Quantitatively Intrinsic Biomimetic Catalytic Activity ... - ACS Publications

Subscriber access provided by EAST TENNESSEE STATE UNIV

Article

Quantitatively Intrinsic Biomimetic Catalytic Activity of Nanocerias as Radical Scavengers and their Ability against H2O2 and Doxorubicin-Induced Oxidative Stress Zhimin Tian, Xuhui Li, Yuanyuan Ma, Tao Chen, Dehui Xu, Bingchuan Wang, Yuan Gao, and Yongquan Qu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b04761 • Publication Date (Web): 23 Jun 2017 Downloaded from http://pubs.acs.org on June 23, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 13

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Quantitatively Intrinsic Biomimetic Catalytic Activity of Nanocerias as Radical Scavengers and their Ability against H2O2 and Doxorubicin-Induced Oxidative Stress Zhimin Tian,† Xuhui Li,‡ Yuanyuan Ma,† Tao Chen,‡,§ Dehui Xu,ǁ Bingchuan Wang,ǁ Yuan Gao*,† and Yongquan Qu*,† †

Center for Applied Chemical Research, Frontier Institute of Science and Technology, and Department of Cardiology, the First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710049, China ‡

Center for Neuron and Disease, Frontier Institute of Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China §

Department of Anatomy, Histology and Embryology and K.K. Leung Brain Research Center, The Fourth Military Medical University, Xi’an 710032, China ǁ

Centre for Plasma Biomedicine, State Key Laboratory of Electrical Insulation and Power Equipment, Xi’an Jiaotong University, Xi’an, 710049, China KEYWORDS: porous nanorods of ceria, nanozymes, reactive oxygen species, radical scavenger, oxidative stress ABSTRACT: Artificial enzymes as radical scavengers show great potentials in treatments on various diseases induced by oxidative stress. Herein, the quantitative analysis indicates the intrinsic activity of nanocerias for degradation of radicals is determined by the concentration of surface defects as well as their morphological features. The surface Ce3+ fraction of the CeO2 nanozymes with the similar morphology can be used as a descriptor to index their catalytic activity as radical scavengers. Defect-abundant porous nanorods of ceria (PN-CeO2) with a large surface area (141 m2/g) and high surface Ce3+ fraction (32.8%) deliver an excellent catalytic capability for degradation of radicals, which is 15.5 time higher than that of Trolox. Results indicate that the PN-CeO2 not only provides more surface catalytic centers also supplies the active site with higher activity. Oxidative stress induced by doxorubicin (Dox), an essential medicine for a wide range of tumors, was used as the model system to evaluate the radical degradation ability of the PN-CeO2. Both in vitro cellar (H9c2 cells) and in vivo animal models revealed that the PN-CeO2 did not affect the cell and rat growth and was able to alleviate the Dox-induced oxidative stress. Results suggest that the artificial PN-CeO2 nanozymes have potentials to function as an adjuvant medicine during tumor chemotherapy.

1. INTRODUCTION Release of a high level of reactive oxygen species (ROS, i.e., O2˙-, OH˙-, H2O2) is considered as the main risk factor of numerous diseases.1-3 In order to maintain the balance of the intracelluar ROS level, the natural enzymes including superoxide dismutase (SOD), catalase and glutathione peroxidase build up an intracelluar self-defense system to regulate the ROS levels at equilibrium. Non-enzymatic natural and/or synthetic compounds (Vitamins C and E, glutathione, Trolox, etc.) as radical scavengers also have been explored to modulate the intracellular ROS levels.4-7 However, none of them can effectively suppress the ROS induced by oxidative stress, which might be attributed to their insufficient ability in the complicated environment of the human beings. Besides, the practical clinical applications of natural products face the high cost in preparation and separation, poor storage stability (pH and temperature sensitivity) and requirements of daily dosing.8 Thus, it is urgent to develop novel artificial enzymes for practical applications, which should satisfy the requirements of costeffective synthesis, easy and stable storage under the ambient conditions and high capability against oxidative stress. On account of those, the artificial nanostructural materials (e.g. CeO2, Fe3O4, V2O5, pol-

ymers) show their potentials to replace natural ones.918

CeO2 with low toxicity and good biocompatibility has been widely investigated as an artificial enzyme against various cellular damages including neuroprotection, radioprotection and anti-inflammatory.19-30 Generally, their activity for degradation of ROS in the biological systems is considered to be originated from their reversible surface Ce3+/Ce4+ redox pair.2,9,11,31 However, the quantitative correlation between their intrinsic biomimetic activity and surface properties hasn't been well established. We also noted that CeO2 has been of great concerns for treatments on various diseases relative to oxidative stress. Chemotherapy as an effective protocol for tumor therapy has been used for clinical applications. However, the patients generally suffer from the increased ROS level during the treatments. The increased ROS level sometimes is fatal. Despite the great potentials of the nanoscale artificial enzymes as the radical scavengers, it's lack of their practical studies on the suppression of oxidative stress induced by anticancer chemotherapeutic drugs. In this work, we evaluate the catalytic activity of nanocerias as radical scavengers and build up the quantitative correlation between their surface properties and intrinsic catalytic capability. With the similar

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 13

morphology, the surface Ce3+ fraction of CeO2 can be used as a descriptor to index their ROS degradation activity. Among them, porous nanorods of ceria (PNCeO2) with the largest surface area (141 m2/g) and highest surface Ce3+ fraction (32.8%) delivered the best catalytic capability. By normalizing the catalytic activity to total amount of the surface Ce3+ species, the PN-CeO2 not only offers more active sites also supply the catalytic centers with higher average intrinsic biomimetic activity. Afterwards, toxicity from oxidative stress induced by doxorubicin32-34 (Dox, a common tumor drug) was used as a model system to investigate the practical applications of the PN-CeO2 for degradation of ROS in both in vitro cellar and in vivo animal models. The PN-CeO2 successfully reduced intracellular ROS levels and alleviated the cell and tissue damage induced by Dox, suggesting the potentials of the PN-CeO2 to construct a self-defense system against the oxidative stress. 2. RESULTS AND DISCUSSION Synthesis and Characterizations of the CeO2 Nanozymes. Figure 1a-c show the transmission electron microscopy (TEM) images of various ceria nanostructures including the PN-CeO2, nonporous nanorods of ceria (NR-CeO2) and ceria nanoparticles (NP-CeO2).35-42 As-synthesized PN-CeO2 shows porous nanorod-like morphology with a diameter of ~ 8 nm, a pore size of 2 ~ 4 nm and a length of ~60 nm (Figure 1a). The NR-CeO2 has an average diameter of ~10 nm and a length of 150~200 nm (Figure 1b). Figure 1c exhibits the aggregated CeO2 nanoparticles. X-ray diffraction (XRD) patterns show a fluorite phase for the three CeO2 materials (Figure 1d). Their surface properties were characterized by X-ray photoelectron spectroscopy (XPS) and Brunauer–Emmett–Teller (BET) analysis. As shown in Figure S1a, the PN-CeO2 presents the largest surface Ce3+ fraction (32.8%), which are much higher than those of the NR-CeO2 (15.9%) and NP-CeO2 (14.4%). The surface areas of various nanocerias show an order of PN-CeO2 (141 m2/g) > NR-CeO2 (107 m2/g) > NP-CeO2 (82.4 m2/g). The zeta potential of PN-CeO2 is −14.0 mV in H2O and −27.8 mV in the culture medium, respectively (Figure S2). The measurements suggest the stable dispersion of PN-CeO2 under physiological conditions. Quantitative Analysis on the Intrinsic Catalytic Activity of the CeO2 nanozymes as Radical Scavengers. Catalytic activity of the CeO2 nanozymes was quantitatively evaluated by calibration from the standard SOD assay kit (Sigma-Aldrich). At the same concentration of the CeO2 nanozymes (1 μM), the PNCeO2 showed the highest SOD mimetic activity, which was 3.6 and 10.2 times higher than that of the NR-CeO2 and NP-CeO2, respectively (Figure 1e).

Figure 1. Structural characterization of the CeO2 nanozymes and their catalytic activity for degradation of ROS. (a) TEM image of the PN-CeO2. Inset is the dark field TEM image. (b) TEM image of the NR-CeO2. (c) TEM image of the NP-CeO2. (d) XRD patterns of CeO2. (e) SOD mimetic activity of the three CeO2 enzymes. (f) Catalytic capacity of the three CeO2 enzymes for ROS degradation.

It is, however, necessary to point out that the surface properties (e.g. surface defects, morphology) of the CeO2 nanozymes were not quantitatively considered in the above evaluations on their activity. Despite the SOD activity of the artificial CeO2 nanozymes is connected with their surface properties,9,19 very little is known about the quantitative correlation between the intrinsic activity of each active surface site of CeO2 and their surface properties. In order to understand the intrinsic biomimetic ability of nanoscaled CeO2, we assume each surface Ce3+ as an active center and normalize their activity on each surface active site. Initially, we didn't account the effects of crystal facets of various CeO2 nanozymes since they all possess the polycrystalline nature. In this way, the correlation between the surface properties of nanocerias and their ability for ROS degradation can be reached quantitatively. Table 1 summarizes the structural information of the CeO2 nanozymes including the surface areas and surface Ce3+ fractions. Derived from those data, the total amounts of the surface Ce3+ in each gram of the cata-


Page 3 of 13

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


lysts can be obtained (detailed calculations in Figure S3). On the basis of above analysis, the PN-CeO2 possesses 0.52 mmol/g of the surface Ce3+ species, which is much larger than those of the NR-CeO2 (0.17 mmol/g) and NP-CeO2 (0.13 mmol/g). Consequently, the fractions of the surface Ce3+ species to total Ce for the three CeO2 nanozymes can be derived (Figure 2a).

Figure 2. Quantitative analysis of SOD mimetic activity of nanocerias. (a, c) The fractions of the surface Ce3+ species to the total Ce in various CeO2 nanozymes. (b, d) The normalized SOD activity on the total amount of the surface Ce3+ species for various CeO2 nanozymes.

Calibrated from the standard SOD kit, the SOD activity of the PN-CeO2 (1 mM) corresponds to that of 90.9 U SOD kit (Figure S3a-b). Similarly, the NRCeO2 (1 mM) and NP-CeO2 (1 mM) deliver the standard SOD activity of 25.2 U and 8.9 U, respectively. Generally, the performance of the mimic SOD enzymes at a single surface reaction site for various CeO2 nanozymes should be similar due to the same type of surface active centers. However, the normalized SOD activity to the total amount of the surface available Ce3+ species doesn’t support this assumption. Detailed normalization of catalytic activity of the CeO2 nanozymes as the radical scavengers is shown in Supporting Information. Based on the amount of the surface Ce3+ species in 1 gram nanozymes, we set the catalytic activity of the PNCeO2 at each surface Ce3+ site as 100 a.u. As shown in Figure 2b, the normalized catalytic activities of the NR-CeO2 and NP-CeO2 at each surface Ce3+ site are 82.0 a.u. and 40.5 a.u., respectively. They are much lower than that of the PN-CeO2. Thus, it can be deduced that the intrinsic SOD activity of various CeO2 nanozymes is also significantly affected by their morphologies. The NP-CeO2 shows no preference for the exposed facet. While, the NR-CeO2 is dominated by the (100) and (110) facets on the sidewalls.37 For the PN-CeO2, the sidewalls are mainly composed of (100) and (110) facets and multiple facets can be found in the pores.35 Generally, the formation of oxygen vacancy is associated with the formation of Ce3+ species. It

has been reported that the formation energy of oxygen vacancy is in the trend of (110) < (100) < (111).41-42 Thus, the structure dependence of catalytic reactions over well-defined faceted CeO2 nanostructures has been observed, in which the CeO2nanorods always delivered the best catalytic activity.35,41,42 Our quantitative analysis shows much higher intrinsic mimetic enzyme activity of the PN-CeO2 and NR-CeO2 over the NP-CeO2, which can be assigned to their dominated (100) and (110) facets. Even higher intrinsic catalytic activity of the PN-CeO2 can be attributed to the ultrathin walls of porous nanorods, in which the exposed surface species with smaller coordination numbers and higher activity as a consequence. To further examine the important roles of the surface defects and morphological features of the CeO2 nanozymes on their activity, the PN-CeO2 was annealed at various temperatures in the presence of air for 4 hours. As shown in Figure S4, the morphology of the PN-CeO2 is well preserved. Thus, the activity of the CeO2 nanozymes is only determined by their surface defects in terms of the surface Ce3+ species. It provides a very straight-forward model to investigate the correlation between the catalytic activity and surface defects of CeO2 by excluding the influence of their morphological features. The obtained nanozymes were named as PN-CeO2-300 and PNCeO2-500 for those calcinated samples at 300 °C and 500 °C, respectively. Importantly, the surface Ce3+ fractions of the PN-CeO2-300 and PN-CeO2-500 are decreased from 32.8 % of as-synthesized PN-CeO2 to 26.1 % and 17.2 %, respectively (Figure S1b). Thus, the derived fractions of the surface Ce3+ species to the total Ce for the PN-CeO2, PN-CeO2-300 and PN-CeO2500 nanozymes are 8.95%, 6.24% and 3.84%, respectively (Figure 2c). When the concentration of the CeO2 nanozymes was fixed at 1 mM, the PN-CeO2, PN-CeO2-300 and PN-CeO2-500 delivered the SOD activity of 90.9 U, 61.5 U and 38.1 U, respectively, based on the calibration from the standard SOD kits (Figure S3a and 3c). Similar to the above quantitative analysis, the values of the normalized activity of the three PN-CeO2 nanozymes as radical scavengers at each surface Ce3+ specie are very close (Figure 2d), in which are 100 a. u., 97.9 a. u. and 98.3 a. u. for the PN-CeO2, PN-CeO2-300 and PN-CeO2-500, respectively. The results confirm that the intrinsic activity of nanocerias with the similar morphology is mainly determined by the concentrations of surface defects: a linearly relationship (Figure 2d and Figure S3d). In this case, the surface Ce3+ fraction can be employed as a descriptor to reveal the catalytic activity of CeO2 for ROS degradation. Combining with Figure 2, the morphological features of nanocerias also affect their SOD activity due to their different formation energies of oxygen vacancy associated with Ce3+ on various facets of the CeO2 nanozymes.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 13

Table 1.Surface characteristics and relative SOD activity of various CeO2nanozymes.

Surface area (m2/g) Ce3+ fraction (%) Surface Ce3+ species (mmol/g) Calibrated SOD activity (U) Normalized SOD activity per total amount of surface Ce3+ species (a.u.)

PN-CeO2

NR-CeO2

NP-CeO2

PN-CeO2-300

PN-CeO2-500

141.1 32.8 0.52 90.9

98.3 15.9 0.17 25.2

77.7 14.4 0.13 8.9

122.5 26.1 0.363 61.5

114.6 17.2 0.223 38.1

100.0

82.0

40.5

97.9

98.3

The capability of the CeO2 artificial enzymes for ROS degradation was examined by measuring their free radical scavenging ability, where the oxygenradical absorbance capacity assay was used to index ROS level. The quantitative catalytic capacity of CeO2 was determined by a comparative measurement on a typical synthetic radical scavenger Trolox (a derivative of Vitamin E). Scavenging ability of the PN-CeO2 on H2O2 was 15.5 times higher than that of Trolox (Figure 1f). While, the catalytic activity of the NRCeO2 and NP-CeO2 were 3.3 and 6.3 times lower than that of the PN-CeO2. Thus, the PN-CeO2 with the highest surface Ce3+ fractions and largest surface area exhibits the highest ability for ROS degradation by providing more surface active sites with higher activity. The regenerative biomimetic activity of the PNCeO2 was also confirmed by UV-Vis spectroscopic studies by repetitively treating the artificial enzyme (2 mg/mL) with H2O2 (50 mM) and subsequent aging procedure (Figure. S5). After H2O2 treatment, a redshift of UV-Vis spectrum indicated the oxidation of surface Ce3+ into Ce4+. After aging in dark for 9 days, the surface Ce3+ regeneration was confirmed by the nearly completed recovery of its UV-Vis spectrum to the initial one. Such a repeatable process suggested the restorable activity of the PN-CeO2 (Figure. S5). Comparison of the reversible autocatalytic activity, the PN-CeO2 is also better than the NP-CeO2. The high catalytic activity and regenerative capability of the PN-CeO2 under oxidative stress indicate the potentials in biological research and biomedicines. Dox as a common drug for tumor chemotherapy has been demonstrated to significantly improve the survival of patients. However, Dox also delivers many adverse effects including skin reactions and heart damage. Among them, myocardial damage induced by the cumulative Dox is the most dangerous side effect during the chemotherapy.32-34 Thus, the construction of adjuvant chemotherapy system to suppress the Dox-induced oxidative stress is urgently desired for practical clinical treatments. As far as we know, the protective capability of nanoscaled artificial enzymes against the Dox-induced cardiotoxicity has not been explored yet. Herein, we investigated

catalytic capability of the PN-CeO2 against the Doxinduced oxidative stress. To investigate the intracellular ROS degradation performance of CeO2 for the Dox-induced cardiotoxicity, H9c2, a subclone of the original clonal cell line derived from embryonic rat heart tissue, was used as a model to investigate the protective functions of the artificial enzymes against oxidative stress.43 Toxicity of the three CeO2nanozymes at various concentrations was investigated on the cultured H9c2 cells in dulbecco’s modified eagle medium (DMEM). With the CeO2 concentrations varying from 0.01 μg/mL to 1000 μg/mL, the survival rates of the cells sustained in a high degree, demonstrating the low toxicity of the three CeO2 artificial enzymes (Figure S6). Endocytosis of the PN-CeO2 in H9c2 cells was also detected by TEM technique. After incubating the PN-CeO2 in H9c2 cells for 24 h, the PN-CeO2 were observed within the cytoplasm (Figure S7a-b), indicating the uptake of the PN-CeO2 by H9c2 cells. Inductively coupled plasma optical emission spectroscopy (ICP-OES) further confirmed the endocytosis of PN-CeO2 by H9c2 cells (Figure. S7c). When the concentration of the PN-CeO2 in cell medium reached 100 μg/mL, the uptake of the PN-CeO2 was saturated. Thus, we chose 100 μg/mL as the optimal concentration for all cell experiments unless otherwise specified. In order to evaluate the activity of the artificial enzymes for ROS degradation, the H9c2 cells were pretreated by the artificial enzymes for 24 hours before exposure to oxidative stress (H2O2 or Dox). In Vitro Cellar Evaluations on Catalytic Activity of the Artificial Enzymes against the Oxidative Stress Induced by H2O2. Intracellular H2O2 level can be enhanced by diffusion of extracellular H2O2 through cell membranes. The increased expression of ROS induced by oxidative stress of intracellular H2O2 will thus oxidize cysteine-rich regions in cytoplasmic proteins and subsequently trigger cell death through the apoptotic pathway.44 We initially tested the ROS degradation activity of CeO2 on H9c2 cells in the presence of various concentrations of H2O2. Trolox was used as the positive control to quantify the ROS scavenging ability of the artificial enzymes. ROS level was judged by the intensity of ROS sensitive DCFH2DA. Thus, the intracellular ROS level in pre-treated


Page 5 of 13

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. ROS degradation activity of the artificial CeO2 nanozymes and Trolox on H2O2 induced oxidative stress. (a) Confocal fluorescent images of H9c2 cells treated with various artificial enzymes and/or H2O2. H9c2 cells were treated with the CeO2 nanozymes or Trolox for 24 hours at 37 °C. Afterwards, the cells were treated with H2O2 (100 mM) for 30 minutes at 37 °C and stained with DCFH-DA dye. (b) Quantification of the relative ROS levels of H2O2 (100 mM) by DCFH-DA probe and fluorimeter in H9c2 cells under various treatments. The concentration of the artificial CeO2 enzymes was 100 μg/mL. The same morality of Trolox was used. (c) Cell viabilities of H9c2 under various treatments were measured by using the Alamar Blue assays. Cells without exposure were used as the control group. All experiments were repeated for six times.

cells under normal or oxidative stress can be compared with control experiments. H2O2 (100 μM) strongly increased intracellular ROS level in living cells, which was effectively inhibited by three CeO2 nanozymes (100 μg/mL) and Trolox (Figure. 3a), demonstrating their activity for ROS degradation. Among them, the PN-CeO2 showed the strongest ability to catalytically degrade ROS (Figure. 3a), in which the intracellular ROS level was reduced near to the control group. Quantitatively, the biomimetic capability of the PN-CeO2 was 1.9, 2.1 and 3.7 times higher than those of the NR-CeO2, NP-CeO2 and Trolox, respectively (Figure.3b). The cell viability treated with the PN-CeO2 in various concentrations of H2O2 (20 - 100 μM) was much higher than those treated with other CeO2 nanozymes and Trolox (Figure. 3c). In Vitro Cellar Evaluations on Catalytic Activity of the Artificial Enzymes against the Oxidative Stress Induced by Dox. All results suggest that the PN-CeO2 as the most effective artificial enzymes could sufficiently protect H9c2 cells from H2O2-

induced oxidative stress. Thus, the PN-CeO2 was chosen as the effective radical scavenger against the Doxinduced cardiotoxicity. Comparing to the control group, Dox (30 μM) stimulated a high level of the intracellular ROS oxidative stress and a high apoptosis in H9c2 cells. In contrast, the PN-CeO2 alone did not raise the intracellular ROS level. When H9c2 cells were pre-treated with the PN-CeO2 (100 μg/mL), the intracellular ROS level induced by Dox was significantly decreased (Figure.4a). Electron paramagnetic resonance (EPR) spectroscopy with 5, 5-Dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap was also performed to quantificationally analyze the capability of the PN-CeO2 and Trolox as radical scavengers in the presence of Dox. Dox alone produced large amount of radials with a strong EPR signal (Figure 4b). Trolox reduced the ROS level of 61.9%. While, 79.5% of radicals were degraded in presence of the PN-CeO2, suggesting the PN-CeO2 with more effective activity against the oxidative stress induced by Dox. We further checked the intracellular ROS levels with DCFH-DA probe by a flow 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 13

Figure 4. ROS degradation efficiency of the PN-CeO2 against Dox-induced intracellular ROS. (a) Confocal fluorescent images of H9c2 cells treated with the nanozymes and/or Dox. H9c2 cells were pre-treated with the PN-CeO2 for 24 hours before the dose of Dox (30 μM) at 37 °C and then stained with DCFH-DA dye. (b) EPR spectra of the hydroxyl radicals captured by DMPO after treatment with various artificial enzymes and/or Dox (30 μM). Untreated H9c2 cells were used as a control. (c) Quantitative analysis of ROS levels by staining cells with DCFH-DA dye. The experiments were repetitively performed by flow cytometry for three times. The concentrations of the PN-CeO2 and Dox were controlled at 100 μg/mL and 30 μM, respectively. (d) Cell viabilities of H9c2 treated with the PN-CeO2 and/or Dox. H9c2 cells were pre-incubated with the PN-CeO2 (100 μg/mL) for 24 hours, and treated with Dox (5, 10, 20, 30μM) for 12 hours. Cells without exposure were used as the control group. All experiments were repeated for six times.

cytometry (Figure. 4c) in the presence of Dox (30 μM). Dox-induced ROS level was 14% higher than that of the control group. In contrast, when coapplication with the PN-CeO2 and Dox, the intracellular ROS level of H9c2 cells was significantly reduced. It's close to or even lower than that of the control group, suggesting the high catalytic activity of the PN-CeO2 for free radical degradation in living cells (Figure. 4c). Compared to the negative control, even lower ROS level of the cells treated with the PNCeO2 indicates the artificial nanozymes CeO2 might deliver an effect on the intracellular signalling pathways to a certain extent, similar to the previous reports.11,23 Despite this, the experiments undoubtedly confirm the capability of the PN-CeO2 to reduce the Dox-induced ROS level. In addition, the PN-CeO2 also dramatically improved the cell viability (Figure. 4d). At a high concentration of Dox (30 μM), only 18% of H9c2 cells survived. In contrast, the PN-CeO2 improved the cell viability to 46.5%. The efficient ROS scavenging activity of the PN-CeO2 in the living H9c2 cells strongly suggests its potentials to adjust the intracellular ROS imbalance and inhibit Doxinduced oxidative damage. Meanwhile, the incubating time of the PN-CeO2 also influenced the cardio-

protective performance, which was optimized at 24 hours (Figure S8). Apoptotic Investigations and Nuclear Damage by Dox-Induced Oxidative Stress. The apoptotic process induced by ROS was also studied to recognize the functions of the PN-CeO2 against the oxidative stress by monitoring the relative level of caspase-3, which has been found to be a frequently activated protease in mammalian cell apoptosis.45 Herein, caspase-mediated apoptosis was indicated by the CellEvent™ Caspase 3/7 Green Ready Probes, which could be cleaved by active caspases 3/7 and emit green fluorescence. In Figure 5a-b, caspases-3 was highly expressed in the H9c2 cells treated with Dox (30 μM). The relative level of caspase-3 reached 259.2% of the control group, indicating a significant cellular apoptosis. However, the increased caspase-3 level could be largely rescued to 120.6% of the control group by the PN-CeO2. The apoptosis rate of the H9c2 cells was also quantified by Annexin V-FITC assay (Figure 5c). The PN-CeO2 alone delivered a low apoptosis rate (0.4%), even significantly lower than that of the control group (4.0%). In the presence of Dox (30 μM), the PN-CeO2 decreased the cell apoptosis from 49.0%

6


Page 7 of 13

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. ROS-induced apoptosis of H9c2 cells under various treatments. (a) Caspases-3/7 confocal microscopy images of H9c2 cells with the PN-CeO2 treatment (100 μg/mL) for 24 hours and then introduction of Dox (30 μM). (b) Relative caspases-3 levels induced by Dox (30 μM) in the presence or absence of the PN-CeO2 (100 μg/mL). Experiments were repeated for six times. Cells without exposure were used as the control group. (c) The PN-CeO2 protects H9c2 cells against Dox induced apoptosis. Cells without exposure were used as the control group.

to 13.9%. In consistent with the toxicity results (Figure. S6), the results suggest a low toxicity and strong antioxidative nature of the PN-CeO2 nanozymes. ROS can induce a serious break of DNA double strands. Therefore, we tested whether the PN-CeO2 could protect the Dox-induced nuclear damage of the Hoechst 3342 stained H9c2 cells. The PN-CeO2 itself showed no obvious nuclear damage (Figure S9). However, Dox (30 μM) led to a serious nuclear damage, which could be largely rescued by the PN-CeO2. Results further illustrate the PN-CeO2’s capability against Dox-induced oxidative stress. In Vivo Animal Evaluations on Catalytic Activity of the Artificial Enzymes against the Oxidative Stress Induced by Dox. After confirming the high capability of the PN-CeO2 against the Dox-induced oxidative stress in cultured cells, we then explored the possible protective functions of the artificial nanozymes on the healthy adult rats. The Dox and PN-CeO2 were delivered intravenously at 20 mg/Kg and 4 mg/Kg, respectively. Elemental analysis by ICPOES indicated the PN- CeO2 could be distributed invarious organs including myocardial tissues (Figure S10). Meanwhile, Figure S10 also showed that the

concentrations of the PN-CeO2 were decreased by 50.1 % (liver) and 42.2 % (spleen) after 2 weeks, indicating the metabolism of the PN-CeO2. When the rates were only treated with Dox, the survival rates were decreased to 40% and 0 % at the 10th and 12th day (Figure 6a). While no rat death was observed over 12 days with the PN-CeO2 treatment. The weight changes showed the similar protection of the PN-CeO2 (Figure 6b). The Dox treatment led to a rapid weight decrease: 10.2% loss of the body weight at the 3rd day after drug injection and 30% body weight loss at death. Encouragingly, neither obvious body weight loss nor noticeable behavioral abnormality was found in the groups with the PN-CeO2 treatments. The in vivo results strongly confirm the potential effect of the PN-CeO2 against the Dox-induced toxicity in rats. To directly observe the myocardial protective effect of the PN-CeO2, we further investigated the relative caspase-3 level in rat’s myocardial tissue. Caspase-3 levels exposed to the Dox and/or PN-CeO2 were detected by using Caspase Activity Kit. Dox stimulated a high level of the intracellular caspsase-3 in myocardial tissue, which reached 317.2% of the control group (Figure 6c). Quantitatively, this value was

7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 13

Figure 6. In vivo protection of the PN-CeO2 against Dox-induced oxidative stress. (a) Body weight changes of rats after various treatments. (b) Survival curves of rats after various treatments. (c) Effect of the PN-CeO2 on the relative caspase-3 level induced by Dox. (d) Electron micrographs of cardiac specimens of rates under various treatments. Note that the vacuolization and marked myofibrillar loss in the cardiomyopathic cells. The PN-CeO2 and Dox were given through caudal vein injection once at the first day.

significantly reduced by the PN-CeO2 to 151.3 % of the control group. We also introduced TEM technique to observe the structural myocardial tissue of the rats (Figure 6d). Partial or total loss of myofibrils, vacuolar de generation, mitochondria swelling and crista fragmentation were observed in the group treated by the Dox only. Meanwhile, the PN-CeO2 itself led to no obvious structural change but obviously alleviated the Dox-induced cellular structural damage, leaving very slight myocardial swelling. 3. CONCLUSIONS In summary, we perform the quantitative analysis on the correlation between the intrinsic catalytic activity of the CeO2 nanozymes as radical scavengers and their surface properties. Our results demonstrate that the intrinsic catalytic activity of nanocerias with the similar morphology for ROS degradation is mainly determined by the concentrations of their surface defects. Their morphological features also significantly influence to the intrinsic catalytic activity. Thus, the PN-CeO2, an artificial nanozymes with abundant surface defects, delivers a strong intrinsic catalytic capability. The artificial PN-CeO2 nanozymes effectively alleviate the Dox-induced oxidative stress in

both in vitro cultured cells and in vivo animal model. The results strongly suggest that the PN-CeO2 has the potential as clinical cardioprotective agent to shield the heart oxidative damage induced by chemotherapy. These findings show the promise of the PN-CeO2 for effective suppression of Dox-induced oxidative stress as well as the potentials for controlling ROS-damage under UV and/or γ irradiation in the future studies. EXPERIMENTAL SECTION Synthesis of the CeO2 nanozymes. Synthesis of the PNCeO2 involved the hydrothermal formation of non-porous rod-like precursor of Ce(OH)3/CeO2 at 100 °C followed by dehydration and oxidation under the second hydrothermal treatment at 160 °C.35 CeO2 nanoparticles were obtained by calcining cerium nitrate at 500 °C for 2 hours.38 Nonporous CeO2 nanorods were synthesized through a hydrothermal process.37 For the annealed PN-CeO2, assynthesized samples were calcinated at the desired temperatures in a tube furnace in the presence of air. Characterization. The CeO2 nanozymes were characterized by TEM (Hatchie HT-7700 at an accelerating voltage of 120 kV), XRD (Shimadzu X-ray diffractometer, Model 6000 with Cu Kα radiation), XPS (Thermo Electron Model

8


Page 9 of 13

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


K-Alpha with Al Kα as the excitation source) and UVvisible spectra (Perkin Elmer UV spectrophotometer, Lambda 35). Nitrogen adsorption and desorption measurements were performed on ASAP 2020 HD88 (Micromeritics). The specific surface areas were derived by the BET method. The Zeta potential measurements was carried out with Zetasizer Nano ZS90 (Malvern). SOD mimetic kinetic assays. SOD mimetic activity was assayed using the Sigma SOD assay kit (Sigma, 19160) by the manufacturer's instructions. Catalytic evaluations of the artificial enzymes. The catalytic evaluation of the artificial enzymes as the radical scavengers was performed according to previously reported method.22 2-2’-azobis (2-amidinopropane)dihydrochloride (AAPH, 0.15 M), fluorescein sodium salt (FL, 0.2 μM), 6-hydroxy-2,5,7,8-tetramethylchromane-2carboxylic acid (Trolox), and CeO2 (PN-CeO2, nanoparticles and nonporous nanorods) were dissolved/dispersed in phosphate buffer saline (PBS, pH 7.4) in a 96-well plate (flat bottom, white polystyrene). The freshly prepared solutions were used within 6 hours. The fluorescence was recorded at 530 nm by 485 nm excitation for each minute using a SpectraMax Paradigm Multi-Mode reader (Molecular Devices). The catalytic capacity was calculated according to the equation (1) by measuring the area under curve (AUC) of the time dependent fluorescence intensity from the nanozymes and the blank (without the nanozymes). This analysis was repeated for 5 times. Catalytic capacity (%) =AUCenzymes − AUCblank / AUCblank × 100 (1)

Cell culture. The rat cardiac myocytes, H9c2 Cell lines were maintained in dulbecco's modified eagle medium (DMEM, GIBCO) supplemented with 10% foetal bovine serum (FBS, GIBCO) and 1% v/v penicillin-streptomycin under standard conditions of culture at 37 °C in humidified atmosphere of 5% CO2 and 95% air (Thermo-Forma, Model 371). Biocompatibility safety assessment. Biocompatibility of the CeO2 (PN-CeO2, nanoparticles and nonporous nanorods) was assessed by using the Alamar Blue assay method. With a seeded 1× 104 cells/mL into 96-well plate, H9c2 cells were incubated 12 hours to adhere and then treated with the CeO2 (PN-CeO2, NP-CeO2 and NR-CeO2) at various concentrations suspended in DMEM for 48 hours. Afterwards, the relative cell viabilities were determined by the standard Alamar Blue assay. Cell uptake assay. After H9c2 cells (1 × 105 cells) cultured in 100mm plates with 5.0 mL DMEM (10 % fetal calf serum) for 24 hours were incubated with various concentrations (10-1000 μg/mL) of the PN-CeO2 for 24 h, respectively, the cells were rinsed by PBS (pH 7.4) and then cen-

trifuged off for three times. Digestion of the H9c2 cells was performed in 1 mL of concentrated nitric acid solution. The uptake of the PN-CeO2 by H9c2 cells was measured by inductively coupled plasma optical emission spectroscopy (ICP-OEC) analysis. Preparation and imaging the H9c2 cells using TEM. H9c2 cells were pre-fixed with cold 2.5% (w/v) glutaraldehyde (Sigma Aldrich) solution in 0.1 M cacodylate buffer (Sigma Aldrich, pH 7.4) for 2 minutes at room temperature. Subsequently, H9c2 cells were rinsed in the same buffer and centrifuged off. The pellet was post-fixed for 30 minutes with a mixture of 1 % osmium tetroxide (OsO4) and 1.5 % potassium ferrocyanide (0.1 M cacodylate, pH 7.4), dehydrated in a graded series of ethanol, and then embedded in EPON 812 (Fluka) according to standard procedures.46 The resin specimen blocks were trimmed. Ultrathin sections of selected areas were cut and mounted on copper grids, then contrasted with 1 % (w/v) uranyl acetate before examination with a transmission electron microscope (Hitachi H-7650) at 80 kV. Viabilities of H9c2 cells after CeO2 preincubation and H2O2 treatment. A total of 1 × 103 cells were seeded into 96-well plates and cultured in DMEM medium with 10% FBS. A total of 5 groups were set: Trolox, PN-CeO2, NRCeO2, NP-CeO2 and negative control. Six replicates for each H2O2 concentration point (20, 40, 60, 80 and 100μM) were performed. At each H2O2 concentration, cells in one well were treated with Alamar Blue solution (10 μL) and incubated for 2 hours at 37 °C. The values were measured in a SpectraMax fluorescence microplate reader (Molecular Devices). Relative cell viabilities were determined by the standard Alamar Blue assay. Viabilities of H9c2 cells after PN-CeO2 preincubation and Dox treatment. A total of 1 × 103 cells were seeded into 96-well plates and cultured in DMEM medium with 10% FBS. A total of 2 groups were set: PN-CeO2 treated and negative control, with six replicates for each doxorubicin concentration (5, 10, 20, and 30μM). At each concentration of Dox, cells in one well were treated with Alamar Blue solution (10 μL) and incubated for 2 hours at 37°C. The values were measured in a SpectraMax fluorescence microplate reader (Molecular Devices). Relative cell viabilities were determined by the standard Alamar Blue assay. Determination of ROS level induced by H2O2. Intracellular ROS levels were investigated by DCFH-DA dyebased assay. H9c2 cells were plated in 96-well plates and incubated with the Trolox and CeO2 for 24 hours prior to H2O2 at various concentrations exposure for 30 minutes. Untreated cells were used as control group to determine the normal level of ROS in cells. Afterwards, H9c2 cells were washed with PBS (pH 7.4) for three times. Then, cells were incubated in PBS containing DCFH-DA dye (10

9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

μM) for 30 min at 37 °C. Quantitative assessment of ROS was carried out on a SpectraMax fluorescence microplate reader (Molecular Devices). Images were taken using the FITC filter of the confocal microscope (Olympus, FV1000). Determination of ROS level induced by Dox. H9c2 Cells were plated in individual 35 mm cell culture dishes and incubated with the PN-CeO2 for 24 hours prior to Dox exposure at various concentrations for 6 hours. Untreated cells were used as negative control to determine the normal level of ROS in cells. Cells were resuspended in PBS (pH 7.4) containing DCFH-DA dye (10 μM) and incubated for 30 min at 37 °C. Images were taken using the FITC filter of the fluorescence microscope (Olympus, FV1000). Quantitative assessment of ROS was carried out with the flow cytometer (BD Accuri C6). For flow analysis, trypsinizated cell was dispersed in PBS (pH 7.4). The fluorescence intensity was measured by using 488 nm excitation laser of the flow cytometer. Electron paramagnetic resonance spectroscopy measurements. A solution with a total volume of 0.6 ml with ~106 cells per ml of H9c2 cells, D-glucose (1 mg/ml), 10 mM DMPO and 0.2 μg/ml of phorbol-12-myristate-13acetate (PMA) was prepared. After that, the solution was transferred to an EPR capillary tube (Bruker). EPR acquisition parameters conditions are: microwave frequency: 9.8 GHz; center field: 3360 G; sweep width: 100 G; total number of scans: 20; sweep time: 5 s; and microwave power: 20 mW. Apoptotic cell ratio. H9c2 Cells were plated in individual 35 mm cell culture dishes and incubated with the PNCeO2 for 24 hours prior to Dox exposure (30 μM) for 6 hours. Untreated cells were used as control group to determine the normal level of ROS in cells. The apoptotic cells were examined using Annexin V, Alexa Fluor® 488 conjugate (ThermoFisher, A13201) according to the manufacturer's instructions. Apoptotic cells were detected by flow cytometer (BD Accuri C6). Animal model. Animal experiments were performed according to the guide for the care and use of laboratory animals, established by the committee on animal research at Xi'an Jiaotong University. For this work, rats (weight = 220 ± 20g, purchased from Xi’an Jiaotong University Laboratory Animal Center) were housed in an airconditioned room with 12-hour light/dark cycles, with temperature (22 ± 2 °C) and relative humidity (65%–70%). The rats were fed standard commercial pellets and water ad libitum. Rats were divided into four groups (n = 5). The PN-CeO2 suspended in PBS (pH 7.4, dose = 4 mg/kg) was introduced through tail intravenous injection into the each rat. After 24 hours, the Dox (dose = 20 mg/kg) was introduced through tail intravenous injection into the each rat. For control groups, rats were either treated with

Page 10 of 13

the same volume of PBS, or injected with PN-CeO2 without Dox. For the positive control groups, rats were only injected with the same dose of Dox. PN-CeO2 and Dox were given by caudal vein injection once at the first day. All rats' weights were measured daily, until the positive control groups died. Each test was repeated for five times. Analysis of caspase-3 activities. Fluorescence images of caspases-3 were recorded by using CellEvent® Caspase-3/7 Green Ready Probes (Invitrogen, R37111) according to the manufacturer’s reference. Caspases activities were measured by using Caspase Activity Kit (Beyotime, C1115) according to the manufacturer’s instructions. Briefly, cells or heart tissue were washed with cold PBS (pH 7.4), resuspended in lysis buffer and left on ice for 15 minutes. The lysate was centrifuged off at 16000 g at 4 °C for 15 minutes. Activities of caspase-3 were measured by using substrate peptides of Ac-DEVD-pNA. The release of pnitroanilide (pNA) was qualified by the absorbance from a SpectraMax fluorescence microplate reader (Molecular Devices) at 405 nm. Preparation and imaging the heart tissue using TEM. For TEM observation, heart tissue was fixed in a cold 2.5% (w/v) glutaraldehyde (Sigma Aldrich) in PBS buﬀer (pH 7.4) overnight and then incubated under the protection from light in 1% osmium tetroxide for 2 hours. After washing in distilled water, specimens were incubated in 2% uranyl acetate for 2 hours at room temperature and then dehydrated in graded ethanol solution. Finally, samples were embedded in molds with fresh resin. Ultrathin sections were examined by a Hitachi H-7650 at 80 kV.

ASSOCIATED CONTENT Supporting Information Experimental details of more characterizations and quantitative analysis on catalytic activity of the CeO2 nanozymes as radical scavengers. Characterization data include: BET, XPS, TEM, UV-Vis, cytotoxicity assessment inH9c2 cells, cellular uptake of the PN-CeO2 in H9c2 cells, nuclear damage by confocal microscopy, and biodistributions of the PN-CeO2 in rats. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author *[email protected]; [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We acknowledge the financial support from NSFC Grants (21401148) and the National 1000-Plan program. Y. Qu is also supported by the Cyrus Tang Foundation through Tang Scholar program.

10


Page 11 of 13

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


REFERENCES (1) Kalyanaraman, B. Teaching the Basics of Redox Biology to Medical and Graduate Students: Oxidants, Antioxidants and Disease Mechanisms. Redox Biol. 2013, 1, 244-257. (2) Karakoti, A.; Singh, S.; Dowding, J. M.; Seal, S.; Self, W. T. Redox-Active Radical Scavenging Nanomaterials. Chem. Soc. Rev. 2010, 39, 4422-4432. (3) Finkel, T.; Holbrook, N. J. Oxidants, Oxidative Stress and the Biology of Ageing. Nature 2000, 408, 239-247. (4) Sena, L. A.; Chandel, N. S. Physiological Roles of Mitochondrial Reactive Oxygen Species. Mol. Cell 2012, 48, 158-167. (5) Valko, M.; Leibfritz, D.; Moncol, J.; Cronin, M. T. D.; Mazur, M.; Telser, J. J. Free Radicals and Antioxidants in Normal Physiological Functions and Human Disease. Int. J. Biochem. Cell Biol. 2007, 39, 44-84. (6) Rzigalinski, B. A.; Meehan, K.; Davis, R. M.; Xu, Y.; Miles, W. C.; Cohen, C. A. Radical Nanomedicine. Nanomedicine 2006, 1, 399-412. (7) Muzykantov, V. R. Targeting of Superoxide Dismutase and Catalase to Vascular Endothelium. J. Controlled Release 2001, 71, 1-21. (8) Clark, S.; Youngman, L. D.; Chukwurah, B.; Palmer, A.; Parish, S.; Peto, R.; Collins, R. Effect of Temperature and Light on the Stability of Fat-Soluble Vitamins in Whole Blood Over Several Days Implications for Epidemiological Studies. Int. J. Epidemiol. 2004, 33, 518525. (9) Heckert, E.; Karakoti, A.; Seal, S.; Self, W. T. The Role of Cerium Redox State in the SOD Mimetic Activity of Nanoceria. Biomaterials 2008, 29, 2705-2709. (10) Karakoti, A. S.; Singh, S.; Kumar, A.; Malinska, M.; Kuchibhatla,S. V. N. T.; Wozniak, K.; Self, W. T.; Seal, S. PEGylated Nanoceria as Radical Scavenger with Tunable Redox Chemistry. J. Am. Chem. Soc. 2009, 131, 14144-14145. (11) Das, S.; Dowding, J. M.; Klump, K. E.; McGinnis, J. F.; Self, W. Seal, S. Cerium Oxide Nanoparticles Applications and Prospects in Nanomedicine. Nanomedicine 2013, 8, 1483-1508. (12) Tian, Z. M.; Li, J.; Zhang, Z. Y.; G, W.; Zhou, X. M.; Qu, Y. Highly Sensitive and Robust Peroxidase-Like Activity of Porous Nanorods of Ceria and their Application for Breast Cancer Detection. Biomaterials 2015, 59, 116124. (13) Zhang, Y.; Wang, Z. Y.; Li, X. J.; Wang, L.; Yin, M.; Wang, Li. H.; Chen, N.; Fan, C. H.; Song, H. Y. Dietary Iron Oxide Nanoparticles Delay Aging and Ameliorate Neurodegeneration in Drosophila. Adv. Mater. 2016, 28, 1387-1393. (14) Vernekar, A. A.; Sinha, D.; Srivastava, S.; Paramasivam, P. U.; D’Silva, P.; Mugesh, G. An Antioxidant Nanozyme that Uncovers the Cytoprotective Potential of Vanadia Nanowires. Nat. Commun. 2014, 5, 5301. (15) Liu, L. Z.; Yu, W.; Luo, D.; Xue, Z. J.; Qin, X. Y.; Sun, X. H.; Zhao, J. C.; Wang, J. L.; Wang, T. Catalase Nanocapsules Protected by Polymer Shells for Scavenging Free Radicals of Tobacco Smoke. Adv. Funct. Mater. 2015, 25, 5159-5165. (16) Huang, Y. Y.; Liu, Z.; Liu, C. Q.; Ju, E.; Zhang, Y.; Ren, J. S.; Qu, X. G. Self-Assembly of Multi-nanozymes to Mimic an Intracellular Antioxidant Defense System. Angew. Chem. Int. Ed. 2016, 55, 6646-6650.

(17) Wang, X. Y.; Hua, Y. H.; Wei, H. Nanozymes in Bionanotechnology: From Sensing to Therapeutics and Beyond. Inorg. Chem. Front. 2016, 3, 41-60. (18) Wang, Y. J.; Dong, H.; Lyu, G. M.; Zhang, H. Y.; Ke, J.; Kang, L. Q.; Teng, J. L.; Sun, L. D.; Si, R.; Zhang, J.; Liu, Y. J.; Zhang, Y. W.; Huang, Y. H.; Yan, C. H. Engineering the Defect State and Reducibility of Ceria Based Nanoparticles for Improved Anti-Oxidation Performance. Nanoscale 2015, 7, 13981-13990. (19) Xia, T.; Kovochich, M.; Liong, M.; Mädler, L.; Gilbert, B.; Shi, H. B.; Yeh, J. I.; Zink, J. I.; Nel, A. E. Comparison of the Mechanism of Toxicity of Zinc Oxide and Cerium Oxide Nanoparticles Based on Dissolution and Oxidative Stress Properties. ACS Nano 2008, 2, 21212134. (20) Pierscionek, B. K.; Li, Y. B.; Yasseen, A. A.; Colhoun, L. M.; Schachar, R. A.; Chen, W. Nanoceria have No Genotoxic Effect on Human Lens Epithelial Cells. Nanotechnology 2010, 21, 035102. (21) Kim, C. K.; Kim, T.; Choi, I. Y.; Soh, M.; Kim, D.; Kim, Y. J.; Jang, H.; Yang, H. S.; Kim, J. Y.; Park, H. K.; Park, S. P.; Park, S.; Yu, T.; Yoon, B. W.; Lee, S. H.; Hyeon, T. Ceria Nanoparticles that can Protect against Ischemic Stroke. Angew. Chem. Int. Ed. 2012, 51, 11039-11043. (22) Lee, S. S.; Song, W. S.; Cho, M. J.; Puppala, H. L.; Nguyen, P.; Zhu, H. G.; Segatori, L.; Colvin, V. L. Antioxidant Properties of Cerium Oxide Nanocrystals as a Function of Nanocrystal Diameter and Surface Coating. ACS Nano 2013, 7, 9693-9703. (23) Pagliari, F.; Mandoli, C.; Forte, G.; Magnani, E.; Pagliari, S.; Nardone, G.; Licoccia, S.; Minieri, M.; Nardo, P. D.; Traversa, E. Cerium Oxide Nanoparticles Protect Cardiac Progenitor Cells from Oxidative Stress. ACS Nano 2012, 6, 3767-3775. (24) Xu, P. T.; Maidment 3rd, B. W.; Antonic, V.; Jackson, I. L.; Das, S.; Zodda, A.; Zhang, X.; Seal, S.; Vujaskovic, Z. Cerium Oxide Nanoparticles: A Potential Medical Countermeasure to Mitigate Radiation-Induced Lung Injury in CBA/J Mice. Radiat. Res. 2016, 185, 516-526. (25) Wei, H.; Wang, E. Nanomaterials with Enzyme-Like Characteristics (nanozymes): Next-Generation Artificial Enzymes. Chem. Soc. Rev. 2013, 42, 6060-6093. (26) Xu, C.; Qu, X. G. Cerium Oxide Nanoparticle: a Remarkably Versatile Rare Earth Nanomaterial for Biological Applications. NPG Asia Mater. 2014, 6, e90. (27) Chen, J. P.; Patil, S.; Seal, S.; McGinnis, J. F. Rare Earth Nanoparticles Prevent Retinal Degeneration Induced by Intracellular Peroxides. Nat. Nanotechnol. 2006, 1, 142-150. (28) Liu, B.; Liu, J. Surface Modification of Nanozymes. Nano Res. 2017, 10, 1125-1148. (29) Song, G.; Chen, Y.; Liang, C.; Yi, X.; Liu, J.; Sun, X.; Shen, S.; Yang, K.; Liu, Z. Catalase-Loaded TaOx Nanoshells as Bio-Nanoreactors Combining High-Z Element and Enzyme Delivery for Enhancing Radiotherapy. Adv. Mater. 2016, 28, 7143-7148. (30) Zhang, C.; Bu, W.; Ni, D.; Zhang, S.; Li, Q.; Yao, Z.; Zhang, J.; Yao, H.; Wang, Z.; Shi, J. Synthesis of Iron Nanometallic Glasses and Their Application in Cancer Therapy by a Localized Fenton Reaction. Angew. Chem. Int. Ed. 2016, 55, 2101-2106. (31) Korsvik, C.; Patil, S.; Seal, S.; Self, W. T. Superoxide Dismutase Mimetic Properties Exhibited by Vacancy Engineered Ceria Nanoparticles. Chem. Commun. 2007, 10, 1056-1058.

11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(32) WHO. 19th WHO Model List of Essential Medicines (April 2015). 19th WHO Model List of Essential Medicines. 2015, Geneva. (33) Singal, P. K.; IIiskovic, N. Doxorubicin-Induced Cardiomyopathy. N. Engl. J. Med. 1998, 339, 900-905. (34) Minotti, G.; Menna, P. Salvatorelli, E.; Cairo, G.; Gianni, L. Anthracyclines: Molecular Advances and Pharmacologic Developments in Antitumor Activity and Cardiotoxicity. Pharmacol. Rev. 2004, 56, 185-229. (35) Li, J.; Zhang, Z. Y.; Tian, Z. M.; Zhou, X. M; Zheng, Z. P.; Ma, Y.; Qu, Y. Low Pressure Induced Porous Nanorods of Ceria with High Reducibility and Large Oxygen Storage Capacity: Synthesis and Catalytic Applications. J. Mater. Chem. A 2014, 2, 16459-16466. (36) Cargnello, M.; Doan-Nguyen, V. V. T.; Gordon, T. R.; Diaz, R. E.; Stach, E. A.; Gorte, R. J.; Fornasiero, P.; Murray, C. B. Control of Metal Nanocrystal Size Reveals Metal-Support Interface Role for Ceria Catalysts. Science 2013, 341, 771-773. (37) Mai, H. X.; Sun, L. D.; Zhang, Y. W.; Si, R.; Feng, W.; Zhang, H. P.; Liu, H. C.; Yan, C. H. Shape-Selective Synthesis and Oxygen Storage Behavior of Ceria Nanopolyhedra, Nanorods, and Nanocubes. J. Phys. Chem. B 2005, 109, 24380-24385. (38) Feng, L.; Hoang, D. T.; Tsung, C. K.; Huang, W. Y.; Lo, S. H. Y.; Wood, J. B.; Wang, H. T.; Tang; J. Y.; Yang, P. D. Catalytic Properties of Pt Cluster-Decorated CeO2 Nanostructures. Nano Res. 2011, 4, 61-71. (39) Zhang, S.; Chang, C. R.; Huang, Z. Q.; Li, J.; Wu, Z. M.; Ma, Y.; Zhang, Z. Y.; Wang, Y.; Qu, Y. High Catalytic Activity and Chemoselectivity of Sub-Nanometric Pd Clusters on Porous Nanorods of CeO2 for Hydrogenation of Nitroarenes. J. Am. Chem. Soc. 2016, 138, 2629-2637.

Page 12 of 13

(40) Zhang, Z.Y.; Li, J.; Gao, W.; Ma, Y.; Qu, Y. Pt/porous Nanorods of Ceria as Efficient High Temperature Catalysts with Remarkable Catalytic Stability for Carbon Dioxide Reforming of Methane. J. Mater. Chem. A 2015, 3, 18074-18082. (41) Zhang S, Huang Z.Q, Ma Y, Gao W, Li J, Cao F, Li L, Chang C.R, Qu Y. Solid Frustrated-Lewis-Pair Catalysts Constructed by Regulations on Surface Defects of Porous Nanorods of CeO2. Nat. Commun. 2017, 8, 15266. (42) Wu, Z. L.; Li, M. J.; Overbury, S. H. On the Structure Dependence of CO Oxidation Over CeO2 Nanocrystals with Well-Defined Surface Planes. J. Catal. 2012, 285, 61-73. (43) Kimes, B. W.; Brandt, B. L. Properties of a Clonal Muscle Cell Line from Rat Heart. Exp. Cell Res. 1976, 98, 367-381. (44) Migliaccio, E.; Giorgio, M.; Mele, S.; Pelicci, G.; Reboldi, P.; Pandolfi, P. P.; Lanfrancone, L.; Pelicci, P. G. shc The p66 Adaptor Protein Controls Oxidative Stress Response and Life Span in Mammals. Nature 1999, 402,309-313. (45) Porter, A. G.; Jänicke, R. U. Emerging Roles of Caspase-3 in Apoptosis. Cell Death Differ. 1999, 6, 99-104. (46) Heckman, K. L.; DeCoteau, W.; Estevez, A.; Reed K. J.; Costanzo, W.; Sanford, D.; Leiter J. C.; Clauss, J.; Knapp, K.; Gomez, C.; Mullen, P.; Rathbun, E.; Prime, K.; Marini, J.; Patchefsky, J.; Patchefsky, A. S.; Hailstone, R. K.; Erlichman, J. S. Custom Cerium Oxide Nanoparticles Protect Against a Free Radical Mediated Autoimmune Degenerative Disease in the Brain. ACS Nano 2013, 7, 10582-10596.

12


Page 13 of 13

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of Contents

13


Quantitatively Intrinsic Biomimetic Catalytic Activity ... - ACS Publications

Recommend Documents