Ag-Activated Metal−Organic Framework with Peroxidase-like Activity Synergistic Ag+ Release for Safe Bacterial Eradication and Wound Healing

Silver nanoparticles (Ag NPs), a commonly used antibacterial nanomaterial, exhibit broad-spectrum antibacterial activity to combat drug-resistant bacteria. However, the Ag NPs often causes a low availability and high toxicity to living bodies due to their easy aggregation and uncontrolled release of Ag+ in the bacterial microenvironment. Here, we report a porous metal−organic framework (MOF)-based Zr-2-amin-1,4-NH2-benzenedicarboxylate@Ag (denoted as UiO-66-NH2-Ag) nanocomposite using an in-situ immobilization strategy where Ag NPs were fixed on the UiO-66-NH2 for improving the dispersion and utilization of Ag NPs. As a result, the reduced use dose of Ag NPs largely improves the biosafety of the UiO-66-NH2-Ag. Meanwhile, after activation by the Ag NPs, the UiO-66-NH2-Ag can act as nanozyme with high peroxidase (POD)-like activity to efficiently catalyze the decomposition of H2O2 to extremely toxic hydroxyl radicals (·OH) in the bacterial microenvironment. Simultaneously, the high POD-like activity synergies with the controllable Ag+ release leads to enhanced reactive oxygen species (ROS) generation, facilitating the death of resistant bacteria. This synergistic antibacterial strategy enables the low concentration (12 μg/mL) of UiO-66-NH2-Ag to achieve highly efficient inactivation of ampicillin-resistant Escherichia coli (Ampr E. coli) and endospore-forming Bacillus subtilis (B. subtilis). In vivo results illustrate that the UiO-66-NH2-Ag nanozyme has a safe and accelerated bacteria-infected wound healing.


Introduction
Nowadays, bacterial infection-induced diseases, such as skin abscesses [1,2], respiratory tract infections [3], and urinary tract infections, have become one of the most serious public problems in the world [4,5]. In the past few decades, many strategies have been made to fight against bacterial infections, and a range of antibiotics have been developed [6,7]. However, owing to the abuse of antibiotics, many bacteria emerged multi-drug resistance (MDR), resulting in increasingly difficult antibiotic treatments [8][9][10][11]. Therefore, it is very urgent to develop innovative antimicrobial systems and therapies as a complement to current antimicrobial treatments [12,13]. Scheme 1. Schematic illustrations of the synthesis process of well-dispersed Ag NPs anchored with porous MOF-based UiO-66-NH2 to form UiO-66-NH2-Ag nanocomposite and their peroxidase-like activity synergistic Ag + release for high efficient and rapid bacterial elimination.
Synthesis of UiO-66-NH2 nanoparticles. UiO-66-NH2 nanoparticles (NPs) were synthesized via a hydrothermal approach in terms of an improved process [53]. In a typical synthesis, 30.3 mg (0.13 mM) ZrCl4 and 23.5 mg (0.13 mM) ATA were dissolved in Scheme 1. Schematic illustrations of the synthesis process of well-dispersed Ag NPs anchored with porous MOF-based UiO-66-NH 2 to form UiO-66-NH 2 -Ag nanocomposite and their peroxidase-like activity synergistic Ag + release for high efficient and rapid bacterial elimination.
Synthesis of UiO-66-NH 2 nanoparticles. UiO-66-NH 2 nanoparticles (NPs) were synthesized via a hydrothermal approach in terms of an improved process [53]. In a typical synthesis, 30.3 mg (0.13 mM) ZrCl 4 and 23.5 mg (0.13 mM) ATA were dissolved in DMF (15 mL) with stirring. Then, the above mixture was shifted to a sealed Teflon-lined stainlesssteel autoclave (25 mL), heat at 200 • C for 1 d. After cooling, the resulting product was washed by centrifugation with anhydrous methanol. The final products were obtained after drying.
Synthesis of UiO-66-NH 2 -Ag nanocomposite. In-situ immobilization strategy was adopted to synthesize the UiO-66-NH 2 -Ag nanocomposite. First, the UiO-66-NH 2 NPs (20 mg) were dispersed in anhydrous methanol (5 mL) under stirring for 20 min. Then, anhydrous methanol solution (5 mL) containing AgNO 3 (10 mg) was added under stirring for another 20 min. The suspension was vigorously stirred in the dark under 50 • C. 20 h later, the obtained product UiO-66-NH 2 -Ag was centrifuged and washed several times with methanol and distilled water, and then dried at room temperature under dark.
Characterizations. A Bruker D8 (Germany) Advanced Power diffractometer (λ = 1.5406 A) was used for powder X-ray diffraction (XRD) analysis of samples. A Hitachi S-4800 (Tokyo, Japan) field emission scanning electron microscope (FE-SEM) and a Tecnai G2 F20 (Eindhoven, The Netherlands) transmission electron microscope (TEM) were used to characterize the morphology and size of samples. Element analysis result was obtained via an energy-dispersive X-ray spectrum (EDX, Oxford x-met 8000, Oxford, UK) attached on the TEM. A JEM-ARM 200F cold field gun (Tokyo, Japan, 200 kV) was used for high-angle annular dark-field scanning transmission electron microscope (HAADF-STEM) to analyze the structure and composition of samples. A Micromeritics Tristar 3000 (Norcross, GA, USA) instrument was used to analyze the pore size distributions and nitrogen adsorption-desorption isotherms at 77 K. The specific surface was calculated based on the relative pressure (P/P0) from 0.01 to 1.00 via the Brunauer-Emmett-Teller (BET) method. Escalab 250 X-ray photoelectron spectrometer (Thermo Fisher Scientific Inc., Waltham, UK) with Al K α radiation was used for X-ray photoelectron spectroscopy (XPS) measurements. UV-Vis-NIR spectrophotometer (VARIAN CARY 50, Shanghai, China) was used to test the absorption peak. A Zeta potential/Particle system (Malvern Zetasizer Nano ZS, Worcestershire, UK) was used to measure Zeta potential.
After filtering the samples, the suspension solutions were shifted to flasks containing HNO 3 (5 mL, 70%), and digested at 180 • C. Afterwards, inductively coupled plasma mass spectrometry (ICP-MS, Thermo Elemental X7, Winsford, UK) was used to detect the digested solutions. The detected samples were repeated three times.
Detection of ·OH. Non-fluorescent terephthalic acid (TA) can be oxidized by ·OH to 2-hydroxyl terephthalic acid (TAOH), which has a characteristic peak at 435 nm under the excitation of 315 nm [54]. By observing the fluorescence intensity of TAOH, we detected the formation of ·OH from H 2 O 2 catalyzed by UiO-66-NH 2 -Ag using the Horiba FluoroLog-3 fluorescence spectrometer (USA). Typically, the solutions to be measured were divided into five groups, including TA, UiO-66-NH 2 -Ag, H 2 O 2 , TA + H 2 O 2 and TA + H 2 O 2 + UiO-66-NH 2 -Ag. The final working concentrations were 10 µg/mL for UiO-66-NH 2 -Ag, 0.1 mM for H 2 O 2 and 0.5 mM for TA. The solution was gently mixed in the dark at 37 • C for 12 h. Then, the fluorescence intensity was recorded.
For hemolysis analysis, fresh blood (1 mL) obtained from BALB/c mice (6 weeks old) obtained from Vital River was mixed with PBS (2 mL) containing EDTA. After centrifugation at 2000 rpm and washing three times, red blood cells (RBCs) were collected and resuspended in PBS. The diluted RBCs suspension solution was then added to PBS (negative control), distilled water (positive control) and different concentrations of UiO-66-NH 2 -Ag dispersions (3.9, 7.8, 15.6, 31.3, 62.5, 125, and 250 µg/mL). These mixtures were kept at 25 • C for 4 h. Finally, UV-Vis spectrophotometry was used to measure the absorbance of the supernatant at 541 nm.
Bacterial solutions. Gram-negative ampicillin-resistant Escherichia coli (Amp r E. coli) and Gram-positive endospore-forming Bacillus subtilis (B. subtilis) were respectively transferred to Luria-Bertani (LB) containing ampicillin (50 µg/mL) and Beef-Peptone-Yeast (BPY) broth. The two bacteria were kept at 37 • C under shaking for 5 h. Then, the bacteria were centrifuged and washed with PBS.
In vitro antibacterial performances. The antibacterial performances of UiO-66-NH 2 -Ag nanocomposite were tested by the plate counting method. Two Gram bacteria (Amp r E. coli and B. subtilis, OD 600 = 0.10) were assayed diluted to 1.0 × 10 5 CFU/mL in PBS. The diluted bacteria (0.4 mL) were mixed with the UiO-66-NH 2 -Ag (0.1 mL) with the final concentration of 0, 3, 6, 12, 25, 50, and 100 µg/mL and oscillated at 37 • C for 4 h. Next, the above mixture (100 µL) was spread on LB or BPY solid culture plates and cultured at 37 • C for 24 h. Moreover, we also performed a live/dead staining experiment to study the antibacterial performance of UiO-66-NH 2 -Ag. First, UiO-66-NH 2 -Ag nanocomposite (0, 3, 6, 12 µg/mL) were incubated with 500 µL of Amp r E. coli or B. subtilis suspension (1.0 × 10 8 CFU/mL) at 37 • C for 4 h. After incubation, propidium iodide (PI) (red) and SYTO 9 (green) were mixed with the suspension of Amp r E. coli or Bacillus subtilis. The final concentrations of PI and SYTO 9 were 30 µM and 20 µM, respectively. The mixture was incubated at 37 • C under dark for 25 min. Finally, laser scanning confocal microscopy (Nikon, Tokyo, Japan) was used to observe the fluorescence images.
ROS detection. DCFH-DA fluorescent probe was used to detect ROS production in bacteria. First, UiO-66-NH 2 -Ag nanocomposite (0, 3, 6, 12 µg/mL) were incubated with 500 µL of Amp r E. coli or B. subtilis suspension (1.0 × 10 8 CFU/mL) for 4 h at 37 • C. After incubation, DCFH-DA was mixed with suspensions of E. coli or B. subtilis. The final concentration of DCFH-DA was 10 µM. The mixture was incubated at 37 • C for 15 min under dark. Finally, the fluorescence images were observed using a laser scanning confocal microscope.
In vivo wound healing. To evaluate the performance of UiO-66-NH 2 -Ag on promoting wound healing, male BALB/c mice (6 weeks) were grouped into three groups (n = 6 in each group): (1) Control PBS; (2) Ag + ; (3) UiO-66-NH 2 -Ag. After anesthetizing, the wound with diameter of 5 mm (∼78 mm 2 ) was surgically obtained on the back of the mice. These skin wounds were then infected with Amp r E. coli suspension (1.0 × 10 5 CFU/mL). 12 h later, 5 µL of PBS, AgNO 3 (0.86 µg/mL) and UiO-66-NH 2 -Ag (12.5 µg/mL with 0.86 µg of Ag loading) solutions were applied to the wounds at corresponding groups, respectively. This process of adding the above solutions to the wound was performed twice at 12-h intervals. The wounds were photographed daily, and the body weight of mice was recorded. During the treatment, wound tissues were photographed, and then the skin tissues (n = 3 per group) were collected and fixed with 4% formaldehyde for Hematoxylin-Eosin (H&E) staining and Masson's staining within day 8. Furthermore, blood samples were collected from each group of mice on day 8 for routine blood analysis. After that, representative H&E-stained images of major organs of mice (n = 6 for each group) after being treated with UiO-66-NH 2 -Ag at day 7, day 15, and day 30 were also used to evaluate long-term safety. All animal experiments were performed and approved in accordance with the Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS guidelines for the care and use of laboratory of Animals Ethics Committee.

Results and Discussion
The porous MOF-based UiO-66-NH 2 NPs were synthesized by a hydrothermal approach. Subsequently, an in-situ immobilization dispersion reaction of Ag + and the UiO-66-NH 2 was used to initiate and fix Ag NPs on the surface of UiO-66-NH 2 , resulting in the formation of MOF-based UiO-66-NH 2 -Ag nanocomposite. FE-SEM image in Figure S1a and TEM image in Figure 1a showed that the UiO-66-NH 2 -Ag had an octahedral-like construction with a diameter of~60 nm. Noteworthy, the Ag NPs with a diameter of about 5 nm were uniformly distributed on the surface of UiO-66-NH 2 . XRD patterns of the UiO-66-NH 2 showed the purity and good crystallinity (Figure 1b) [56]. The high agreement between UiO-66-NH 2 and UiO-66 on the 2θ peak indicated that the introduction of the -NH 2 group had no impact on the skeletal structure of UiO-66. After in-situ immobilization of the Ag NPs, typical peaks (111 and 200) of cubic-phased Ag appeared (JCPDS No. 87-0719), implying the successful anchoring of Ag NPs on the surface of UiO-66-NH 2 . To evaluate the porosity of UiO-66-NH 2 , N 2 adsorption/desorption isotherm was measured, and the specific surface area was calculated as 473 m 2 g −1 ( Figure S2a). According to the Barrett-Joyner-Halenda (BJH) model, the pore size distribution from the isothermal adsorption branch was estimated to be approximately 1.5 nm ( Figure S2b). Furthermore, we found that UiO-66-NH 2 had a low zeta potential in water (−5.5 ± 0.47 mV). However, the zeta potential of UiO-66-NH 2 -Ag changed to +8.06 ± 0.16 mV, further implying a successful immobilization of Ag to the UiO-66-NH 2 -Ag surface (Figure 1c).   The element mapping of Zr, N, Ag, and O in UiO-66-NH 2 -Ag nanocomposite was illustrated by HAADF-STEM images (Figure 1d). The EDX spectrum of UiO-66-NH 2 -Ag nanocomposite ( Figure S3) exhibited a similar result with the elemental mapping analysis. Next, we analyzed the surface elemental compositions and chemical states of the obtained nanocomposite. As shown in Figure 1e, XPS survey spectrum indicated the coexistence of Zr, C, N, Ag and O in the UiO-66-NH 2 -Ag. Moreover, the XPS spectrum of Ag 3d was shown in Figure 1f, where the Ag 3d 5/2 (368.5 eV) and Ag 3d 3/2 (374.6 eV) peaks implied the existence of Ag in the zero-valent form [57]. The XPS Zr 3d peak could be further split into Zr 3d 5/2 (182.6 eV) and Zr 3d 3/2 (185.0 eV), which were attributed to Zr-O in UiO-66 ( Figure S4a) [58]. The N 1s XPS spectrum showed a typical peak located at 399.4 eV, corresponding to -NH 2 and -NH groups ( Figure S4b). UV-Vis-NIR spectrum of the UiO-66-NH 2 -Ag well inherited the feature of UiO-66-NH 2 and had wide absorbance from UV-Vis to NIR regions. Especially, UiO-66-NH 2 -Ag had a stronger absorbance than UiO-66-NH 2 at 400-750 nm on account of the successful anchoring of Ag NPs (Figure 1g).
Nanomaterials especially metal-based nanocomposites have the ability as nanozyme with improved POD-like catalytic activity, which can catalyze H 2 O 2 to produce highly toxic ·OH for killing bacteria [16,17]. We then attempted to evaluate the POD-like activity of UiO-66-NH 2 -Ag nanocomposite by the catalytic oxidation of colorless TMB to blue oxidized TMB (oxTMB) in the presence of H 2 O 2 . In Figure 2a, compared with other groups, the TMB + H 2 O 2 + UiO-66-NH 2 -Ag had an intense absorption at 652 nm with a significant blue color within 10 min, confirming that UiO-66-NH 2 -Ag had a high POD-like activity. Meanwhile, the blue gradually deepened with the increased UiO-66-NH 2 -Ag concentration, indicating the concentration-dependent POD-like activity (Figure 2b). The catalytic activity also depended on H 2 O 2 concentration, pH values and temperature. As shown in Figure 2c, the POD-like activity increased with the increased H 2 O 2 concentration. Moreover, we found that the optimal pH value and temperature were~pH 5.0 and 37-42 • C, which were very close to the pH value of real bacterial microenvironment and living body temperature (Figure 2d,e). It is reasonable to refer that the enhanced POD-like activity of UiO-66-NH 2 -Ag after being activated by the Ag NPs could be due to the accelerated electron transfer from TMB to H 2 O 2 compared with the UiO-66-NH 2 NPs.
Due to the enhanced POD-like activity of UiO-66-NH 2 -Ag, it is necessary to further investigate the formation of ·OH. TA was used as a typical fluorescence (FL) probe to detect the ·OH with a maximum peak at 435 nm upon the reaction with ·OH. As shown in Figure S5, besides its inherent FL peak [59], the characteristic peak at 435 nm was significantly enhanced in TMB + H 2 O 2 + UiO-66-NH 2 -Ag group compared to the TMB + UiO-66-NH 2 -Ag group, implying that H 2 O 2 could be efficiently changed into ·OH catalyzed by UiO-66-NH 2 -Ag. The enhanced POD-like activity provided a possibility for the subsequent synergy with controllable Ag + release to kill bacteria.
It was reported that the bacteria-infected acute wound microenvironment is weak acidity [60]. To investigate the pH-dependent Ag + release capacity of UiO-66-NH 2 -Ag nanocomposite responsive to acute wound microenvironment, the Ag content on the surface of UiO-66-NH 2 -Ag was determined to be 4.396 wt% by ICP-MS. In addition, the cumulative Ag + release capacity of UiO-66-NH 2 -Ag over 10 d is shown in Figure 2f. The release of Ag + increased significantly within the first 2 h both in pH 7.4 and 5.0. Then, 47.33 µg/mL of Ag + was released from UiO-66-NH 2 -Ag into 20 mL media within 1 d, and 56.12 µg/mL of cumulative release of Ag + within 10 d was calculated. Especially, the cumulative Ag + release capacity of UiO-66-NH 2 -Ag over 10 d can reach up to~84.12 µg/mL, which was very higher than that in the neural condition. This process proves the pHresponsive release ability of UiO-66-NH 2 -Ag, which can be explained as follow. First, the porous UiO-66-NH 2 scaffolds as a relatively stable nanocarrier make the Ag NPs welldispersed in the biological environment and difficult to reunite. Second, the well-dispersed Ag NPs with large surface area fixed on the surface of UiO-66-NH 2 -Ag scaffolds could easily release the relatively large number of Ag + . At the same time, the positive surface charge of UiO-66-NH 2 -Ag (shown in Figure 1c) could also easily repel the released Ag + to the surface of bacteria, and then more easily meets the release requirement into acute acidic wound microenvironment. The pH-responsive Ag + release of the UiO-66-NH 2 -Ag offers great potential for chemical sterilization.  Due to the enhanced POD-like activity of UiO-66-NH2-Ag, it is necessary to further investigate the formation of ·OH. TA was used as a typical fluorescence (FL) probe to detect the ·OH with a maximum peak at 435 nm upon the reaction with ·OH. As shown in Figure S5, besides its inherent FL peak [59], the characteristic peak at 435 nm was significantly enhanced in TMB + H2O2 + UiO-66-NH2-Ag group compared to the TMB + UiO-66-NH2-Ag group, implying that H2O2 could be efficiently changed into ·OH catalyzed by UiO-66-NH2-Ag. The enhanced POD-like activity provided a possibility for the subsequent synergy with controllable Ag + release to kill bacteria.
It was reported that the bacteria-infected acute wound microenvironment is weak acidity [60]. To investigate the pH-dependent Ag + release capacity of UiO-66-NH2-Ag nanocomposite responsive to acute wound microenvironment, the Ag content on the surface of UiO-66-NH2-Ag was determined to be 4.396 wt% by ICP-MS. In addition, the cumulative Ag + release capacity of UiO-66-NH2-Ag over 10 d is shown in Figure 2f. The release of Ag + increased significantly within the first 2 h both in pH 7.4 and 5.0. Then, 47.33 μg/mL of Ag + was released from UiO-66-NH2-Ag into 20 mL media within 1 d, and 56.12 μg/mL of cumulative release of Ag + within 10 d was calculated. Especially, the cumulative Ag + release capacity of UiO-66-NH2-Ag over 10 d can reach up to ~84.12 μg/mL, which was very higher than that in the neural condition. This process proves the pH-responsive release ability of UiO-66-NH2-Ag, which can be explained as follow. First, the porous UiO-66-NH2 scaffolds as a relatively stable nanocarrier make the Ag NPs well-dispersed in the biological environment and difficult to reunite. Second, the well-dispersed Ag NPs with large surface area fixed on the surface of UiO-66-NH2-Ag scaffolds could easily release the relatively large number of Ag + . At the same time, the positive surface charge of UiO-66-NH2-Ag (shown in Figure 1c) could also easily repel the released Ag + to the surface of bacteria, and then more easily meets the release re- Inspired by the ability of controllable release of Ag + and the remarkable POD-like activity, we next evaluated the antibacterial effects of UiO-66-NH 2 -Ag nanocomposite. Typical Gram-negative ampicillin-resistant Escherichia coli (Amp r E. coli) was selected as a model bacterial strain, and the antibacterial ability was determined by plate counting method. Interestingly, the UiO-66-NH 2 -Ag had superior antibacterial efficacy and led to a very low survival rate of 2.2% for Amp r E. coli after co-incubated with UiO-66-NH 2 -Ag (6 µg/mL) for 4 h (Figure 3a,c). However,~98% survival rate of Amp r E. coli was observed for UiO-66-NH 2 without Ag NPs loading under the same incubation condition ( Figure S6), suggesting the negligible antimicrobial effect of low concentration UiO-66-NH 2 . The large gap in antimicrobial effect of UiO-66-NH 2 before and after loading of Ag NPs suggested the importance of the synergy of Ag + release and the increased POD-like activity of UiO-66-NH 2 after loading Ag NPs with uniform distribution. And the UiO-66-NH 2 -Ag can promote the production of a large amount of ROS with a concentration-dependent effectin bacterial microenvironment ( Figure S7). To further investigate the antibacterial performance of UiO-66-NH 2 -Ag against other bacteria, endospore-forming Bacillus subtilis (B. subtilis) as a typical Gram-positive bacterium was selected for antibacterial test. As shown in Figure 3b,d, after 4 h of incubation, the bacterial survival rate decreased sharply with the increased concentration of UiO-66-NH 2 -Ag. Notably, the two bacteria died with >99.99% of killing rate when the concentration of UiO-66-NH 2 -Ag was 12 µg/mL. can promote the production of a large amount of ROS with a concentration-dependent effectin bacterial microenvironment ( Figure S7). To further investigate the antibacterial performance of UiO-66-NH2-Ag against other bacteria, endospore-forming Bacillus subtilis (B. subtilis) as a typical Gram-positive bacterium was selected for antibacterial test. As shown in Figure 3b,d, after 4 h of incubation, the bacterial survival rate decreased sharply with the increased concentration of UiO-66-NH2-Ag. Notably, the two bacteria died with >99.99% of killing rate when the concentration of UiO-66-NH2-Ag was 12 μg/mL. Next, live/dead staining was used to distinguish living cells from dead cells by SYTO 9 (for dead and live cells, green) and propidium iodide (PI for dead cells, red) staining for further understanding its concentration-dependent killing ability to Amp r E. coli and B. subtilis. Similar to the previous plate counting results, more dead bacteria were observed in the 12 μg/mL of UiO-66-NH2-Ag group, while a few dead bacteria were observed when treated with 6 μg/mL and 3 μg/mL of UiO-66-NH2-Ag ( Figure S8). The Next, live/dead staining was used to distinguish living cells from dead cells by SYTO 9 (for dead and live cells, green) and propidium iodide (PI for dead cells, red) staining for further understanding its concentration-dependent killing ability to Amp r E. coli and B. subtilis. Similar to the previous plate counting results, more dead bacteria were observed in the 12 µg/mL of UiO-66-NH 2 -Ag group, while a few dead bacteria were observed when treated with 6 µg/mL and 3 µg/mL of UiO-66-NH 2 -Ag ( Figure S8). The above results showed that this Ag-loaded nanocomposite exhibited remarkable antibacterial properties for Amp r E. coli and B. subtilis at very low doses.
FE-SEM images in Figure 4a were used to further study the antibacterial behavior of UiO-66-NH 2 -Ag. As expected, the Amp r E. coli incubated with control group was smooth. The rod-like morphology and the cell membrane structure of the Amp r E. coli were intact. However, 6 µg/mL of UiO-66-NH 2 -Ag treated for 20 min resulted in slight wrinkles and some disruption on the surface of bacterial cell wall (red arrows). As the concentration of UiO-66-NH 2 -Ag rose to 12 µg/mL, the damage of bacterial surface was more intense with more wrinkles and incomplete surfaces. As the concentration increased to 50 µg/mL, the membrane of bacteria was severely damaged, and the bacterial morphology was changed and fused together. Similar results were verified on B. subtilis (Figure 4b). Therefore, UiO-66-NH 2 -Ag with low dose use of Ag NPs has a powerful antibacterial effect due to the high POD-like activity synergistic Ag + release, which can produce large amounts of ROS to directly disrupt bacteria. This efficient antibacterial performance and low dose use of Ag NPs offer great possibilities for bacterial disinfection. more wrinkles and incomplete surfaces. As the concentration increased to 50 μg/mL, the membrane of bacteria was severely damaged, and the bacterial morphology was changed and fused together. Similar results were verified on B. subtilis (Figure 4b). Therefore, UiO-66-NH2-Ag with low dose use of Ag NPs has a powerful antibacterial effect due to the high POD-like activity synergistic Ag + release, which can produce large amounts of ROS to directly disrupt bacteria. This efficient antibacterial performance and low dose use of Ag NPs offer great possibilities for bacterial disinfection. Good biocompatibility is a prerequisite for biological applications. Therefore, we performed hemolysis to assess the effect of UiO-66-NH2-Ag nanocomposite on RBCs. Interestingly, no obvious erythrocyte hemolysis was observed after incubation in RBCs with different concentrations of UiO-66-NH2-Ag ( Figure S9). Furthermore, in vitro cytotoxicity studies of UiO-66-NH2-Ag nanocomposite on HUVEC and U87 cells were performed by CCK-8 assay. Even at 250 μg/mL of UiO-66-NH2-Ag, the two cells remained highly active ( Figure S10). These results clearly revealed that UiO-66-NH2-Ag has good biocompatibility at the tested dosage.
To observe the in vivo wound healing effect of UiO-66-NH2-Ag nanocomposite, Amp r E. coli was selected to infect the wounds on the epidermis of BALB/c mice (Figure Good biocompatibility is a prerequisite for biological applications. Therefore, we performed hemolysis to assess the effect of UiO-66-NH 2 -Ag nanocomposite on RBCs. Interestingly, no obvious erythrocyte hemolysis was observed after incubation in RBCs with different concentrations of UiO-66-NH 2 -Ag ( Figure S9). Furthermore, in vitro cytotoxicity studies of UiO-66-NH 2 -Ag nanocomposite on HUVEC and U87 cells were performed by CCK-8 assay. Even at 250 µg/mL of UiO-66-NH 2 -Ag, the two cells remained highly active ( Figure S10). These results clearly revealed that UiO-66-NH 2 -Ag has good biocompatibility at the tested dosage.
To observe the in vivo wound healing effect of UiO-66-NH 2 -Ag nanocomposite, Amp r E. coli was selected to infect the wounds on the epidermis of BALB/c mice (Figure 5a). The mice were divided into (1) Control, (2) Ag + , and (3) UiO-66-NH 2 -Ag groups. Figure 5b showed representative photographs of different treated wounds in mice during the treatment. All wounds shrank with the prolonged time. Notably, the UiO-66-NH 2 -Ag treated group showed better wound healing than the control and Ag + groups, which can be attributed to the effective Ag + release synergetic POD-like activity on the wound surface. The maximum percentage of wound area in each group further demonstrated its effect in accelerating wound healing (Figure 5c). There was no obvious change in the body weight of mice for UiO-66-NH 2 -Ag treated group compared with the control group, indicating that UiO-66-NH 2 -Ag had no significant toxicity to mice (Figure 5d). After treatment, the wound tissues were analyzed by H&E staining and Masson trichrome staining (Figures 5e and S11). On day 3, H&E staining showed epidermal damage in each group. On day 5, repair of damaged skin tissues was observed in all treatment groups. On day 8, complete reepithelialization and differentiated epithelium were clearly observed in the UiO-66-NH 2 -Ag group compared to the other groups. Masson trichrome staining (blue) was used to distinguish collagen fibers from muscle fibers during wound healing. On days 3 and 5, no obvious collagen fibers were observed in each group. On day 8, collagen fibers in control group had no obvious recovery effect, while collagen fibers in Ag + group had some recovery. The UiO-66-NH 2 -Ag group showed the optimal recovery of collagen fiber. In addition, major organs of mice were stained with H&E and blood indexes of mice were used to evaluate the biocompatibility of the UiO-66-NH 2 -Ag. As shown in Figure S12, no obvious damage or inflammatory reaction was observed in the H&E staining, indicating that the UiO-66-NH 2 -Ag had good histocompatibility within 30 days. Meanwhile, the nine blood routine indexes were all within the normal range, indicating that UiO-66-NH 2 -Ag was biologically safe ( Figure S13). Therefore, this synergistic antibacterial system can not only achieve rapid wound disinfection, but also has good biosafety.

Conclusions
In summary, we constructed a biocompatible MOF-based UiO-66-NH2-Ag nanocomposite consisting of well-dispersed Ag NPs anchored on the porous UiO-66-NH2. The nanocomposite can avoid the aggregation of Ag NPs while enhancing dispersion of Ag NPs. Meanwhile, UiO-66-NH2-Ag not only improved the utilization of Ag NPs, but also provided a pH-responsive Ag + release in acidic wound environment. In addition, the UiO-66-NH2-Ag can act as nanozyme with enhanced POD-like catalytic activity after loading Ag NPs, which allowed it to break down H2O2 within bacteria to produce highly toxic ·OH to destroy bacteria integrity. This POD-like activity synergistic Ag + release greatly facilitated the production of more ROS in bacteria, resulting in highly effective bactericidal activity. UiO-66-NH2-Ag at a low concentration of 12 μg/mL could effectively kill Amp r E. coli and B. subtilis with >99.99% bactericidal activity. Moreover,

Conclusions
In summary, we constructed a biocompatible MOF-based UiO-66-NH 2 -Ag nanocomposite consisting of well-dispersed Ag NPs anchored on the porous UiO-66-NH 2 . The nanocomposite can avoid the aggregation of Ag NPs while enhancing dispersion of Ag NPs. Meanwhile, UiO-66-NH 2 -Ag not only improved the utilization of Ag NPs, but also provided a pH-responsive Ag + release in acidic wound environment. In addition, the UiO-66-NH 2 -Ag can act as nanozyme with enhanced POD-like catalytic activity after loading Ag NPs, which allowed it to break down H 2 O 2 within bacteria to produce highly toxic ·OH to destroy bacteria integrity. This POD-like activity synergistic Ag + release greatly facilitated the production of more ROS in bacteria, resulting in highly effective bactericidal activity. UiO-66-NH 2 -Ag at a low concentration of 12 µg/mL could effectively kill Amp r E. coli and B. subtilis with >99.99% bactericidal activity. Moreover, UiO-66-NH 2 -Ag maintained low cytotoxicity over a wide concentration range (0-250 µg/mL). In vivo results revealed that UiO-66-NH 2 -Ag exhibited high antibacterial effect and significantly accelerated bacteriainfected wound healing without any influence on organ and blood indicators. This work provides a new strategy for safe wound disinfection broadens the prospect of antimicrobial nanomaterials for broad-spectrum bactericide application.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/nano12224058/s1, Figure S1: FE-SEM image of UiO-66-NH 2 -Ag nanocomposite; Figure S2: N 2 adsorption/desorption isotherms of UiO-66-NH 2 and BJH pore size distribution curves of UiO-66 and UiO-66-NH 2 ; Figure S3: EDX of UiO-66-NH 2 -Ag nanocomposite. Inset is the mass ratio of different elements; Figure S4: XPS data of high-resolution Zr 3d and N 1s of UiO-66-NH 2 -Ag nanocomposite; Figure S5: FL spectra of TAOH after different treatments; Figure S6: Photographs of bacterial colonies formed by Amp r E. coli exposed to different concentrations of UiO-66-NH 2 . And Amp r E. coli killing ratio treated with different concentrations of UiO-66-NH 2 ; Figure S7: Fluorescence images of Amp r E. coli and B. subtilis incubated with different concentrations of UiO-66-NH 2 -Ag using DCFH-DA probe for ROS detection; Figure S8: Fluorescence images of Amp r E. coli and B. subtilis treated with different concentrations of UiO-66-NH 2 -Ag incubated with SYTO-9 and PI; Figure S9: Hemolysis experiment of UiO-66-NH 2 -Ag at different concentrations; Figure S10: Cell viabilities of HUVEC and U87 cells after incubation with different concentrations of UiO-66-NH 2 -Ag for 24 h; Figure S11: Percentage of collagen fiber area after different treatments; Figure S12: Representative H&E stained images of major organs of mice after treated with UiO-66-NH 2 -Ag for different days; Figure S13: Routine blood analysis of mice after 8 days of treatment. Institutional Review Board Statement: All animal experiments were performed and approved in accordance with the Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS guidelines for the care and use of laboratory of Animals Ethics Committee.

Conflicts of Interest:
The authors declare no conflict of interest.