Preparation method and application of nano-enzyme with efficient bacterium capturing capacity and dual-catalytic activity

Abstract

The invention relates to a preparation method and application of a nano-enzyme with efficient bacterium capturing capacity and dual-catalytic activity, and aims to realize remarkable catalytic sterilization and effective resistance to bacterial infection without causing bacterial drug resistance. The preparation method of the nano-enzyme with the efficient bacterium capturing capacity and the dual-catalytic activity comprises the following steps: 1) synthesis of HOF; (2) synthesizing PdCu; (3) synthesis of HBioNzyme is carried out; the method is simple and easy to operate, the prepared nano-enzyme can capture bacteria to generate ROS (reactive oxygen species) and antibacterial active molecules in situ through high-efficiency bacteria capture capability and dual-catalytic activity, has an obvious killing effect on the bacteria, causes surface shrinkage, rupture and even death of the bacteria, realizes remarkable catalytic sterilization and effectively resists bacterial infection, and has a wide application prospect. Meanwhile, the problem of bacterial drug resistance cannot be caused, and the preparation method and application of the nano-enzyme are innovated.

Description

1. Technical Field

[0001] The present invention relates to the field of nanomaterials, and in particular to a preparation method and application of a nanozyme with high-efficiency bacteria capturing ability and dual catalytic activity. 2. Background Technology

[0002] Bacterial infections continue to threaten people's lives and health and have become one of the most serious threats to global public health. In particular, with the overuse of antibiotics in recent years, bacterial resistance to antibiotics has continued to increase, significantly reducing their antibacterial efficacy. Once bacteria develop resistance, repeated infections and persistent inflammation will lead to slow healing, wound deterioration, and further complications such as shock, sepsis, and lung infection, and even death. Therefore, in order to combat this serious threat, there is an urgent need to develop a broad-spectrum antibacterial strategy that can replace traditional antibiotics and avoid the problem of bacterial resistance, in order to solve problems such as unsatisfactory treatment effects, long-term toxicity, and the induction of antibiotic resistance in bacteria.

[0003] Nanozymes are nanomaterials that can produce reactive oxygen species. They have attracted widespread attention due to their low cost, adjustable catalytic centers, good biocompatibility, and high stability. The broad-spectrum antibacterial strategy of nanozyme-mediated ROS generation destroys bacterial cell walls and cell membranes through oxidation, leading to DNA breakage, protein inactivation, lipid peroxidation, and ultimately bacterial death. This method does not cause the development of bacterial resistance and shows broad application prospects in the biomedical field. However, current nanozymes have poor selectivity, and the catalytically generated ROS have problems such as short lifespan and limited transmission distance, which limit their antibacterial efficiency. How to accurately capture bacteria and release active bactericidal substances in situ to eliminate bacteria without causing damage to surrounding healthy tissues is a technical problem that needs to be solved in the design of an ideal nanozyme antibacterial strategy. III. Summary of the invention

[0004] In view of the above situation, in order to solve the defects of the prior art, the purpose of the present invention is to provide a method for preparing a nanozyme with efficient bacterial capture ability and dual catalytic activity and its application, which can achieve significant catalytic bactericidal effects and effectively combat bacterial infection problems without causing bacterial resistance problems.

[0005] The technical solution provided by the present invention is that the preparation method of the nanozyme having high-efficiency bacteria capture ability and dual catalytic activity comprises the following steps:

[0006] 1) Synthesis of HOF

[0007] Weigh terephthalamidine hydrochloride and dissolve it in water to form solution A, weigh tetrakis (4-carboxyphenyl) methane and dissolve it in water, continue to add NaOH solution to deprotonate to form solution B, transfer solution A to a round-bottom flask, slowly add solution B dropwise to solution A, and under stirring conditions at room temperature, a precipitate can be observed to form immediately, stir and centrifuge, collect the product, discard the supernatant, continue to centrifuge and wash three times under the same conditions to remove any unreacted precursors, and the product after washing is evenly dispersed with water and stored in a refrigerator to obtain HOF;

[0008] 2) Synthesis of PdCu

[0009] Weigh KBr solid and add it into the round-bottom flask of polyethylene glycol under stirring, then add H 2 PdCl 4 and CuCl 2 ·2H 2 O, stir and mix slightly, then continue to add HCl and ascorbic acid solution, stir evenly, and carry out oil bath reaction without any disturbance. After the reaction is completed, collect the material by centrifugation, centrifuge, wash three times, disperse in water and place in a refrigerator to obtain PdCu;

[0010] 3) Synthesis of HBioNzyme

[0011] The HOF prepared in step 1) is added to the PdCu prepared in step 2) and then H 2 O, stirring at room temperature to fully mix, after the stirring is completed, the above mixture is ultrasonically homogenized, and then the oil bath reaction is carried out without any disturbance. When black precipitate is observed on the wall of the flask and the solution is almost colorless, it indicates that the reaction is over, the reaction is stopped and the material is collected by centrifugation, washed three times, stored in water and placed in a refrigerator to obtain HBioNzyme.

[0012] The invention relates to the use of the nanozyme with high-efficiency bacteria capture capability and dual catalytic activity in the preparation of anti-bacterial infection drugs.

[0013] The method of the present invention is simple and easy to operate. The prepared nanozyme can capture ROS and antibacterial active molecules generated in situ by bacteria through its efficient bacteria capture ability and dual catalytic activity, causing obvious killing effect on bacteria, causing the bacterial surface to shrink, rupture or even die, achieving significant catalytic sterilization, effectively combating bacterial infection problems, and at the same time will not cause bacterial resistance problems. It is an innovation in the preparation method and application of nanozymes. IV. Description of the drawings

[0014] Figure 1 This is a scanning electron microscope (SEM) image of the nanozyme prepared in the present invention.

[0015] Figure 2Transmission electron microscopy and corresponding element distribution map of the nanozyme prepared in the present invention.

[0016] Figure 3 This is a diagram of the capture of bacteria by the nanozyme prepared in the present invention.

[0017] Figure 4 Schematic diagram of the click reaction of molecule 6 of the present invention.

[0018] Figure 5 LC-MS analysis of molecules 4 and 5 in LB medium in the presence of HBioNzyme according to the present invention.

[0019] Figure 6 Agar plate counts of Escherichia coli and Staphylococcus aureus after different treatments of the nanozymes prepared in the present invention.

[0020] Figure 7 Live-dead staining images of Escherichia coli and Staphylococcus aureus after different treatments of the nanozymes prepared in the present invention.

[0021] Figure 8 This is a diagram showing the cytotoxicity experiment of the nanozyme prepared in the present invention on NIH3T3 cells.

[0022] Fig. 9 The figures are as follows: wound area size and healing status of each group of mice after treatment in the present invention.

[0023] Fig.10 These are H&E and Masson staining images of mice in each group after treatment. V. Specific implementation methods

[0024] The specific implementation of the present invention is further described in detail below with reference to the accompanying drawings and examples.

[0025] Example 1

[0026] In the specific implementation of the present invention, the preparation method of the nanozyme with efficient bacteria capture ability and dual catalytic activity comprises the following steps:

[0027] 1) Synthesis of HOF

[0028] Weigh 0.5-1 mg of terephthalamidine hydrochloride (1:2) and dissolve it in 1 mL of water to form solution A. Weigh 1-2 mg of tetrakis(4-carboxyphenyl)methane and dissolve it in 1-3 mL of water. Continue to add 5-10 μL of NaOH (2M) solution to deprotonate and form solution B. Transfer solution A to a 25 mL round-bottom flask, and slowly add solution B dropwise to solution A. Under room temperature stirring conditions, a precipitate can be observed to form immediately. After stirring for 5-10 minutes, centrifuge at 5000-7000 rpm for 4-6 minutes to collect the product, discard the supernatant, and continue to centrifuge and wash three times under the same conditions to remove any unreacted precursors. After washing, the product is evenly dispersed with 1 mL of water and stored in a refrigerator at 3-5°C to obtain HOF.

[0029] 2) Synthesis of PdCu

[0030] Weigh 120-150 mg of KBr solid and add it to a round-bottom flask containing 4-6 mL of polyethylene glycol (Mw600, 0.01 wt%) under stirring, and then add 1.25-2 mL of H 2 PdCl 4 (20 mM) and 100-500 μL CuCl 2 ·2H 2 O (20mM), stir slightly and then add 80-120μL HCl (6M) and 1.2-1.5mL ascorbic acid solution (0.1M), stir evenly, react in an oil bath at 90-95°C for 30-40min without any disturbance, collect the material by centrifugation after the reaction, wash three times by centrifugation at 5000-7000rpm for 4-6min, disperse in water and place in a refrigerator at 3-5°C to obtain PdCu;

[0031] 3) Synthesis of HBioNzyme

[0032] Add 25-35 mg HOF prepared in step 1) to 15-20 mg PdCu prepared in step 2) and 3-5 mL H 2 O, stir at room temperature for 0.5-1h to mix thoroughly. After stirring, the mixture is homogenized by ultrasonication, and reacted in an oil bath at 80-85°C for 30-40min without any disturbance. When black precipitate appears on the wall of the flask and the solution is almost colorless, it indicates that the reaction is over. Stop the reaction and collect the material by centrifugation at 5000-7000rpm for 4-6min, wash three times, store in water and place in a refrigerator at 3-5°C to obtain HBioNzyme.

[0033] Example 2

[0034] In the specific implementation of the present invention, the preparation method of the nanozyme with efficient bacteria capture ability and dual catalytic activity comprises the following steps:

[0035] 1) Synthesis of HOF

[0036] Weigh 0.78 mg of terephthalamidine hydrochloride (1:2) and dissolve it in 1 mL of water to form solution A. Weigh 1.56 mg of tetrakis(4-carboxyphenyl)methane and dissolve it in 1 mL of water. Continue to add 5 μL of NaOH (2M) solution to deprotonate and form solution B. Transfer solution A to a 25 mL round-bottom flask, and slowly add solution B dropwise to solution A. Under room temperature stirring conditions, a precipitate can be observed to form immediately. After stirring for 8 minutes, centrifuge at 6000 rpm for 5 minutes to collect the product, discard the supernatant, and continue to centrifuge and wash three times under the same conditions to remove any unreacted precursors. After washing, the product is evenly dispersed with 1 mL of water and stored in a refrigerator at 4°C to obtain HOF.

[0037] 2) Synthesis of PdCu

[0038] 138 mg of KBr solid was weighed and added into a round-bottom flask containing 5 mL of polyethylene glycol (Mw 600, 0.01 wt%) under stirring, followed by 1.25 mL of H 2 PdCl 4 (20 mM) and 500 μL CuCl 2 ·2H 2 O (20mM), stir slightly and then add 100μL HCl (6M) and 1.5mL ascorbic acid solution (0.1M), stir evenly, react in an oil bath at 95℃ for 30min without any disturbance, collect the material by centrifugation after the reaction, wash three times by centrifugation at 6000rpm for 5min, disperse in water and place in a refrigerator at 4℃ to obtain PdCu;

[0039] 3) Synthesis of HBioNzyme

[0040] 30 mg HOF prepared in step 1) was added to 15 mg PdCu prepared in step 2) and 4 mL H 2 O, stirred at room temperature for 1 hour to fully mix. After the stirring was completed, the above mixture was ultrasonically homogenized and reacted in an 80°C oil bath for 30 minutes without any disturbance. When black precipitate was observed on the wall of the flask and the solution was almost colorless, it indicated that the reaction was over. The reaction was stopped and the material was collected by centrifugation at 6000 rpm for 5 minutes, washed three times, and stored in water in a 4°C refrigerator to obtain HBioNzyme.

[0041] The nanozyme with efficient bacterial capture ability and dual catalytic activity prepared by the present invention is based on a hydrogen-bonded organic framework (HOF) as a framework, and PdCu is reduced and grown in its cavity in situ. HOF itself is a type of linear material with high biocompatibility and can be interwoven into a network. Its building blocks have rich amino and carboxyl groups that can form hydrogen bond interactions with the bacterial surface, so that it has the bacterial capture ability of neutrophil extracellular traps (NETs), and PdCu has a spike-like morphology that can pierce bacteria, further enhancing the bacterial capture ability of the nanozyme. Therefore, the nanozyme prepared by the present invention has excellent POD-like catalytic activity and click chemistry catalytic activity as a dual catalytic platform, simulates the phagosome of NETs, ​​generates ROS and antibacterial agents in situ at the bacterial capture site, exerts a synergistic antibacterial effect, and achieves efficient bacterial removal. At the same time, it can continuously generate ROS in situ at the bacterial capture site at physiological H2O2 levels to overcome the problems of short ROS life and limited diffusion distance. In addition, the bioorthogonal click chemistry catalytic ability of the nanozyme can generate antibacterial active molecules in situ without affecting other biochemical reactions and reducing the off-target effects of antibacterial active molecules, which has been proven by experiments. The relevant experimental data are as follows: 1. Morphological characterization

[0042] SEM

[0043] HOF, PdCu and HBioNzyme were dispersed in water respectively, and then dropped on a silicon wafer after ultrasonic homogenization. Gold was sprayed to increase conductivity, and the morphology of HBioNzyme was observed using SEM with appropriate voltage and magnification.

[0044] TEM

[0045] The HBioNzyme solution was ultrasonically homogenized and then dripped onto a copper grid. After the sample dried, the TEM was used to select the appropriate voltage and magnification to observe the morphology of the nanomaterial. A high-angle annular dark-field scanning transmission electron microscope (HAADF-STEM) was used in a surface scanning mode within a suitable field of view to collect and record images of the main elements contained in the HBioNzyme nanozyme.

[0046] The morphological characteristics of HOF, PdCu and HBioNzyme were observed by SEM. Figure 1 As shown. Figure a shows the uniformly distributed needle-like structure, confirming the successful synthesis of HOF. Figure b shows the successful synthesis of sea urchin-like PdCu. Figure c confirms that HBioNzyme is a nanofiber rod-like material with HOF as a template and PdCu in situ reduced and grown on its surface, which has the characteristics of both HOF and PdCu.

[0047] like Figure 2As shown in the figure, the morphological characteristics of HBioNzyme nanozyme were observed by TEM, and the results confirmed that the morphology of HBioNzyme was a barbed nanofiber rod. HAADF-STEM element mapping showed that C, Cu, N, O, and Pd elements were evenly distributed in the HBioNzyme nanomaterial, further proving the successful synthesis of the material.

[0048] 2. Bacteria capture test

[0049] Due to the inherent shortcomings of ROS such as short life span and limited diffusion distance, before studying the in vitro antibacterial ability of HBioNzyme, the bacterial capture ability of HBioNzyme was first studied. First, E. coli and S. aureus were revived to the logarithmic growth phase, 100 μL of bacterial solution was added to a sterile 1.5 mL EP tube, 50 μg HBioNzyme was added, LB medium was supplemented to make the reaction system 1 mL, and it was transferred to a bacterial incubator for 3 hours. After the culture is completed, 0.5 mL of bacterial solution and 0.5 mL of sterile PBS were pipetted into a sterile 1.5 mL EP tube, centrifuged at 3000 rpm for 5 minutes, and the supernatant was removed. Wash three times with sterile PBS. After washing, the PBS was discarded and 1 mL of 4% paraformaldehyde was added for fixation for 40 minutes. After fixation, centrifugation was completed and the fixative was discarded. Sterile H 2 O for three times, and then dehydrated with gradient alcohol (30%, 50%, 75%, 100%). Each dehydration treatment needs to be left to stand for 10 minutes, and after dehydration is completed, it is stored in anhydrous ethanol. Then a small amount of bacterial suspension is added dropwise to the silicon wafer, dried, and gold-sprayed, and the capture of E. coli and S. aureus by HBioNzyme is observed under a scanning electron microscope. Figure 3 As shown, the SEM image clearly demonstrates that the spiky structure of HBioNzyme and the network formed by each other can almost completely capture E. coli and S. aureus, providing favorable conditions for its in situ POD-like activity and click chemistry catalytic activity to generate ROS and activate antibacterial agents.

[0050] 3. Agar plate count method to evaluate the in vitro antibacterial ability of HBioNzyme

[0051] First, the precursor molecules 4 and 5 were synthesized, with 3-nitrobenzaldehyde and ethylenediamine as reaction substrates. In the presence of other reagents, the reaction was heated to generate 2-(3-nitrophenyl)-4,5-dihydro-1H-imidazole (NBDI). The imino group on dihydroimidazole was protected by the Boc group to obtain N-boc-2-(3-nitrophenyl)-4,5-dihydro-1H-imidazole. Next, Boc-NBDI was reduced by Pd / C catalyst to obtain the intermediate product N-boc-2-(3-aminophenyl)-4,5-dihydro-1H-imidazole (Boc-ABDI). Boc-ABDI reacted with 4-alkynylbenzoic acid and 4-azidobenzoic acid through carboxyl-amino condensation to obtain Boc-protected 4 and 5, respectively. The product was purified and deprotected with TFA. The synthesis of 4 and 5 was first analyzed by 1H NMR and LC-MS results, and then molecules 4 and 5 were added in the presence of HBioNzyme. The results of LC-MS were used to analyze whether new compounds were generated to detect the click chemistry catalytic activity of HBioNzyme.

[0052] After the precursor molecules 4 and 5 were synthesized and purified, 1H NMR was performed on them to prove the successful synthesis of molecules 4 and 5. 1H NMR of molecule 4: 1H NMR (400 MHz), δ (ppm): 10.69 (s, 1H), 10.54 (s, 2H), 8.51 (s, 1H), 7.99-7.92 (dd, 3H), 7.69-7.61 (m, 4H), 4.44 (s, 1H), 4.02 (s, 4H). 1H NMR of molecule 5: 1H NMR (400 MHz), δ (ppm): 10.62 (s, 1H), 8.50 (s, 1H), 8.07 (d, 2H), 7.93-7.91 (dd, 1H), 7.84 (m, 1H), 7.64-7.63 (m, 2H), 7.32-7.30 (d, 2H), 4.02 (s, 4H). The above results confirmed the successful synthesis of molecules 4 and 5.

[0053] After completing the 1H NMR experiment, LC-MS experiments were continued on molecules 4 and 5 to further prove the successful preparation of the target product. At the same time, molecules 4 and 5 were added in the presence of HBioNzyme for LC-MS experiments. Figure 4 The schematic diagram of the click reaction of molecule 6. In the presence of HBioNzyme, molecules 4 and 5 can be catalyzed to produce active molecule 6 (596.2493), confirming the click catalytic activity of HBioNzyme. Figure 5 shown.

[0054] Pipette 10 μL of bacterial culture in the logarithmic growth phase into a sterile EP tube containing LB medium, and add materials from different groups to make the reaction system 1 mL. Incubate at 37°C in a bacterial incubator for 3 h. After the incubation, dilute the bacterial culture in a sterile EP tube by gradient dilution of 1×10 -6 Take the agar plate, remove the sealing film, burn the plate spreader with an alcohol lamp, let it cool, add 100 μL of diluted bacterial solution to the center of the agar plate, spread it evenly with the plate spreader, put it in a 37°C bacterial incubator, culture it upright for 0.5 hours, and then culture it upside down overnight. The experiment set up six reaction conditions: Control, HBioNzyme, 4+5, HBioNzyme+H 2 O 2 ,HBioNzyme+4+5,HBioNzyme+H 2 O 2 +4+5, where 4, 5, H 2 O 2 The concentrations of HBioNzyme and HBioNzyme were 100 μg mL -1 , 100 μg·mL -1 , 100 μM and 50 μg mL -1 After the reaction is completed, the agar plates with colonies are photographed and counted.

[0055] The in vitro antibacterial activity of HBioNzyme was evaluated by agar plate counting method. Figure 6 As shown in the figure, the control group and HBioNzyme group could hardly inhibit bacterial growth, and a large number of colonies existed. The 4+5 group had a slight inhibitory effect on bacterial growth. 2 O 2 The inhibitory effects of HBioNzyme+4+5 group on E. coli and S. aureus were slightly stronger, and the colony reduction was observed, which proved that the POD-like activity and click chemistry catalytic activity of HBioNzyme inhibited bacterial growth by generating ROS and antibacterial active molecules 6. 2 O 2 The +4+5 group significantly inhibited bacterial growth, with almost no colonies present, which was consistent with the results of the bacterial growth curve, further proving that the POD-like activity and click chemistry catalytic activity of HBioNzyme have a synergistic effect and can effectively kill bacteria.

[0056] IV. Live / dead staining to evaluate the in vitro antibacterial ability of HBioNzyme

[0057] The in vitro antibacterial effect of HBioNzyme was further evaluated by live / dead staining experiments. In this experiment, SYTO-9 (green, representing live bacteria) and PI (representing dead bacteria) were selected as fluorescent dyes for live / dead staining experiments. After the bacteria were cultured to the logarithmic growth phase according to the above method, the bacteria were inoculated into 24-well plates and divided into different groups (Control, HBioNzyme, 4+5, HBioNzyme+H 2 O 2 , HBioNzyme+4+5 and HBioNzyme+H 2 O 2 +4+5) Add materials to make the final volume of each group 1mL. 2 O 2 The concentrations of HBioNzyme and HBioNzyme were 100 μg mL -1 , 100 μg·mL -1 , 100 μM and 50 μg mL -1 . After incubation in a 37℃ bacterial incubator for 3h, the bacterial solution was pipetted into a sterile 1.5mL EP tube, centrifuged at 3500rpm for 5min to remove the supernatant, and washed twice with sterile saline to remove impurities to reduce the impact on fluorescent staining. Resuspend with 1mL sterile saline, add SYTO-9 and PI fluorescent dyes (1:1) for a total of 4μL, mix well, and stain in the dark for 15-20min. After staining, centrifuge at 3500rpm for 5min, remove the supernatant, wash twice with sterile saline, and then resuspend the precipitate with 100μL saline, pipet 10μL of bacterial solution onto a slide and gently cover it with a coverslip, observe and photograph using a fluorescent inverted microscope and save. The excitation wavelength of SYTO-9 is 480nm and the emission wavelength is 500nm. The excitation wavelength of PI is 490nm and the emission wavelength is 635nm.

[0058] In order to further evaluate the in vitro antibacterial properties of HBioNzyme, live / dead staining experiments were used to verify the survival of bacteria after different treatments. SYTO-9 (green) can penetrate the intact cell membrane to label bacteria, emitting green fluorescence. However, PI (red) cannot penetrate the bacterial cell membrane. When the bacteria are damaged or dead, the cell membrane permeability increases, and PI can enter the bacteria, bind to the DNA in the bacterial nucleus, and emit red fluorescence. Figure 7 As shown, a large amount of green fluorescence can be observed in untreated bacteria and bacteria treated with HBioNzyme alone, indicating that almost no bacteria died. 2 O 2Weaker red fluorescence was observed in the HBioNzyme+4+5 treatment group, confirming that the POD-like activity and click chemistry catalytic activity of HBioNzyme can cause certain killing effects on bacteria, leading to the death of some bacteria. 2 O 2 After +4+5 treatment, stronger red fluorescence was observed, proving that the capture effect of HBioNzyme can synergize with POD-like activity and click chemistry catalytic activity to cause significant killing effect on bacteria.

[0059] 5. Cytotoxicity test to evaluate the biocompatibility of HBioNzyme

[0060] The CCK-8 method was used to evaluate the cytotoxicity of HBioNzyme on mouse embryonic fibroblast NIH3T3 cells. After NIH3T3 cells were cultured to the logarithmic growth phase, they were digested with trypsin and resuspended in DMEM medium. The cells were centrifuged at 1000rpm for 3min, the supernatant was discarded, and fresh DMEM medium was replaced and the cells were counted after being blown evenly. A 96-well plate was taken and 2000 cells were plated per well to make the final volume of 100uL per well. After being placed in a 37°C cell culture incubator for 24h, different concentrations of HBioNzyme (0, 12.5, 25, 50, 100, 150, 200μg·mL -1 ), cultured at 37°C for 24 h, discarded the culture medium, added 100 μL of fresh DMEM culture medium and 10 μL of CCK-8 solution to each well, incubated for 3-4 h, measured the absorbance at 450 nm using a microplate reader, and calculated the cell viability.

[0061]

[0062] OD (drug added): absorbance of wells with CCK-8 solution, cells, drug solution and culture medium.

[0063] OD (blank): absorbance of wells with CCK-8 solution, drug solution, culture medium, and no cells.

[0064] OD (0 drug addition): absorbance of wells with CCK-8 solution, cells, culture medium, and no drug.

[0065] After completing the in vitro antibacterial experiment and before applying HBioNzyme to mice, it is necessary to test its biocompatibility, which is an important indicator of biomedical materials. The CCK-8 experiment was used to evaluate the effect of HBioNzyme on the viability of NIH3T3 cells. Figure 8As shown, even when the concentration of HBioNzyme reached 150 μg / mL, the cell survival rate remained above 80% after incubation with NIH3T3 for 48 h, proving that HBioNzyme had no obvious toxicity to NIH3T3 cells and had good biocompatibility.

[0066] VI. Establishment of Mouse Wound Infection Model and Wound Healing Evaluation In Vivo Antibacterial Activity Test of HBioNzyme

[0067] In order to evaluate the antibacterial effect of HBioNzyme in vivo, a full-thickness skin defect infection model was established on the back of BALB / c mice for animal experiments. The mice were anesthetized, the hair on the back of the mice was removed with a depilatory cream, and a full-thickness wound with a diameter of about 8 mm was created on the back of the mice through skin puncture. Subsequently, 30 μL of 1×10 8 CFU mL -1 E. coli suspension was used to construct a wound infection model. After 24 hours of bacterial infection, all mice were randomly divided into four groups (PBS, HBioNzyme, HBioNzyme+H 2 O 2 HBioNzyme+H 2 O 2 +4+5), and treated the wounds of mice according to the groups. The materials were replaced every three days, and the changes in the wound area and the recovery of the mice during the treatment were measured and recorded.

[0068] like Fig. 9 As shown in the figure, by analyzing the size and area of ​​mouse skin wounds and healing conditions recorded during the treatment, and quantitatively analyzing the wound area of ​​mice using ImageJ, it can be seen that the control group still has larger wounds and large scabs after the treatment, proving that the wound recovery is poor and there is still infection and inflammation. The wound area of ​​the HBioNzyme group was slightly reduced, and scabs were present, indicating that HBioNzyme alone has limited inhibitory effect on bacteria in the wound, while HBioNzyme+H 2 O 2 The wound area of ​​the group was further reduced and there was no obvious scab, which proved that this group of treatment had a certain promoting effect on the recovery of mouse skin wounds. 2 O 2 The wounds in the +4+5 group were almost completely healed, which fully demonstrated that it can effectively fight bacterial infection in the body and promote wound healing in mice.

[0069] 8. H&E and Masson staining of mouse wound tissue to evaluate inflammation and wound healing

[0070] H&E staining steps:

[0071] (1) Dewaxing and hydrating sections: bake the prepared paraffin sections at 65°C for 30 min, wash them three times in xylene solution for 5 min, wash them three times in anhydrous ethanol for 5 min each, then wash them in 95%, 90%, 80%, and 70% ethanol for 5 min each, and wash them twice in distilled water for 5 min.

[0072] (2) Staining: Add hematoxylin staining solution, stain for 3 minutes, rinse with running water for 2 minutes. Remove the tissue sections and shake dry, add differentiation solution for 3 minutes, rinse with running water for 2 minutes. Then add 0.5% ammonia water to turn blue for 2 minutes, rinse with running water for 2 minutes. Dehydrate twice with 95% ethanol, 2 minutes each time. Add eosin staining solution for 1 minute, rinse with running water for 2 minutes.

[0073] (3) Dehydration and sealing: Rinse the slices quickly in 70%, 80%, 90%, and 95% ethanol in sequence, then wash three times in anhydrous ethanol for 5 minutes, and finally dehydrate in xylene solution twice, each time for 5 minutes. After the slices are dry, seal them with neutral resin.

[0074] (4) Observation under a microscope: Observe the tissue morphology under a microscope and scan the slides for preservation.

[0075] Masson staining steps:

[0076] (1) Dewaxing and hydrating the sections were performed in the same manner as the H&E staining procedure above.

[0077] (2) Staining: Place the slices in the mordant solution and incubate overnight at room temperature. Then place the slices in a 60°C incubator for 1 hour for mordant dyeing, and then rinse with running water for 10 minutes. Add lapis lazuli blue staining solution for 2 minutes, rinse with water twice, 15 seconds each time. Add Mayer's hematoxylin staining solution for 2 minutes, rinse with distilled water twice, 15 seconds each time. Differentiate with acidic differentiation solution for a few seconds, rinse with water to terminate differentiation, and rinse with running water for 10 minutes. Drip with Ponceau-fuchsin staining solution for 10 minutes, rinse with distilled water twice, 10-15 seconds each time. Treat with phosphomolybdic acid solution for about 10 minutes. Discard the upper solution, do not wash the slices with water, and directly add aniline blue staining solution for 5 minutes. After washing away the aniline blue with 1% glacial acetic acid dilution, add 1% glacial acetic acid to cover the slices and treat for 2 minutes.

[0078] (3) Dehydrate and seal the slides for H&E staining.

[0079] (4) Observation under a microscope: Observe the tissue morphology under a microscope and scan the slides for preservation.

[0080] In order to further analyze the effects of different treatment groups on mouse wounds, the skin wound tissues of mice in each group were collected for histological analysis by H&E staining and Masson staining. Fig.10As shown in the figure, obvious skin structural damage and a large number of inflammatory cell infiltration were observed in the Control group and HBioNzyme, indicating that the wound recovery was poor. 2 O 2 The skin structure of the group was relatively intact, but some inflammatory cells still existed, indicating that the wound had not fully recovered. 2 O 2 The skin structure of mice treated with HBioNzyme+H was intact, new hair follicles were observed, and no obvious inflammatory cell infiltration was found, confirming the efficacy of HBioNzyme+H 2 O 2 The treatment effect of the +4+5 group was the best, which could effectively inhibit bacterial growth and promote wound recovery. From the Masson staining results, it can be seen that compared with the other treatment groups, the HBioNzyme+H 2 O 2 +4+5 The skin tissue structure after treatment was intact, and more and darker blue could be observed, indicating that there was a larger area of ​​new and continuous collagen fibers in the skin tissue of this group, showing the HBioNzyme+H 2 O 2 The +4+5 group had stronger wound repair ability, which was consistent with the recovery of wound tissue.

[0081] IX. Conclusion

[0082] The preparation method of the present invention is simple and easy to operate. The prepared nanozyme can capture ROS and antibacterial active molecules generated in situ by bacteria through its efficient bacteria capture ability and dual catalytic activity, causing obvious killing effect on bacteria, resulting in wrinkling, rupture and even death of the bacterial surface. HBioNzyme has efficient enzyme-mimicking catalysis and wound healing performance, can be used to inhibit bacterial growth, destroy biofilm and promote wound healing, and will not cause problems such as bacterial resistance. It has practical application value, broadens the scope of biomedical applications, provides new ideas for the antibacterial application of bionic nanozymes, and has significant economic and social benefits.

[0083] It should be pointed out that the above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession can make changes or modify the technical contents disclosed above into equivalent embodiments with equivalent changes without departing from the scope of the technical solution of the present invention, which all fall within the protection scope of the present invention.

Claims

1. A method for preparing a nanozyme with high-efficiency bacteria capture capability and dual catalytic activity, characterized in that: The following steps are involved: 1) Synthesis of HOF Weigh terephthalamidine hydrochloride and dissolve it in water to form solution A. Weigh tetrakis(4-carboxyphenyl)methane and dissolve it in water. Continue to add NaOH solution to deprotonate and form solution B. Transfer solution A to a round-bottom flask. Slowly add solution B dropwise into solution A. Under stirring conditions at room temperature, a precipitate can be observed to form immediately. Stir and centrifuge to collect the product. Discard the supernatant. Continue to centrifuge and wash three times under the same conditions to remove any unreacted precursors. After washing, the product is evenly dispersed with water and stored in a refrigerator to obtain HOF. 2) Synthesis of PdCu Weigh KBr solid and add it to a round-bottom flask of polyethylene glycol under stirring, then add H2PdCl4 and CuCl2•2H2O respectively, stir and mix well, then continue to add HCl and ascorbic acid solution, stir well, and react in an oil bath without any disturbance. After the reaction is completed, collect the material by centrifugation, centrifuge, wash three times, disperse in water and place in a refrigerator to obtain PdCu; 3) Synthesis of HBioNzyme The HOF prepared in step 1) is added to the PdCu prepared in step 2) and H2O, and stirred at room temperature to make them fully mixed. After stirring, the mixture is homogenized by ultrasonication and then subjected to oil bath reaction without any disturbance. When a black precipitate is observed on the wall of the flask and the solution is almost colorless, it indicates that the reaction is over. The reaction is stopped and the material is collected by centrifugation, washed three times, and stored in water in a refrigerator to obtain HBioNzyme.

2. The method for preparing the nanozyme with high-efficiency bacteria capture ability and dual catalytic activity according to claim 1, characterized in that: The following steps are involved: 1) Synthesis of HOF Weigh 0.5-1 mg of terephthalamidine hydrochloride and dissolve it in 1 mL of water to form solution A. Weigh 1-2 mg of tetrakis(4-carboxyphenyl)methane and dissolve it in 1-3 mL of water. Continue to add 5-10 μL of 2M NaOH solution to deprotonate and form solution B. Transfer solution A to a 25 mL round-bottom flask. Slowly add solution B dropwise to solution A. Under room temperature stirring conditions, a precipitate can be observed to form immediately. After stirring for 5-10 minutes, centrifuge at 5000-7000 rpm for 4-6 minutes to collect the product. Discard the supernatant and continue centrifugation and washing three times under the same conditions to remove any unreacted precursors. After washing, the product is evenly dispersed with 1 mL of water and stored in a refrigerator at 3-5°C to obtain HOF. 2) Synthesis of PdCu Weigh 120-150 mg of KBr solid, add it to a round-bottom flask containing 4-6 mL of polyethylene glycol 600 with a mass percentage concentration of 0.01% under stirring, then add 1.25-2 mL of H2PdCl4 with a molar concentration of 20 mM and 100-500 μL of CuCl2•2H2O with a molar concentration of 20 mM, respectively, stir and mix, then continue to add 80-120 μL of HCl with a molar concentration of 6 M and 1.2-1.5 mL of ascorbic acid solution with a molar concentration of 0.1 M, stir evenly, react in an oil bath at 90-95°C for 30 minutes without any disturbance, after the reaction is completed, collect the material by centrifugation, wash it three times by centrifugation at 5000-7000 rpm for 4-6 minutes, disperse it in water and place it in a refrigerator at 3-5°C to obtain PdCu; 3) Synthesis of HBioNzyme Add 25-35 mg HOF prepared in step 1) to 15-20 mg PdCu and 3-5 mL H2O prepared in step 2), and stir at room temperature for 0.5-1 h to mix them thoroughly. After stirring, ultrasonically homogenize the mixture, and react it in an oil bath at 80-85°C for 30-40 min without any disturbance. When a black precipitate appears on the wall of the flask and the solution is almost colorless, the reaction is completed. Stop the reaction and collect the material by centrifugation at 5000-7000 rpm for 4-6 min, wash it three times, and store it in water in a refrigerator at 3-5°C to obtain HBioNzyme.

3. The method for preparing the nanozyme with high-efficiency bacteria capture ability and dual catalytic activity according to claim 1, characterized in that: The following steps are involved: 1) Synthesis of HOF Weigh 0.78 mg of terephthalamidine hydrochloride and dissolve it in 1 mL of water to form solution A. Weigh 1.56 mg of tetrakis(4-carboxyphenyl)methane and dissolve it in 1 mL of water. Continue to add 5 μL of 2M NaOH solution to deprotonate and form solution B. Transfer solution A to a 25 mL round-bottom flask. Slowly add solution B dropwise to solution A. Under room temperature stirring conditions, a precipitate can be observed to form immediately. After stirring for 8 minutes, centrifuge at 6000 rpm for 5 minutes to collect the product. Discard the supernatant and continue centrifugation and washing three times under the same conditions to remove any unreacted precursors. After washing, the product is evenly dispersed with 1 mL of water and stored in a refrigerator at 4°C to obtain HOF. 2) Synthesis of PdCu Weigh 138 mg of KBr solid and add it to a 5 mL round-bottom flask containing 0.01% polyethylene glycol 600 under stirring. Then, add 1.25 mL of H2PdCl4 with a molar concentration of 20 mM and 500 μL of CuCl2•2H2O with a molar concentration of 20 mM, respectively. After stirring, add 100 μL of HCl with a molar concentration of 6 M and 1.5 mL of ascorbic acid solution with a molar concentration of 0.1 M. After stirring, react in an oil bath at 95°C for 30 min without any disturbance. After the reaction is completed, collect the material by centrifugation, wash it three times by centrifugation at 6000 rpm for 5 min, disperse it in water and place it in a refrigerator at 4°C to obtain PdCu. 3) Synthesis of HBioNzyme Add 30 mg HOF prepared in step 1) to 15 mg PdCu and 4 mL H2O prepared in step 2), and stir at room temperature for 1 h to mix them thoroughly. After stirring, ultrasonically homogenize the mixture, and react it in an oil bath at 80°C for 30 min without any disturbance. When a black precipitate appears on the wall of the flask and the solution is almost colorless, the reaction is completed. Stop the reaction and collect the material by centrifugation at 6000 rpm for 5 min, wash it three times, and store it in water in a refrigerator at 4°C to obtain HBioNzyme.

4. Use of the nanozyme with efficient bacteria capture ability and dual catalytic activity prepared by any one of the preparation methods of claims 1 to 3 in the preparation of anti-bacterial infection drugs.

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