Enzyme Hybrid Nanocatalyst as Benign

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2D Metal-Organic Framework/Enzyme Hybrid Nanocatalyst as Benign and Self-Activated Cascade Reagent for in Vivo Wound Healing Xinping Liu, Zhengqing Yan, Yan Zhang, Zhengwei Liu, Yuhuan Sun, Jinsong Ren, and Xiaogang Qu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b09501 • Publication Date (Web): 19 Apr 2019 Downloaded from http://pubs.acs.org on April 19, 2019

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2D Metal-Organic Framework/Enzyme Hybrid Nanocatalyst as Benign and Self-Activated Cascade Reagent for in Vivo Wound Healing Xinping Liu,1,2 Zhengqing Yan,1,3 Yan Zhang,1,3 Zhengwei Liu,1,3 Yuhuan Sun,1,2 Jinsong Ren,1 Xiaogang Qu*,1 1

Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization,

Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China *E-mail: [email protected]. 2

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

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University of Chinese Academy of Sciences, Beijing 100039, P. R. China

ABSTRACT Metal-organic frameworks (MOFs)-based peroxidase mimics have been seldom applied in biomedical field, especially in vivo. One of the main reasons is their optimum reactions occur in strong acidic environment with pH of 3-4, severely limiting their applications in living system where neutral pH is usually required. Other types of peroxidase mimics also suffer such a fatal defect. Additionally, the direct introduction of relative high concentrated and toxic reaction reagent H2O2 would induce undesired damage to normal tissues. Herein, a MOFbased hybrid nanocatalyst as a benign and self-activated cascade reagent has been constructed.

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Owing to better catalytic performance compared with 3D bulk MOF, ultrathin 2D MOF (2D CuTCPP(Fe)) nanosheet is chosen as a model of peroxidase mimic to physically adsorb glucose oxidase (GOx) for fabricating such a hybrid nanocatalyst. Nontoxic glucose can be continuously converted into abundant gluconic acid and H2O2 by GOx, avoiding the direct use of relative high concentrated and toxic H2O2 and minimizing the harmful side effect. The generated gluconic acid can decrease pH value from 7 to 3-4, dramatically activating the peroxidase-like activity of 2D Cu-TCPP(Fe) nanosheet. Meanwhile, the produced H2O2 is used for subsequent catalysis of activated 2D Cu-TCPP(Fe) nanosheet, leading to efficient generation of extremely toxic hydroxyl radial and antibacterial capacity. In vitro and in vivo results illustrate the designed benign and self-activated cascade reagent possesses robust antibacterial effect with negligible biotoxicity.

KEYWORDS MOF-based enzyme mimic, nanozyme, wound healing, self-activated, cascade reaction

As emerging porous materials, metal-organic frameworks (MOFs) have received considerable research interest.1 Their highly ordered pore structure, large surface area, and tunable structure and function make them ideal candidates for diverse applications.2-13 More recently, MOFs have been explored to exhibit robust peroxidase-like activity by introducing copper or iron as metal node or employing iron-porphyrin structure, the heme-like active center, as organic ligand.14-21 For example, Zhou et al. prepared a series of 3D bulk MOFs (PCN-600 (M), M =Mn, Fe, Co, Ni, and Cu), among which PCN-600 (Fe) could serve as a peroxidase mimic to catalyze the cooxidation reaction.18 Another promising biomimetic catalyst constructed by incorporating 2D MOF nanosheet with Au nanoparticle has been engineered by Zhang and coworkers, where Au


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nanoparticle possesses glucose oxidase-mimicking activity and 2D MOF nanosheet presents peroxidase-like activity.17 Significantly, the large surface area of 2D MOF nanosheets favors the accessibility of substrate molecules to the active sites on their surface with smaller diffusion barriers while the majority of active sites in 3D bulk MOF are hidden within the framework, thus 2D MOF nanosheets possess better potential in the biocatalysis application compared to conventional 3D bulk MOF.17, 22-24 Although MOFs with peroxidase-like activity have been extensively studied, the research is mostly concentrated on the catalysis and biosensing in vitro,14-21 and the activity has seldom been applied in biomedical field, especially in vivo. One of the main reasons is that the best enzyme-like activity requires a strong acidic environment with a pH of 3-4, this restricts their practical uses in biological system where a neutral pH is usually required.14-21 Similar phenomenon also occurs in other types of nanomaterials with peroxidaselike activity such as Fe3O4 nanoparticle, V2O5 nanowire, and N-doped porous carbon nanosphere, etc.25-37 Therefore it becomes a ubiquitous and fatal defect to prevent these nanozymes from being applied in living system. Furthermore, the introduction of toxic and relative high concentrated H2O2 would induce undesired damage to normal tissues.25-39 Taking bacterial therapy as an example, severe limitations of peroxidase mimics applied in the biological system remain unsolved for a long time. Infection caused by bacteria has become a common and frequently encountered disease that seriously threatens human’s health.41, 42 Though bacteria produce protons and induce local acidification during the process of growth and metabolism, the pH of bacterial system is still close to neutral.43 For combating bacterial infection, a wide variety of nanomaterials with the peroxidase-mimic activity converted H2O2 into extremely toxic •OH for antimicrobial application.26, 28, 30-32, 38 Unfortunately, the further development of peroxidaselike systems are still restricted by (1) since the best enzyme-like activity of these nanomaterials


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requires a strong acidic environment with a pH of 3-4, these peroxidase mimics usually show low catalytic activity at a neutral pH; (2) almost all of peroxidase mimic systems use toxic and relative high concentrated H2O2 to generate •OH. These severe drawbacks significantly limited the antibacterial efficiency of peroxidase mimics and induced undesired damage to normal tissues.26, 28, 30-32, 38 Hence, we conceived that if naturally nontoxic and biocompatible substance could be transformed into toxic •OH with the help of catalyst (natural enzyme or inorganic catalyst (2D Cu-TCPP(Fe) nanosheet as model)), and meanwhile the designed system could self-activate the peroxidase-like activity of 2D Cu-TCPP(Fe) nanosheet by decreasing pH of whole systems to 34, the significant antibacterial effect will be achieved without or with minimized potentially harmful side effect. This strategy, if applicable, hopefully results in the concurrent satisfactory therapeutic effect and negligible damage to normal tissues. Toward these goals, we developed a MOF-based hybrid nanocatalyst (2D Cu-TCPP(Fe)/GOx) as a benign and self-activated cascade reagent for efficient bacterial therapy in vivo, which was constructed by the physisorption of natural GOx (enzyme catalyst) on 2D Cu-TCPP(Fe) nanosheet (inorganic catalyst) (Scheme 1a). Upon implantation to the wound of the mice (Scheme 1b), GOx, serving as the starting enzyme catalyst, was competent to continuously convert nontoxic glucose into abundant gluconic acid and H2O2,44-47 avoiding the direct use of relative high concentrated and toxic reaction reagent H2O2 and minimizing the harmful side effect. Meanwhile, the generated gluconic acid during the reaction would decrease pH of the whole system to 3-4,47 and dramatically activate the peroxidase-like activity of 2D Cu-TCPP(Fe) nanosheet.15, 17 Then the elevated H2O2 would be catalyzed by 2D Cu-TCPP(Fe) nanosheet with


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significantly activated peroxidase-like activity to liberate extremely toxic •OH, which could lead to bacterial death (Scheme 1c).26, 28, 31, 32 RESULTS AND DISCUSSION Prior to the preparation of 2D Cu-TCPP(Fe) nanosheet (2D MOF)/GOx, we first synthesized 2D MOF nanosheet according to the previous report.17, 48 Transmission electron microscopy (TEM) and atomic force microscopy (AFM) images (Figure 1a, 1b) presented that the sheet-like structure of the obtained 2D MOF. AFM image further showed the thickness of 2D MOF nanosheet was 3-5 nm, proving the ultrathin nature of 2D MOF nanosheet (Figure 1b and Table S1). The hydrodynamic size of 2D MOF nanosheet was around 645 nm as measured by dynamic light scattering (DLS) (Figure S1, Supporting Information), which was consistent with TEM and SEM statistical results (Table S1).40 To execute precise elemental analysis of nanosheet, corresponding TEM element mapping of 2D MOF nanosheet was performed (Figure 1c), which displayed the homogeneous distributions of Cu, Fe, C, N, O in nanosheet. The porosity of 2D MOF nanosheet was examined by N2 adsorption experiments (Figure S2). In addition, the X-ray diffraction (XRD) pattern of 2D Cu-TCPP(Fe) nanosheet shows three characteristic peaks, indexed as (110), (001), and (002), respectively, which are identical with that of previous study (Figure S3).17, 22 X-ray photoelectron spectroscopy (XPS) results also confirmed the successful fabrication of 2D MOF nanosheet (Figure S4).17, 22 Then the 2D MOF/GOx hybrid nanocatalyst was synthesized by the physisorption of GOx on 2D MOF nanosheet. TEM image (Figure 1d) showed the sheet-like structure remained in the 2D MOF/GOx. Meanwhile, the adsorption of GOx had negligence influence on the crystal structure of 2D MOF nanosheet (Figure S5). The zeta potential decreased from -15.6 mV to -21.8 mV indicated the adsorption of GOx on the 2D MOF nanosheet (Figure 1g). Bicinchoninic acid (BCA) assays demonstrated the amount of GOx


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assembled on 2D MOF was about 2.5 ± 0.03 wt%. More importantly, after the adsorption of GOx, the suspension (Figure 1f) presented the enhanced stability compared to that of 2D MOF alone (Figure 1e). Altogether, all the aforementioned results confirmed the successful adsorption of GOx on 2D MOF. Since the prepared 2D MOF/GOx hybrid nanocatalyst included two catalytic species, in which GOx catalyzed the decomposition of glucose, while 2D MOF nanosheet owned intrinsic peroxidase-like activity, we expected that such a catalytic cascade reaction could be achieved by 2D MOF/GOx. We first evaluated the peroxidase-like activity of 2D MOF nanosheet by using 3, 3, 5, 5-tetramethylbenzidine (TMB) as peroxidase substrate. As shown in Figure S6, 2D MOF nanosheet could catalyze the oxidation of TMB to produce the deep blue product oxTMB in the presence of H2O2, which verified that the 2D MOF nanosheet exhibited excellent peroxidase-like activity. In contrast, control experiments indicated that neither 2D MOF nanosheet+TMB nor H2O2+TMB generated the deep blue product under our experimental conditions. Furthermore, the catalytic activity was dependent on temperature (Figure S7a), pH values (Figure S7b), the concentrations of 2D MOF nanosheet (Figure S7c) and H2O2 (Figure S7d), similarly to previously reported nanomaterials with peroxidase-like activity.14, 16-20 However, a fatal defect of these nanozymes was that their optimum reactions occurred in strong acidic environment with a pH of 3-4, which severely limited their applications in bacterial system where a neutral pH was required (Figure S7b). Fortunately, glucose, naturally nontoxic and biocompatible substance, could be catalytically oxidized by GOx to generate substantial gluconic acid and H2O2, and the generation of gluconic acid dramatically decreased the ambient pH and greatly activated the peroxidase-like activity of 2D MOF nanosheet. In our experiments, we used a laboratory pH meter to directly monitor pH changes in the solution. As shown in Figure S8a, a significant


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decrease of pH from 7.4 to 3.0 was observed when glucose and GOx were coexisted. Additionally, it was not surprised that the generated acid concentration increased in response to the elevated glucose level (Figure S8b).47 Meanwhile, to monitor pH changes of the solution easily, methyl red (yellow at pH over 6.2 and red at pH under 4.4) was used as a pH indicator in the following experiments.49 As shown in Figure S8c, the indicator presented a yellow color in the control experiments, while an obvious color change from yellow to red was observed after incubation of GOx with glucose, suggesting a pH drop from 7.4 to below 4.4. These results confirmed that GOx-catalyzed the decomposition of glucose could significantly decrease the pH of the whole system. As mentioned above, having demonstrated the peroxidase-like activity of 2D MOF nanosheet and the significant pH decrease caused by GOx-catalyzed decomposition of glucose, we further explored the cascade reaction catalyzed by 2D MOF/GOx. It is noteworthy that the optimum reaction condition (pH=3-4) of 2D MOF-based peroxidase activity has hardly changed after the adsorption of GOx on 2D MOF (Figure S9). As shown in Figure 2a, the solution 6 showed intense characteristic absorbance at 652 nm while negligible absorbance in the UV-visible spectrum was observed in the control experiments (1-5), demonstrating that the cascade reaction could occur only when glucose and 2D MOF/GOx coexisted. Meanwhile, it was also demonstrated the obvious decrease of pH in the solution 6 (Figure 2b). Besides, the hybrid catalyst presented superior stability or resistance ability compared with free GOx (Figure S10). In order to analyze the catalytic mechanism of 2D MOF/GOx, we carried out fluorescence experiments. As shown in Figure 2c, the as-prepared 2D MOF/GOx could catalyze nonfluorescent terephthalic acid (TA) into a highly fluorescent 2-hydroxy terephthalic acid (TAOH) in the presence of glucose,28, 31 indicating the formation of •OH. For further confirming the peroxidase-mimic activity of 2D MOF/GOx enhanced by the production of gluconic acid, the


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effect of oxidation of glucose in buffers with different buffer capacity on the catalytic activities of 2D MOF/GOx was studied. Dramatic improvement of catalytic activity of hybrid nanocatalyst in PBS buffer (pH=7.4, 0.5 mM) could be observed over time (0 min-240 min) while it remained more or less unchanged in PBS buffer (pH=7.4, 25 mM) (0 m-240 m) (Figure 2d). This phenomenon was ascribed to the fact that the generated gluconic acid played significant role in enhancing the catalytic activity of 2D MOF/GOx. The introduction of methyl red also confirmed the production of gluconic acid (Figure 2e, f). Considering that the designed hybrid nanocatalyst could convert the naturally nontoxic and biocompatible glucose into significantly toxic •OH and the excellent antibacterial activity of •OH, we next evaluated the antimicrobial activity of 2D MOF/GOx in the presence of glucose against Gram-negative E. coli and Gram-positive S. aureus. As compared to (1) PBS, (2) glucose, (3) glucose+2D MOF and (4) 2D MOF/GOx, despite the generation of toxic H2O2, the bacteria viabilities were still above 55% and 50% for E. coli and S. aureus, respectively, for the (5) glucose+GOx (Figure 3a, 3b), indicating that H2O2 generated by oxidation of glucose could not guarantee effectively elimination of these two bacterial species. Expectedly, the introduction of (6) glucose+2D MOF/GOx led to apparent reduction of bacteria survival percentages, and the bacteria inactivation rates reached 88% and 90% for E. coli and S. aureus, respectively, implying 2D MOF significantly enhanced the antibacterial activity of generated H2O2 by oxidation of glucose. All these results were in agreement with that of plate counting method (Figure S11, S12). To further decipher the excellent antibacterial capacity of the system, SEM was used to study the morphological transformation of the two bacterial strains. As suggested in Figure 3c, untreated E. coli cells presented typical rod with smooth surface and possessed intact cell walls (Figure 3c (1)). After treatment with control materials (Figure 3c (2-4)), few disruptions were


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observed on the surface of cells, which indicated that glucose or hybrid nanocatalyst 2D MOF/GOx alone showed negligible toxicity against E. coli. However, when exposed to the glucose+GOx, the bacterial surface became partially wrinkled owing to the generation of H2O2 (Figure 3c (5)). Obviously, after treatment with glucose+2D MOF/GOx, the cell walls got rough and damaged on account of the excellent antibacterial capacity of generated toxic •OH (Figure 3c (6)). For S. aureus, morphological changes were consistent with that of E. coli (Figure 3d). Moreover, a significant decrease of pH from 7.4 to around 4.4 was observed when bacteria were incubated with glucose+2D MOF/GOx (Insets in the Figure 3c, 3d), which undoubtedly activated the peroxidase-like activity of 2D MOF nanosheet to a great extent and further dramatically enhanced antibacterial performance of hybrid nanocatalyst. In short, the enhanced •OH catalyzed by the self-activated 2D MOF/GOx resulted in the improved antibacterial activity, which was caused by the decrease of pH from 7.4 to around 4.4. In addition, a comparison of the antibacterial performance of H2O2 alone, a standard treatment, and that of our hybrid nanocatalyst in the presence of glucose was performed. As presented in Figure 3e, 3f, even at the same concentration of glucose and H2O2, the bacterial viabilities of glucose+2D MOF/GOx was reduced significantly compared with that of H2O2, which was also ascribe to that the decrease of pH enhanced the antibacterial effect of hybrid nanocatalyst. More importantly, continuously generated H2O2 by oxidation of glucose perfectly avoided the direct introduction high concentrated and toxic reaction reagent H2O2 and minimized the harmful side effect, which confirmed our designed hybrid nanocatalyst was a benign antibacterial system. To assess the antibacterial property of our benign and self-activated cascade reagent in vivo, we first constructed the wound model on the back of Kunming mice and fabricated 2D MOF/GOxBand-Aid (Figure S13). Then the mice were divided into: Blank-Band-Aid, glucose+Blank-


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Band-Aid, glucose+2D MOF-Band-Aid, 2D MOF/GOx-Band-Aid, glucose+GOx-Band-Aid and glucose+2D MOF/GOx-Band-Aid with three mice in each group. Compared with controls, the wounds of mice treated with glucose+2D MOF/GOx-Band-Aid formed scabs gradually and scars became abnormally smaller at the third day. In addition, the mice in experimental group did not appear any erythema in the process of wound healing, while the wounds of the control groups presented various degrees of erythema (Figure 4a). To evaluate the sterilization efficiency, the wounds of mice were excised at the third day to determine the number of bacteria (Figure 4b, 4c). The results in Figure 4c showed that the bacteria of glucose+2D MOF/GOx-Band-Aid could be decreased to 9.1%, presenting the most effective bactericidal action. In parallel, the group of the glucose+GOx-Band-Aid also exhibited a degree of antibacterial activity, whereas the bacteria were just decreased to 56.5%. To further evaluate the wound healing ability, we performed histological analysis using hematoxylin and eosin (H&E) staining. At the third day, significant amounts of inflammatory cells and incomplete epidermal layers were observed for the control groups. On the other hand, a mostly intact epidermis structure and just a few inflammatory cells appeared on the wounds of glucose+2D MOF/GOx-Band-Aid group (Figure 4d). Consequently, our hybrid nanocatalyst presented excellent antibacterial activity and further promoted wound healing in vivo. After confirming the excellent antibacterial property of 2D MOF/GOx in the presence of glucose, we further evaluated in vitro and in vivo biosafety of the designed hybrid nanocatalyst. The toxicity of MOF nanocomposites is mainly derived from the release of toxic metal ion.50, 51 Therefore, the amount of Cu2+ and TCPP(Fe) released from 2D MOF/GOx after 7 days of incubation in water, PBS buffer (pH 7.4, 25 mM), PBS buffer (pH 4.0, 25 mM) and LB broth was investigated by inductively coupled plasma-mass spectrometry (ICP-MS). As presented in


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Table S2, even under acidic condition, total amount of Cu2+ and TCPP(Fe) released from 2D MOF/GOx only reached 4.7 µM and 3.3 µM, respectively. It was found that Cu2++TCPP(Fe) under these concentrations presented significantly low contribution on the antimicrobial activity (Figure S14). Owing to the generation of H2O2 during the catalysis of hybrid nanocatalyst, the stability of 2D MOF/GOx in the presence of different concentration of H2O2 was examined by TEM and DLS. Corresponding results in the Figure S15-S16 illustrated that the structure of 2D MOF/GOx was destructed gradually with the increase of H2O2 concentration and no intact nanosheet could be found in the condition of 10 mM H2O2. Although the degradation of 2D MOF/GOx in the presence of relative high concentration H2O2, we found that 2D MOF/GOx was stable under the used condition (Figure S17), which was ascribed to the continuously generated H2O2 by oxidation of glucose. Meanwhile, the morphology of 2D MOF/GOx remained unchanged in different media after incubation for 7 days (Figure S18). DLS data showed that the 2D MOF/GOx presented excellent dispersion with an average size of around 600 nm, which remained essentially the same during incubation (Figure S19). XRD pattern was still in good agreement with that of 2D MOF nanosheet (Figure S20). All these results indicated their longterm biological stability. Then methyl thiazolyl tetrazolium (MTT) assay was employed to evaluate the cytotoxicity of the nanosheet. The result revealed that negligible toxicity was displayed in NIH 3T3 and L929 cells even with concentration of Cu2+ up to 20 µM (Figure S21), illustrating the negligible cytotoxicity of released Cu2+. In addition, the MTT assay associated with NIH 3T3 and L929 cells in the presence of 2D MOF at various concentrations (0-500 μg mL−1) further confirmed its good biocompatibility (Figure S22). Next, hematological and pathological examinations on mice were performed to assess whether 2D MOF/GOx caused serious in vivo toxicity. The 2D MOF/GOx was intravenously injected to Kunming mice. Blood


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biochemistry was tested for evaluating the kidney and liver function at seventh day after administration. Indeed, compared to the untreated mice, no appreciable differences in these indicators were observed (Figure 5a). Additionally, the administration of 2D MOF/GOx had negligible effect on the normal growth of mice (Figure 5b). Further H&E staining assay of major organs confirmed the excellent histocompatibility of 2D MOF/GOx (Figure 5c). The biodistribution of hybrid nanocatalyst was also studied and the result indicated that most 2D MOF/GOx nanosheets accumulated in the liver and spleen and only a small amount of 2D MOF/GOx accumulated in the heart, kidney and lung. Meanwhile, it was found that the hybrid catalyst could be cleared out from the body as time prolonging, indicating the good biocompatibility of 2D MOF/GOx (Figure S23). Besides, there was no discernible difference or damage of major tissues after treating with glucose+2D MOF/GOx-Band-Aid dressing compared with normal mice (Figure S24). These results revealed that our hybrid nanocatalyst 2D MOF/GOx displayed negligible in vitro and in vivo toxicity. In brief, this hybrid nanocatalyst possessed outstanding advantages of low-cost (without noble metal), good biocompatibility, significantly high catalytic activity and inactivation percentage (Gram-negative bacteria E. coli and Gram-positive bacteria S. aureus), which has superior or comparable performance to other systems applied in this field (Table S3). CONCLUSION In summary, we develop a MOF-based hybrid nanocatalyst as a benign and self-activated cascade reagent for in vivo wound healing. Owing to better catalytic performance compared to 3D bulk MOF, ultrathin 2D MOF nanosheet (inorganic catalyst) is chosen as a model of peroxidase mimic to physically adsorb natural GOx (enzyme catalyst). GOx in hybrid nanocatalyst can effectively convert the glucose into abundant gluconic acid to self-activate the


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peroxidase-like activity of 2D MOF nanosheet, and meanwhile the generated considerable amount of H2O2 can be used for the subsequent catalysis of 2D MOF nanosheet, resulting in enhanced production efficiency of extremely toxic •OH and antibacterial capacity. This system employs naturally nontoxic and biocompatible glucose to continuously generate H2O2, completely avoiding the direct use of relative high concentrated and toxic reaction reagent H2O2 and minimizing the harmful side effect. Furthermore, the designed system can self-activate the peroxidase-like activity of 2D MOF nanosheet owing to the formation of gluconic acid, which dramatically improves the generation rate of highly toxic •OH and enhanced the antibacterial effect. More importantly, in vitro and in vivo results suggest that this designed benign and selfactivated cascade reagent possesses robust antibacterial effect with negligible biotoxicity, and would promote MOF-based nanozymes applied in biochemical and biomedical fields.

MATERIALS AND METHODS Chemicals. Methyl 4-formylbenzoate, propionic acid and pyrrole were obtained from SigmaAldrich. Tetramethylbenzidine (TMB) was achieved from BBI (Ontario, Canada). Glucose and polyvinylpyrrolidone (PVP, average mol wt 40,000) were directly purchased from Alfa Aesar. Trifluoroacetic acid (CF3COOH), copper (II) nitrate trihydrate (Cu(NO3)2•3H2O) and iron(II) chloride tetrahydrate (FeCl2•4H2O) were purchased from Beijing Chemicals (Beijing, China). Glucose oxidase (GOx) was achieved from Sangon Biotechnology Inc. (Shanghai, China). Ultrapure water (18.2 MΩ; Millpore Co., USA) was used throughout the experiment. Instruments. TEM images and TEM element mapping were obtained using a FEI TECNAI G2 20 high resolution transmission electron microscope operating at 200 kV. AFM measurement


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was analyzed using Nanoscope V multimode atomic force microscope. XRD measurement was carried out on a D8 Focus diffractometer (Bruker) using Cu Kα radiation. XPS was recorded using a Perkin Elmer PHI 5600. UV-vis spectra were recorded with a JASCO V-550 UV-vis spectrophotometer. Fluorescence spectra were measured on a JASCO FP-6500 spectrofluorometer. The pH measurement was carried out using a PHS-3C portable pH meter. Synthesis of 2D MOF nanosheet. The synthesis of the ligand has been previously described.48 Typically, Cu(NO3)2•3H2O (2.4 mg), trifluoroacetic acid (40 µL, 1.0 M) and PVP (10 mg) were dissolved directly in DMF (9 mL) and ethanol (3 mL) mixed solvents in a 50 mL roundbottomed flask. Then, the TCPP(Fe) (4.4 mg) dissolved in DMF (3 mL) and ethanol (1 mL) mixed solvents was added dropwisely to the above system under stirring. Then the reaction mixture was sonicated for 20 min and heated at 80°C for 4 h. The resulting product were washed with ethanol for two times and then collected by centrifuge at 12,000 r.p.m for 10 min. Finally, the obtained 2D nanosheet was dispersed in water. Preparation of 2D MOF/GOx. The prepared 2D MOF nanosheet was dissolved into 5 mL water under mild magnetic stirring, followed by the addition of GOx (5 mg). After another 12 h, the obtained 2D MOF/GOx could be acquired by centrifugation and and the supernatant was collected. The amount of GOx assembled on 2D MOF was quantified using BCA protein assay kit (Beyotime Biotechnology, China). Peroxidase-mimic activity of 2D MOF/GOx enhanced by the production of gluconic acid. The experiment was performed as follows: (1) 10 mM glucose and 20 μg mL-1 2D MOF/GOx in PBS buffer (pH=7.4, 0.5 mM) or PBS buffer (pH=7.4, 25 mM) were incubated at 37°C. (2) 15 µL of


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1.2 mM TMB, 25 µL of the 5 μg mL-1 2D MOF/GOx and 20 µL of 4 mM H2O2 were added into the above 450 µL supernatant every 30 minutes. Bacterial Culture. Single colony of E. coli and S. aureus on the solid Luria-Bertani (LB) agar plate was transferred to liquid LB broth and shaked for 8 h at 37°C. The bacteria were then diluted with broth to 3×106 CFU mL-1. Antibacterial Experiments. For growth-inhibition assay in liquid medium, six groups of asprepared bacterial suspensions treated with: (1) PBS, (2) glucose, (3) glucose+2D MOF nanosheet, (4) 2D MOF/GOx, (5) glucose+GOx and (6) glucose+2D MOF/GOx were incubated separately with at 37 °C under 180 rpm. The concentrations of glucose and 2D MOF/GOx are 15 mM and 100 µg mL-1. After 5 h incubation, the absorbance at 600 nm was recorded. For plate counting method, a certain concentration of bacterial suspensions were treated with: (1) PBS, (2) glucose, (3) glucose+2D MOF nanosheet, (4) 2D MOF/GOx, (5) glucose+GOx and (6) glucose+2D MOF/GOx. After 5 h incubation, the bacteria suspensions were separately diluted 10,000 times with LB broth. Then 100 μL of the diluted bacterial suspension of 1-6 were spread on the solid medium and incubated at 37°C for 12 h. For SEM images, the bacteria of six groups were collected by centrifugation and fixed with 4% formaldehyde for 0.5 h. The bacteria were further dehydrated using 30, 50, 70, 85 and 100% of ethanol. Finally, the dried bacteria were observed under SEM after sputter-coating with gold. Preparation of 2D MOF/GOx-Band-Aid. 2D MOF/GOx (100 µg mL-1, 200 µL) was dropped into one square cotton fabric (1 cm×1 cm) until all 2D MOF/GOx solution was completely absorbed by cotton fabric. Then the square fabric was taken out and vacuum-dried at 37°C. Finally, the Fabric-2D MOF/GOx was fixed on the dressing purchased from drugstore, and the 2D


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MOF/GOx-Band-Aid was obtained. The square cotton fabric of the same size absorbed GOx or 2D nanoosheet or PBS was fixed on the dressing as control groups. Mouse Wound Model. To assess the antibacterial property of glucose+2D MOF/GOx for wound healing application, we successfully built the wound model. The Kunming male mice (6-8 weeks) mice with wound on the back were divided into: (1) Blank-Band-Aid, (2) glucose+Blank-BandAid, (3) glucose+2D MOF-Band-Aid, (4) 2D MOF/GOx-Band-Aid, (5) glucose+GOx-Band-Aid and (6) glucose+2D MOF/GOx-Band-Aid with three mice in each group. The wounds were injected with S. aureus (3×107 CFU, 50 µL) and different Band-Aids were covered on the wounds of mice and changed at 24 h interval. The concentration and volumn of glucose is 10 mM and 50 µL, respectively. Meanwhile, photos of the wounds were taken every other day. The wounds of mice were excised at the third day and placed in sterile saline (1 mL). Then the bacterial samples were collected from the wounds after incubation for 24 h at 37°C. All animal procedures were in compliance with the guidelines of the Institutional Animal Care and Use Committee. Histology. For histological analysis, the wounds of mice were excised at the third day and fixed in 10% formaldehyde, embedded into paraffin, made sections and stained with H&E. In Vivo Biosafety. 2D MOF/GOx (12 mg kg-1) was administrated into mice via tail vein injection. The blood samples were collected from mice at the seventh day of treatment for blood biochemistry examination. Major organs were collected from mice treated with hybrid nanocatalyst or PBS buffer after seven days treatment for pathological examination. In addition, the control mice (treated with PBS buffer dressing) and the experimental mice (treated with


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glucose+2D MOF/GOx dressing) were also sacrificed after 3 days of therapy. The major organs were harvested from both groups. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. Characterization and catalytic performance of as-synthesized 2D MOF and 2D MOF/GOx; resistance ability of 2D MOF/GOx; photo of prepared 2D MOF/GOx-Band-Aid; amount of released Cu2+ and TCPP(Fe) and their antibacterial activity; stability and degradation of 2D MOF/GOx in different media; cytotoxicity of 2D MOF and Cu2+; in vivo biodistribution and biocompatibility evaluation of 2D MOF/GOx; comparison between the antibacterial performance of current work and that of other nanomaterials AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. ORCID Jinsong Ren: 0000-0002-7506-627X Xiaogang Qu: 0000-0003-2868-3205 ACKNOWLEDGMENT We thank Prof. Dapeng Wang for providing some data analysis. Financial support was provided by the National Natural Science Foundation of China (Grants 21431007, 21533008, 91856205, 21871249, and 21820102009), the Key Program of Frontier of Sciences (CAS QYZDJ-SSWSLH052) and 20190701028GH from Jilin Province.


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Scheme 1. Design and antibacterial mechanism of the 2D MOF/GOx hybrid nanocatalyst as a benign and self-activated cascade reagent.a

a

(a) The composition of the 2D MOF/GOx hybrid nanocatalyst. (b) The 2D MOF/GOx-Band-

Aids used for wound healing of mice. (c) The antibacterial mechanism of the 2D MOF/GOx hybrid nanocatalyst as benign and self-activated cascade reagent.


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Figure 1. (a) TEM image of 2D MOF nanosheet. (b) AFM image of 2D MOF nanosheet and their thickness distribution. (c) Dark-field TEM image of typical 2D MOF nanosheet and the corresponding TEM element mapping of Cu K-edge, Fe K-edge, C K-edge, N K-edge, and O Kedge signals.(d) TEM image of 2D MOF/GOx. Images of water dispersion of (e) 2D MOF nanosheet and (f) 2D MOF/GOx. (g) Zeta potential of 2D MOF nanosheet and 2D MOF/GOx.


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Figure 2. (a) UV-visible spectra and photographs of different reaction systems in the presence of TMB or in the presence of methyl red (b) after 4 h incubation in PBS buffer (pH=7.4, 0.5 mM). (1) PBS; (2) glucose; (3) glucose+2D MOF nanosheet; (4) 2D MOF/GOx; (5) glucose+GOx; (6) glucose+2D MOF/GOx. (5 mM glucose, 20 µg mL-1 2D MOF/GOx, 1.2 mM TMB or 0.001% methyl red). (c) Fluorescence spectra of different reaction systems in PBS buffer (pH=7.4, 0.5 mM) after 12 h reaction. (1) glucose+GOx; (2) only TA; (3) glucose+GOx+TA; (4) 2D MOF nanosheet; (5) 2D MOF nanosheet+TA; (6) glucose+2D MOF/GOx; (7) glucose+2D MOF/GOx+TA. The concentrations of TA, glucose and 2D MOF/GOx were 0.5 mM, 5 mM and 20 μg mL-1, respectively. (d) Time-dependent absorbance changes at 652 nm of TMB reaction solutions catalyzed by the 2D MOF/GOx every 30 minutes in PBS buffer (pH=7.4, 0.5 mM) (0


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min-240 min) or PBS buffer (pH=7.4, 25 mM) (0 m-240 m). Photographs of above reaction systems in (e) PBS buffer (pH=7.4, 0.5 mM) (0 min-240 min) and (f) PBS buffer (pH=7.4, 0.5 mM) (0 m-240 m) upon the addition of 0.001% methyl red.


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Figure 3. Viability analyses of (a) E. coli and (b) S. aureus. SEM images of (c) E. coli and (d) S. aureus. *Significantly different (P

Enzyme Hybrid Nanocatalyst as Benign

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