A nano-enzyme hydrogel with controlled release hydrogen performance and a preparation method and application thereof

Abstract

The present application belongs to the technical field of medical devices, and particularly relates to a nano-enzyme hydrogel with hydrogen control release performance, a preparation method and application thereof. The present application takes palladium nano-enzyme and hydrogen as the core of nano-drug, loads hydrogen on the palladium nano-enzyme to obtain palladium hydride nanoparticles, mixes the nanoparticles with a hydrogel to prepare a composite hydrogel loaded with palladium hydride nanoparticles. The palladium nanoparticles in the present application have catalase and superoxide dismutase activities, can remove ROS, and regulate immune inflammation. The hydrogen loaded by the palladium nano-enzyme acts as a signal molecule, blocks downstream inflammatory pathways, and the two synergistically inhibit inflammation and promote wound healing. The hydrogel in the present application changes the traditional injection administration mode, and simultaneously plays a role in regulating immune inflammation and restoring normal immune microenvironment. The composite hydrogel can be used for treating diabetic skin wounds, and becomes a new strategy for promoting diabetic wound healing.

Description

Technical Field

[0001] This invention belongs to the field of medical device technology, and in particular relates to a nanoenzyme hydrogel with controlled hydrogen release properties, its preparation method and application. Background Technology

[0002] In recent years, the prevalence of diabetes in my country has shown a significant upward trend, and diabetes and its complications, as a chronic disease, have gradually affected all aspects of people's lives. Diabetic foot (DFU) is the most common complication of diabetes, with high morbidity, mortality, and recurrence rates, and is a leading 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. Furthermore, a high pro-inflammatory state inhibits granulation tissue formation, collagen deposition, and angiogenesis. Therefore, effectively inhibiting the sustained 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 activities have achieved 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 molecule that makes up hydrogen gas is the smallest molecule in nature. Therefore, hydrogen gas has strong diffusivity and penetrability, and can efficiently pass through various biological barriers. In-situ release of hydrogen gas can not only remove excess ROS, but also inhibit inflammatory pathways, thereby reducing the expression of pro-inflammatory cytokines and inflammation in wound tissue. However, most documented wound healing methods have significant limitations. For example, hydrogen-rich water (HRW) and hydrogen-rich saline (HRS) have limited tissue penetration capabilities due to the low solubility of hydrogen gas. Hydrogel materials composed of biocompatible macromolecules are considered to have significant advantages in wound treatment. They provide a hydrated microenvironment for the wound, and this moist healing environment promotes the formation and growth of granulation tissue, thereby accelerating the wound healing process and providing a physical barrier against microbial infection and moist environments. It is worth noting that traditional hydrogels suffer from problems such as limited functionality and difficulty in fixation; therefore, existing technologies still require improvement and development. Summary of the Invention

[0004] The purpose of this invention is to provide a nanoenzyme composite hydrogel with controlled hydrogen release properties, its preparation method and application, to restore the normal immune microenvironment of DFU-affected areas.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for preparing a nanoenzyme hydrogel with controlled hydrogen release properties includes the following steps:

[0007] S1. Synthesis of palladium nanozyme solution: 44-45 mg of palladium chloride was dissolved in 0.02 M hydrochloric acid solution at 45-55℃ to obtain 0.01 M sodium tetrachloropalladate solution for later use; under stirring, 0.55-0.65 ml of 0.01 M sodium borohydride solution was added to a mixture of 9.7-9.8 ml of sodium cetyl sulfate solution and 0.2-0.3 ml of sodium tetrachloropalladate solution, and allowed to stand at room temperature for 3-5 h to obtain seed solution; the seed solution was added to a mixture of 9.5-10 ml of 4.5 mM CTAC solution and 0.25-0.35 ml of 0.01 M sodium tetrachloropalladate solution, and 0.1-0.15 ml of 0.1 M ascorbic acid solution was added, stirred for 10-20 s, and allowed to stand at room temperature for 1-1.5 h. h, centrifuge, wash, and disperse the obtained palladium nanozyme in deionized water to obtain palladium nanozyme solution, which is stored in the dark for later use;

[0008] S2. Synthesis of palladium hydride nanoparticle solution: 1.5-2.5 mL of palladium nanozyme solution was placed in container A and sealed with a rubber stopper. 95-105 mg of sodium borohydride was placed in container B and sealed with a rubber stopper. The two containers were connected by a pipe. A needle connected to the atmosphere was placed inside the rubber stopper of container A. 2-3 mL of sulfuric acid solution with pH = 4.5-5.5 was injected into container B to generate hydrogen gas, which was then bubbled into the palladium nanozyme solution in container A. After 20-30 minutes, the solution containing the final product, palladium hydride nanoparticles, was sealed and stored away from light.

[0009] S3. Preparation of nanoenzyme hydrogel: 1.2-1.4 g acrylamide, 0.31-0.33 g alginate and 0.25-0.75 g trehalose were dissolved in 10 ml of 200 μg / ml palladium hydride nanoparticle solution to prepare a mixed solution. After stirring the solution for 6 h, 0.0010-0.0020 g MBA, 0.0040-0.0060 g TMEDA, 0.0135-0.0235 g APS and 0.02-0.03 g CaSO4∙2H2O were added. After degassing, the solution was placed in a mold and allowed to stand at room temperature for 22-26 h to obtain nanoenzyme hydrogel.

[0010] Furthermore, in step S1, centrifugation is performed at 8000-10000 r / min for 10-15 min.

[0011] Furthermore, in step S3, the degassing is performed by allowing the mixture to stand at 25-28°C for 1-2 hours.

[0012] A method for preparing a nanoenzyme hydrogel with controlled hydrogen release properties, and its application in wound dressings for treating skin wounds in diabetic patients.

[0013] Mechanism of action: Studies have shown that palladium is a single-atom nanozyme with excellent catalase (CAT) and superoxide dismutase (SOD) activities comparable to normal enzymes. It can eliminate excessive ROS accumulation and inhibit the production and secretion of inflammatory factors at chronic wound sites. Simultaneously, palladium is also an excellent hydrogen storage material, enabling large-scale hydrogen loading and controlled (NIR-responsive) release. Hydrogen, with its reducing properties, is an endogenous signaling molecule in the human body, capable of inhibiting the expression of inflammatory factor receptors. It synergistically blocks downstream inflammatory pathways with palladium, inhibiting the release of inflammatory factors and restoring a normal immune microenvironment. This hydrogel dressing not only exhibits high mechanical properties and good adhesion for various skin wounds but also rapidly promotes wound healing, with no irritation, safety, or toxic side effects. Compared to the positive control group and other prescription hydrogel groups, its effects are superior.

[0014] The advantages of this invention are:

[0015] 1. The palladium nanozyme hydrogel prepared in this invention plays a dual role in dressings. First, it is an enzyme catalyst. Due to its catalase-like and superoxide dismutase-like activities, it can scavenge ROS, inhibit downstream inflammatory pathways, and restore the normal immune microenvironment. Second, it can serve as a hydrogen carrier to achieve controlled release of hydrogen.

[0016] 2. The palladium hydride nanoparticles prepared in this invention can achieve near-infrared controllable hydrogen release, inhibit the expression of inflammatory factor receptors, synergistically block inflammatory pathways with palladium nanozymes, regulate inflammation, and restore the normal immune microenvironment;

[0017] 3. The hydrogel prepared by this invention has good biocompatibility, high safety, good adhesive strength and mechanical properties. The large number of hydroxyl groups in trehalose can repair defects in the hydrogel network, making it better suited for DFU. Attached Figure Description

[0018] Figure 1 ESR spectra of CAT and SOD enzyme activities in Pd solutions of different concentrations;

[0019] Figure 2 A schematic diagram showing the hydrogen release rates of PdH and PdH + NIR in hydrogen-rich water.

[0020] Figure 3 This is a SEM scan of the freeze-dried hydrogel of the present invention;

[0021] Figure 4 These are the experimental results of the mechanical properties and adhesion of the hydrogel of this invention;

[0022] Figure 5 These are the results of the hydrogel cytotoxicity experiment of this invention;

[0023] Figure 6 These are the experimental results of the hydrogel used in this invention to scavenge ROS in vitro;

[0024] Figure 7 Results of wound healing experiments on mice in different groups;

[0025] Figure 8 Immunofluorescence images of CD86, CD206, and TNF-α in wound tissue sections from different groups on day 10;

[0026] Figure 9 This diagram illustrates the formation mechanism and therapeutic mechanism of the hydrogel of this invention. Detailed Implementation

[0027] Example 1

[0028] A method for preparing a nanoenzyme hydrogel with controlled hydrogen release properties includes the following steps:

[0029] 1. 44.5 mg of palladium chloride (PdCl2) was dissolved in 25 ml of 0.02 M hydrochloric acid solution at 50 °C to obtain a 0.01 M sodium tetrachloropalladate solution. Under vigorous stirring at 1000 rpm / min, 0.60 ml of fresh 0 °C 0.01 M sodium borohydride solution was added to a mixture of 9.75 ml of sodium hexadecyl sulfate solution and 0.25 ml of sodium tetrachloropalladate solution. The mixture was allowed to stand at room temperature for 3 h to obtain a seed solution. 0.025 ml of the seed solution was added to a mixture of 9.7 ml of 4.5 mM sodium hexadecyl sulfate solution and 0.3 ml of 0.01 M sodium tetrachloropalladate solution, along with 0.1 ml of 0.1 M ascorbic acid solution. The mixture was stirred for 10 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.

[0030] 2. Transfer 2 mL of palladium nanoparticle solution into a 10 mL vial A and seal it with a rubber stopper. Add 100 mg of sodium borohydride to vial B and seal it with a rubber stopper as well. Connect the two vials with a thin rubber tubing. Insert the needle of a 1 mL syringe into the rubber stopper of vial A, connecting it to the atmosphere. Inject 2 mL of pH = 5 sulfuric acid solution into vial B to generate hydrogen gas, which is then slowly bubbled into the palladium nanoparticle solution. After 25 minutes, seal the solution containing the final product, palladium hydride nanoparticles, and store it protected from light.

[0031] 3. Dissolve 1.35 g of polyacrylamide (Paam), 0.325 g of alginate (Alg), and 0.5 g of trehalose in 10 ml of 200 μg / ml palladium hydride (PdH) solution to form a mixed aqueous solution. After stirring the solution for 6 h, add 0.0015 g of MBA, 0.005 g of TMEDA, 0.0235 g of APS, and 0.02 g of CaSO4∙2H2O. After degassing, place the solution in a mold and let it stand at room temperature for 24 h to obtain a PATP hydrogel.

[0032] Example 2

[0033] 45 mg of palladium chloride (PdCl2) was dissolved in 26 ml of 0.02 M hydrochloric acid solution at 55 °C to obtain a 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 mixture of 9.8 ml of sodium hexadecyl sulfate solution and 0.3 ml of sodium tetrachloropalladate solution. 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 mixture of 10 ml of 4.5 mM sodium hexadecyl sulfate solution and 0.35 ml of 0.01 M sodium tetrachloropalladate solution, followed by 0.15 ml of 0.1 M ascorbic acid solution. The mixture was stirred 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.

[0034] 2. Transfer 2.5 mL of palladium nanoparticle solution into a 10 mL vial A and seal it with a rubber stopper. Add 105 mg of sodium borohydride to vial B and seal it with a rubber stopper as well. Connect the two vials with a thin rubber tubing. Insert the needle of a 1 mL syringe into the rubber stopper of vial A, connecting it to the atmosphere. Inject 3 mL of pH = 5 sulfuric acid solution into vial B to generate hydrogen gas, which is then slowly bubbled into the palladium nanoparticle solution. After 30 minutes, seal the solution containing the final product, palladium hydride nanoparticles, and store it away from light.

[0035] 3. Dissolve 1.4 g of polyacrylamide (Paam), 0.33 g of alginate (Alg), and 0.75 g of trehalose in 10 ml of 200 μg / ml palladium hydride (PdH) solution to form a mixed aqueous solution. After stirring the solution for 6 h, add 0.0020 g of MBA, 0.006 g of TMEDA, 0.0235 g of APS, and 0.03 g of CaSO4∙2H2O. After degassing, place the solution in a mold and let it stand at room temperature for 26 h to obtain a PATP hydrogel.

[0036] Example 3

[0037] 44 mg of palladium chloride (PdCl2) was dissolved in 24 mL of 0.02 M hydrochloric acid at 45 °C to obtain a 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 mixture of 9.7 mL of sodium hexadecyl sulfate solution and 0.2 mL of sodium tetrachloropalladate solution. 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 mixture of 9.5 mL of 4.5 mM sodium hexadecyl sulfate solution and 0.25 mL of 0.01 M sodium tetrachloropalladate solution, along with 0.1 mL of 0.1 M ascorbic acid solution. The mixture was 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.

[0038] 2. Transfer 1.5 mL of palladium nanoparticle solution into a 10 mL vial A and seal it with a rubber stopper. Add 95 mg of sodium borohydride to vial B and seal it with a rubber stopper as well. Connect the two vials with a thin rubber tubing. Insert the needle of a 1 mL syringe into the rubber stopper of vial A, connecting it to the atmosphere. Inject 2 mL of pH = 5 sulfuric acid solution into vial B to generate hydrogen gas, which is then slowly bubbled into the palladium nanoparticle solution. After 25 minutes, seal the solution containing the final product, palladium hydride nanoparticles, and store it protected from light.

[0039] 3. Dissolve 1.2 g of polyacrylamide (Paam), 0.31 g of alginate (Alg), and 0.25 g of trehalose in 10 ml of 200 μg / ml palladium hydride (PdH) solution to form a mixed aqueous solution. After stirring the solution for 6 h, add 0.001 g of MBA, 0.004 g of TMEDA, 0.0135 g of APS, and 0.02 g of CaSO4∙2H2O. After degassing, place the solution in a mold and let it stand at room temperature for 24 h to obtain a PATP hydrogel.

[0040] Performance testing

[0041] Using the PATP hydrogel prepared in Example 1 as the experimental object, polyacrylamide hydrogel (P), polyacrylamide-alginate hydrogel (PA), and polyacrylamide-alginate-trehalose hydrogel (PAT) were prepared as controls. All tests were conducted under the same conditions.

[0042] Electron spin resonance (ESR) detection was used to determine the catalase and superoxide dismutase activities of palladium nanozymes. A series of palladium nanozyme solutions at different concentrations (0, 25, 50, 100, 50, and 200 μg / mL) were prepared at 37 °C for ESR analysis. The results are shown below. Figure 1 As can be seen from the figure, palladium nanozymes exhibit high peroxidase (3.44 ± 0.22 U / ml) and superoxide dismutase (5.17 ± 0.41 U / ml) activities, and the peroxidase and superoxide dismutase activities of palladium are concentration-dependent.

[0043] Measurement of hydrogen release from palladium hydride: Hydrogen release was measured using the method reported in (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, methyl methacrylate (MB) can be reduced by hydrogen to colorless leucom vinyl blue (leucoMB), 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 using a UV spectrophotometer. Hydrogen release experiments were conducted under near-infrared and non-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 all groups were observed for 24 h. The hydrogen release concentration was calculated based on the final absorbance of MB and the standard curve formula. Each sample was measured three times, and the average value was taken. The results are as follows: 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 gas; under near-infrared irradiation, palladium hydride nanoparticles significantly enhance the reduction of MB, releasing a large amount of hydrogen gas (12.56 ± 0.33 µm).

[0044] Depend on Figure 3 It can be seen that the synthesized hydrogel exhibits a loose and porous gel network structure. Further SEM observation of the PATP hydrogel complex at higher magnifications shows that PdH nanoparticles are uniformly distributed within the hydrogel pore network, demonstrating the uniform loading of PdH nanoparticles in the PATP hydrogel complex.

[0045] The mechanical properties and adhesion properties of the hydrogel to the pigskin surface were tested using the methods described in the references (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, fast-adhesive nano-hybrid hydrogel ‘band-aid’, Acta Polymerica Sinica, 2019, 50(6): 613-622). The results are as follows: Figure 4The addition of trehalose improved the mechanical properties of the hydrogel. The final PATP hydrogel formulation had an elongation at break of 597.23% and a tensile strength of 9.49 kPa, which better prevents the hydrogel from deforming or breaking after being applied 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.

[0046] Cytotoxicity assay: The cytotoxicity of the hydrogel was tested using the CCK8 assay. The effect of the hydrogel on the proliferation of HUVECs and RAW 264.7 cells was also investigated. HUVECs and RAW 264.7 cells were cultured at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates and incubated at 37°C for 24 hours. After 24 hours of incubation, the cell culture medium was replaced with fresh medium, and the cells were treated with control (Negative), hydrogen peroxide (Active), hydrogen peroxide + PATP hydrogel (Group 1), and hydrogen peroxide + PATP + NIR (Group 2), respectively. Then, the cell culture medium was removed, and the obtained hydrogel extract was added to 96-well plates along with fresh medium containing 10% CCK-8. After 4 hours of incubation, the absorbance of the solution was measured at 450 nm. Cell viability was calculated using the formula: Cell viability = Ah / Ac × 100%, where Ah represents the absorbance of the hydrogel extract treatment group, and Ac represents the absorbance of the control group. Results are as follows: 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.

[0047] In vitro antioxidant capacity of the hydrogel: The ROS scavenging performance of the hydrogel was assessed using a ROS fluorescent probe assay. RAW 264.7 cells were seeded in confocal culture dishes. Cell culture medium was extracted 24 hours after seeding, and cells were then treated with culture media including hydrogen peroxide solutions treated with different hydrogels. After adding DCFH-DA, cells were observed using a laser scanning confocal microscope. Results are shown below. 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 enzyme activity of palladium in scavenging ROS. In the PATP + NIR group, palladium and hydrogen worked synergistically, resulting in the lowest fluorescence intensity. These results demonstrate the superior antioxidant capacity of PATP hydrogel and highlight its potential application in wound healing.

[0048] Application examples

[0049] The efficacy of the PATP hydrogel prepared in Example 1 was verified in a diabetic wound healing experiment.

[0050] Diabetic mouse modeling: Twenty-one male C57 / BL6J mice, weighing 20-25g, were selected and gradually fed a high-fat diet for one week, followed by continuous feeding with the high-fat diet for one month under a specific pathogen-free environment. A pre-prepared streptozotocin (STZ) solution (50 mg / kg dissolved in 0.1 mol / L, pH 4.2-4.5 sodium citrate buffer) was administered intraperitoneally for five consecutive days. One week later, blood glucose levels were measured via tail vein. Successful modeling was confirmed by monitoring fasting blood glucose levels above 11.1 mmol / L for one week.

[0051] A full-thickness resection wound model of the back in diabetic mice: Mice were randomly divided into 7 groups (Normal, Diabetic, PG, PAG, PATG, PATPG, and PATPG + NIR), with 3 mice in each group. 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 created on the back of each mouse. Different samples were then applied to the wound site. On postoperative days 0, 3, 5, 7, and 10, the wound was photographed using a digital camera, and the results were analyzed using image processing software. Mouse weight was also recorded. Results are as follows: Figure 7 Compared with the diabetic 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 in the PATPG+NIR group was reduced to 18.7%. By day 10, the diabetic positive control group still had 45% of the original wound area remaining large, while the PATPG group had 11.9% of the original wound area remaining. In contrast, the PATPG+NIR group had the smallest remaining wound area, at only 7.3%.

[0052] Anti-inflammatory properties of the hydrogel of this invention

[0053] Macrophages play a crucial role in the local immune response following tissue injury, particularly in diabetes-related wounds. The gradual transition of macrophages from an inflammatory-inducing (M1) state to an anti-inflammatory (M2) state negatively impacts tissue regeneration. Therefore, we used immunofluorescence assays to assess macrophage polarization. The results are as follows: Figure 8 Compared to the untreated group, the percentage of CD86 (M1 biomarker) was decreased in all hydrogel treatment groups, with the PATPG + NIR group exhibiting the weakest immunofluorescence intensity. In contrast, the mean fluorescence intensity of CD206 (M2 biomarker) showed an increase in all groups treated with different hydrogels. We also assessed the expression of tumor necrosis factor (TNF-α) at ​​the wound site, an important cytokine involved in the immune response. Results are as follows: Figure 8Among all experimental groups, the expression level of TNF-α in the diabetic positive control group was the highest, which was twice that of the PATPG+ NIR group.

[0054] The above results indicate that the nanozyme composite hydrogel with controlled hydrogen release properties prepared in this invention has good biocompatibility, adhesion properties, and diabetic wound healing properties. The Pd nanozyme has catalase and superoxide dismutase activities, which can remove excess ROS at the wound site and reduce oxidative stress. The hydrogen released from Pd synergistically inhibits downstream inflammatory pathways with Pd, reduces the expression of inflammatory factors, alleviates inflammation, restores the normal immune microenvironment, and efficiently promotes the healing of diabetic wounds.

Claims

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

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

3. The method for preparing the nanoenzyme hydrogel with controlled hydrogen release properties as described in claim 1, characterized in that: In step S3, degassing is performed by allowing the mixture to stand at 25-28℃ for 1-2 hours.

4. A nanoenzyme hydrogel with controlled hydrogen release properties prepared by the preparation method according to any one of claims 1-3.

5. The application of a nanoenzyme hydrogel with controlled hydrogen release properties as described in claim 4 in the preparation of a skin wound dressing for diabetic patients.

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