Ultrasmall Nanozymes Isolated within Porous Carbonaceous

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Ultrasmall Nanozymes Isolated within Porous Carbonaceous Frameworks for Synergistic Cancer Therapy: Enhanced Oxidative Damage and Reduced Energy Supply Fangfang Cao, Yan Zhang, Yuhuan Sun, Zhenzhen Wang, Lu Zhang, Yanyan Huang, Chaoqun Liu, Zhen Liu, Jinsong Ren, and Xiaogang Qu Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.8b03348 • Publication Date (Web): 09 Oct 2018 Downloaded from http://pubs.acs.org on October 10, 2018

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Chemistry of Materials

Ultrasmall Nanozymes Isolated within Porous Carbonaceous Frameworks for Synergistic Cancer Therapy: Enhanced Oxidative Damage and Reduced Energy Supply Fangfang Cao,a,b Yan Zhang,a,c Yuhuan Sun,a,b Zhenzhen Wang,a,c Lu Zhang,a,c Yanyan Huang,a,c Chaoqun Liu,a,c Zhen Liu,a Jinsong Ren,*,a and Xiaogang Qu*,a State Key Laboratory of Rare Earth Resources Utilization and Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China a

b

University of Science and Technology of China, Hefei, Anhui 230029, P.R. China

c

Graduate School of the Chinese Academy of Sciences, Beijing 100039, P. R. China

ABSTRACT: Due to the robust stability and ultralow cost, nanozymes have been considered as one of the most promising alternatives to natural enzymes in recent years. Generally, shrinking the sizes of nanozymes can generate a large active surface area for catalytic reactions in various practical usages. However, the concomitant increase of surface free energy will intensify the risk of nanozymes’ aggregation and further cause the loss of the catalytic ability. To overcome these limitations, we rationally design and fabricate uniformly dispersed ultrasmall nanozymes for the first time by using wellordered crystalline metal organic frameworks (MOFs) as precursors in this study. Typically, nano-sized cerium-based MOFs (Ce-MOFs) are thermally converted into homogeneous cerium oxide nanoparticles (CeO2 NPs) isolated within porous carbonaceous frameworks with a high density via a one-pot facile approach. As expected, excellent characters of these MOF-derived CeO2 NPs including oxidase-like activity, ATP deprivation capacity, and porous structure endow them with admirable oxidative damage effect, specially reduced energy supply ability, and high drug loading capacity. Both in vitro and in vivo results indicate the great promise of these well-prepared nanostructures in synergistic cancer therapy with negligible side effects. Thus, our study paves a new way for the development of high-performance MOFs-derived nanozymes particularly useful for the safe and efficient cancer therapy.

INTRODUCTION As one of the emerging nanomaterials, nanozymes have garnered tremendous interest in various usages ranging from biosensor to nanomedicine because of their higher stability and lower cost than natural enzymes.1-16 Recently, a series of nanomaterials have been explored to possess inherent oxidase,17-21 peroxidase,1,22-26 superoxide 27-29 30 dismutase, catalase and laccase mimicking activities.31 However, their catalytic efficiency is still far from satisfactory. To improve their catalytic performance in various practical usages, one of the most common strategies is to generate a sufficiently larger surface area for catalytic reactions via shrinking the sizes of nanozymes.1,28,32 Although promising, the surface free energy of nanozymes boosts significantly while these particles become smaller, which thus lead to their serious aggregation and further loss of their catalytic ability.17,33 To overcome these limitations, tremendous efforts have been devoted to dispersing these highly active nanozymes by using polymers as surface modification reagents and inorganic materials as deposition matrixes.4,17,34,35 A high fraction of nanozymes have been embedded into various catalytic systems, however these approaches can’t totally avoid their aggregation and usually loss their accessible active sites. More importantly, the distribution of

nanozymes in substrate materials is neither homogeneous nor dense, which fails to further enormously improve the catalytic efficacy. Thereby, it is still a great challenge to construct nanozymes featuring precise distributions and high activities. Metal-organic frameworks (MOFs) with high surface areas, tunable porosity, as well as inherent existence of coordinated metal and heteroatoms have become one of the best examples of materials fabricated from molecular engineering.36-40 Currently, MOFs can serve as sacrificed precursors to construct MOF-derived porous metal oxides, which have been widely verified in the fields of catalysis, energy, adsorption, and gas sensor.41-45 During the thermolysis process, periodically arranged metal ions together with organic ligands in MOFs can be directly converted into uniformly distributed metal oxide nanoparticles, serving as the high-performance catalyst. Meanwhile, the organic linkers in MOFs can be transformed into porous carbonaceous structures, avoiding the potential aggregation from metal oxide nanocrystals. Taking together, MOF-derived metal oxide catalysts with uniform components distributions and porous structures possess higher catalytic efficacy as compared with traditional metals oxide-based catalysts. For example, hierarchically nanoporous magnesia has

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been demonstrated to exhibit exceptional CO2 adsorption capacity.42 Moreover, 3D porous carbon and cobalt oxide asymmetric supercapacitors have been shown to produce much higher energy density.44 Consequently, the rational design and fabrication of MOF-derived nanomaterials can provide a new routine to uniformly disperse metal oxidebased nanocrystals within porous carbonaceous substrate materials and enhance their related catalytic performance. Inspired by these unique features, herein, for the first time, we employed cerium-contained MOFs (Ce-MOFs) as sacrificed precursors to prepare uniformly dispersed ultrasmall CeO2-based nanozymes isolated within porous carbonaceous frameworks with high enzyme-like activity, which were defined as n-CeO2 NSs. As a common oxidasemimic, cerium oxide (CeO2) stand out with higher activity and can induce irreversible cellular apoptosis and necrosis in acidic microenvironments via the harmful oxidation stress.46-49 Meanwhile, lanthanide-based materials including cerium-contained ones usually hold high deprivation abilities towards ATP and lead to serious cellular autophagy and death.50 However, most of the current CeO2 NPs tend to exist in aggregated states with limited catalytic activity.17 Our present design by using Ce-MOFs as templates could overcome above shortcomings, and these well-defined n-CeO2 NSs could be achieved via a facile thermolysis process. Typical synthesis of n-CeO2 NSs and their usages as multifunctional nanomedicine for cancer theranostics were illustrated in Scheme 1. As expected, these wellprepared n-CeO2 NSs held admirable oxidase-like activity and high metal-phosphate coordination ability, which could strongly enhance the oxidation damage towards cancer cells and reduce the energy supply of malignant tumor. In addition, these porous n-CeO2 NSs could serve as excellent nanocarriers in chemotherapy owing to the interconnected pores of MOF-derived nanostructures. By virtue of their high oxidase-like activities, efficient ATPdeprivation abilities, and good drug loading capacity, our n-CeO2 NSs would blaze a new path for efficient synergistic cancer therapy both in vitro and in vivo. RESULTS AND DISCUSSION Prior to the preparation of CeO2 NSs, monodispersed CeMOFs were first synthesized via a fast coordination by using cerium ammonium nitrate ((NH4)2Ce(NO3)6) and terephthalic acid (TA) as initial raw materials.51 Both SEM image and TEM image revealed that Ce-MOFs held approximate spherical morphology with an average diameter of 220 nm, which were quite similar with those of previously reported studies (Figure S1-S2). In order to ensure the formation process of Ce-MOFs to homogeneous CeO2 NPs, calcination temperature and related atmosphere were explored in detail. To benefit the understanding of readers, we defined the calcinated product of Ce-MOFs in N2 as n-CeO2 and that in air as aCeO2. As shown in Figure S3, thermogravimetric analysis

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(TGA) revealed that a sharp decrease in weight of CeMOFs was found after they were calcined in air at 400 °C, Scheme 1. Schematic illustration of the rational design and synthesis of n-CeO2 NSs with enhanced oxidase-like activity (a), as well as their usage as an efficient multifunctional platform for synergistic cancer therapy (b).

and there was only 40% weight loss of Ce-MOFs after calcination in N2 upon the same calcination temperature. FT-IR spectra indicated that multiple bands assigned to the vibrational modes of -COO- nearly totally disappeared in a-CeO2 (Figure S4). However, these essential bonds still remained in the sample of n-CeO2 NSs due to the presence of carbonaceous frameworks after the incomplete transformation of Ce-MOFs in N2. Wide-angle XRD patterns further confirmed that CeMOFs could be converted into highly crystallized a-CeO2 after the calcination in air at 400 °C (Figure S5). Owing to the abundant existence of carbonaceous frameworks and potential loose structure, CeO2 NSs exhibited related weakly crystallized. Quantificationally, the mass ratio of CeO2 to carbonaceous frameworks was about 7:5. In addition, N2 adsorption and desorption isotherms indicated that the Brunauer–Emmett–Teller (BET) specific surface areas of n-CeO2 NSs and a-CeO2 were 401 and 65 m2 g-1, respectively (Figure S6). Compared with aCeO2, more porous structures could be found in n-CeO2 NSs, which could enhance their potential adsorption of various guest molecules. All these results suggested that our design of n-CeO2 NSs was reasonable, which could provide the protection for untrasmall CeO2 NPs against aggregation via well-prepared carbonaceous frameworks as efficient support. Detailed structural and component information of n-CeO2 NSs was listed below. As shown in


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Figure 1. (a) SEM image of n-CeO2 NSs (a), TEM images and HR-TEM image of n-CeO2 NSs under different magnifications (b-e), SAED image of n-CeO2 NSs (f), as well as dark-field TEM image of n-CeO2 NSs and corresponding EDS mapping (g-catalytic performance of these 7 kinds of CeO2-based nanozymes were investigated in detail. Wide-angle j). Figure 1a, the resultant n-CeO2 NSs retained the original morphology of Ce-MOFs precursors with an average diameter of 200 nm. Significantly, lots of CeO2 NPs with an average diameter of 4 nm were homogeneously dispersed in porous carbonaceous frameworks and separated from each other with apparent pore structures (Figure 1b-d, Figure S7). We could ascribed the lattice spacing of 0.31 nm in HR-TEM images to the (111) facet of cubic phase CeO2 (JCPDS card no. 65-2975), which was also consistent with related XRD pattern and selected area electron diffraction (SAED) image (Figure 1e-f). Compared with n-CeO2 NSs, a-CeO2 prepared in air showed serious agglomeration of larger CeO2 NPs with an average diameter of 7 nm, which was mainly caused by the total loss of porous carbonaceous frameworks and the formation of collapse structure during the pyrolyzation process (Figure 2a). We further verified the element distributions of n-CeO2 NSs and related valence state condition of Ce in n-CeO2 NSs. As expected, results based on energy dispersive spectroscopic (EDS) mapping and Xray photon spectroscopy (XPS) demonstrated that elements including Ce, O, and C were found in n-CeO2 NSs and n-CeO2 NSs was existed in a mixed valence state with a Ce(III)/Ce(IV) ratio of 1.07 (Figure 1g-j, Figure S8). Overally, all these exciting information of n-CeO2 NSs promoted us to further explore their enzyme-like activity. In order to fully understand the high enzyme-like activity of n-CeO2 NSs, other 5 kinds of frequently used polymer-coated CeO2 NPs including d-CeO2, β-CeO2, BSA-CeO2, PAA-CeO2, and T-CeO2 were prepared at first. All of the five controls hadn’t undergo any thermal treatment and they had different sizes, dispersities as well

Figure 2.TEM images of various CeO2 NPs under different magnifications (a). Time-dependent absorbance changes at 652 nm of TMB reaction solutions catalyzed by 25 μg/mL different CeO2 NPs in Tris buffer (10 mM, pH 4.0) (b). Schematic illustration of the existent morphology effect of CeO2 NPs on their oxidase-like activities (c). as Ce contents. Together with a-CeO2 and n-CeO2 NSs, the close relationship between the structure and the catalytic performance of these 7 kinds of CeO2-based nanozymes were investigated in detail. Wide-angle XRD patterns indicated the formation of cubic phase CeO2 with various crystallization degrees upon different experimental designs (Figure S9). TEM images and related size distribution of these newly synthesized CeO2 NPs were illustrated in Figure 2a and Figure S10. Typically, 4 kinds of CeO2-based nanozymes including dCeO2, β-CeO2, BSA-CeO2, and T-CeO2 exhibited severe aggregation of CeO2 NPs compared to that of n-CeO2 NSs while PAA-CeO2 with homogeneous dispersion of CeO2 NPs was similar to that of n-CeO2 NSs. As mentioned above, a-CeO2 showed significant agglomeration of larger CeO2 NPs with an average diameter of 7 nm in comparison with n-CeO2 NSs. The contents of Ce in different samples were evaluated by ICP-MS in the experiments, and the contents were high in a-CeO2 and n-CeO2 NSs. Information focused on the oxidase-like activity of these 7 kinds of CeO2-based nanozymes was listed in Figure 2b. As expected, n-CeO2 NSs held the best catalytic activity towards 3,3’,5,5’-tetramethylbenzidine (TMB) among all these 7 kinds of nanozymes, which was able to catalyze the oxidation of TMB and produce a deep blue color (Figure S11). However, d-CeO2, β-CeO2, BSACeO2, T-CeO2, and a-CeO2 exhibited extremely low enzyme-like activity upon the same experimental conditions, which could be ascribed to the serious particles’ aggregation in these nanostructures with a reduction of exposed active sites. Accordingly, nanoparticles’ surface-to-volume ratio and related aggregation state might be considered as one of the most important factor to describe the enzyme-like activity. Moreover, PAA-CeO2 showed a higher enzyme-like activity compared with other nanozymes instead of nCeO2 NSs, which could be attributed to the intrinsic



porous structure of n-CeO2 NSs and their improved mass transportation process for catalytic reaction.44,45 Generally, high content of Ce(III) usually went together with abundant oxygen vacancies in CeO2-based nanozymes, which was conducive to easier oxygen exchange and redox reactions.52-55 Therefore, we further explored the Ce(III) contents in various samples. As shown in Figure S12 and Figure S13, the Ce(III) contents in d-CeO2, β-CeO2, BSA-CeO2, T-CeO2, and a-CeO2 were lower than 50% while PAA-CeO2 and n-CeO2 NSs held higher Ce(III) contents over 50%. All these exciting results indicated more oxygen vacancies could be found in PAA-CeO2 and n-CeO2 NSs, which once more confirmed the intrinsic relationship between enzyme-like activity and high dispersity of CeO2 NPs. As summarized in Figure 2c, uniformly dispersed CeO2 usually possessed enhanced oxidizability towards TMB while the aggregated ones exhibited limited oxidizability. More importantly, high surface-to-volume ratio together with interconnected pore structure might highly enhance the enzyme-like activity. Overall, n-CeO2 NSs held the highest enzyme-like activity among all these CeO2-based nanozymes by using porous carbonaceous frameworks as the support of ultrasmall CeO2 NPs against aggregation and the efficient mass transportation medium for catalytic reaction. In order to use these highly active nanozymes in biorelated systems, we further explored their enzyme-like properties upon different temperatures, pH values, as well as ATP concentrations. As shown in Figure S14, n-CeO2 NSs held excellent catalytic activity around weak acid conditions and negligible ability at neutral condition, indicating that these well-prepared nanozymes could efficiently kill cancer cells while they were accumulated in acidic lysosomes after efficient cellular uptake.46,47 Figure S15 revealed the temperature-dependent enzyme-like activity of these n-CeO2 NSs. Moreover, both the enzymelike activity and the ROS sensitization ability of n-CeO2 NSs held concentration-dependent manners (Figure 3ab). In common, compounds based on lanthanide series including CeO2 usually possessed strong coordination ability towards phosphates, which suggested their ability to hydrolyze ATP to release phosphate groups and adenosine.48,50 The amount of inorganic phosphate liberated into the solution during hydrolysis was measured by malachite green-ammonium molybdate assays.48 The results (Figure S16) showed that the nanoceria had catalytic hydrolysis activities in response to ATP. As expected, the catalytic activity of n-CeO2 NSs could be improved by increasing the concentrations of ATP and this enhancement held a concentrationdependent manner (Figure 3c). We assumed that this promotion was derived from the couple of the oxidative reaction with the ATP hydrolysis. To confirm above summing-up, solutions containing ATP with a certain concentration were further co-incubated with n-CeO2 NSs. 1 d later, the ATP contents decreased dramatically with a concentration-dependent manner. In detail, nCeO2 NSs with a concentration of 200 μg/mL were found

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to induce nearly 80% reduction in ATP content (Figure 3d). All of

Figure 3. Time-dependent absorbance changes at 652 nm of TMB solutions catalyzed by different concentrations of n-CeO2 NSs in Tris buffer (10 mM, pH 4.0) (a). Fluorescence spectra of DCFH incubated with different concentrations of n-CeO2 NSs for 1 h in dark (b). Timedependent absorbance changes at 652 nm of TMB solutions catalyzed by 25 μg/mL n-CeO2 NSs in the presence of different concentrations of ATP (c). The contents of ATP after various treatments with n-CeO2 NSs for 24 h (d). Schematic illustration of the enhanced oxidative damage activity and reduced energy supply ability of n-CeO2 NSs (e). results demonstrated that n-CeO2 NSs could distinctly consume ATP and show their promising in the reduction of energy supply during the catalytic process. It is known that ATP binding to the particles surfaces changes the redox potential of Ce(III)/Ce(IV) couple, favouring the TMB oxidation. That is to say in Figure 3e, the energy released from the ATP hydrolysis can further improve the oxidase-like activity of nanoceria in return.48 More importantly, the values of their released energies in catalytic process would determine the improvement efficiencies of enzyme-like activity. All of these results demonstrated that n-CeO2 NSs owned high-performance ATP-deprivation ability and enhanced oxidase-like activity. Encouraged by the highly enhanced oxidative activity and excellent ATP-deprivation ability of n-CeO2 NSs, we then investigated their stability in different physiological solutions such as saline and PBS. Figure S17 demonstrated the colloid stability of bare n-CeO2 NSs. Figure S18 indicated that the n-CeO2 NSs haven’t changed their morphology after incubation in different media. Figure S19 further verified that the ultrasmall CeO2 NPs in nCeO2 NSs sample were well dispersed without


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aggregation in physiological solutions. Benefit from their good stability, we then explored their synergetic anticancer effect in vitro. As well known, ATP as essential energy supply played a vital role in tumor metabolism and survival, and efficient ATP deprivation could extremely promote the starving-like therapy.50,56 Typically, we evaluated the energy-consuming therapeutic effect by using standard methyl thiazolyl tetrazolium (MTT) assay and ATP level measurement(Figure 4a-b ， Figure S20). In detail, the cellular viability decreased to approximately 30% and the intracellular ATP level of Hela cells reduced to 60% upon the treatment with n-CeO2 NSs at a concentration of 200 μg/mL for 72 h. Meanwhile, the cellular ATP contents declined dramatically as a function of the concentration and incubation period of n-CeO2 NSs. These results indicated that n-CeO2 NSs could reduce the energy supply for tumor cells, which validated their negative impact on cell growth and proliferation. Besides, as a pH value-dependent oxidase-mimic, n-CeO2 NSs could induce cytotoxicity by converting oxygen into ROS without any external stimuli. Before the investigation of cytotoxicity induced by n-CeO2 NSs, fluorescence imaging was used to investigate their intracellular localization process. As shown in Figure 4c, most of n-CeO2 NSs were localized in lysosomes, in which n-CeO2 NSs behaved as an oxidase-mimic to generate excess ROS and induced distinct cytotoxicity. To verify the oxidative damage of Hela cells, the levels of intracellular ROS after various treatments were then evaluated via fluorescence imaging and flow cytometry (Figure 4d-f, and Figure S21). A negligible green fluorescence of 1.7% was found in the control group while an increased fluorescence of 13.9% could be detected in the group of n-CeO2 NSs with a treated concentration of 200 μg/mL. These results confirmed the cellular oxidative damage of n-CeO2 NSs and their potential as a promising nanozyme-based therapeutic platform. In addition, the porous structure of n-CeO2 NSs inherited from Ce-MOFs endowed them with the ability as excellent nano-sized drug-delivery vectors. Owing to the porosity of n-CeO2 NSs and the coordination of rare-earth ion with carboxylic group, DOX, a common anticancer drug, could be loaded into n-CeO2 NSs with an encapsulation efficiency of 2%, which exhibited a pH value-dependent release manner. As shown in Figure S22, faster release could be detected upon the treatment in acid environment. Although the loading of DOX on the CeO2based nanozymes indeed lead to the loss of accessible active sites and reduced their oxidase-like activity. However, with the release of DOX, the oxidase-like activity would restore (Figure S23). Surprisingly, the loading of DOX hasn’t influenced the ATP deprivation capacity (Figure S23d). The gradual release of drugs and the recovery of enzyme-like activity as well as insusceptible ATP-deprivation capacity endowed DOX@n-CeO2 NSs with much enhanced anticancer effect compared with DOX and n-CeO2 NSs (Figure S24). In vitro time-dependent drug release assay was then applied to investigate the therapeutic effects of DOX@n-CeO2

NSs. As illustrated in Figure S25, only small amounts of DOX was released within the first 1 h while an obvious enhanced red fluorescence could be detected 4 h later. When the incubation period increased to 10 h, most of DOX could be released from the porous pores of n-CeO2 NSs. These results indicated that n-CeO2 NSs could serve as a potential nanocarriers for anticancer drug. After understanding the excellent synergistic anticancer therapy of DOX@n-CeO2 NSs in vitro, we then explored their anticancer performance in vivo. First, biodistribution of n-CeO2 NSs was evaluated by intravenous injection of n-CeO2 NSs into H22 tumor-bearing mice. 24 h later, mice were sacrificed, and major organs and tumors were collected to determine the content of Ce by using inductively coupled plasma mass spectrometer. As shown in Figure 5a, most of n-CeO2 NSs were accumulated in liver and spleen as a result of the efficient capture by the reticuloendothelial system. The relative

Figure 4. Viabilities (a) and ATP levels (b) of Hela cells after treatments with n-CeO2 NSs. Spatial distribution of DOX-contained n-CeO2 NSs (10 μg/mL) in Hela cells (c). Visible intracellular ROS production by n-CeO2 NSs with different concentrations: 0 μg/mL (d) and 200 μg/mL (e). Average DCF fluorescence intensities of Hela cell treated with n-CeO2 NSs (f). The scale bar was 50 μm.. distributed amounts of n-CeO2 NSs within tumor could be calculated as 2.47% ID/g, which was attributed to the enhanced permeability and retention effect. Stimulated by the relative effective tumor accumulation of n-CeO2 NSs, we elevated the therapeutic efficacy of our system. Four groups of H22 tumor-bearing mice (n=6) were used in our present experiment. As shown in Figure 5b, mice after injection of n-CeO2 NSs and DOX@n-CeO2 NSs showed enhanced tumor regression compared to that of



0.9% NaCl solution or DOX 2 weeks after various treatments. Significantly, DOX@n-CeO2 NSs held the best anticancer effect. Tumor volume variations were consistent with the digital photograph of tumors (Figure 5c-d). In addition, the therapeutic effect was also explored by histopathological examination. As shown in Figure 5e, hematoxylin and eosin (H&E)-stained results indicated that tumors treated with n-CeO2 NSs and DOX@ n-CeO2 NSs revealed an intensive condensation of chromatin and nuclear fragmentations compared with other groups, suggesting the aggravated necrosis and apoptosis of cancer cells.57-60 More importantly, no significant body weight variations were observed in all test groups during the whole therapeutic period (Figure S26). Also, no obvious tissue damages and side effects about the major organs of mice were found after various treatments, which were revealed by digital photograph

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carbonaceous frameworks with enhanced enzyme-like activity and reduced energy supply ability. Compared with traditional surface-coating strategy towards highperformance CeO2-based nanozymes, our approach could not only avoid the possible aggregation, but also homogeneously disperse CeO2 NPs with a high density by using a facile synthesis. Our results indicated that these MOF-derived n-CeO2 NSs featured with oxidase-like activity, ATP deprivation capacity, and porous structure held great oxidative damage effect, efficient reduced energy supply ability, and high drug loading capacity. Both in vitro and in vivo results demonstrated the great promise of these well-prepared nanostructures in synergistic cancer therapy with negligible side effects. In detail, n-CeO2 NSs could localize into lysosome and reinforce the oxidation damage of cancer cells, promote the ATP-consuming processes and disturb the metabolism of cancer cells, as well as deliver anticancer drug and kill cancer cells. Overall, our present study might provide new insight into the development of highly reactive MOFs-derived nanozymes in the future particularly useful for the safe and efficient cancer therapy. EXPERIMENTAL SECTION Chemicals. Cerium ammonium nitrate ((NH4)2Ce(NO3)6), terephthalic acid (TA) and ammonium hydroxide (NH3·H2O, 25%) were achieved from Beijing Chemicals (Beijing, China). Dextran, 3,3’,5,5’tetramethylbenzidine (TMB) and polyacrylic acid (PAA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Adenosine 5′-triphosphate disodium salt hydrate (ATP), cerium nitrate (Ce(NO3)6·6H2O), β-cyclodextrin (β-CD) and tartaric acid were purchased from Aladdin. Bovine serum albumin (BSA), Kits for ROS assay and ATP level determination were all ordered from Shanghai Sangon Biotechnology Development Co., Ltd. Ultrapure water (18.2 MU; Millpore Co., USA) was obtained by using a Milli-Q water system and used throughout the experiments.

Figure 5. Bio-distribution of n-CeO2 NSs in tumor-bearing mice (n=3) determined by ICP-MS (a). Relative tumor volume after various treatments (b). Photographs of tumor-bearing mice before treatment and on day 14 after various treatments (c). Representative photographs of the tumor dissection (d). Hematoxylin and eosin (H&E) staining of tumor sections collected from the mice after 1 day treatment (e). Microscopy images were acquired under magnification of 40. and H&E staining (Figure S27-Figure 28). Taking together, in vivo results demonstrated that n-CeO2 NSs showed great promise in synergetic cancer treatment with negligible side effects to healthy tissue. CONCLUSION In summary, for the first time, we have utilized Ce-MOFs as precursors to successfully fabricate uniformly dispersed ultrasmall CeO2-based nanozymes isolated within porous

Instruments. Scanning electron microscope (SEM) images were obtained on Hitachi S-4800 FE-SEM at working voltage of 10 kV and working current of 10 A. Transmission electron microscope (TEM) measurements were carried out on a TECNAI G2 equipped with energy dispersive spectroscopic (EDS) at 200 kV. X-ray diffractometer (XRD) measurements were performed on a Bruker D8 Focus and D/max 2500pc power X-ray diffractometer using Cu Kα radiation. Fourier transform infrared (FTIR) analyses were measured on a Bruker Vertex 70 FT-IR Spectrometer. X-ray photon spectroscopy (XPS) datas were recorded with an ESCALab220i-XL electron spectrometer from VG Scientific using 300 W Al Kα radiation. Thermogravimetric analysis (TGA) were carried out on a PerkinElmer Pyris Diamond TG/DTA analyzer, using an oxidant atmosphere (Air) with a heating program consisting of a dynamic segment (10 °C min−1) from 293 to 1073 K. N2 adsorption–desorption isotherms were obtained using a Micromeritics ASAP


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2020 automated sorption analyzer. UV–vis spectroscopy was carried out by using a JASCO V-550 UV/vis spectrometer. Fluorescence measurements were carried out on a JASCO FP-6500 spectrofluorometer (Jasco International Co., Japan). Fluorescence images were captured with an Olympus BX-51 optical equipped with a CCD camera. All the photos were taken by a Canon camera. Synthesis of nanoscale Ce-MOFs. Nanoscale Ce-MOFs were prepared based on a modified approach.51 Briefly, 283.2 mg TA was dissolved in 9.6 mL DMF solution and then mixed with an aqueous solution of ((NH4)2Ce(NO3)6) (400 µL, 0.5333 M). The mixture was stirred for 15 min at 100 °C and then transformed into white precipitate. The obtained Ce-MOFs was washed with DMF for 3 times and dried in the oven at 70 °C. Synthesis of n-CeO2 NSs and a-CeO2. Ce-MOFs were ground into powders and then heated at 5 °C/min under nitrogen (N2). After reaching the target temperature (400 °C), the materials were maintained at the temperature for 4 h and cooled down to 20 °C naturally. The resultant nCeO2 NSs were yellow-brown, whereas Ce-MOFs were obtained as white powders. a-CeO2 was prepared with the same procedure except that N2 was replaced by air. The content of Ce in n-CeO2 NSs was 483.8 μg/mg and in aCeO2 was 748.9 μg/mg, which were determined by ICPMS. Synthesis of d-CeO2. d-CeO2 was prepared referring to previous literature.48 Briefly, 2.17g Ce(NO3)3·6H2O was dissolved in 5.0 mL H2O, and mixed with 1.0 M dextran. Then, the solution was added to 30.0 mL 25% NH3·H2O by dropwise. After stirring for 24 h at 25 °C, the suspension was centrifuged, washed and freeze-dried. The content of Ce in d-CeO2 NSs was 250.6 μg/mg, which was determined by ICP-MS. Synthesis of β-CeO2. β-CeO2 was prepared referring to previous literature.47 Briefly, 92.5 mg β-CD was dissolved in 5 mL H2O, and then added 69.4 mg Ce(NO3)3·6H2O. The solution was added to 5 mL 25% NH3·H2O solution by dropwise. After stirring for 24 h at 25 °C, the solution was centrifuged at 4000 rpm to get rid of large agglomerates. Lastly, the obtained solution was dialyzed and freezedried . The content of Ce in β-CeO2 NSs was 223.6 μg/mg, which was determined by ICP-MS. Synthesis of BSA-CeO2. 43 mg BSA was dissolved in 1 mL H2O, and then added 75 mg Ce(NO3)3·6H2O. Immediately, the white precipitate appeared, then the above mixture was added to 5 mL 25% NH3·H2O solution. After stirring for 24 h at 25 °C, the obtained precipitate was washed and freeze-dried. The content of Ce in BSA-CeO2 NSs was 218.2 μg/mg, which was determined by ICP-MS. Synthesis of T-CeO2. 75 mg tartaric acid was dissolved into 1 mL H2O, and then added 434 mg Ce(NO3)3·6H2O. The above solution was added to 6 mL 25% NH3·H2O solution by dropwise. After stirring for 24 h at 25 °C, the solution was centrifuged at 4000 rpm to get rid of large agglomerates. Lastly, the obtained solution was dialyzed

and freeze-dried. The content of Ce in T-CeO2 NSs was 256.8 μg/mg, which was determined by ICP-MS. Synthesis of PAA-CeO2. PAA-CeO2 was synthesized according to the previous methods.46 Briefly, 4.5 g PAA was dissolved into 5 mL H2O, and added 2.17 g Ce(NO3)3·6H2O. Then, the above solution was addedto 30 mL 25% NH3·H2O solution by dropwise. After stirring for 24 h at 25 °C, the mixture was centrifuged at 4 000 rpm to get rid of large agglomerates. Lastly, the centrifuged solution was dialyzed and freeze-dried. The content of Ce in PAA-CeO2 NSs was 234.7 μg/mg, which was determined by ICP-MS. Oxidase-like activity of n-CeO2 NSs. The oxidase-like activity of n-CeO2 NSs was studied by the catalytic oxidation of the TMB in Tris buffer. All the reactions were incubated in Tris buffer and measured spectrophotometrically at 652 nm. Typically, certain amounts of the n-CeO2 NSs and 10 µL 40 mM TMB were added into 380 µL Tris buffer solution (10 mM , pH 4.0) under 37 oC, unless otherwise stated. The C, pH, Tdependent and ATP-promoted oxidase-like activity were investigated by the changing of nanoparticles’ concentration, pH, T and ATP contents. The ATP deprivation of n-CeO2 NSs. Different concentrations of n-CeO2 NSs were mixed with 1 mM ATP at 37 oC in dark for 24 h. Then, the mixture was centrifuged at 12 000 rpm to remove n-CeO2 NSs and obtain the supernatant. Finally, the concentration of ATP was determined by ATP Assay Kit and recorded by SpectraMax M2 Molecular Devices. Detection of reactive oxygen species (ROS). DCFH, transformed from DCFH-DA, was used as a ROS probe. Firstly, 0.5 mL of DCFH-DA in DMSO was deesterified to DCFH with 2 mL 0.01 M NaOH. After incubation in the dark for 30 min at 25 oC, the reaction was stopped by 10 mL Tris buffer (25 mM, pH 7.2). The obtained DCFH was kept from light on ice before use. In a typical test, different concentrations of n-CeO2 NSs were mixed with DCFH (10 μM). After reacting at 37 oC for 1 h in dark, they were centrifuged at 12 000 rpm to remove the nanoparticles. Finally, the produced ROS was determined by the the fluorescence of the supernatants. Stability of n-CeO2 NSs in different physiological solutions. The bare n-CeO2 NSs were suspended in the saline and PBS buffers for 3 days. The solutions were taken photos every day. After three days, the nanoparticles were washed with H2O, then the morphology of n-CeO2 NSs was observed by SEM as well as the dispersity of ultrasmall CeO2 was determined by TEM. Loading of DOX into n-CeO2 NSs. For DOX loading, 10 mg n-CeO2 NSs were dispersed into 10 mL ultrapure water, then 2 mg DOX was added into above reaction system. After stirring for 24 h at 25 oC, the residual free DOX was removed through centrifuging and washing. Finally, the loading content of DOX was calculated via the standard concentration curve of DOX established by UVVis spectroscopy.



pH-response drug release experiments. The assynthesized DOX@n-CeO2 NSs samples were suspended in the PBS buffers of different pH and the final concentration was 1 mg/mL. At given time intervals, they were centrifuged. The obtained solid was redispersed in equal fresh PBS. The centrifugal supernatant was measured through UV-Vis spectroscopy to evaluate the released DOX. Oxidase-like activity and ATP deprivation capacity of DOX@n-CeO2 NSs in different release time. During release, the DOX@n-CeO2 NSs was obtained everday day and washed with water. Then, their oxidase-like activity, ROS generation and ATP deprivation capacity were estimated. The measurement method was consistent with the above n-CeO2 NSs. Cell culture. The HeLa cells were cultured in Dulbecco's Modified Dulbecco's medium (DMDM) (Gibco) supplemented with 10% heat-inactivated FBS under 5% CO2 at 37 ℃.The medium was replaced every three days, and the cells were digested with trypsin and resuspended in fresh complete medium before plating. MTT assay. 100 μL of HeLa cell solution was pipetted into the wells of a 96-well plate at a density of 5 000 cells/well for 24 h. Then, the samples with an indicated concentrations (6.25, 12.5, 25, 50, 100, 200 μg/mL) were added to the cell culture medium. Cells were incubated for another 24 h, 48 h, 72 h. After removal of the cell medium, 10 μL of MTT solution was added to each well of the microtiter plate and incubated for additional 4 h. Then, the media was removed and 100 μL DMSO was added into each well. After gently swirled for 2 min at room temperature at dark, the absorbance values of 490 nm were determined with Bio-Rad model-680 microplate reader. They were corrected for background absorbance at 630 nm. The cell viability was estimated referring to the following equation: Cell Viability (%) = (ODtreated / ODcontrol) ×100%. ODcontrol was measured in the absence of samples, whereas ODtreated obtained in the presence of samples. ATP assay. 1 mL HeLa cell solution was pipetted into the wells of a 6-well plate at a density of 1×106 cells/well for 12 h. Then, the as-prepared n-CeO2 NSs with different concentrations (0, 6.25, 12.5, 25, 50, 100, 200 μg/mL) were added to the cell culture medium and incubated for 24 h, 48 h, 72 h respectively. Finally, the ATP levels were determined by ATP Assay Kit and recorded by SpectraMax M2 Molecular Devices. Determination of ROS Generation in vitro. 1 mL HeLa cell solution was pipetted into the wells of a 6-well plate at a density of 1×106 cells/well for 12 h. Then cells were incubated with n-CeO2 NSs for 24 h. The treated cells were washed with PBS twice and incubated with 10 μM DCFH-DA at darkness for 1 h. Finally, the fluorescence intensity of the cells was measured by flow cytometry and fluorescence microscopy (excitation at 488 nm, emission at 530 nm). Cellular uptake and subcellular distribution of nCeO2 NSs. HeLa cells were seeded on 24-well culture

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plates and grown overnight. Then, the cell medium was removed, washed, and 0.5 mL fresh cell medium containing DOX@n-CeO2 NSs (10 μg/mL) was added to the wells and incubated different times. After that, the medium was washed with PBS and cells were stained by 50 nM Lysotracker Green DND-26. After staining for 30 min at 37 oC, the cells were washed with PBS and imaged using fluorescence microscopy. The pictures were taken with an Olympus digital camera. Flow cytometry analyses. 1 mL HeLa cell solution was pipetted into the wells of a 6-well plate at a density of 1×106 cells/well for 12 h. Then, the as-prepared n-CeO2 NSs with different concentrations were added to the cell culture medium. After incubation for 24 h, the cells were digested with trypsin, centrifuged, washed and then redispersed in the buffer. The mean fluorescence was measured by counting 10 000 events via BD LSRFortessa cytometer. Animal Experiments. Animal experiments were in accordance with the direction of the Regional Ethics Committee for Animal Experiments. Healthy Kunming mice with an average weight of 25 g were acquired from the Laboratory Animal Center of Jilin University (Changchun, China). Tumor Models. Hepatoma 22 (H22) tumor-bearing mice (tumor size ≈ 100 mm3) were used to evaluate the in vivo tumoricidal potentials. The Kunming mice (25 g) were subcutaneously inoculated in the oxter region with 0.1 mL H22 cells (2×106 cells/mL) suspended in saline and further raised until the tumors growed to ~100 mm3 before experiment (7d after tumor inoculation, the average weight of mice reached 35 g). Biodistribution. Biodistribution was assessed in tumorbearing mice. n-CeO2 NSs dispersed in physiological saline (400 μL, 200 μg/mL) were intravenously injected into each mouse (n=3). The Ce contents in different tissues injection were measured through the ICP-MS method. After 24 h post-injection of n-CeO2 NSs, the tissues (heart, liver, spleen, lung, kidneys, and tumor) were surgically removed. These tissues were dissolved in aqua regia (2 mL for liver and 1 mL for all others) under oil bath (80 oC) for 2 day. Then, the acquired liquid was diluted and subjected to ICP-MS analysis. The percentage injected dose per gram (% ID g-1) tissue (D) of Ce was calculated according to the following formula: D=

m / morgan ×100% mID

where m (μg) represents the content of Ce in the measured tissue, morgan (g) is the mass of tissue, and mID (μg) is the totally injected dose of Ce. Antitumor activity in vivo. Tumor bearing mice (tumor size ≈ 100 mm3 ) were randomly assigned to four groups (n=6 mice/group) for different treatment. Mice were intravenously injected with 200 μL saline (group 1, 0.9% NaCl ), DOX (group 2, 2 μg/mL), n-CeO2 NSs (group 3, 200 μg/mL) or DOX@n-CeO2 NSs in saline (group 3, 200 μg/mL, the amount of contained DOX was about 0.8 μg,


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the released DOX was about 0.5 μg), respectively. The tumor dimensions (maximum length L and maximum width W) were measured every other day with a caliper (n=6 for each group) and the tumor volume (V) was calculated as V=L × W 2 /2 (mm 3). The relative tumor volume was normalized to their initial size (V0) before administration. The mice were sacrificed after 2 weeks and the tumors were harvested for taking photo and histology. Histology. For histology, major organs (heart, liver, spleen, lung and kidney) and tumors were obtained from above four groups after treatment. After these tissues fixing in 10% neutral buffered formalin for 24 h, they were embedded into paraffin, sectioned into ~4 μm thickness, as well as stained with hematoxylin and eosin (H&E). The histology was conducted in college of Basic Medical Sciences of Jilin University. The samples were elevated through an Olympus BX-51 microscope in bright field. Statistical Analysis. All the tests were performed at least three times, and the datas were expressed as means ± standard deviation (SD). The statistical analysis was conducted with Origin 8.0 software.

ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Characterization of materials (SEM, TEM, TGA, FTIR, XRD, BET, XPS, oxidase-like performance, ATP-deprivation assays, drug-release result, in vitro and in vivo results)

AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected].

Notes

The authors declare no competing financial interests.

ACKNOWLEDGMENT Financial support was provided by Natural Science Foundation of China (Grants 21533008, 21673223, 21431007, 21601175, 21871249 and 21820102009), the Key Program of Frontier of Sciences (CAS QYZDJ-SSW-SLH052) and the Jilin Province Science and Technology Development Plan Project (20160520129JH, 20170101184JC).

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Ultrasmall Nanozymes Isolated within Porous Carbonaceous

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