Facet Energy versus Enzyme-like Activities: The Unexpected

Facet Energy versus Enzyme-like Activities: The Unexpected Protection of Palladium Nanocrystals against Oxidative Damage Cuicui Ge,†,‡,# Ge Fang,†,# Xiaomei Shen,∥ Yu Chong,†,‡ Wayne G. Wamer,‡,⊗ Xingfa Gao,*,∥ Zhifang Chai,† Chunying Chen,*,⊥ and Jun-Jie Yin*,‡ †

School for Radiological and Interdisciplinary Sciences (RAD-X), Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215123, China ‡ Division of Bioanalytical Chemistry and Division of Analytical Chemistry, Office of Regulatory Science, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, Maryland 20740, United States ⊥ Key Laboratory For Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology of China and Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100190, China ∥ College of Chemistry and Chemical Engineering, Jiangxi Normal University, Nanchang 330022, China S Supporting Information *

ABSTRACT: To develop nanomaterials as artificial enzymes, it is necessary to better understand how their physicochemical properties affect their enzyme-like activities. Although prior research has demonstrated that nanomaterials exhibit tunable enzyme-like activities depending on their size, structure, and composition, few studies have examined the effect of surface facets, which determine surface energy or surface reactivity. Here, we use electron spin-resonance spectroscopy to report that lower surface energy {111}-faceted Pd octahedrons have greater intrinsic antioxidant enzymelike activity than higher surface energy {100}-faceted Pd nanocubes. Our in vitro experiments found that those same Pd octahedrons are more effective than Pd nanocubes at scavenging reactive oxygen species (ROS). Those reductions in ROS preserve the homogeneity of mitochondrial membrane potential and attenuate damage to important biomolecules, thereby allowing a substantially higher number of cells to survive oxidative challenges. Our computations of molecular mechanisms for the antioxidant activities of {111}- and {100}-faceted Pd nanocrystals, as well as their activity order, agree well with experimental observations. These findings can guide the design of antioxidant-mimicking nanomaterials, which could have therapeutic or preventative potential against oxidative stress related diseases. KEYWORDS: palladium nanocrystals, surface facet, enzyme-like activity, antioxidants, computational chemistry, oxidative stress

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the production of reactive oxygen species (ROS) exceeds the antioxidant capacity of cellular antioxidants in a biological system, oxidative stress occurs, which results in damage to biomolecules such as lipids, proteins, and nucleic acids.8,9 Such cellular damage and tissue inflammation may contribute to

eactive oxygen species (ROS), including superoxide anion (O2•−), hydrogen peroxide (H2O2), singlet oxygen, and hydroxyl radicals (•OH),1,2 are byproducts of various metabolic processes caused by leakage in the electron-transport chain inside mitochondria and other sources;3 these play important roles in cellular signaling processes and contribute to different intracellular functions.4,5 Under normal physiological conditions, the cellular redox balance is maintained by antioxidant systems.6,7 However, when © 2016 American Chemical Society

Received: September 18, 2016 Accepted: November 7, 2016 Published: November 7, 2016 10436

DOI: 10.1021/acsnano.6b06297 ACS Nano 2016, 10, 10436−10445

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Figure 1. TEM and HRTEM images of the Pd nanocubes (a,c,e) and octahedrons (b,d,f).

Figure 2. Enzyme-like activity of the Pd nanocrystals. (a) CAT-like activity of Pd nanocrystals. ESR spectra of samples containing 0.1 mM 15NPDT, 10 mM H2O2, without (control) and with 25 μg/mL Pd nanocrystals vs 10 U/mL CAT. (b) Steady-state kinetic assay of Pd nanocrystals with H2O2.Km is the Michaelis−Menten constant, Vmax is the maximal reaction velocity. (c) SOD-like activity of Pd nanocrystals. ESR spectra of O2•− in the Xan/XOD generating system, without (control) and with 25 μg/mL Pd nanocrystals vs 1 U/mL SOD. (d) The quantification of oxygen production from superoxide turnover by Pd nanocrystals vs 1 U/mL SOD in KO2/18-crown-6-ether system.

limited by their lack of stability and loss of activation in harsh environments (e.g., nonphysiological pH, high temperature, or in the presence of inhibitors).14 In contrast, artificial enzymes created from nanomaterials (nanoenzymes) exhibit high stability, are inexpensive, and can be developed easily.15−17 Over the past several years, a wide variety of inorganic nanomaterials have been explored as nanoenzymes such as metal oxides,18−20 noble metals,21−23 carbon nanostructures,24−26 and other substances.27−29 Among these, the noble metal-based nanoenzymes, particularly gold, platinum, palladium, and iridium, have received tremendous interest due

conditions such as neuro-degeneration, cancer, diabetes, atherosclerosis, and arthritis.10−12 Robust and highly efficient ROS scavengers that could counteract these detrimental effects and maintain the physiologically appropriate balance of ROS formation and reduction would, therefore, be extremely valuable therapeutic agents. For these reasons, natural enzymes capable of detoxifying ROS, such as the superoxide dismutase (SOD) family converting superoxide anion to hydrogen peroxide and catalase converting H2O2 to water, have been considered as potential antioxidant drugs.13 However, those applications have been 10437


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Menten curves were received in a certain range of H2O2 concentration. With the Lineweaver−Bark equation, the important enzyme kinetic parameters including Michaelis− Menten constant (Km) and maximal reaction velocity (Vmax) were obtained. As Km values are indicators of enzyme affinity to substrates, a low Km value represents a strong affinity.49 The Km value of Pd octahedrons with H2O2 as the substrate was significantly higher than that observed for Pd nanocubes, suggesting that Pd nanocubes have a higher affinity for H2O2. This agrees with the calculated adsorption energies for H2O2 on Pd, which are −0.31 and −0.19 eV on the {100} and {111} facets, respectively, and is also consistent with the understanding that Pd {100} has a higher surface energy than Pd {111}. Nevertheless, the Vmax value of H2O2 decomposition catalyzed by Pd octahedrons is more than twice as fast as that catalyzed by Pd nanocubes, indicating that {111}-faceted Pd octahedrons have excellent catalytic efficiency. Another important enzyme in the cellular defense against ROS is superoxide dismutase (SOD), which can catalyze the dismutation of the superoxide anion radical (O2•−) into ordinary O2 and H2O2. Substances that could safely and reliably act as SOD mimetics could be used as therapeutic agents against various oxidative stress-related diseases.50,51 To study the O2•− scavenging activity by Pd nanocrystals, the xanthine/xanthine oxidase system (Xan/XOD) and BMPO were chosen as the generator and detector of O2•−, respectively. The characteristic ESR spectrum for the spin adducts BMPO/•OOH, under neutral conditions, has four lines with relative intensities of 1:1:1:1 (shown in Figure 2c). The addition of SOD resulted in a significant decrease of ESR signal intensity due to SOD’s strong ability to dismutate superoxides. A similar phenomenon was observed when Pd nanocrystals were added, suggesting that Pd nanocrystals potentially have SOD-like activity. Consistently, Pd octahedrons exhibited much higher SOD-like activity than Pd nanocubes, demonstrated by scavenging significantly more O2•−. We infer that this reaction may also be accompanied by the decomposition of H2O2, due to the catalase-like activity of Pd nanocrystals; in such reactions, superoxides are finally decomposed into O2 and H2O. To verify that mechanism, we identified the intermediates of the reaction, specifically O2. As shown in Figure 2d, the activity of Pd octahedrons enhanced the turnover of O2•− to O2 nearly 7-fold over the amount produced in the presence of Pd nanocubes. The Pd nanocubes did not even match the performance of the SOD under these conditions. Pd Nanocrystals Rescue Cell Viability via Efficient ROS Scavenging upon Oxidative Stress. Previous studies have demonstrated the antioxidant potential of nanomaterials with enzyme-like activities.19,52,53 The successful verification of facetdependent enzyme-like activities inspired us to explore the effects of facet number on the antioxidant functions of Pd nanocrystals added to cultures of human cell lines. To mimic conditions of oxidative stress, we treated human umbilical vein endothelial cells (HUVEC) with H 2O 2 at micromolar concentrations, which are known to induce ROS in the cells. First, we studied the kinetics of H2O2-mediated cytotoxicity to determine the appropriate treatment concentration; 60 μM H2O2 was selected with just over 50 ± 1% viability (Figure S1). The cell viability assays demonstrated that these Pd nanocrystals, even in concentrations up to 200 μg/mL (Figure S2), showed no obvious cytotoxicity, indicating good biocompatibility.

to their excellent catalytic activity and good biocompatibility.30−32 Nonetheless, there is still much important work to do to improve the catalytic activity of nanoenzymes as the enzyme mimetic systems.33 Previous studies have demonstrated that the enzyme-like activities of nanomaterials can be fine-tuned by controlling their size,34−36 chemical composition,23,37−40 and surface coating.41 Surface atoms also play an important role in the catalytic activity of nanoparticles;42−45 nanomaterials enclosed by different facets possess different surface energy, and therefore, each facet structure may exhibit markedly varied reactivity. However, these facet-dependent activities have been little understood. Here, we demonstrate how the surface facets of nanomaterials affect their performance as antioxidant enzyme mimics. For our investigation, we prepared two types of nanocrystals having cubic and octahedral morphologies: cubes enclosed by {100} facets and octahedrons enclosed by {111} facets. We then systematically investigated their potential enzyme-like activities, antioxidant behavior in vitro, and property−activity relationships, and then the specific molecular mechanisms were identified. This allows us to demonstrate how surface facets affect the enzyme-like activities of metal-based nanomaterials.

RESULTS AND DISCUSSION Preparation and Characterization of Pd Nanocrystals. The synthesis of palladium (Pd) nanocrystals, enclosed by either {111} or {100} facets, was carried out using a hydrothermal method, as described previously.46,47 We used transmission electron microscopy (TEM) to characterize the morphology of these Pd nanocrystals. The standard TEM images clearly show that these synthesized Pd nanocrystals have either cubic or octahedral shapes with an average edge length of 10 nm (Figure 1a,b). The high-resolution TEM (HRTEM) images of Pd nanocrystals fully confirm that these nanocubes and octahedrons are enclosed by {100} and {111} facets, respectively (Figure 1c−f). Enzyme-Like Activity of Pd Nanocrystals. Next, we employed electron spin resonance (ESR) to study how the surface facets might affect the antioxidant enzyme-like activity of these Pd nanocrystals. We selected catalase (CAT), a common enzyme present in nearly all living organisms exposed to O2 as the naturally occurring model antioxidant, as it has the well-documented ability to catalyze the decomposition of H2O2 into O2, thus protecting cells and tissues from oxidative damage.48 To determine the catalytic activity of Pd nanocrystals upon H2O2 decomposition, we monitored O2 production using ESR oximetry. As shown in Figure 2a, the ESR signal in the control condition has a narrow line width but high peak intensity, indicating a small amount of O2 dissolved in the solution and slow decomposition of H2O2. However, the addition of Pd nanocrystals results in an increased line width and decreased peak intensity of the ESR signal, indicating more rapid oxygen formation. These are similar to the changes in the ESR spectrum observed when catalase is added to the samples containing the spin probe and H2O2, providing evidence that Pd nanocrystals have catalase-like activity. Interestingly, the dissolved oxygen rate increases more rapidly in the presence of Pd octahedrons than Pd nanocubes, suggesting that Pd octahedrons possess much higher catalase-activity than Pd nanocubes under the same experimental conditions. Figure 2b shows the divergence between the behaviors of cubic and octohedral PD nanocrystals. Typical Michaelis− 10438


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Figure 3. Pd nanocrystals rescue cell viability via efficient ROS-scavenging, thus reducing oxidative stress. (a) Representative fluorescence images of live (green) and dead (red) cells. After exposure to 60 μM H2O2, cells were either untreated (UT) or treated with 25 μg/mL Pd nanocrystals. Scale bar: 20 μm. (b) Fluorescent images of ROS levels in cells after different treatments. Scale bar: 20 μm. (c) Quantitative analysis of the ROS levels by flow cytometry. Data is represented as the mean fluorescence intensity. These data are the means and standard deviations from three experiments. P values compared to that of H2O2-treated cells were calculated by Student’s t test: **p < 0.01, ***p < 0.001.

irreversible step of apoptosis, as shown by the decreased number of fluorescent red-stained mitochondria and an increased number of fluorescent green-stained mitochondria. The addition of Pd nanocrystals prevented the loss of red fluorescence aggregate and the increase of the green fluorescence monomer forms, suggesting that Pd nanocrystals attenuated H2O2-induced mitochondria dissipation. More specifically, compared to Pd nanocubes, cells incubated with Pd octahedrons showed predominantly red fluorescence aggregates, indicating that Pd octahedrons provided much better protection against H2O2-induced mitochondrial dysfunction than Pd nanocubes. Pd Nanocrystals Protect Damage to Important Biomolecules under Oxidative Stress. Overproduction of ROS within cells has been associated with detrimental consequences for important biomolecules, such as lipids, proteins, and DNA. Oxidation byproducts of these biological molecules are important oxidative stress biomarkers. The lipid peroxides in the cell can be quantified by measuring the final oxidative product in the lipid peroxidation reaction, malondialdehyde (MDA), using the thiobarbituric acid (TBA) assay.55 Oxidative stress has been shown to increase protein oxidation, and high levels of protein carbonyl (CO) groups have been observed in several human diseases.56 Therefore, our next assays determined the levels of oxidative products. After exposure to H2O2, levels of MDA increased by more than twice over that of untreated control cells (Figure 5a). With the assistance of Pd nanocrystals, that lipid peroxidation was significantly inhibited, and as expected, Pd octahedrons were more effective for inhibiting lipid peroxidation than Pd nanocubes. A similar phenomenon was observed for protein oxidation: the presence of Pd octahedrons significantly reduced the H2O2-induced oxidative damage to proteins (p < 0.01), again outperforming Pd nanocubes (p < 0.05) (Figure 5b). ROS-induced oxidative DNA damage encompasses both DNA strand breaks and nucleotide modifications.57 As such, γ-H2AX foci mark potential DNA damage sites, and γ-H2AX immunohistochemistry has been established as a reliable marker for measuring damage to DNA.58 As shown by the

To understand the antioxidant potential of these Pd nanocrystals, we performed the LIVE/DEAD cell viability assay (Thermo Fisher Scientific) and examined the resulting fluorescent images. Controls clearly showed a uniform confluent layer of viable cells, whereas cells exposed to 60 μM H2O2 alone showed a dramatic increase in dead cells and surviving cells showed a more rounded structure, a change in morphology that is characteristic of cells in the process of becoming apoptotic or dying from toxicity (Figure 3a). The addition of Pd nanocrystals appeared to preserve viability and normal morphology. Of the two nanocrystal types, Pd octahedrons were more effective for preventing the loss of cell viability, as can be seen from the increased density of viable cells. Next, we used DCFH−DA as a fluorescence probe to measure intracellular levels of ROS. As shown in Figure 3b, the strong fluorescence signal indicates that the peroxide level inside H2O2-treated HUVEC cells was remarkably enhanced in comparison to that in the untreated cells. However, this induction of intracellular ROS formation in H2O2-stimulated cells was significantly inhibited when 25 μg/mL Pd nanocrystals were added, as indicated by much weaker fluorescence signals. Quantitative analysis showed that 35% of the ROS in H2O2-stimulated cells could be scavenged by Pd nanocubes compared to 65% ROS scavenged by Pd octahedrons (Figure 3c). This is consistent with the other findings of superior performance by Pd octahedrons in enzyme-like and antioxidant activities. Pd Nanocrystals Reduce Mitochondria Injury upon Oxidative Stress. Excessive ROS are known to induce the collapse of mitochondrial membrane potential (MMP), which is an important event in mitochondrial dysfunction.54 To explore whether Pd nanocrystals could prevent or mitigate such damage, we first measured the MMP of HUVEC cells by confocal microscopy after staining with JC-1. The fluorescence images reveal that control cells presented predominantly red fluorescent aggregates indicating the preservation of the MMP (Figure 4). However, exposure to H2O2 induced depolarization of the mitochondrial membrane, considered to be an initial and 10439


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Figure 4. Pd nanocrystals reduce mitochondrial injury caused by oxidative stress, as demonstrated using the JC-1 reagent, which enters the mitochondria and forms aggregates displaying red fluorescence. When the mitochondrial membrane potential collapses, this dye reagent can no longer accumulate within the mitochondria and instead exists in a monomeric form that fluoresces green. Thus, we can observe the changes in mitochondrial membrane potential of HUVEC cells exposed to 60 μM H2O2 and then either untreated (UT) or treated with 25 μg/ mL Pd nanocrystals having different surface facets. Left and middle columns show the fluorescence of green fluorescent J-monomer and red fluorescent J-aggregate, respectively. The rightmost column of images shows the overlay of both images. Scale bar: 20 μm.

provides a convincing demonstration of the facet-dependent effects of Pd nanocrystals that can protect cells against oxidative challenges. Theoretical Simulations. Based on these consistent results across multiple experiments, we find that Pd octahedrons exhibit higher enzyme-like activity and greater antioxidant behavior than Pd nanocubes. However, this is surprising because the {111} facet structure which encloses Pd octahedrons has lower surface energy than the {100}, which encloses Pd nanocubes. Our ESR experiments have also indicated that {111}-faceted Pd octahedrons apparently have a lower affinity for H2O2 than {100} faceted Pd nanocubes. To unravel the mechanism for the unexpectedly greater antioxidant activity of Pd octahedrons, we computationally studied the H2O2 and O2•− scavenging reactions on Pd {111} and {100} facets, respectively. Reportedly, the H2O2 scavenging activity of metals originates from the following reactions occurring on the metal surfaces.

red dots in in Figure 5c, we detected a high level of expression of γ-H2AX in the H2O2-only treatment group. Addition of Pd nanocrystals alleviated oxidative damage to DNA, indicated by the decreased amount of γ-H2AX foci in the presence of Pd nanocrystals. Further, results demonstrate Pd octahedrons were more effective in decreasing the expression of γ-H2AX than Pd nanocubes, consistent with results of our other assays. Taken together, these results clearly demonstrate that Pd nanocrystals can effectively suppress the generation of ROS and protect important biomolecules and cell organelles from the effects of oxidative stress. These results also demonstrate the superiority of octahedral Pd nanocrystals having {111} facets. An additional apoptosis assay verified the facet-dependent cytoprotective effect of Pd nanocrystals. As shown in Figure 6, exposure to 60 μM H2O2 induced apoptosis in almost half of the cells, compared to the unexposed control cells; however, addition of Pd nanocrystals significantly reduced that level of H2O2-induced apoptosis. The percentage of apoptotic cells was reduced to 37% (p < 0.05) in the presence of Pd nanocubes and to 20% in the presence of Pd octahedrons (p < 0.01). This

H 2O2 * + OH* = HO2•* + H 2O* 10440

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Figure 5. Pd nanocrystals prevent damage to lipid, protein and DNA caused by oxidative stress. (a) The content of malondialdehyde (MDA), as a general indicator of the extent of lipid peroxidation. Cells were left untreated (UT) or treated with 25 μg/mL Pd nanocrystals after exposure to 60 μM H2O2. (b) The content of carbonylated proteins of cells after different treatments using protein carbonyl calorimetric assay. These data are the means and standard deviations from three experiments. P values comparing treatment performance to that of cells treated only with H2O2 were calculated by using Student’s t test: *p < 0.05, **p < 0.01. (c) Imaging of the marker (γ-H2AX) for double-strand DNA breaks in cells after different treatments. Under laser confocal scanning microscope, the γ-H2AX foci appear in red fluorescence, and cell nuclei, stained by Hoechst, show as blue fluorescence. Scale bar: 20 μm.

Figure 6. Protective effects of Pd nanocrystals against H2O2-induced apoptosis in HUVEC cells. (a) Flow cytometric profiles of PI-stained apoptotic cells left untreated (UT) or treated with Pd nanocrystals after exposure to H2O2. (b) The percentage of apoptosis/necrosis cells determined by flow cytometry. These data are the means and standard deviations from three experiments. P values compared to cells treated only with H2O2 were calculated using Student’s t test: *p < 0.05, **p < 0.01.

H 2O2 * + HO2•* = O2 * + OH* + H 2O*

scavenging activity of Pd octahedrons than that of Pd nanocubes. The following two reactions play a key role in a given metal’s ability to scavenge O2•−.

(2)

In the above reactions, the asterisk designates an adsorbate; OH* is the hydroxyl preadsorbed on the metal surface that assists the reactions. Equation 2 is the rate-limiting step, whose reaction energy (Er) can be used as the descriptor of the metal surface, a more negative Er means greater H2O2 scavenging activity.59 According to our calculations, the lowest energy adsorption structures of H2O2 and HO2• on the Pd {111} facet before and after the reaction of eq 2 are shown in parts a and b, respectively, of Figure 7; those on Pd {100} are shown in parts e and f, respectively, of Figure 7. According to these structures, the Er on Pd {111} and Pd {100} were calculated to be 2.81 and 2.64 eV, respectively. This suggests the Pd {111} facet has greater H2O2 scavenging activity than the Pd {100}, which is consistent with our experimental observations of greater H2O2

O2•− + H+ = HO2•

(3)

2HO2•* = O2 * + H 2O2 * •−

(4) •

First, the O2 is protonated to HO2 in an equilibrium reaction (eq 3); then the HO2• adsorbs on the metal surface and is converted to O2 and H2O2 via the disproportionation reaction (eq 4). Reaction eq 4 is the rate-limiting step, whose Er can be used as the descriptor of the metal surface; a more negative Er means greater O2•− scavenging activity.60 The lowest energy adsorption structures of two HO2• groups on the Pd {111} facet before and after the reaction of eq 4 are shown 10441


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Figure 7. Lowest-energy adsorption structures and reaction energies (in eV) for the reactions on structures having either Pd {111} or {100} facets. peroxide (H2O2) solution was mixed with 0.1 mM 15N-PDT in the presence of 25 μg/mL Pd nanocrystals or 10 U/mL catalase (CAT). The kinetic experiments of H2O2 decomposition catalyzed by Pd nanocrystals were carried out by using varied concentrations of H2O2. The following instrument settings were used for collecting ESR spectra: 0.04 G field modulation, 3 G scan range, and 1 mW microwave power. To verify the ability of Pd nanocrystals to scavenge superoxide anions (O2•−), xanthine and xanthine oxidase (Xan/XOD) were mixed in PBS buffer (pH 7.4) to generate a superoxide and BMPO was used to trap the superoxide. The reaction mixture contained 25 mM BMPO, 25 μg/mL Pd nanocrystals, or 1 U/mL SOD, 1 mM xanthine, 0.05 mM DTPA, 0.1 U/mL XOD. The ESR spectra were recorded at 5 min after initiating the generation of superoxide by adding XOD. The following instrument settings were used for collecting ESR spectra: 1 G field modulation, 100 G scan range, and 20 mW microwave power. The KO2/18-crown-6 ether system was used to verify the turnover of O2•− to O2 by Pd nanocrystals by detecting the production of oxygen. The reaction mixture contained 2 mM 15N-PDT, 0.35 mM 18crown-6, 20% (v) DMSO, 25 μg/mL Pd nanocrystals or 10 U/mL SOD, and 2.5 mM KO2. The reaction was initiated by the addition of KO2, and the ESR spectra were recorded at 10 min. Cell Culture and Viability Assay. Human umbilical vein endothelial cells (HUVEC, ATCC Number: CRL-1730) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM, HyClone), supplemented with 10% fetal bovine serum (FBS, Gibco BRL) and 1% penicillin/streptomycin (pen/strep, HyClone) at 37 °C with 5% CO2 in a humidified incubator. HUVEC cells were seeded at a density of 1 × 104 cells/well in a 96-well plate and grown in complete culture medium for 24 h. HUVEC cells were exposed to 60 μM H2O2, followed by 25 μg/mL Pd nanocrystals for 2 h. Cells treated only with H2O2 were used as the positive control. After treatments, cells were washed with PBS and stained with a Live/Dead Cell Double Staining Kit (Sigma, USA) for 15 min. After being stained and washed twice with phosphate buffer solution (PBS), inverted luminescence microscopy (OLYMPUS X73, Japan) was applied to take the luminescence images. Measurement of Intracellular ROS. The oxidant-sensitive dye CM-H2DCFDA (Life Technologies, USA) was used to measure the intracellular ROS level. HUVEC cells were seeded in confocal microscope dishes at a density of 1 × 105 cells/well and respectively treated with Pd nanocrystals and H2O2 as described above. After treatments, cells were washed with PBS and labeled with 5 μM DCFH−DA at 37 °C for 30 min in the dark. After being washed three times with PBS, intracellular ROS levels were determined by a confocal laser microscope (FV1200, OLYMPUS, Japan), with the excitation wavelength set at 488 nm, analyzed by a flow cytometer (BD

in parts c and d, respectively, of Figure 7; those on Pd {100} facet are shown in parts g and h, respectively, of Figure 7. Based on these structures, the Er’s on Pd {111} and {100} facets were calculated to be 0.60 and 0.13 eV, respectively. This means the disproportionation of HO2• on Pd {111} facet is more thermodynamically favorable than that on Pd {100}, indicative of the greater O2•− scavenging activity of Pd {111} than {100}. This computational result displays excellent agreement with our experimental observations of superior O2•− scavenging activity by Pd octahedrons over that by Pd nanocubes.

CONCLUSIONS In summary, we have found an important correlation between the surface energy of Pd nanocrystals and their antioxidant enzyme-like activities. Lower surface energy Pd octahedrons {111} exhibit higher antioxidant enzyme-like activities than higher surface energy Pd nanocubes {100}. Our in vitro experiments demonstrated that Pd octahedrons were more effective than Pd nanocubes for protecting cells against oxidative challenges by suppressing the generation of ROS, resulting in the protection of important biomolecules and cell organelles. The detailed mechanism has been computationally rationalized at the molecular level. This study will open a door to designing nanomaterials-based enzyme mimics having high catalytic activities, which could be used as the basis of future therapeutics for clinical conditions associated with oxidative stress. MATERIALS AND METHODS Synthesis and Characterization of Pd Nanocrystals. Pd nanocrystals were synthesized according to the published protocol.46 The morphologies of nanocrystals were examined using a FEI TECNAI G2 transmission electron microscopy (TEM), operating at 200 kV. High-resolution TEM (HRTEM, Tecnai F20, FEI, USA) images were taken on a field-emission high-resolution transmission electron microscope operated at 200 kV. The concentrations of Pd nanocrystals were measured with an inductively coupled plasma mass spectrometry (ICP-MS, Element 2, Thermo Finnigan, Germany) after the nanocrystals were dissolved with a mixture of HCl and HNO3 (3:1, volume ratio). Electron Spin Resonance Spectroscopic Measurements. All ESR measurements were carried out at ambient temperature (27 °C) using a Bruker EMX ESR spectrometer (Billerica, MA). ESR spin label oximetry is a quantitative approach for measuring oxygen content using the water-soluble spin label 15N-PDT. The 10 mM hydrogen 10442


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Following expression was used to calculate the adsorption energies (Eads)

FACSverse, San Jose, CA). Data are representative of three experiments. Measurement of Mitochondrial Membrane Potential. Mitochondrial membrane potential was measured using the JC-1 mitochondrial membrane potential assay kit (Invitrogen, Carlsbad, CA). The reagent, JC-1, enters the mitochondria and forms aggregates showing red fluorescence. However, when the mitochondrial membrane potential collapses, this dye reagent can no longer accumulate within the mitochondria and instead exists in a monomeric form which fluoresces green. After treatments, the mitochondria were stained with JC-1 in DMEM medium for 30 min. After washing with PBS three times, the cells were imaged using a confocal laser scanning microscope (FV1200, Olympus, Japan). The JC-1 monomers and aggregates were respectively detected at 530 and 590 nm emission wavelengths. Lipid Peroxidation and Protein Carbonylation. The content of malondialdehyde (MDA) was quantified using a commercial kit (Beyotime Biotechnology, China). In brief, after different treatments, HUVEC cells were harvested and lysed at 4 °C. Trichloroacetic acid and TBA were added to the mixture of lysed cells and mixed thoroughly, and then this solution was heated for 15 min in a boiling water bath. After cooling, the solution was centrifuged at 12000 rpm for 10 min, and the absorbance of the supernatant was measured at 535 nm using a UV−vis spectrophotometer (UV-3600, Shimadzu). The standard solution was diluted with H2O to prepare the standard curve, and the MDA content of the sample can be calculated according to the standard curve. Carbonylated proteins were measured using protein carbonyl calorimetric assay (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s protocol. Briefly, cell lysates were prepared, and proteins were extracted with 2,4-dinitrophenylhydrazine. After centrifugation, the protein−hydrozone pellets were dissolved. Finally, the absorbance of solution was measured at 370 nm, and protein carbonyl amounts were calculated according to the formula. DNA Double-Strand Breaks. After different treatments, HUVEC cells were fixed with 4% paraformaldehyde, and subsequently, 0.2% Triton X-100 was added to permeate the cells for 10 min. After incubation in 1% bovine serum albumin for 1 h to prevent nonspecific protein interactions, fixed cells were then immunostained with anti-γH2AX antibody (ab81299, Abcam) overnight at 4 °C. Cells were washed with PBS and incubated with a second antibody for 1 h at 37 °C. After washing with PBS, cell nuclei were stained with Hoechst (Invitrogen, CA) at room temperature in dark for 5 min. The cells were visualized with a confocal laser microscopy (FV1200, OLYMPUS, Japan). Cell Apoptosis Detection. Cell apoptosis was determined by Annexin V/PI staining using the Annexin V Apoptosis Detection Kit (BD Pharmingen, San Diego, CA). After treatments, HUVEC cells were harvested and washed with ice-cold PBS buffer and stained with Annexin V and PI. Stained cells were analyzed by a flow cytometry (BD FACSverse, San Jose, CA). Calculations. The Vienna ab initio Simulation Package61−63 was used to perform density functional theory calculations. The projector augmented wave method was employed to describe the electron−ion interactions.64 The exchange-correlation functional of Perdew-Burke− Ernzerhof65 with generalized gradient approximation was used. Geometry optimizations and energy calculations were performed using an energy cutoff with 400 eV and a first order Methfessel− Paxton66 smearing with 0.2 eV. Four-layered slabs in (111) and (100) directions were employed with (4 × 4) and (2 × 2) unit cells in the lateral direction to model Pd(111) and Pd(100) surfaces, respectively. A vacuum height was set to 15 Å. The (3 × 3 × 1) Monkhorst−Pack mesh k-points67 for (4 × 4) and (2 × 2) unit cells were selected for the calculations. With regard to geometry optimization, the top one layer of the (111) and (100) surfaces was fully relaxed, and the remaining layers were kept fixed. The conjugated-gradient algorithm was employed to optimize the structures. Electronic structures and forces were converged at the criteria of 10−6 eV and 0.02 eV/Å, respectively.

Eads = Eslab + mol − (Eslab + Emol ) where Eslab+mol denotes the total energy of the chosen surface with adsorbate on it and the Eslab denotes the energy of bare metal surface. Emol denotes the energy of adsorbate. Statistical Analysis. Mean and standard deviation (SD) were calculated. Results were expressed as mean ± SD. Comparisons within each group were conducted by a Student’s t test.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b06297. Details of the experimental procedures (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Xingfa Gao: 0000-0002-1636-6336 Chunying Chen: 0000-0002-6027-0315 Author Contributions #

These authors contributed equally to the work.

Notes

The authors declare no competing financial interest. ⊗ (W.G.W.) Deceased July 29, 2016.

ACKNOWLEDGMENTS Dedicated to the memory of Wayne G. Wamer. This work is partially supported by the National Basic Research Program of China (973 Program Grant Nos. 2014CB931900 and 2016YFA0201600), National Natural Science Foundation of China (Nos. 21207164, 11575123, and 21373226), Jiangsu Provincial Key Laboratory of Radiation Medicine and Protection, a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and a regulatory science grant under the FDA Nanotechnology CORES Program. C.C. appreciates support from the NSFC Distinguished Young Scholars (11425520).We thank Dr. Lili Fox Vélez for editorial and scientific writing support. This article is not an official U.S. FDA guidance or policy statement. No official support or endorsement by the U.S. FDA is intended or should be inferred. REFERENCES (1) Lambeth, J. D. NOX Enzymes and the Biology of Reactive Oxygen. Nat. Rev. Immunol. 2004, 4, 181−189. (2) Wirth, T. Small Organoselenium Compounds: More than just Glutathione Peroxidase Mimics. Angew. Chem., Int. Ed. 2015, 54, 10074−10076. (3) Winterbourn, C. C. Reconciling the Chemistry and Biology of Reactive Oxygen Species. Nat. Chem. Biol. 2008, 4, 278−286. (4) Sauer, H.; Wartenberg, M.; Hescheler, J. Reactive Oxygen Species as Intracellular Messengers during Cell Growth and Differentiation. Cell. Physiol. Biochem. 2001, 11, 173−186. (5) Sena, L. A.; Chandel, N. S. Physiological Roles of Mitochondrial Reactive Oxygen Species. Mol. Cell 2012, 48, 158−167. (6) Gechev, T. S.; Van Breusegem, F.; Stone, J. M.; Denev, I.; Laloi, C. Reactive Oxygen Species as Signals that Modulate Plant Stress 10443


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