Nano-enzyme hydrogel with hydrogen controlled release performance as well as preparation method and application of nano-enzyme hydrogel

Abstract

The invention belongs to the technical field of medical instruments, and particularly relates to nano-enzyme hydrogel with hydrogen controlled release performance and a preparation method and application thereof.The nano-enzyme hydrogel with the hydrogen controlled release performance is characterized in that palladium nano-enzyme and hydrogen form a core of a nano-drug, and the hydrogen is loaded on the palladium nano-enzyme to obtain palladium hydride nanoparticles; mixing the nanoparticles with hydrogel to prepare composite hydrogel loaded with palladium hydride nanoparticles; the palladium nanoparticles have the activity of catalase and superoxide dismutase, and can remove ROS (reactive oxygen species) and regulate immune inflammation; the palladium nano-enzyme loaded hydrogen is used as a signal molecule to block a downstream inflammatory pathway, and the palladium nano-enzyme loaded hydrogen and the signal molecule have a synergistic effect to inhibit inflammation and promote wound healing; according to the hydrogel, the traditional injection administration mode is changed, meanwhile, the effects of regulating immune inflammation and restoring a normal immune microenvironment are achieved, and the composite hydrogel can be used for treating diabetic skin wounds and becomes a new strategy for promoting healing of the diabetic wounds.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical devices, and in particular relates to a nanoenzyme hydrogel with controlled hydrogen release performance, and a preparation method and application thereof. Background Art

[0002] In recent years, the prevalence of diabetes in my country has shown a clear upward trend. As a chronic disease, diabetes and its complications have gradually affected all aspects of people's lives. Diabetic foot (DFU) is the most common complication in diabetic patients. It has high morbidity, mortality and recurrence rates and is the main cause of non-traumatic amputation worldwide. Wound healing in diabetes is hindered by the continuous release of inflammatory factors, excessive accumulation of free radicals (ROS) and inflammatory cascade reactions. In addition, the high pro-inflammatory state inhibits the formation of granulation tissue, the deposition of collagen and the maturation of blood vessels. Therefore, effectively inhibiting the continuous release of inflammatory cytokines is one of the most promising methods to promote functional recovery and healing of diabetic wounds.

[0003] Nanozymes with natural enzyme-like activity have made new breakthroughs in the field of biomedical engineering due to their stability and high catalytic activity, and have been applied in various fields. In particular, nanomaterials with both superoxide dismutase-like and catalase-like catalytic activities can remove ROS through nanozyme cascade reactions, and their research has received widespread attention. Hydrogen is a colorless, odorless, and harmless gas that is widely present in nature. The hydrogen molecules that make up hydrogen are the smallest molecules in nature. Therefore, hydrogen has strong diffusivity and penetration and can efficiently pass through various biological barriers. The in situ release of hydrogen can not only remove excessive ROS, but also inhibit inflammatory pathways, thereby reducing the expression of pro-inflammatory cytokines and inflammation of wound tissue. However, most of the documented wound healing methods have major limitations, such as hydrogen-rich water (HRW) and hydrogen-rich saline (HRS), because the low solubility of hydrogen limits its ability to penetrate tissues. Hydrogel materials composed of biocompatible macromolecules are considered to have significant advantages in wound treatment. They provide a hydrated microenvironment for the wound. This moist healing environment is conducive to the formation and growth of granulation tissue, thereby accelerating the wound healing process and providing a physical barrier to microbial infection and moist environment. It is worth noting that traditional hydrogels have problems such as single function and difficulty in fixation. Therefore, the existing technology still needs to be improved and developed. Summary of the invention

[0004] The purpose of the present invention is to provide a nanoenzyme composite hydrogel with controlled hydrogen release performance and a preparation method and application thereof, so as to restore the normal immune microenvironment of the DFU affected area.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: A method for preparing a nanozyme hydrogel with controlled hydrogen release performance comprises the following steps: S1. Synthesis of palladium nanozyme solution: 44 ~ 45 mg palladium chloride was dissolved in 0.02 M hydrochloric acid solution at 45 ~ 55 ° C to obtain 0.01 M sodium tetrachloropalladate solution for use; 0.55 ~ 0.65 ml 0.01 M sodium borohydride solution was added to a mixed solution of 9.7 ~ 9.8 ml sodium hexadecyl sulfate solution and 0.2 ~ 0.3 ml sodium tetrachloropalladate solution under stirring, and the mixture was allowed to stand at room temperature for 3 ~ 5 h to obtain a seed solution; the seed solution was added to a mixed solution of 9.5 ~ 10 ml 4.5 mM CTAC solution and 0.25 ~ 0.35 ml 0.01 M sodium tetrachloropalladate solution, and 0.1 ~ 0.15 ml 0.1 M ascorbic acid solution was added, the mixture was stirred for 10 ~ 20 s, and the mixture was allowed to stand at room temperature for 1 ~ 1.5 h. h, centrifuging, washing, dispersing the obtained palladium nanozyme in deionized water to obtain a palladium nanozyme solution, and storing it in the dark for later use; S2. Synthesis of palladium hydride nanoparticle solution: 1.5 ~ 2.5 mL of palladium nanozyme solution is placed in container A and sealed with a rubber stopper, 95 ~ 105 mg of sodium borohydride is placed in container B and sealed with a rubber stopper, the two containers are connected by a pipe, a needle connected to the atmosphere is set in the rubber stopper of container A, 2 ~ 3 ml of sulfuric acid solution with pH = 4.5 ~ 5.5 is injected into container B, hydrogen is generated and blown into the palladium nanozyme solution in container A, after 20 ~ 30 minutes, the solution containing the final product palladium hydride nanoparticles is sealed and stored away from light; S3. Preparation of nanozyme hydrogel: 1.2 ~ 1.4g acrylamide, 0.31 ~ 0.33g alginate and 0.25 ~ 0.75g trehalose were dissolved in 10ml 200μg / ml palladium hydride nanoparticle solution to prepare a mixed solution. After stirring the solution for 6h, 0.0010 ~ 0.0020g MBA, 0.0040 ~ 0.0060g TMEDA, 0.0135 ~ 0.0235g APS and 0.02 ~ 0.03g CaSO4∙2H2O were added. After degassing, the solution was placed in a mold and allowed to stand at room temperature for 22 ~ 26h to obtain the nanozyme hydrogel.

[0006] Furthermore, in step S1, the centrifugation is performed at 8000-10000 r / min for 10-15 min.

[0007] Furthermore, the degassing in step S3 is performed by standing at 25-28° C. for 1-2 h.

[0008] A method for preparing a nanozyme hydrogel with controlled hydrogen release performance and application of the nanozyme hydrogel with controlled hydrogen release performance. The nanozyme hydrogel is used in wound dressings to treat skin wounds of diabetic patients.

[0009] Therapeutic mechanism: According to research, palladium is a single-atom nanozyme with good catalase (CAT) and superoxide dismutase (SOD) activities, which are equivalent to normal enzymes. It can eliminate excessive accumulation of ROS and inhibit the production and secretion of inflammatory factors in chronic wounds. At the same time, palladium is also a good hydrogen storage material, which can achieve large-scale hydrogen loading and controllable (NIR response) release. Hydrogen has a certain reducing property and is an endogenous signal molecule in the human body. It can inhibit the expression of inflammatory factor receptors and synergize with palladium to block downstream inflammatory pathways, inhibit the release of inflammatory factors, and restore the normal immune microenvironment. This hydrogel dressing has high mechanical properties and good adhesion for various skin injuries, and can quickly promote wound healing. It is non-irritating, safe, and has no toxic side effects on the wound. Compared with the positive control group and other prescription hydrogel groups, the effect is more excellent.

[0010] The present invention has the advantages that: 1. The palladium nanoenzyme hydrogel prepared by the present invention plays a dual role in the dressing. First, it is an enzyme catalyst. Due to its catalase-like activity and superoxide dismutase-like activity, it can remove ROS, inhibit downstream inflammatory pathways, and restore the normal immune microenvironment; second, it can be used as a carrier of hydrogen to achieve controlled release of hydrogen; 2. The palladium hydride nanoparticles prepared by the present invention can achieve near-infrared controllable hydrogen release, inhibit the expression of inflammatory factor receptors, and cooperate with palladium nanozymes to block inflammatory pathways, regulate inflammation, and restore the normal immune microenvironment; 3. The hydrogel prepared by the present invention has good biocompatibility, high safety, good bonding strength and mechanical properties, and can be better applied to DFU due to the repair effect of a large number of hydroxyl groups in trehalose on hydrogel network defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The ESR spectra of CAT and SOD enzyme activities in Pd solutions with different concentrations; Figure 2 Schematic diagram of hydrogen release from hydrogen-rich water, PdH and PdH + NIR; Figure 3 is a SEM scan of the freeze-dried hydrogel of the present invention; Figure 4 The mechanical properties and adhesion test results of the hydrogel of the present invention; Figure 5 The results of the cytotoxicity test of the hydrogel of the present invention are as follows; Figure 6 The results of the in vitro ROS removal experiment of the hydrogel of the present invention; Figure 7 The results of the wound healing experiment on mice in different groups; Figure 8 Immunofluorescence images of CD86, CD206 and TNF-α in wound tissue sections of different groups on day 10; Fig. 9 This is a diagram of the formation mechanism and treatment mechanism of the hydrogel of the present invention. DETAILED DESCRIPTION

[0012] Example 1 A method for preparing a nanozyme hydrogel with controlled hydrogen release performance comprises the following steps: 1. Dissolve 44.5 mg of palladium chloride (PdCl2) in 25 ml of 0.02 M hydrochloric acid solution at 50°C to obtain 0.01 M sodium tetrachloropalladate solution. Add 0.60 ml of fresh 0°C 0.01 M sodium borohydride solution to a mixed solution of 9.75 ml of sodium hexadecyl sulfate solution and 0.25 ml of sodium tetrachloropalladate solution under vigorous stirring at 1000 rpm / min, and let stand at room temperature for 3 h to obtain a seed solution. Add 0.025 ml of the seed solution to a mixed solution of 9.7 ml 4.5 mM sodium hexadecyl sulfate solution and 0.3 ml 0.01 M sodium tetrachloropalladate solution, add 0.1 ml 0.1 M ascorbic acid solution, stir and mix for 10 s, let stand at room temperature for 1 h, centrifuge, and wash. Finally, disperse the obtained palladium nanozyme in deionized water and store it away from light for later use.

[0013] 2. Place 2 mL of palladium nano solution into a 10 mL vial A and seal it with a rubber stopper; add 100 mg of sodium borohydride into vial B, also seal it with a rubber stopper, and connect the two vials with a rubber cap. Insert the needle of a 1 mL syringe into the rubber stopper of vial A, connect it to the atmosphere, and inject 2 ml of pH = 5 sulfuric acid solution into vial B to generate hydrogen, which is slowly blown into the palladium nano solution. After 25 minutes, seal the solution containing the final product, palladium hydride nanoparticles, and store it away from light.

[0014] 3. Dissolve 1.35 g acrylamide (Paam), 0.325 g alginate (Algnate, Alg) and 0.5 g trehalose in 10 ml 200 μg / ml palladium hydride (PdH) solution to form a mixed aqueous solution. Stir the solution for 6 h, add 0.0015 g MBA, 0.005 g TMEDA, 0.0235 g APS and 0.02 g CaSO4∙2H2O, degas and place in a mold, and let stand at room temperature for 24 h to obtain PATP hydrogel.

[0015] Example 2 45 mg of palladium chloride (PdCl2) was dissolved in 26 ml of 0.02 M hydrochloric acid solution at 55 °C to obtain 0.01 M sodium tetrachloropalladate solution. Under vigorous stirring (1000 rpm / min), 0.65 ml of fresh ice-cold (0 °C) 0.01 M sodium borohydride solution was added to a mixed solution of 9.8 ml sodium hexadecyl sulfate solution and 0.3 ml sodium tetrachloropalladate solution, and the mixture was allowed to stand at room temperature for 5 h to obtain a seed solution. 0.03 ml of the seed solution was added to a mixed solution of 10 ml 4.5 mM sodium hexadecyl sulfate solution and 0.35 ml 0.01 M sodium tetrachloropalladate solution, and 0.15 ml of 0.1 M ascorbic acid solution was added, stirred and mixed for 20 s, allowed to stand at room temperature for 1.5 h, centrifuged, and washed. Finally, the obtained palladium nanozyme was dispersed in deionized water and stored in the dark for later use.

[0016] 2. Place 2.5 mL of palladium nano solution into a 10 mL vial A and seal it with a rubber stopper; add 105 mg of sodium borohydride into vial B, also seal it with a rubber stopper, and connect the two vials with a rubber cap. Insert the needle of a 1 mL syringe into the rubber stopper of vial A, connect it to the atmosphere, and inject 3 ml of pH = 5 sulfuric acid solution into vial B to generate hydrogen, which is slowly blown into the palladium nano solution. After 30 minutes, seal the solution containing the final product, palladium hydride nanoparticles, and store it away from light.

[0017] 3. Dissolve 1.4 g acrylamide (Paam), 0.33 g alginate (Algnate, Alg) and 0.75 g trehalose in 10 ml 200 μg / ml palladium hydride (PdH) solution to form a mixed aqueous solution. Stir the solution for 6 h, add 0.0020 g MBA, 0.006 g TMEDA, 0.0235 g APS and 0.03 g CaSO4∙2H2O, degas and place in a mold, and let stand at room temperature for 26 h to obtain PATP hydrogel.

[0018] Example 3 44 mg of palladium chloride (PdCl2) was dissolved in 24 ml of 0.02 M hydrochloric acid solution at 45 °C to obtain 0.01 M sodium tetrachloropalladate solution. Under vigorous stirring (1000 rpm / min), 0.55 ml of fresh ice-cold (0 °C) 0.01 M sodium borohydride solution was added to a mixed solution of 9.7 ml sodium hexadecyl sulfate solution and 0.2 ml sodium tetrachloropalladate solution, and the mixture was allowed to stand at room temperature for 4 h to obtain a seed solution. 0.02 ml of the seed solution was added to a mixed solution of 9.5 ml 4.5 mM sodium hexadecyl sulfate solution and 0.25 ml 0.01 M sodium tetrachloropalladate solution, and 0.1 ml of 0.1 M ascorbic acid solution was added, stirred for 15 s, allowed to stand at room temperature for 1 h, centrifuged, and washed. Finally, the obtained palladium nanozyme was dispersed in deionized water and stored in the dark for later use.

[0019] 2. Place 1.5 mL of palladium nano solution into a 10 mL vial A and seal it with a rubber stopper; add 95 mg of sodium borohydride into vial B, also seal it with a rubber stopper, and connect the two vials with a rubber cap. Insert the needle of a 1 mL syringe into the rubber stopper of vial A, connect it to the atmosphere, and inject 2 ml of pH = 5 sulfuric acid solution into vial B to generate hydrogen, which is slowly blown into the palladium nano solution. After 25 minutes, seal the solution containing the final product, palladium hydride nanoparticles, and store it away from light.

[0020] 3. Dissolve 1.2 g acrylamide (Paam), 0.31 g alginate (Algnate, Alg) and 0.25 g trehalose in 10 ml 200 μg / ml palladium hydride (PdH) solution to form a mixed aqueous solution. Stir the solution for 6 h, add 0.001 g MBA, 0.004 g TMEDA, 0.0135 g APS and 0.02 g CaSO4∙2H2O, degas and place in a mold, and let stand at room temperature for 24 h to obtain PATP hydrogel.

[0021] Performance Testing The PATP hydrogel prepared in Example 1 was used as the experimental object, and polyacrylamide hydrogel (P), polyacrylamide-alginate hydrogel (PA), and polyacrylamide-alginate-trehalose hydrogel (PAT) were prepared as controls. Each group of tests was carried out under the same conditions.

[0022] Electron spin resonance (ESR) detection technology was used to determine the catalase and superoxide dismutase activities of palladium nanozymes. A series of palladium nanozyme solutions with different concentrations (0, 25, 50, 100, 50 and 200 μg / mL) were prepared at 37°C for ESR detection. The results are shown in Figure 1 : As can be seen from the figure, palladium nanozyme exhibited higher peroxidase (3.44 ± 0.22 U / ml) and superoxide dismutase activities (5.17 ± 0.41U / ml), and the peroxidase and superoxide dismutase activities of palladium were concentration-dependent.

[0023] Measurement of hydrogen release from palladium hydride: The release of hydrogen was carried out using the method reported by (P. Zhao, Z. Jin, Q. Chen, T.Yang, D. Chen, J. Meng, X. Lu, Z. Gu, Q. He, Nat. Commun. 2018, 9, 4241.). In the presence of a palladium catalyst, MB can be reduced to colorless MB (leucomethylene blue, leucoMB) by hydrogen, and its absorbance at 664 nm is linearly correlated with the amount of hydrogen. Palladium hydride was dispersed in the MB probe solution in a cuvette and monitored in real time by an ultraviolet spectrophotometer. Hydrogen release experiments were carried out under near-infrared irradiation and without near-infrared irradiation. The near-infrared irradiation group was irradiated with an 808 nm laser (1.2 W cm-2) for 2 min at 0 h and 6 h, and each group was observed for 24 h. The concentration of hydrogen release can be calculated based on the final absorbance of MB and the standard curve formula. Each sample was measured 3 times and the average value was taken. The results are shown in Figure 2. Figure 2 :Hydrogen-rich water (1.20 ± 0.41 µm) and palladium hydride nanoparticles (1.83 ± 0.05 µm) can release a small amount of hydrogen; under near-infrared irradiation, palladium hydride nanoparticles significantly enhanced the reduction of MB and released a large amount of hydrogen (12.56 ± 0.33 µm).

[0024] Depend on Figure 3It can be seen that the synthesized hydrogel presents a loose and porous gel network structure. Further increasing the SEM observation magnification of the PATP hydrogel complex, it can be seen that the PdH nanoparticles are evenly distributed in the hydrogel pore network, proving the uniform loading of the PdH nanoparticles in the PATP hydrogel complex.

[0025] The mechanical properties and adhesion properties of the hydrogel to the pig skin surface were tested by the methods in the reference literature (Y. Cai, C. Liu, K. Gong, H. Li, H. Song, Y. Zhang, D. Ding, J. Liu, J. Guo, L. Fang, Chemical Engineering Journal. 2023, 465,142942; Cui Chunyan et al. "A high-strength and fast-adhesive nanohybrid hydrogel "Band-Aid", Journal of Polymer Science, 2019, 50 (6): 613-622). The results are as follows Figure 4 The addition of trehalose improved the mechanical properties of the hydrogel. The elongation at break of the final formulated PATP hydrogel was 597.23%, and the tensile strength was enhanced to 9.49 KPa, which can better prevent the hydrogel from deforming or breaking after being attached to the skin. At the same time, the addition of trehalose also improved the adhesion of the hydrogel. The rolling ball stopping distance of the PATP hydrogel was 3.43 cm, and the shear adhesion strength reached 10.58 KPa, proving that it has good adhesion and is more suitable for DFU.

[0026] Cytotoxicity test: The CCK8 method was used to test the cytotoxicity of the hydrogel; the experiment tested the effect of the hydrogel on the proliferation of HUVECs cells and RAW 264.7 cells. HUVECs and RAW 264.7 cells were cultured at 5×10 3 The cells were seeded into a 96-well plate at a density of and incubated at 37°C for 24 hours. After 24 hours of incubation, the cell culture medium was replaced with fresh culture medium and treated with control (Negative), hydrogen peroxide (Active), hydrogen peroxide + PATP hydrogel (Group 1) and hydrogen peroxide + PATP + NIR (Group 2), respectively. Then, the above cell culture medium was removed, and the obtained hydrogel extract was added to a 96-well plate, and a new culture medium containing 10% CCK-8 was added. After incubation for 4 hours, the absorbance of the solution was measured at 450 nm. The formula for calculating cell viability is cell viability = Ah / Ac×100%, where Ah represents the absorbance of the hydrogel extract-treated group, and Ac represents the absorbance of the control group. The results are shown in Figure 5 : Within 3 days, the cell viability of the experimental group was basically similar to that of the negative control group, indicating that the toxicity of PATP hydrogel was negligible.

[0027] In vitro antioxidant capacity of hydrogels: The ability of hydrogels to remove ROS in vitro was tested by ROS fluorescent probe detection. RAW 264.7 cells were inoculated in confocal culture dishes. Cell culture medium was extracted 24 hours after inoculation, and then the cells were treated with culture medium, including hydrogen peroxide solution treated with different hydrogels. After adding DCFH-DA, the cells were observed using a laser scanning confocal microscope. The results are shown in Figure 6 : The hydrogen peroxide group showed severe oxidative damage and the highest fluorescence intensity. The PATP group had a lower fluorescence intensity due to the scavenging effect of palladium's enzymatic activity on ROS. In the PATP + NIR group, palladium and hydrogen worked synergistically, so it had the lowest fluorescence intensity. These results demonstrated the superior antioxidant capacity of PATP hydrogel and highlighted its potential for application in wound healing.

[0028] Application Examples In the diabetic wound healing test, the effect of the PATP hydrogel prepared in Example 1 was verified.

[0029] Diabetic mouse modeling: 21 male C57 / BL6J mice weighing 20-25g were selected. After gradually adding high-fat diet for 1 week, they were continuously fed with high-fat diet for 1 month in a specific pathogen-free environment. The pre-prepared streptozotocin (STZ) solution was intraperitoneally injected at 50 mg / kg for 5 consecutive days (STZ was dissolved in 0.1 mol / L, pH 4.2-4.5 sodium citrate buffer). After one week, the blood glucose level of mice was measured through the tail vein. The fasting blood glucose of mice above 11.1 mmol / L was monitored for one week to confirm the success of the modeling.

[0030] Back full-thickness excisional wound model of diabetic mice: The mice were randomly divided into 7 groups (Normal, Diabetic, PG, PAG, PATG, PATPG and PATPG + NIR), with 3 mice in each group. The mice were anesthetized in a device containing 1.5% isoflurane and 60% oxygen. A circular full-thickness wound with a diameter of 0.8 cm was made on the back of the mice. Subsequently, different samples were applied to the wound site. On the 0th, 3rd, 5th, 7th and 10th days after surgery, the wounds were photographed with a digital camera, and the photographic results were analyzed using image processing software. At the same time, the weight of the mice was recorded. Results Figure 7 : Compared with the diabetes positive control group, the PATPG group and the PATPG + NIR group showed a significant reduction in wound area after a 3-day treatment period. After 7 days, the wound area of ​​the PATPG + NIR group was reduced to 18.7%. On the 10th day, the diabetes positive control group still had 45% of the original wound area, while the wound area of ​​the PATPG group was 11.9%. In contrast, the PTAPG + NIR group had the smallest remaining wound area, only 7.3%.

[0031] Anti-inflammatory properties of the hydrogel of the present invention Macrophages play a key role in the local immune response after tissue injury, especially in diabetes-related wounds. The gradual shift of macrophages from an inflammation-inducing (M1) state to an anti-inflammatory (M2) state negatively affects tissue regeneration. Therefore, we used immunofluorescence assays to assess the polarization of macrophages. Figure 8 : The percentage of CD86 (M1 biomarker) decreased in the different hydrogel-treated groups compared to the group that did not receive any treatment, with the PATPG + NIR group having the weakest immunofluorescence intensity. In contrast, the mean fluorescence intensity of CD206 (M2 biomarker) showed an increase in the groups treated with different hydrogels. We also evaluated the expression of tumor necrosis factor (TNF-ɑ), an important cytokine involved in the immune response, at the wound site. The results are shown in Figure 2. Figure 8 : Among all experimental groups, the expression level of TNF-ɑ in the diabetes positive control group was the highest, which was twice that of the PATPG+ NIR group.

[0032] The above results show that the nanozyme composite hydrogel with controlled hydrogen release performance prepared by the present invention has good biocompatibility, adhesion performance and diabetic wound healing performance. The Pd nanozyme has catalase and superoxide dismutase activity, which can remove excess ROS in the wound and reduce oxidative stress. The hydrogen released from Pd synergizes with Pd to inhibit downstream inflammatory pathways, reduce the expression of inflammatory factors, alleviate inflammation, restore the normal immune microenvironment, and effectively promote diabetic wound healing.

Claims

1. A method for preparing a nanozyme hydrogel with controlled hydrogen release performance, characterized in that: The following steps are involved: S1. Synthesis of palladium nanozyme solution: 44-45 mg palladium chloride was dissolved in 0.02 M hydrochloric acid solution at 45-55°C to obtain 0.01 M sodium tetrachloropalladate solution for use; 0.55-0.65 ml 0.01 M sodium borohydride solution was added to a mixed solution of 9.7-9.8 ml sodium hexadecyl sulfate solution and 0.2-0.3 ml sodium tetrachloropalladate solution under stirring, and the mixture was allowed to stand at room temperature for 3-5 h to obtain a seed solution; The seed solution was added to a mixed solution of 9.5-10 ml 4.5 mM CTAC solution and 0.25-0.35 ml 0.01M sodium tetrachloropalladate solution, and 0.1-0.15 ml 0.1M ascorbic acid solution was added, and the mixture was stirred for 10-20 s, and allowed to stand at room temperature for 1-1.5 h, and then centrifuged and washed. The obtained palladium nanozyme was dispersed in deionized water to obtain a palladium nanozyme solution, and stored in the dark for later use; S2. Synthesis of palladium hydride nanoparticle solution: 1.5 ~ 2.5 mL of palladium nanozyme solution is placed in container A and sealed with a rubber stopper, 95 ~ 105 mg of sodium borohydride is placed in container B and sealed with a rubber stopper, the two containers are connected by a pipe, a needle connected to the atmosphere is set in the rubber stopper of container A, 2 ~ 3 ml of sulfuric acid solution with pH = 4.5 ~ 5.5 is injected into container B, hydrogen is generated and blown into the palladium nanozyme solution in container A, after 20 ~ 30 minutes, the solution containing the final product palladium hydride nanoparticles is sealed and stored away from light; S3. Preparation of nanozyme hydrogel: 1.2 ~ 1.4g acrylamide, 0.31 ~ 0.33g alginate and 0.25 ~ 0.75g trehalose were dissolved in 10ml 200μg / ml palladium hydride nanoparticle solution to prepare a mixed solution. After stirring the solution for 6h, 0.0010 ~ 0.0020g MBA, 0.0040 ~ 0.0060g TMEDA, 0.0135 ~ 0.0235g APS and 0.02 ~ 0.03g CaSO4∙2H2O were added. After degassing, the solution was placed in a mold and allowed to stand at room temperature for 22 ~ 26h to obtain the nanozyme hydrogel.

2. The method for preparing the nanozyme hydrogel with controlled hydrogen release performance according to claim 1, characterized in that: In step S1, the centrifugation is performed at 8000-10000 r / min for 10-15 min.

3. The method for preparing the nanoenzyme hydrogel with controlled hydrogen release performance according to claim 1, characterized in that: The degassing in step S3 is performed by standing at 25-28° C. for 1-2 h.

4. An application of a nanozyme hydrogel with controlled hydrogen release performance obtained by the preparation method of a nanozyme hydrogel with controlled hydrogen release performance as claimed in any one of claims 1 to 3, characterized in that: The nanoenzyme hydrogel is used in wound dressings to treat skin wounds in diabetic patients.

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