A kind of peroxide-like nanoscale enzyme hydrogel and its preparation method and application

Abstract

The application belongs to the technical field of biological medical materials, and relates to a kind of peroxide-like nanoscale enzyme hydrogel and its preparation method and application. It is composed of temperature-sensitive hydrogel and peroxide-like nanoscale enzyme, the peroxide-like nanoscale enzyme is loaded in the temperature-sensitive hydrogel, the peroxide-like nanoscale enzyme is formed by coating MPN coating on the surface of silver nanoparticles, EGF is connected on the surface of the MPN coating, and the MPN coating is formed by complexing ferric ion with GA. The peroxide-like nanoscale enzyme hydrogel provided by the application integrates the functions of improving hypoxic environment, antibacterial, anti-inflammatory, antioxidant, promoting vascular regeneration and promoting cell proliferation, so as to promote diabetic wound healing.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and relates to a hydrogen peroxide-like nanoenzyme hydrogel, its preparation method and application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] According to the inventor's research, the chronic slow healing of diabetic wounds is caused by a combination of factors. First, chronic hyperglycemia disrupts local metabolism at the wound site, creating a microenvironment conducive to chronic inflammation. This damages blood vessels and nerves around the wound, hindering healing due to insufficient blood supply and nutrition. Second, persistent open wounds are often accompanied by difficult-to-control bacterial infections, especially drug-resistant bacteria, making wound infection treatment challenging. Uncontrolled infections can further develop into chronic ulcers, leading to adverse outcomes such as amputation or even death. Currently used clinical treatments include repeated debridement and drainage of pus, but these methods are insufficient to simultaneously promote wound healing and combat bacterial infection. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a hydrogen peroxide-like nanoenzyme hydrogel, its preparation method, and its application. The hydrogen peroxide-like nanoenzyme hydrogel provided by the present invention integrates functions such as improving hypoxic environment, antibacterial, anti-inflammatory, antioxidant, promoting angiogenesis, and promoting cell proliferation, thereby promoting the healing of diabetic wounds.

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

[0006] On one hand, a hydrogen peroxide-like nanozyme hydrogel is composed of a thermosensitive hydrogel and a hydrogen peroxide-like nanozyme. The hydrogen peroxide-like nanozyme is loaded in the thermosensitive hydrogel. The hydrogen peroxide-like nanozyme is formed by coating a metal polyphenol network (MPN) structure on the surface of silver nanoparticles. Surface growth factor (EGF) is connected to the surface of the MPN coating. The MPN coating is formed by the complexation of ferric ions and gallic acid (GA).

[0007] On the other hand, a method for preparing a hydrogen peroxide nanoenzyme hydrogel involves adding a silver nanoparticle solution to a container containing a solid ferric iron source, then adding a gallic acid solution, and reacting under conditions of solution disturbance to allow ferric iron and GA to complex on the surface of the silver nanoparticles to form an MPN coating, thereby obtaining GAP.

[0008] EGF was added to the GAP solution and reacted at 1–10 °C with stirring to allow the ferric iron in the MPN coating to bind with the histidine and methionine-rich structural domains inside the EGF, thus obtaining a hydrogen peroxide-like nanozyme.

[0009] Add hydrogen peroxide-like nanozymes to the thermosensitive hydrogel solution and mix thoroughly to obtain the final product.

[0010] Thirdly, the application of the aforementioned hydrogen peroxide nanozyme hydrogel in the preparation of a drug that promotes wound healing in diabetes.

[0011] The hydrogen peroxide-like nanoenzyme hydrogel provided by this invention forms a core-shell structured nanoparticle GA / Fe@AP (GAP) through the complexation of GA and iron (Fe III). Further, iron ions directly coordinate with histidine and methionine on the surface of EGF, ultimately forming a multifunctional nanodrug EGF+GA / Fe@AP (EGAP). EGAP exhibits good antibacterial, antioxidant, anti-inflammatory, oxygen-supplying, and angiogenesis-promoting activities. Furthermore, to achieve local application in wounds, this invention uses a temperature-sensitive hydrogel with good biocompatibility, high safety, and shape adaptability as a carrier to encapsulate the therapeutic EGAP, forming an in-situ hydrogel drug therapy system EGAP@HG. This therapeutic system provides a comfortable microenvironment for cell proliferation, angiogenesis, granulation tissue formation, and epithelial regeneration, significantly accelerating the healing of diabetic wounds both in vivo and in vitro.

[0012] The beneficial effects of this invention are as follows:

[0013] 1. The hydrogen peroxide-like nanozyme prepared in this invention has a stable structure, and the EGAP@HG hydrogen peroxide-like nanozyme hydrogel can be used for local injection into diabetic wounds. The prepared EGAP-like hydrogen peroxide nanozyme particles have a distinct core-shell structure. DLS dynamic light scattering (DLS) determined the average diameter of EGAP to be 137.3 nm, and the zeta potential was -26.87 ± 1.44 mV. The prepared EGAP@HG hydrogen peroxide-like nanozyme hydrogel is in a fluid state at room temperature and can be injected locally into the skin wound using a syringe. After injection, it transforms into a gel state as the temperature rises, adhering to the wound surface and forming a protective physical barrier and maintaining a moist healing environment.

[0014] 2. The hydrogen peroxide-like nanoenzyme hydrogel provided by this invention has good biocompatibility and high safety. In vitro hemolysis experiments, cell viability staining experiments, and in vivo toxicity tests have demonstrated its good biocompatibility and that it will not cause damage to cell viability or the skin and internal organs of mice.

[0015] 3. The hydrogen peroxide-like nanoenzyme hydrogel provided by this invention enhances therapeutic efficacy through the synergistic effect of drugs, creatively combining MPN, AP, and EGF. MPN exerts a catalase-like effect, converting local H2O2 in the wound into O2, alleviating the hypoxic environment of the wound, while also exerting anti-inflammatory and antioxidant effects. AP effectively kills wound bacteria, while EGF promotes wound cell proliferation and angiogenesis. In vivo experiments show that compared with the control group or the single-factor treatment group (simple antibacterial group or simple healing promotion group), EGAP@HG significantly improves wound re-epithelialization, collagen deposition, and angiogenesis, significantly accelerating the healing speed of diabetic wounds. Attached Figure Description

[0016] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0017] Figure 1 A schematic diagram illustrating the EGAP@HG preparation for promoting diabetic wound healing in an embodiment of the present invention;

[0018] Figure 2 The continuous assembly process of GAP (A) and the molecular structure diagram of MPN network (B) are shown in the embodiments of the present invention.

[0019] Figure 3 The following diagrams illustrate the synthesis and characterization of EGAP in this invention: A is a photograph of the GAP assembly process; B is a schematic diagram of EGAP manufacturing; C is the UV-Vis-NIR absorption spectrum of GA, AP, GAP, and EGAP; D is a representative TEM image of AP; E is a representative TEM image of EGAP; F is the size distribution of AP, GAP, and EGAP; G is the zeta potential of AP, GAP, and EGAP; H is the binding rate of EGF to GAP; and I is the reaction efficiency of EGAP coexisting with hydrogen peroxide for different times.

[0020] Figure 4 The diagrams show the synthesis process and property characterization of HG and EGAP@HG in this embodiment of the invention. A is a schematic diagram of HG fabrication, B is a visualization of the states of HG and EGAP@HG at different temperatures (25°C and 37°C). At 37°C, HG and EGAP@HG are clearly in a gel state, while at 25°C, they are in a sol state. C shows injectable EGAP@HG, D shows the strain test of HG and EGAP@HG, and E shows the self-healing test of HG and EGAP@HG, applying alternating strains from 1% to 100% over two cycles.

[0021] Figure 5To illustrate the in vitro therapeutic effect of EGAP@HG in this embodiment of the invention, Figures A and B show the photographs and quantitative statistics of the inhibition zone of Staphylococcus aureus in the inhibition zone experiment, respectively; Figures C and D show the photographs and quantitative statistics of Staphylococcus aureus viability in the colony counting experiment, respectively; Figure E shows the viability of HaCaT cells after culturing in PBS and different conditioned media for 24 hours; Figures F and G show the quantitative analysis and morphology of the migration region of HaCaT cells after culturing with EGAP@HG for 0 hours, 12 hours, 18 hours, 24 hours and 36 hours, respectively.

[0022] Figure 6 The figures show the experimental results of biocompatibility, in vitro toxicity, and in vivo toxicity of EGAP@HG in the embodiments of the present invention. A and B are photographs and hemolysis rates after 1 hour of incubation with Triton X-100, PBS, HG, GAP solution, EGAP solution, GAP@HG extract, or EGAP@HG extract, respectively. C is a confocal microscope image of HaCaT cells after 24 hours of culture in different culture media to detect live / dead cells. D is a representative image of the wound in the in vivo toxicity experiment.

[0023] Figure 7 Figure A shows the experimental results of EGAP@HG on diabetic wound healing in an embodiment of the present invention. A is a schematic diagram of the experimental design for inducing diabetes and subsequent inducing and healing of infected wounds. B is a representative image of the wound on days 3, 5, 7 and 10 after trauma and treatment. C is a schematic diagram of the wound area of ​​different groups from day 3 to day 10. D is the quantification of the residual wound on days 3, 5, 7 and 10.

[0024] Figure 8 The images shown are histological evaluation results of EGAP@HG promoting diabetic wound healing in this embodiment of the invention. A is a representative image of H&E staining of wound tissue sections from different groups on day 10, B is a representative image of Masson trichrome staining of wound tissue sections from different groups on day 10, and C is an immunohistochemical image of CD31 in wound tissue sections from different groups on day 10. Detailed Implementation

[0025] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0026] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0027] Given that existing drugs are ineffective in promoting wound healing in diabetic patients, this invention proposes a hydrogen peroxide-like nanoenzyme hydrogel, its preparation method, and its application.

[0028] In a typical embodiment of the present invention, a hydrogen peroxide-like nanozyme hydrogel is provided, which is composed of a thermosensitive hydrogel and a hydrogen peroxide-like nanozyme. The hydrogen peroxide-like nanozyme is loaded in the thermosensitive hydrogel. The hydrogen peroxide-like nanozyme is formed by coating the surface of silver nanoparticles with an MPN coating. The surface of the MPN coating is connected to EGF. The MPN coating is formed by the complexation of trivalent iron ions and GA.

[0029] In some embodiments, the MPN coating surface is linked to the EGF via ferric iron binding to histidine- and methionine-rich domains within the EGF.

[0030] In some embodiments, the silver nanoparticles are amino-modified silver nanoparticles.

[0031] In some embodiments, the diameter of the hydrogen peroxide-like nanozyme is 120–140 nm.

[0032] In some embodiments, the diameter of the silver nanoparticles is 100–120 nm.

[0033] In some embodiments, the thermosensitive hydrogel is a poloxamer hydrogel. Specifically, the poloxamer hydrogel is prepared by mixing poloxamer 407 and poloxamer 188 at a mass ratio of 3.0 to 4.0:1.

[0034] Another embodiment of the present invention provides a method for preparing a hydrogen peroxide nanoenzyme hydrogel, wherein a silver nanoparticle solution is added to a container containing a solid ferric iron source, and then gallic acid solution is added. The reaction is carried out under the condition of solution disturbance, so that ferric iron and GA complex on the surface of silver nanoparticles to form an MPN coating, thereby obtaining GAP.

[0035] EGF was added to the GAP solution and reacted at 1–10 °C with stirring to allow the ferric iron in the MPN coating to bind with the histidine and methionine-rich structural domains inside the EGF, thus obtaining a hydrogen peroxide-like nanozyme.

[0036] Add hydrogen peroxide-like nanozymes to the thermosensitive hydrogel solution and mix thoroughly to obtain the final product.

[0037] The solid ferric iron source described in this invention is a solid substance containing ferric iron that is insoluble in solvent, such as rusty nails. When using rusty nails as the solid ferric iron source, the nails are obtained by sequentially removing grease and acid washing. Specifically, for grease removal, a degreasing solution is first used for washing, and then acetone is used to wash away the degreasing solution adhering to the surface of the nails. Specifically, 5-15% hydrochloric acid is used for acid washing.

[0038] In some embodiments, the mass ratio of silver nanoparticles to GA is 4 to 6:1.

[0039] In some embodiments, the method of agitating the solution is to continuously shake the solution at a frequency of 30 to 50 rpm.

[0040] In some embodiments, the reaction time under solution disturbance conditions is 50–70 min.

[0041] In some embodiments, after the reaction is carried out under conditions of solution disturbance, the solid ferric source is removed, the solution is centrifuged, the precipitate is collected, the precipitate is dispersed in ultrapure water, and then centrifuged again to obtain GAP. The GAP solution is obtained by dispersing GAP in ultrapure water.

[0042] In some embodiments, the reaction is carried out at 1–10°C with stirring, and the stirring speed is 550–650 rpm.

[0043] In some embodiments, the mass ratio of EGF to GA is 1:550 to 650.

[0044] In some embodiments, the reaction is carried out at 1–10°C for 50–70 min under stirring conditions.

[0045] In some embodiments, the thermosensitive hydrogel is prepared by adding poloxamer 407 and poloxamer 188 to water at 1-10°C in a mass ratio of 2.0-4.0:1 (preferably 2.0-3.0:1) and mixing them evenly.

[0046] A third embodiment of the present invention provides the application of the above-mentioned hydrogen peroxide-like nanoenzyme hydrogel in the preparation of a drug that promotes the healing of diabetic wounds.

[0047] The drug is a topical preparation, such as an ointment, liniment, or patch.

[0048] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0049] Example

[0050] Preparation of EGAP-type hydrogen peroxide nanozyme particles:

[0051] Solid iron nails (approximately 3-4 cm in length) were first washed with a degreasing solution to remove surface grease, then thoroughly rinsed with Milli-Q water and air-dried. Next, they were soaked in acetone for 10 minutes, rinsed with Milli-Q water, and air-dried again. Then, the iron nails were placed in a beaker containing 50 mL of 10% hydrochloric acid. After 24 hours, the rusted nails were removed from the beaker, cleaned, and air-dried for later use. 3 mL of commercially available amino-modified amino acid (AP) at a concentration of 10 mg / mL was added to a 15 mL centrifuge tube containing the rusted nails, followed by 3 mL of GA solution at a concentration of 2 mg / mL (to adjust the pH to approximately 5). During GAP formation, the centrifuge tube was continuously shaken at a frequency of 40 rpm. After 1 hour, the iron nails were removed, the solution was collected, and centrifuged at 10000g for 5 minutes. The precipitate was collected and resuspended in 1 mL of Milli-Q water, centrifuged at 6000g for 5 minutes, and this process was repeated three times. 10 μg of EGF was added to 1 mL of GAP solution and mixed at 4 °C and 600 rpm for 1 hour. The mixture was then centrifuged at 10000 g for 5 minutes, and the precipitate was collected. The resulting GAP was then washed four times with 1×PBS and finally resuspended in 1 mL of PBS solution.

[0052] Preparation of EGAP@HG type hydrogen peroxide nanozyme hydrogel:

[0053] Weigh out a certain amount of sterile poloxamer 407 (Pol-407, average molecular weight 12600, PEO101-PPO66-PEO101) and poloxamer 188 (Pol-188, average molecular weight 8400, PEO80-PPO27-PEO80), and add them to ice water at 4°C with Pol-407 concentration of 17.9% (w / w) and Pol-188 concentration of 8% (w / w) to obtain a hydrogel (HG). After complete dissolution, place it in a refrigerator at 4°C overnight to obtain a clear solution. Dissolve 1 mg of EGAP completely in 1 mL of HG to obtain EGAP@HG.

[0054] Results and Discussion

[0055] The mechanism of this embodiment is as follows: Figure 1 As shown, the combination of MPN, AP, and EGF, the sequential assembly process of GAP, and the molecular structure of the MPN network are illustrated. Figure 2 As shown, MPN acts like a catalase, converting local H2O2 into O2 to alleviate the hypoxic environment of the wound, while also exerting anti-inflammatory and antioxidant effects. AP release effectively kills wound bacteria, while EGF promotes wound cell proliferation and angiogenesis.

[0056] 1. Preparation and characterization analysis of EGAP:

[0057] This embodiment uses amino-modified AP as a template because it possesses potent and broad-spectrum antimicrobial properties. Rusty iron nails were used as a solid iron source. Simultaneously, a metal-phenol network (MPN) film was formed on the AP through the coordination of phenolic and iron ions. The thickness of the MPN film increased with increasing reaction time, and the solution color gradually changed from transparent to blue-purple. During the formation of the MPN coating, the centrifuge tube was continuously shaken, and core-shell structured nanoparticles were obtained through centrifugation and washing. These nanoparticles were named GA@AP (GAP). Figure 3 A). The obtained GAP particles were resuspended, and EGF was added to the resuspended solution. By binding iron (Fe III) with the histidine and methionine-rich domains inside EGF, EGAP nanoparticles were finally prepared. Figure 3 B). EGAP has a diameter of approximately 110 nanometers. EGAP modified with MPN coating and EGF shows a distinct core-shell structure, with an outer shell diameter of 20 nanometers and EGAP nanoparticles having a diameter of 130 nanometers. Figure 3 D, 3E). The hydrodynamic diameters of the molecules were determined by dynamic light scattering (DLS), and the diameters of AP, GAP, and EGAP were 110.1, 132.0, and 137.3 nm, respectively. Figure 3 F). Amino-modified AP exhibited a positive surface potential of 21.00 ± 1.28 mV, while the zeta potentials of GAP and EGAP after MPN coating and EGF modification were -14.80 ± 1.06 and -26.87 ± 1.44 mV, respectively. Figure 3 G). The binding rate of EGF to GAP was measured using the BCA method. By binding different concentrations of EGF to 1 mg / mL of GAP, it was found that the binding rate increased with increasing EGF concentration, and reached a plateau at approximately 1 μg / mL; therefore, 10 μg / mL of EGF was selected for binding to GAP. Figure 3 H). This embodiment further evaluates the effect of EGAP in promoting hydrogen peroxide decomposition by measuring the hydrogen peroxide conversion rate. It was found that after the addition of EGAP, the hydrogen peroxide conversion rate gradually increased over time. Figure 3 I).

[0058] 2. Preparation and characterization analysis of HG:

[0059] Generally, hydrogels used as medical dressings for wound treatment need to possess certain mechanical properties to meet the requirements of wound healing. The critical hydrogel temperature of Pol-407 at a 25% concentration is 15°C, while that of Pol-188 at a 25% concentration is approximately 50°C. In aqueous solution, the amphiphilic properties of the PEO-PPO-PEO copolymer lead to the self-polymerization of macromolecules into micelles, with an inner core composed of PPO and an outer shell composed of hydrophilic PEO units. Figure 4 A). In this embodiment, by adding Pol-188 to Pol-407, it was observed that when the concentration of Pol-407 was 17.9% (w / w) and the concentration of Pol-188 was 8% (w / w), the critical gelation temperature of the resulting hydrogel was 37°C. At the critical gelation temperature, with the addition of Pol-408 to Pol-407, the mixture gradually became gel-like ( Figure 4 B). Therefore, the fluid state of the gel can adapt to the irregularity of the wound to a great extent. When the temperature of the hydrogel in contact with the skin rises to 37°C, the hydrogel reaches the gel state and adheres to the wound site. In addition, EGAP@HG samples can be continuously injected into a culture dish with a 10 mL syringe, and the formed "SDU" can maintain the gel state, confirming the injectability of the hydrogel. Figure 4 C). Strain scanning analysis of HG and EGAP@HG showed that G′ and G″ intersected at strains below 100%, which is an important indicator of hydrogel network collapse. Figure 4 D). Under high dynamic strain (100%), the value of G′ decreases and falls below that of G″, indicating damage to the gel network. Once a lower strain (1%) is applied, the values ​​of G′ and G″ almost recover to their original values. Notably, this phase transition can even be repeated after two alternating cycles, demonstrating the autonomous self-healing capabilities of HG and EGAP@HG. Figure 4 E).

[0060] 3. In vitro antibacterial activity of EGAP@HG:

[0061] Due to high blood sugar and a poor immune system response, diabetic wounds are often accompanied by persistent bacterial infections, with Staphylococcus aureus being the most common infecting bacteria. The antibacterial effect of the EGAP@HG dressing prepared in this embodiment is primarily driven by AP, which induces local membrane perforation; therefore, NPs are internalized, leading to further damage, ultimately resulting in bacterial death due to their interaction with intracellular proteins (especially sulfur-rich proteins) and DNA. The in vitro antibacterial activity of EGAP@HG against Gram-positive Staphylococcus aureus was first evaluated using a zone of inhibition (ZOI) assay. Figure 5The inhibition zone assays shown in (A, B) indicate that EGAP@HG exhibits increased antibacterial activity with increasing EGAP concentration. It can be observed that at low concentrations (<25 μg / mL), the inhibition zone is not significant, but its diameter increases with increasing EGAP concentration. The average diameters of the inhibition zones formed at EGAP concentrations of 25 μg / mL, 50 μg / mL, and 100 μg / mL were 10.67 ± 1.53 mm, 17.67 ± 2.52 mm, and 27.67 ± 0.58 mm, respectively. Comparison of EGAP@HG with different EGAP contents revealed a positive correlation between antibacterial activity and EGAP content. Therefore, the antibacterial performance can be adjusted by changing the EGAP content. In this embodiment, a hydrogel with an EGAP concentration of 100 μg / mL was subsequently selected for the experiment. Bacteria were further co-cultured with GAP solution, EGAP solution, GAP@HG extract, and EGAP@HG extract, and the in vitro antibacterial performance was evaluated by colony counting. Figure 5 As shown in C and 5D, compared with the PBS negative control and pure HG, GAP solution, EGAP solution, GAP@HG extract, and EGAP@HG extract all showed good antibacterial effects, killing almost all bacteria. This indicates that the antibacterial activity of GAP modified with EGF encapsulated in HG was not affected, and it can achieve a good bactericidal effect.

[0062] 4. Effects of EGAP@HG on cells:

[0063] The effects of EGAP@HG on cells were tested using cell viability assays, such as... Figure 5 As shown in Figure E. After 24 hours, the survival rate of HaCaT cells cultured in different conditioned media was not significantly different from the control group, indicating that the EGAP@HG system can maintain cell growth and proliferation. This example further performed an in vitro cell scratch assay to evaluate the effect of EGAP@HG on HaCaT cell migration, which largely determines the efficiency of wound healing. As cells on both sides of the scratch gradually migrated towards the center, the distance between cells gradually decreased, and the wound closure speed of the EGAP@HG group was faster than that of the control group. Figure 5(F, 5G). The wound area of ​​the two groups was quantitatively compared at 12h, 18h, 24h, and 36h. It was found that the wound area of ​​the EGAP@HG group was significantly smaller than that of the control group at 12h, accounting for 75.70±1.72% and 94.67±5.26% of the initial area, respectively. Furthermore, the difference between the two groups became more pronounced with increasing culture time, at 61.94±1.77%, 40.73±1.79%, 25.45±2.79% and 79.25±2.18%, 61.58±3.44%, 51.58±3.13%, respectively. This finding indicates that EGAP@HG significantly promotes the migration of HaCaT cells. In summary, the EGAP@HG drug-loaded hydrogel prepared in this embodiment can significantly improve the growth and migration of epidermal cells, thereby promoting wound healing.

[0064] 5. Biocompatibility and safety of EGAP@HG:

[0065] In the clinical application of wound dressings, good biocompatibility and safety are crucial. Therefore, this embodiment evaluates the biocompatibility and safety of EGAP@HG through blood compatibility tests, cell death tests, and in vivo toxicity tests. First, using Triton X-100 solution as a positive control, the supernatant of the treated red blood cells was bright red, while the supernatant of the PBS, HG, GAP, EGAP, GAP@HG, and EGAP@HG groups was colorless and transparent. Figure 6 A). The absorbance (OD) of the supernatant of each group at 542 nm was measured using an ELISA reader. It can be seen that the OD values ​​of the PBS, HG, GAP, EGAP, GAP@HG, and EGAP@HG groups were significantly lower than those of the Triton X-100 treatment group, and the calculated hemolysis rate was less than 2%, which can be considered a safe level for the hemostatic material. These data reflect that the hemolytic effect of the nanomedicine combined with the hydrogel dressing designed in this embodiment on red blood cells is negligible. Figure 6 B). The cytotoxicity of the designed nanomedicine and hydrogel was then further investigated using cell live / dead staining techniques. HaCaT cells were cultured in media under HG-, GAP-, EGAP-, GAP@HG-, and EGAP@HG conditions, and cell live / dead staining was performed after 24 hours of culture. Figure 6 As shown in Figure C, HaCaT cells showed very little cell death (red fluorescence) after 24 hours of culture under HG-, GAP-, EGAP-, GAP@HG-, and EGAP@HG conditions, indicating that the cytotoxicity of the nanomedicine and hydrogel in this embodiment is very low or even negligible. This embodiment further conducted toxicity tests on mice, creating a 6 cm diameter total skin defect model on the back of mice. Figure 6D), and HG, GAP, EGAP, GAP@HG, and EGAP@HG were applied every other day. The control group was treated with PBS, and no skin damage was observed in any group. In a normal mouse model, the EGAP@HG group showed a good effect in promoting wound healing compared with the control group.

[0066] 6. EGAP@HG effectively promotes wound healing in diabetic patients.

[0067] To determine whether the EGAP@HG system can effectively promote the healing of diabetic wounds, this embodiment established a diabetic wound model in mice to evaluate the in vivo antibacterial and healing-promoting effects of the EGAP@HG system. Figure 7 A is a schematic diagram of establishing and treating a whole-skin wound model infected with Staphylococcus aureus. Figure 7 B shows representative photographs of wounds and wound closure marks in different groups (control group, HG group, AP group, GAP group, AP@HG group, EGAP group, GAP@HG group, EGAP@HG group) at infection 3 days before treatment and on days 3, 5, 7, and 10 after treatment. The recorded photographs show that after Staphylococcus aureus infection, the mice's wound skin was red and swollen, and the local skin temperature increased. Figure 7 C and D represent schematic diagrams and calculated results for open wounds, respectively. From day 3 of treatment, the wound healing rate in the EGAP@HG group was significantly faster than in the other groups. By day 10, the wounds treated with EGAP@HG showed the smallest open wound rate (5.95%), with almost complete wound healing. In contrast, the open wound rates in the control group, HG, AP, GAP, AP@HG, EGAP, and GAP@HG groups were 24.17%, 18.81%, 18.93%, 15.83%, 13.33%, 12.26%, and 11.90%, respectively. These results indicate that the EGAP@HG group exhibited the best wound healing performance compared to the other groups.

[0068] 7. Histological evaluation of EGAP@HG in promoting diabetic wound healing

[0069] On day 10, mice were euthanized, and wounds and surrounding tissues were collected. A series of histopathological analyses were performed on wound tissue sections using H&E and Masson staining to assess wound healing. Reepithelialization, collagen deposition, and angiogenesis are commonly used indicators for assessing skin wound healing. Figure 8 A shows the H&E staining of the wound tissue. Compared with other groups, EGAP@HG showed the best wound repair on day 10, with completely continuous regenerated tissue and complete epithelialization at the wound site. Furthermore, collagen is an important repair material for wound tissue, so its content was determined using Masson staining to assess treatment efficacy. Figure 8B). Compared to all other groups, the EGAP@HG group showed a deeper blue color, indicating an increased amount of collagen. The control group exhibited sparse, disordered, low-level collagen deposition, while the EGAP@HG group showed densely and orderly arranged collagen fibers, forming mature collagen deposition bundles; therefore, this significant difference indicates that EGAP@HG can significantly accelerate collagen deposition. Achieving angiogenesis is also one of the main goals of effective wound healing in diabetic patients. To further demonstrate whether the application of EGAP@HG leads to enhanced angiogenesis, this example investigated the presence of CD31 signaling positive cells in the wound regeneration tissue. Figure 8 C). The results showed that, compared with the control group and the single-factor treatment group, the expression level of CD31 signaling positive cells in the wound treated with EGAP@HG was significantly increased, and the newly formed blood vessels were larger and more mature, with significantly enhanced angiogenesis. In summary, compared with the control group and the single-factor treatment group, the EGAP@HG group showed more complete epithelialization, more collagen deposition, granulation tissue formation and more mature angiogenesis, effectively promoting wound healing in diabetic mice.

[0070] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A hydrogen peroxide-like nanozyme hydrogel, characterized in that, The product is composed of a thermosensitive hydrogel and a hydrogen peroxide-like nanozyme. The hydrogen peroxide-like nanozyme is loaded within the thermosensitive hydrogel. The hydrogen peroxide-like nanozyme is formed by coating a metal polyphenol network structure onto the surface of silver nanoparticles. Surface growth factors are connected to the surface of the metal polyphenol network structure coating, which is formed by the complexation of ferric ions and gallic acid. The thermosensitive hydrogel is a poloxamer hydrogel. The poloxamer hydrogel is prepared by mixing poloxamer 407 and poloxamer 188 at a mass ratio of 2.0~3.0:

1.

2. The hydrogen peroxide-like nanoenzyme hydrogel as described in claim 1, characterized in that, The surface of the metal polyphenol network structure coating is connected to the surface growth factor by binding trivalent iron to the histidine and methionine-rich structural domains inside the surface growth factor.

3. The hydrogen peroxide-like nanoenzyme hydrogel as described in claim 1, characterized in that, The silver nanoparticles are amino-modified silver nanoparticles.

4. The hydrogen peroxide-like nanoenzyme hydrogel as described in claim 1, characterized in that, The diameter of the hydrogen peroxide-like nanozyme is 120-140 nm; the diameter of the silver nanoparticles is 100-120 nm.

5. A method for preparing a hydrogen peroxide-like nanozyme hydrogel, characterized in that, Silver nanoparticle solution was added to a container containing a solid ferric iron source, and then gallic acid solution was added. The reaction was carried out under the condition of solution disturbance, so that ferric iron and gallic acid complexed on the surface of silver nanoparticles to form a metal polyphenol network structure coating, thus obtaining GAP. Surface growth factor was added to GAP solution and reacted at 1~10 °C with stirring to allow the ferric iron in the metal polyphenol network structure coating to bind with the histidine and methionine-rich structural domains inside the surface growth factor, thus obtaining a hydrogen peroxide-like nanozyme. Add hydrogen peroxide-like nanozymes to the thermosensitive hydrogel solution and mix well to obtain the final product; The thermosensitive hydrogel is prepared by adding poloxamer 407 and poloxamer 188 in a mass ratio of 2.0~3.0:1 to water at 1~10 °C and mixing them evenly.

6. The method for preparing the hydrogen peroxide-like nanoenzyme hydrogel as described in claim 5, characterized in that, The mass ratio of silver nanoparticles to gallic acid is 4~6:1; The method for agitating the solution is to continuously shake it at a frequency of 30-50 rpm. The reaction time under solution disturbance conditions is 50-70 minutes; After the reaction is carried out under turbulent solution conditions, the solid ferric source is removed, the solution is centrifuged, the precipitate is collected, the precipitate is dispersed with ultrapure water, and then centrifuged again to obtain GAP.

7. The method for preparing the hydrogen peroxide-like nanoenzyme hydrogel as described in claim 5, characterized in that, The reaction was carried out at 1~10℃ with stirring at a speed of 550~650 rpm. The reaction time is 50-70 min under stirring conditions at 1-10 °C.

8. The use of a hydrogen peroxide-like nanoenzyme hydrogel according to any one of claims 1 to 4 or a hydrogen peroxide-like nanoenzyme hydrogel obtained by any one of claims 5 to 7 in the preparation of a drug for promoting the healing of diabetic wounds.

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