effect and toxicity were obtained. Nanoceria with

2. MATERIALS AND METHODS 2.1. Materials and Reagents. Hydrochloric acid (HCl), MV, hexamethylenetetramine (HMT, Sinopharm Chemical Reagent Co., Ltd.; Beijing, China), sodium hydroxide (NaOH), iron(II) sulfate heptahydrate (FeSO4·7H2O), hydrogen peroxide (H2O2, 35%), (hydroxymethyl)aminomethane (Tris), and cerium(III) nitrate hexahydrate (Ce(NO3)3·6H2O) were purchased from Sigma-Aldrich. All reagents were of analytical purity and used without further purification. The water used in all experiments was Millipore Milli Q (18 MΩcm). 2.2. Instruments. The morphology and surface of nanoparticles were assessed using an American FEI TECAI G2F20 transmission electron microscope (TEM) and Thermo Scientific ESCALAB 250XI X-ray photoelectron spectrometer (XPS), respectively. The base pressure during XPS analysis was approximately 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 CeO 2 were subsequently fitted using PeakFit (version 4.0) software. X-ray diffraction (XRD) patterns were obtained with a Bruker D8 Advance X-ray diffractometer using Cu KR radiation (λ = 1.5418 Å). The data were recorded at a scan rate of 2°/min. The UV−vis absorption spectra were collected with a UV-2550 spectrophotometer. Electron spin resonance analysis (ESR) was used to identify •OH with the spin trap DMPO. The ESR spectra were acquired using a JEOL-JES FA200 ESR spectrometer operating in the X-band. The ESR settings used to detect DMPO spin-trap adducts are described as follows: modulation frequency = 100 kHz, X-band microwave frequency = 9.44 GHz, microwave power = 4 mW, modulation amplitude = 1.0 gauss (G), time constant = 0.03 s, sweep time = 4 min, and center field = 3378 G. The test solutions were Fenton systems with different proportions of H2O2 and FeSO4. After a 5 min reaction, DMPO (300 mM) was added into the Fenton system and transferred to a capillary tube for ESR spectral analysis at room temperature. 2.3. Preparation of Ceria Nanoparticles and Nanocubes. The nanomaterials were synthesized using hydrothermal treatment with slight modification.50−52 Ceria nanoparticles were synthesized with 0.125 M HMT and 0.025 M Ce(NO3)3·6H2O. HMT was added dropwise to a stirred Ce(NO3)3·6H2O solution. All solution was transferred into a 50 mL autoclave for 2 h at 100 °C or 12 h at 150 °C. The ceria nanoparticle powder was obtained by centrifugation and B

DOI: 10.1021/acsami.6b08219 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 2. TEM (top) and HRTEM (down) images of CeO2: 20−25 nm nanocubes (a, e), 30−40 nm nanocubes (b, f), 5−10 nm nanoparticles (c, g), and 15−20 nm nanoparticles (d, h).

respectively. Therefore, a greater amount of Ce3+ is present at the surface of the smaller-sized particles, which is consistent with the literature.38 As shown in Figure 2, the TEM images for a and b depict nanocubes with sizes of 20−25 and 30−40 nm, respectively, whereas the images of c and d are nanoparticles with sizes of 5−10 and 15−20 nm, respectively. The size of b is greater than that of a, and the size of d is greater than that of c. These results are consistent with the results from the XRD patterns. The nanoceria of b and d were obtained with high temperature, long reaction time, or high reactant concentration, which could promote the Ostwald ripening process, and thus, larger nanocubes or nanoparticles could be formed.38,51 The exposed crystal planes of these four nanoceria were studied in additional detail using high resolution TEM (HRTEM) images (e−h). As shown in images e and f, the (100) plane can be identified according to the interplanar spacing (0.28 nm) for nanocubes. The interplanar spacing of 0.32 nm (images g and h) indicates the dominant presence of the (111) plane for nanoparticles.52,55 3.2. Hydroxyl Radical Concentration Effect on the Conversion. As reported previously, nanoceria are able to scavenge •OH and possess antioxidant activity.38,53,56 However, nanoceria’s toxicity has been reported as well. These two opposite properties of nanoceria are closely related to ROS.53 Until now, the impact of ROS on the properties of nanoceria has been seldom reported. Hence, the behaviors of nanoceria in response to various hydroxyl radical systems are explored in this work. The method used to evaluate the •OH scavenging ability of nanoceria is a simple UV−vis spectroscopic method with the MV-Fenton system, as used in previous work with certain modifications.38,43,48 In this method, purple MV reacts with •OH in the Fenton reagent and fades to a colorless appearance; its maximum absorbance (582 nm) decreases. The change of absorbance (denoted as ΔA) indirectly indicates the amount of generated •OH. The larger the value of ΔA, the more •OH is present in the system. If nanoceria can scavenge •OH, a portion of MV is protected from attack, and the maximum absorbance is expected to increase after nanoceria are added. The change in absorbance after the addition of nanoceria is signified by Δa. If nanoceria have the ability to scavenge •OH, Δa is less than ΔA. For this effect to be clarified, the meanings of Δa and ΔA are provided in Figure S2. The ratio of Δa/ΔA

washing after the reaction solution was cooled to room temperature. Nanocubes were synthesized as follows:53 1 or 1.5 g Ce(NO3)3·6H2O was dissolved in water, and a NaOH solution (16 g) was rapidly added with stirring. After 30 min, the solution was transferred into a 50 mL autoclave for 24 or 36 h and hydrothermally treated at 200 °C. After the solution was cooled to room temperature, powders of 20−25 nm or 30−40 nm ceria nanocubes were obtained by centrifugation and washing. 2.4. UV−Vis Photometric Experiments. The nanoceria stock solutions were prepared by dispersion in 0.1 M Tris−HCl buffer at pH 4.7 and sonication prior to use. The solution used in photometric determination contained 1.2 × 10−5 M MV, 0.075−0.60 mM FeSO4, CeO2 (concentration range of 10−500 μM), H2O2 (35%) (0.1 or 1.0 M), and 0.1 M Tris−HCl buffer with a final volume of 5 mL, and all reagents were added to Tris−HCl buffer in this order. The absorbance of the tested solution was determined after incubation for 5 min at room temperature.

3. RESULTS AND DISCUSSION 3.1. Characterization of Nanoceria. The sizes, morphologies, and crystal planes of the synthesized nanoceria were examined, and the XRD patterns of CeO2 are shown in Figure 1A. The typical fluorite cubic structure (JCPDS card: 34-0394) of cerium oxide was clearly observed for all nanoceria. The peaks of curve b are wider than those of a, which indicates that the nanocube size of b should be greater than that of a, and the size of the nanoceria particles of d is also greater than that of c. Considering the synthesis conditions used during the preparation of these nanoceria, we concluded that a larger size of the nanoceria could be obtained with high temperature, long reaction time, or high concentration of the reaction reagent. This observation was consistent with the results obtained from the Debye−Scherrer formula, which showed that crystallite size was inversely proportional to the full width at half-maximum (Fwhm).54 The XPS results for four nanomaterials are shown in Figure 1B, which reveals the presence of a mixed valence state (Ce3+ and Ce4+). The binding energy peaks at 884.5 and 903.1 eV belong to Ce3+, and the peaks at 881.5, 887.8, 897.2, 899.8, 906.4, and 915.60 eV are indicative of the presence of Ce4+. To further determine the amount of Ce3+ in the four samples, the peak positions were fit using PeakFit (version 4.0) software (Figure S1). According to calculations, the Ce3+concentrations at the particle surfaces are 30.74 and 27.55% for the 5−10 and 15−20 nm particles, respectively, and 26.45 and 25.14% for the 20−25 and 30−40 nm cubes, C

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Figure 3. Variation tendency of Δa/ΔA with increasing concentrations of FeSO4 in different MV (1.2 × 10−5 M)-Fenton reagent systems: 10 μM 15−20 nm nanoparticles with 0.1 M H2O2 (A) or 1.0 M H2O2 (B), 10 μM 20−25 nm nanocubes with 0.1 M H2O2 (C). Relative standard deviations (percentage of RSD) are all less than 6.12%.

system when the concentration of •OH approached a high level. Furthermore, this phenomenon was confirmed by another nanoceria experiment in which 20−25 nm nanocubes were tested in the Fenton reagent system with 0.1 M H2O2 and 0.075 mM to 0.75 mM FeSO4 (Figure 3C). Figure 3C shows that ceria nanocubes could protect MV by scavenging •OH in a lower hydroxyl radical system, but the value of Δa/ΔA also exceeded 1 when the Fe2+ concentration was greater than 0.45 mM, which meant that nanocubes also produce •OH in a high hydroxyl radical system. Therefore, the three comparative experiments described above demonstrated that the amount of •OH has an impact on nanoceria. Nanoceria showed antioxidant activity in systems with low concentrations of •OH but displayed oxidant activity in systems with a high level of •OH content. This observation might be a contributing reason for the conflicting results regarding nanoceria for medicinal use. Certain studies verified that nanoceria were nontoxic to selected organisms but toxic to others.37,57−59 For example, Park et al. found that ceria nanoparticles could induce oxidative stress in cultured BEAS2B cells, but they did not show significant cytotoxicity in cultured T98G cells.59 Leung et al. used three different CeO2 nanoparticle samples to test toxicity in two cell lines. They found that one CeO2 nanoparticle sample exhibited the highest toxicity to E. coli but exhibited negligible toxicity to S. costatum, whereas the other two samples had negligible toxicity to E. coli and exhibited high toxicity to algae at the same concentration.60 These behaviors of nanoceria might be due to the different intracellular concentrations of •OH in different cells,61,62 and as noted above, different concentrations of •OH could result in different activities of nanoceria, as shown in Figure 3. Thus, the opposite results of antioxidant and oxidant activities that nanoceria displayed in certain cases might be ascribed to the different organisms used in various experiments. 3.3. Effect of the Nanoceria’s Concentration on the Conversion. On the basis of the data in Figure S3, four hydroxyl radical systems were used to study the effect of nanoceria concentration: T1, 0.1 M H2O2 + 0.45 mM FeSO4; T2, 0.1 M H2O2 + 0.30 mM FeSO4; T3, 1.0 M H2O2 + 0.15 mM FeSO4; and T4, 1.0 M H2O2 + 0.09 mM FeSO4. The ESR spectra of these four systems were recorded (Figure S4) to demonstrate the amount of •OH in each system. The peak signal strength of curve T1 is greater than that of curve T2,

can be used to demonstrate the protective ability of nanoceria. The lower the value of Δa/ΔA, the stronger the •OH scavenging ability of the nanoceria. Nanoparticles (10 μM) with sizes of 15−20 nm were used to explore the antioxidant activity of nanoceria in several Fenton reagents with different concentrations of •OH. The first system contained 0.1 M H2O2, and the amount of FeSO4 varied from 0.075 to 0.75 mM. For a certain concentration of H2O2, the greater the amount of FeSO4 added, the more •OH was produced in the system and the more dramatically the MV appeared to fade (Figure S3). For the protective ability of nanoceria to be displayed more clearly, the ratio of Δa/ΔA was plotted on the Y-axis to show the relationship between the antioxidant activity of nanoceria and the concentration of Fe2+ (Figure 3A). From this figure, it can be observed that Δa/ΔA first decreased at a low concentration of Fe2+ and subsequently increased with increasing concentrations of Fe2+. However, if the concentration of Fe2+ approached 0.60 mM, the value of Δa/ΔA increased greatly and even exceeded 1, indicating that Δa was larger than ΔA. As noted above, the lower the Δa/ΔA value, the stronger the •OH scavenging ability of nanoceria. In most cases, nanoceria have the ability to scavenge •OH and protect MV from becoming colorless, but when the concentration of Fe2+ reached 0.60 mM, nanoceria promote the generation of •OH instead of protecting MV from being attacked by •OH and result in additional MV turning colorless. When the concentration of Fe2+ continued to increase, the value of Δa/ΔA increased continually, i.e., a greater amount of •OH is produced by nanoceria in a higher •OH content system. For the phenomenon noted above to be verified, another series of Fenton reagent systems (1.0 M H2O2 and 0.075 mM to 0.60 mM of FeSO4) was used, and the results are shown in Figure 3B. The same variation tendency of Δa/ΔA that is shown in Figure 3A was obtained due to addition of the same nanoceria (15−20 nm nanoparticles). With increasing concentrations of Fe2+, the ratio of Δa/ΔA decreased at the beginning, but when the concentration of Fe2+ approached 0.50 mM, the value of Δa/ΔA exceeded 1 as well. This result indicated that ceria nanoparticles could scavenge •OH in a lower •OH concentration system, but in a high •OH environment, the addition of nanoceria could significantly increase the amount of •OH, i.e., the oxidant activity of nanoceria was displayed in this D

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Figure 4. UV−vis absorption spectra of MV in different Fenton reagent systems: T1 (A) or T2 (B) system with different concentrations of 15−20 nm nanoparticles (b, 20; c, 30; d, 10; e, 0; f, 5; g, 50 μM). T3 (C) or T4 (D) solution with different concentrations of CeO2 (b, 50; c, 20; d, 10; e, 0; f, 5 μM). Variation tendency of Δa/ΔA with increasing concentrations of 15−20 nm nanoparticles in different Fenton reagents: T1 and T2 systems (E), T3 and T4 systems (F). Curve a in figures (A−D) is the 1.2 × 10−5 M MV’s UV−vis absorption spectrum. Relative standard deviations (percentage of RSD) are all less than 7.98% in E and F.

which indicates that the yield of •OH generated in system T1 is greater than the yield in system T2. This result also demonstrated the large amount of •OH in the system with a greater amount of Fe2+, which is consistent with the result from Figure S3. Because the intensity of the ESR peak of curve T3 was larger than that of curve T4, the yield of •OH generated in system T3 was greater than the yield in system T4. Subsequently, 5−50 μM 15−20 nm ceria nanoparticles was added to the T1 system, and the results are shown in Figure 4A and curve T1 of Figure 4E. When 5 μM ceria nanoparticles was added, no obvious protection effect occurred, but with increasing concentration of nanoceria, the antioxidant activity notably increased and reached a peak at 20 μM, which was

consistent with the conclusion that additional CeO2 can scavenge additional •OH.38,40 Subsequently, the antioxidant effect was decreased, as shown in Figure 4A, in which the absorbance spectra value of the 30 μM nanoparticles was smaller than that of the 20 μM nanoparticles. Antioxidant activity disappeared and oxidant activity appeared when the concentration reached 50 μM, and the value of Δa/ΔA was larger than 1. When 5−50 μM 15−20 nm ceria nanoparticles were used in another Fenton reagent system (T2 system), the same result was obtained and is shown in Figure 4B and curve T2 of Figure 4E. At the beginning, the protection effect also increased with increasing concentrations of ceria nanoparticles, and 20 μM was the optimal concentration. At that point, 15− E

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Figure 5. ESR spectra of the (A) MV/0.1 M H2O2/0.75 mM FeSO4 system after 0 μM (a) or 10 μM (b) 15−20 nm CeO2 nanoparticles was added or the (B) MV/0.1 M H2O2/0.30 mM FeSO4 system after 0 μM (a), 50 μM (b), or 10 μM (c) 15−20 nm CeO2 was added.

only in a certain concentration range, which means that the concentration effect varies for different systems. These results further proved that •OH affects the properties of nanoceria, and therefore, this topic should receive additional attention. Similar to this result, Atual et al. found that nanoceria could oxidize a series of organic substrates and showed oxidizing activity at high concentrations.63 In addition, it was reported that other antioxidants could scavenge ROS at a low concentration but induce stress at high concentrations.64−67 Although the mechanisms are not well understood, the results suggest that disruption of the delicate balance between antioxidants and ROS could produce antioxidant-induced stress if the antioxidants overwhelm the physiological production of reactive species. In addition, if the concentration of ceria nanoparticles exceeds 50 μM, it could interfere with the absorption of MV, and thus 50 μM was the highest concentration of ceria nanoparticles used in these experiments. For further verifying that a high concentration of nanoceria could convert antioxidant activity to oxidant activity, small ceria nanoparticles were used (5−10 nm) in systems T1−T4, and the results are shown in Figure S5. In the four systems (T1− T4), the values of Δa/ΔA were all greater than 1 when the concentration of 5−10 nm nanoparticles was greater than 5 μM. Therefore, a concentration range exists in which ceria nanoparticles display the protection effect, and in this range, the protection effect increases with increasing concentration of nanoceria. Otherwise, these conditions could instead promote the production of •OH and show toxic effects at a much high concentration. These results again prove that the concentration of nanoceria can affect the properties of •OH. To confirm our results, ESR spectral analysis was used to test the change in the amount of •OH after the addition of nanoceria in different hydroxyl radical systems (Figure 5). The systems of MV/0.1 M H2O2/0.75 mM FeSO4 (Figure 5A) and MV/0.1 M H2O2/0.30 mM FeSO4 (Figure 5 B) were used as high and low •OH content systems to determine the effect of nanoceria according to the result of Figure S3. The ESR signal intensity of curve a (Figure 5A) is obviously stronger than that of curve b, indicating that the concentration of •OH becomes

20 nm ceria nanoparticles showed the largest antioxidant activity, but after that point, the protection effect decreased. When the concentration increased to 50 μM, the opposite result was observed, as the value of Δa/ΔA exceeded 1, which indicated that nanoparticles promoted the production of •OH and exhibited an oxidant effect. This trend was consistent with that of the 15−20 nm ceria nanoparticles in the T1 system, i.e., with increasing concentration, nanoceria exhibited an antioxidant effect at first but showed an oxidant effect at higher concentrations. In certain reports on the toxicity of nanoceria,5,24,25,28 the concentrations of nanoceria used were higher than those in the protective effect reports.14,15,18 But several different points must be considered for these two Fenton reagents. For example, 5 μM ceria nanoparticles showed no effect in the T2 system but showed a slight oxidant effect in the T1 system, whereas the 30 μM ceria nanoparticles showed an obvious antioxidant effect in the T1 system but showed an oxidant effect in the T2 system. As shown in Figure S4, the peak signal strength of curve T1 was larger than that of curve T2, which indicated that the yield of •OH generated in system T1 was greater than the yield in system T2. This result proved again that the concentration of •OH has a large effect on the properties of nanoceria, as noted above. These phenomena showed that the nanoceria concentration had a significant impact on their properties. For this conclusion to be further verified, two other systems (T3 and T4) were used to explore the concentration effect of nanoceria in which the ratios of H2O2:FeSO4 were quite different from those in systems T1 and T2. The same concentration range for 15−20 nm ceria nanoparticles was used in the T3 and T4 systems, and the results are shown in Figure 4C, D, and F. Although the ΔA values of T3 were larger than those of T4 due to a larger amount of catalyst (FeSO4) in T3, the changes in the property trend of the 15−20 nm ceria nanoparticles were almost the same in these two systems. Over the entire range of 5−50 μM, ceria nanoparticles showed good antioxidant ability in the T3 and T4 systems, and this ability increased greatly with an increasing concentration of nanoceria. However, in the T1 and T2 systems, ceria nanoparticles showed antioxidant activity F

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Figure 6. Variation tendency of Δa/ΔA with increasing concentration of nanoceria in different systems. Nanoparticles in the T1 system (A) or the T3 system (B): 15−20 nm (a) and 5−10 nm (b). Nanocubes in the T1 system (C): 20−25 nm (a), 30−40 nm (b). (D) 15−20 nm ceria nanoparticles (a) and 20−25 nm ceria nanocubes (b) in the T1 system. Relative standard deviations (percentage of RSD) are all less than 9.04%.

activity, which may be due to the size effect of nanoceria,40 i.e., the stronger antioxidant ability of the smaller nanoparticles might be due to larger amounts of Ce3+ on the surface of nanoceria, as demonstrated by XPS. Additionally, the smaller ceria nanoparticle sizes had notable oxidant activity at 10 μM, as shown by Δa/ΔA values that exceeded 1 (curves a and b). These results indicate that different sizes of nanoceria exhibited antioxidant activities in different concentration ranges. For smaller particles, a lower concentration was required. For other systems (T3 and T4), the property trends were quite different for 15−20 nm (Figure 4F) and 5−10 nm (Figure S5F) ceria nanoparticles. As mentioned, 15−20 nm ceria nanoparticles showed good antioxidant ability over the entire concentration range in systems T3 and T4, but the 5−10 nm nanoparticles exhibited conversion from antioxidant activity to oxidant activity. As shown in Figure 6B, at the same concentration of 10 μM, the 15−20 nm nanoceria had an obvious protection effect, whereas the smaller nanoceria (5−10 nm) behaved in the opposite manner in the T3 system. These results indicated that materials of different sizes behaved differently even at the same concentration in the same system, i.e., the size of the nanoceria greatly affected their properties, and different sizes of nanoceria could have opposite impacts on the same cell. For instance, Auffan et al. found that 7 nm CeO2 nanoparticles could induce DNA damage in human dermal fibroblasts,5 and Culcasi et al. confirmed the production of ROS in human dermal fibroblasts after exposure to 7 nm CeO2 nanoparticles.53 However, Karakoti et al. reported the antioxidant activity of 4 nm CeO2 nanoparticles in human

much higher than that of the same hydroxyl radical system without nanoceria (curve a) after the addition of 10 μM nanoceria (curve b), which means that nanoceria can increase the amount of •OH in a high hydroxyl radical system. In a lower hydroxyl radical system, as shown in Figure 5B, the ESR signal intensity of curve c is obviously weaker than that in curve a, and the intensity of curve b is slightly stronger than that of curve a. Therefore, in a low hydroxyl radical system, the addition of 10 μM nanoceria could reduce the amount of •OH in the system, whereas the amount of •OH increases after the addition of a greater amount of nanoceria. This result again proved that nanoceria display antioxidant activity at low concentrations of •OH and ceria but exhibit oxidant activity at high concentrations of •OH and ceria. 3.4. Effects of Nanoceria Size and Morphology on the Conversion. On the basis of the results described above, we found that the external factors of •OH and nanoceria concentration have large effects on the antioxidant activity of nanoceria and could result in conversion between the antioxidant and oxidant activities of nanoceria. In addition, the size and morphology of nanoceria have important effects on the properties of nanoceria. For example, from a comparison of 15−20 and 5−10 nm nanoparticles (Figure 6A), the optimal concentration point for smaller nanoparticles in the T1 system was 0.20 μM (curve b), whereas for the larger size, this concentration was 20 μM (curve a). In addition, the concentration applied for smaller nanoparticles was much lower than that of the 15−20 nm nanoparticles, and even a 50 nM concentration of smaller nanoceria has obvious antioxidant G

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Figure 7. UV−vis absorption spectra of MV in the T1 system with different concentrations of 20−25 nm nanocubes (A) or 30−40 nm nanocubes (B) (b, 50; c, 20; d, 0; e, 10; f, 80 μM). Curve a in both panels is the 1.2 × 10−5 M MV UV−vis absorption spectrum.

dermal fibroblasts.9 These three research studies used the same cell line and concentration of nanoceria but different sizes to investigate the properties of nanoceria and obtained conflicting results. In the same manner, Park et al. found that 30 nm ceria nanoparticles showed toxic effects toward BEAS-2B cells,59 whereas Xia et al. used the same concentration and revealed that 13 nm ceria nanoparticles did not induce cytotoxicity in the same cells.27 Therefore, the different sizes of nanoceria used in different studies might explain the conflicting results of nanoceria properties. For further investigating the size impact of nanoceria on their properties, nanoceria with another morphology (nanocubes) and of different sizes were used. Nanocubes have different morphologies due to the different crystal planes exposed (Figure 2e and f). Concentrations of 10−80 μM 20−25 and 30−40 nm nanocubes were used in the T1 system, and the signals were recorded with minimal differences (Figure 7). Both of these sizes of nanocubes showed the same trend for changes in properties. The nanoceria showed antioxidant ability from 20 to 50 μM and began to promote production of •OH at a concentration of 80 μM in the T1 system (Figure 6C). From the weak discrepancy between the two sizes of nanocubes, we conclude that the size effect of the nanocubes (Figure 6C) was quite small compared with that of the nanoparticles (Figure 6A) in the same system (T1), which might be ascribed to the different morphologies of the nanocubes and nanoparticles. Additionally, it was found from XPS that the Ce3+ content was approximately the same for these two nanocubes, which could explain the minimal difference in properties for these two sizes of nanocubes. However, this explanation does not apply to the ceria nanoparticles for which the Ce3+ contents display great differences upon changes in size, which might result in different behaviors of the different morphologies of nanocubes and nanoparticles. From Figure 6D, it can also be observed that the behavior of 15−20 nm ceria nanoparticles (curve a) was quite different from that of 20−25 nm ceria nanotubes (curve b) in the T1 system, although the sizes of the two nanomaterials were nearly the same. First, we note that the optimal concentration point of antioxidant activity for the nanoparticles was 20 μM, but for nanocubes this value was 50 μM. At their respective optimal concentration points, the antioxidant activity of the nanoparticles was notably stronger than that of the nanocubes. At 50 μM, the nanoparticles showed oxidant activity, but the nanocubes behaved as antioxidants. Thus, it can

be inferred that the different morphologies of these two nanomaterials are the main reason for the variation in Δa/ΔA in various hydroxyl radical systems (Figure 3A,C). These observations indicate that morphology is another important factor that affects the antioxidant and oxidant activities of nanoceria and could result in contradictory results in studies of their properties. For the differences among these four nanomaterials to be examined in additional detail, the UV−vis absorption spectra of MV in the T1 system after the addition of four 10 μM nanomaterials are compared in Figure S6. In this figure, only the 15−20 nm nanoparticles showed antioxidant activity, and the 5−10 nm nanoparticles behaved as oxidants. However, the 20−25 and 30−40 nm nanocubes had no obvious effect on the absorbance value. This result proved again that both size and morphology affect the properties of nanoceria and could lead to contradictory effects at the same concentration in the same system. Considering the results obtained in this work, the process used by nanoceria to scavenge •OH is sensitive to many factors, such as the concentrations of •OH and nanoceria and the size and morphology of nanoceria. All of these factors might result in conversion from antioxidant activity to oxidant activity of nanoceria, which could explain conflicting reports on the properties of nanoceria because the nanoceria used in studies differ in cell types, media environment, and variety (such as different sizes, shapes, and concentrations).22,27,28,59 3.5. Mechanism Discussion. Previous results demonstrated that nanoceria could scavenge •OH at low concentrations of •OH and nanoceria, but show oxidant activity and make the amount of •OH increase in the system at higher concentrations of •OH and nanoceria. These findings will aid in developing a better understanding of the conflicting results on nanoceria in medicinal applications. However, the mechanism of this conversion is still unclear. As reported, the nanoceria’s antioxidant and oxidant properties are closely related to the amount of Ce3+ on their surface.13,40−42As reported, UV−vis has been used to characterize the change of Ce3+ and Ce4+ on the surface of nanoceria; the peak of Ce3+ is blue shifted compared with that of Ce4+.8,9 For further studying the mechanism of the property conversion of nanoceria, the surface chemistry of CeO2 after reaction with different concentrations of hydroxyl radical systems was studied using UV−vis spectroscopic analysis (Figure 8). Curve a showed a clear H

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Fenton-like reaction. Therefore, •OH and superoxide anions could be produced from a series of reactions analogous to the Fenton/Haber Weiss reaction28 with the equation Ce3 + + H 2O2 + H+ → Ce 4 + + H 2O + ·OH

In addition, nanoceria could enhance the catalyst activity of Fe2+ because they are capable of interacting with iron oxides and changing the iron valence state in the composite. The Ce(III) in nanoceria could transmit electrons to the iron oxide and thus enhance the overall activity of the catalyst.68 Thus, Ce3+ in ceria could accelerate the production of •OH in the system. Therefore, nanoceria not only act as •OH producers but also as catalyst promoters during the redox reaction, explaining why notable oxidant ability of nanoceria is observed, using the value of Δa/ΔA (larger than 1), when the concentration of nanoceria is greater than 10 μM in the high hydroxyl radical systems (Figure 3). We suggest that nanoceria convert antioxidant activity to oxidant activity primarily due to the increasing amount of Ce3+. As Eric and Talib et al. have noted, the Ce3+/ Ce4+ ratio is critical for the biomimetic activity of nanoceria, then the properties of nanoceria could be controlled by the Ce3+ content.11,18 This work offers a better understanding of the major conflicts in nanoceria’s antioxidant activity reports. The concentrations of •OH and nanoceria can induce conversion between these two properties, and at the same time, the size and morphology of nanoceria can affect the concentration conversion point, thereby affecting these properties.

Figure 8. UV−vis absorption spectra of 15−20 nm CeO2 nanoparticles (a); CeO2 added to the 0.1 M H2O2/0.30 mM FeSO4 (b) or 0.1 M H2O2/0.75 mM FeSO4 system (c).

absorption peak at 318 nm. After interaction with a low hydroxyl radical system, the peak of Ce4+ became weaker and broader (curve b). Additionally, a new peak with a maximum absorbance at 280 nm appeared (curve c) after interaction with a high hydroxyl radical system, and the peak at 318 nm nearly disappeared simultaneously. According to the XPS result described above, Ce4+ should be the main component on the surface of nanoceria; then, the peak at 318 nm should be the UV−vis adsorb of Ce4+, and the new peak at 280 nm is ascribed to Ce3+ considering the blue shift of Ce3+ compared with the peak of Ce4+.8,9 These experiment phenomena indicate that the increase in the Ce3+ state of nanoceria was induced by the increasing concentration of •OH. The interference of FeSO4 and H2O2 was excluded by UV−vis spectroscopic analysis (Figure S7) because we know that Fe2+ or H2O2 interacts with nanoceria but creates no obvious change on the surface of nanoceria. This information proves that the change of Ce3+ on the surface of the nanoceria is likely due to the •OH in the system. With the interaction with •OH, the Ce3+ content could increase, and the higher amount of Ce3+ could scavenge additional •OH, thus strengthening the antioxidant activity of nanoceria.13,34,40−42 However, if the amount of Ce3+ is too high, nanoceria show oxidant activity. These phenomena are consistent with the result shown in Figure 3, i.e., nanoceria can scavenge •OH in a lower •OH concentration system but displays oxidant activity in a high •OH environment. Therefore, we infer that nanoceria show opposite properties in systems with different •OH concentrations, primarily because of the changing amount of Ce3+. Our study found that a particular concentration range is necessary for nanoceria to display a protection effect, and in this range, the protection effect increased with increasing concentration of nanoceria. However, the nanoceria’s antioxidant activity disappeared and oxidant activity appeared when the concentration increased further. We note that, after addition of a high concentration of nanoceria, the amount of Ce3+ also increased in the system, i.e., an increasing concentration of nanoceria actually increased the amount of Ce3+ in the system. High concentrations of •OH and nanoceria can induce a high amount of Ce3+ in the reaction system, and at this moment, Ce3+ could act as a catalyst similar to Fe2+ to induce a

4. CONCLUSIONS This work explored several factors that could affect the conversion of nanoceria from antioxidant to oxidant activity. The concentration of •OH and the sizes, morphologies, and concentrations of nanoceria could result in conversion between antioxidant and oxidant activity. Nanoceria show antioxidant activity at low concentrations of •OH but show oxidant activity at higher •OH levels. A specific concentration range of nanoceria exists in which they show antioxidant activity. In most cases, the antioxidant activity increases at first with increasing concentrations of nanoceria, but as the concentration of nanoceria continues to increase, nanoceria display oxidant activity. The morphologies and sizes of nanoceria can also affect their properties, and the concentration conversion points vary for nanoceria in different systems. It was found that •OH and nanoceria can create a high amount of Ce3+ in the system, and when the amount of Ce3+ reaches a certain amount, nanoceria convert their antioxidant activity to oxidant activity, primarily due to an increase in the amount of Ce3+. At this moment, nanoceria act as •OH producers and as catalyst promoters during the redox reaction. These conclusions explain why many contradictory results have been reported in medicinal applications of nanoceria and promote the practical application of nanoceria.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b08219. Ce 3d3/2,5/2 XPS spectra of nanoceria and the calculative process, UV−vis absorption spectra of MV, ESR images I

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of four hydroxyl radical systems, and UV−vis absorption spectra of 15−20 nm CeO2 nanoparticles (PDF)

AUTHOR INFORMATION

Corresponding Author

*Fax: +86 10 69672552. Tel: +86 10 69672552. E-mail: yaox@ ucas.ac.cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Nature Science Foundation of China (21271184), the Ministry of Science and Technology of China (973 program 2014CB931900 and 2012CB932504), and Beijing Municipal Science and Technology Commission.

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