Composite hydrogel with pH-responsive dual-enzyme activity switching property and preparation method and application thereof

Abstract

This invention discloses a composite hydrogel with pH-responsive dual-enzyme activity switching properties, its preparation method, and its applications, belonging to the field of wound healing materials technology. The composite hydrogel comprises a nanozyme and a hydrogel. The nanozyme is a gold-platinum nanozyme, which is porous and dendritic, exhibiting different enzyme-like activities under different pH conditions. In an acidic environment, it exhibits oxidase-like and peroxidase-like activities, while in a neutral environment, it exhibits superoxide dismutase-like and catalase-like activities. This invention can exhibit reversibly switchable multi-enzyme mimicry activities under different pH conditions, thereby achieving dynamic synergistic regulation of antibacterial and antioxidant effects, overcoming the problem of difficulty in simultaneously achieving both in existing technologies. Furthermore, this invention has good biocompatibility, adhesion, and sustained-release properties, enabling adaptive functional regulation at different stages of wound healing, significantly promoting chronic wound healing and tissue repair, and showing promising clinical application prospects.

Description

Technical Field

[0001] This invention relates to the field of wound healing materials technology, and in particular to a composite hydrogel with pH-responsive dual enzyme activity switching properties, its preparation method and application. Background Technology

[0002] Diabetes mellitus is a metabolic disease characterized primarily by hyperglycemia, which easily leads to a variety of serious complications. Among these, chronic wounds are one of the most common and difficult to heal clinically. Diabetic chronic wounds are usually accompanied by complex pathological changes such as bacterial infection, persistent inflammatory response, local hypoxia, and oxidative stress caused by abnormal accumulation of reactive oxygen species (ROS). These factors interact to form a vicious cycle of "infection-inflammation-oxidative stress," severely hindering the wound healing process. Specifically, wound exposure provides a favorable environment for bacterial adhesion and proliferation. Bacterial infection induces a sustained increase in the inflammatory response, leading to elevated ROS levels. Excessive ROS further damages surrounding normal tissue cells and inhibits angiogenesis, affecting the transport of oxygen and nutrients, thereby hindering cell proliferation and migration, keeping the wound in a chronic, difficult-to-heal state. Therefore, in the treatment of chronic wounds, achieving efficient antibacterial activity, alleviating the inflammatory response, and regulating ROS levels simultaneously is key to improving wound repair outcomes.

[0003] Currently, treatments for chronic wounds mainly include antibiotic therapy and tissue regeneration-promoting drugs. However, these methods still have significant limitations: long-term use of antibiotics has led to increasingly serious bacterial resistance, reducing their effectiveness in treating infected chronic wounds; while cell growth-promoting drugs can promote tissue repair, their ability to inhibit bacterial infection is limited, making it difficult to fundamentally improve the wound microenvironment, resulting in persistent inflammation.

[0004] In recent years, based on the enzyme-like activity of metal nanozymes, antibacterial strategies centered on reactive oxygen species (ROS) have been developed, such as photodynamic therapy (PDT), sonodynamic therapy (SDT), and chemodynamic therapy (CDT). These methods achieve highly efficient sterilization by enhancing ROS generation, but they rely on strong oxidative effects, which may cause oxidative damage to normal tissues while killing bacteria, posing biosafety risks. Conversely, antioxidant strategies targeting ROS scavenging, while helping to alleviate inflammatory responses and protect cells, are insufficiently effective in bacterial infection environments. Therefore, existing technologies generally suffer from the following key technical contradictions: enhancing ROS is beneficial for antibacterial activity but can exacerbate tissue damage; scavenging ROS is beneficial for tissue repair but weakens antibacterial capabilities. Summary of the Invention

[0005] To address the aforementioned issues, this invention aims to provide a composite hydrogel with pH-responsive dual-enzyme activity switching properties, its preparation method, and its applications.

[0006] The technical solution of the present invention is as follows: On one hand, a composite hydrogel with pH-responsive dual-enzyme activity switching characteristics is provided, comprising a nanozyme and a hydrogel loaded with the nanozyme, wherein the nanozyme is a gold-platinum nanozyme, and the gold-platinum nanozyme is porous and dendritic, capable of exhibiting different enzyme-like activities under different pH conditions, and the different enzyme-like activities can be reversibly switched under different pH conditions; the gold-platinum nanozyme exhibits oxidase-like and peroxidase-like activities in an acidic environment, and superoxide dismutase-like and catalase-like activities in a neutral environment; the hydrogel is a hydrogel with a dynamic Schiff base crosslinking network.

[0007] Preferably, the gold-platinum nanozyme has a particle size of 100~1000 nm, and the hydrogel has a pore size of 10~20 μm.

[0008] Preferably, the loading of the gold-platinum nanozyme is greater than or equal to 300 μg / mL.

[0009] Preferably, the hydrogel is a hydrogel with a dynamic Schiff base crosslinking network constructed from aldehyde-modified polysaccharides and amino-modified polysaccharides, wherein the aldehyde-modified polysaccharides are any one or more of oxidized hyaluronic acid, oxidized sodium alginate, oxidized cellulose, oxidized starch, and oxidized traditional Chinese medicine polysaccharides, and the amino-modified polysaccharides are water-soluble chitosan derivatives.

[0010] Preferably, the gold-platinum nanozyme is prepared by the following steps: S1: Prepare chloroauric acid solution, potassium chloroplatinate solution and tannic acid solution respectively; S2: Mix the chloroauric acid solution and the potassium chloroplatinate solution, then add the tannic acid solution under stirring, and stir the reaction at room temperature for 4-8 h; S3: Wash, centrifuge, and sonicate the product to obtain the gold-platinum nanozyme.

[0011] Preferably, in step S1, the concentration of the chloroauric acid solution is 4-6 mM, the concentration of the potassium chloroplatinate solution is 4-6 mM, and the concentration of the tannic acid solution is 15-30 mg / mL; in step S2, the volume ratio of the chloroauric acid solution to the potassium chloroplatinate solution is 1.8-2.2:1, and the volume of the tannic acid solution is the sum of the volumes of the chloroauric acid solution and the potassium chloroplatinate solution.

[0012] On the other hand, a method for preparing a composite hydrogel with pH-responsive dual-enzyme activity switching characteristics as described in any one of the above claims is also provided, comprising the following steps: dispersing gold-platinum nanozymes in a hydrogel, mixing and allowing to stand to obtain the composite hydrogel.

[0013] Preferably, when the hydrogel is an oxidized hyaluronic acid-carboxymethyl chitosan hydrogel, the preparation method includes the following steps: S1': Prepare oxidized hyaluronic acid solution and carboxymethyl chitosan solution respectively; S2': Disperse the gold-platinum nanozyme in the oxidized hyaluronic acid solution, so that the gold-platinum nanozyme is uniformly suspended in the oxidized hyaluronic acid solution, and obtain an oxidized hyaluronic acid solution coated with gold-platinum nanozyme. S3': The oxidized hyaluronic acid solution coated with gold-platinum nanozyme is mixed with the carboxymethyl chitosan solution, and after mixing, it is allowed to stand to obtain the composite hydrogel.

[0014] Preferably, in step S1', the oxidized hyaluronic acid solution is prepared by the following steps: oxidizing hyaluronic acid with sodium periodate, then adding ethylene glycol and stirring to terminate the reaction, dialyzing the reaction solution in deionized water, and finally freeze-drying to obtain the oxidized hyaluronic acid.

[0015] Furthermore, the invention also provides the application of the composite hydrogel with pH-responsive dual enzyme activity switching properties described in any one of the above-mentioned embodiments in the preparation of a chronic wound healing agent for diabetes.

[0016] The beneficial effects of this invention are: The gold-platinum nanozyme of this invention exhibits reversibly switchable multi-enzyme mimicry activity under different pH conditions: in an acidic environment, it shows significantly enhanced oxidase-like and peroxidase-like activities, thereby catalyzing the generation of reactive oxygen species (ROS) to efficiently kill bacteria; while in a neutral environment, it exhibits superoxide dismutase-like and catalase-like activities, efficiently scavenging excess ROS and generating oxygen, thereby alleviating oxidative stress and improving the local hypoxic microenvironment. This functional switching behavior enables the present invention to achieve dynamic synergistic regulation of antibacterial and antioxidant effects, overcoming the problem of difficulty in simultaneously achieving both in existing technologies. Furthermore, the composite hydrogel of this invention has good biocompatibility, adhesion, and sustained-release properties, enabling adaptive functional regulation at different stages of wound healing, significantly promoting chronic wound healing and tissue repair, and showing promising clinical application prospects. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1This is a schematic diagram showing the comparison of the oxidase-like activities of the nanozymes prepared in step (1) of Example 1 and Comparative Example 1. Figure 2 This is a schematic diagram showing the comparison of the oxidase-like activities of the nanozymes prepared in step (1) of Example 1 and Comparative Example 2. Figure 3 This is a schematic diagram comparing the hydrogel adhesion test results of Example 1 and Comparative Example 3; where A represents the test results of Comparative Example 3 and B represents the test results of Example 1. Figure 4 This is a schematic diagram comparing the hydrogel rheological test results of Example 1 and Comparative Example 3; Figure 5 This is a transmission electron microscope schematic diagram of the gold-platinum nanozyme prepared in step (1) of Example 1; Figure 6 The particle size distribution frequency histogram of the gold-platinum nanozyme prepared in step (1) of Example 1; Figure 7 This is a photograph of the composite hydrogel from Example 1. Figure 8 Here is a scanning electron microscope image of the composite hydrogel from Example 1; Figure 9 The graph shows the performance results of ROS generation by the composite hydrogel in Example 1; where A represents the pH response of oxidase-like enzymes and peroxidases, and B represents the temperature correlation of the two enzymes. Figure 10 This is a schematic diagram of the enzyme kinetics test results of the gold-platinum nanozyme and the oxidase-like enzyme-like enzyme in Example 1; where A is a double reciprocal plot of the reaction rate v of the oxidase-like enzyme of the gold-platinum nanozyme and the TMB concentration c, and B is a double reciprocal plot of the reaction rate v of the peroxidase of the gold-platinum nanozyme and the TMB concentration c. Figure 11 This is a schematic diagram showing the performance of the composite hydrogel in removing ROS in Example 1; where A represents the catalase activity of the gold-platinum nanozyme, and B represents the superoxide dismutase-like activity of the gold-platinum nanozyme. Figure 12 This is a schematic diagram showing the antibacterial properties of the composite hydrogel in Example 1; Figure 13 This is a schematic diagram of the cell compatibility results of the composite hydrogel in Example 1; where A is a schematic diagram of the cell viability statistics after AuPt / Gel was co-incubated with cells for 12 h and 24 h, and B is a schematic diagram of the staining results of live / dead cells after AuPt / Gel was co-incubated with cells for 24 h. Figure 14 This is a schematic diagram of the anti-inflammatory results of the composite hydrogel in Example 1; Figure 15This is a schematic diagram showing the cell migration-promoting properties of the composite hydrogel in Example 1; where A is a comparison image of the control group and the AuPt / Gel group after 24 h, and B is the migration rate of the control group and the AuPt / Gel group after 24 h. Figure 16 This is a schematic diagram showing the wound healing performance of the composite hydrogel in Example 1; where A is a graph showing the changes in wound healing in diabetic mice with AuPt / Gel, and B is the wound healing rate of diabetic mice. Figure 17 This is a schematic diagram showing the histological changes in the wound surface caused by the composite hydrogel in Example 1. Figure 18 This is a graph showing the effect of the composite hydrogel in Example 1 on promoting the change of collagen content in wound tissue. Detailed Implementation

[0019] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and technical features described in this application can be combined with each other. It should also be pointed out that, unless otherwise indicated, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The terms "comprising" or "including" and similar words used in this invention refer to elements or objects preceding the word that encompass the elements or objects listed following the word and their equivalents, without excluding other elements or objects.

[0020] On one hand, the present invention provides a composite hydrogel with pH-responsive dual-enzyme activity switching characteristics, comprising a nanozyme and a hydrogel loaded with the nanozyme, wherein the nanozyme is a gold-platinum nanozyme, and the gold-platinum nanozyme is porous and dendritic, capable of exhibiting different enzyme-like activities under different pH conditions, and the different enzyme-like activities can be reversibly switched under different pH conditions; the gold-platinum nanozyme exhibits oxidase-like and peroxidase-like activities in an acidic environment, and superoxide dismutase-like and catalase-like activities in a neutral environment; the hydrogel is a hydrogel with a dynamic Schiff base crosslinking network.

[0021] In this invention, when the gold-platinum nanozyme is in an acidic environment, it exhibits oxidase-like and peroxidase-like activities, promoting the generation of reactive oxygen species (ROS) to achieve highly efficient sterilization. When the gold-platinum nanozyme is in a neutral environment, it again exhibits oxidase-like and peroxidase-like activities, scavenging excess ROS and generating oxygen, thereby alleviating oxidative stress and improving the local hypoxic microenvironment. When using this invention, the mechanism by which it regulates ROS for sterilization and anti-inflammation is as follows: (1) Antibacterial mechanism: When bacteria infect a wound, they proliferate on the wound surface and their metabolic activities produce organic acids such as pyruvate and lactic acid, making the wound microenvironment acidic. The oxidase-like and peroxidase-like activities of gold-platinum nanozymes are enhanced in the acidic environment, producing ROS to kill bacteria.

[0022] (2) Anti-inflammatory regulation: Chronic wounds have a long inflammatory period and contain excessive ROS; gold-platinum nanozymes have good superoxide dismutase-like activity in a neutral environment, which can clear excessive ROS and thus reduce the expression of inflammatory factors (TNF-α, IL-6) in cells.

[0023] (3) Relieve hypoxia: Chronic wounds contain excessive ROS containing H2O2. The catalase-like activity of gold-platinum nanozymes enables them to decompose H2O2 to generate O2, which relieves local hypoxia in chronic wounds, promotes angiogenesis and cell migration, and thus promotes wound healing.

[0024] In summary, when the pH of the environment in which this invention is used changes, the active function can reversibly switch in the opposite direction according to the pH change. This makes this invention different from the single enzyme activity or unidirectional regulation mode in the prior art. It can achieve both antibacterial and antioxidant effects, thereby enhancing antibacterial ability during the infection stage and reducing oxidative damage during the repair stage.

[0025] In existing technologies, enhancing reactive oxygen species (ROS) is beneficial for sterilization but can easily damage normal tissues, while scavenging ROS, although helpful for anti-inflammatory repair, weakens the antibacterial effect; it is difficult to achieve both simultaneously. This invention, through a pH-responsive regulation mechanism, enables the composite hydrogel to enhance ROS generation to inhibit bacteria during the infection phase and reduce ROS levels to protect cells during the repair phase, achieving a dynamic match between antibacterial and tissue repair functions. This represents a functional breakthrough with significant unpredictability, overcoming the functional conflict problem in existing technologies and demonstrating inventiveness.

[0026] Furthermore, this invention does not simply introduce nanozymes into hydrogels, but rather achieves coupled regulation of nanozyme activity and microenvironment response through the synergistic effect of porous dendritic gold-platinum nanozymes and hydrogel networks. The hydrogel provides a carrier and sustained-release function, and its cross-linked network forms a "structure-function integrated" composite system with the porous dendritic gold-platinum nanozymes, significantly improving overall performance.

[0027] In one specific embodiment, the gold-platinum nanozyme has a particle size of 100-1000 nm, and the hydrogel has a pore size of 10-20 μm. In this embodiment, using gold-platinum nanozymes with this particle size allows them to have abundant contact sites and a large specific surface area, enabling them to fully contact bacterial cell membranes for sterilization or contact ROS substrates for removal. Using hydrogels with this pore size provides sufficient deformation space; these pores can absorb the stress from tensile, compressive, and torsional deformations of the hydrogel, maintaining good adhesion even when the wound skin moves and deforms.

[0028] In one specific embodiment, the loading of the gold-platinum nanozyme is greater than or equal to 300 μg / mL. Optionally, the loading of the gold-platinum nanozyme is 400~600 μg / mL.

[0029] In the above embodiments, when the loading of gold-platinum nanozymes is greater than or equal to 300 μg / mL, the sterilization rate reaches over 95%. When the loading is less than this, the sterilization effect is insufficient. The higher the loading, the stronger the antibacterial performance. When the loading is 400 μg / mL, the sterilization rate can reach as high as 99%. To reduce cell damage and avoid material waste, a loading of 400-600 μg / mL is selected for loading.

[0030] In one specific embodiment, the hydrogel is a hydrogel with a dynamic Schiff base crosslinking network constructed from aldehyde-modified polysaccharides and amino-modified polysaccharides. The aldehyde-modified polysaccharide is any one or more selected from oxidized hyaluronic acid, oxidized sodium alginate, oxidized cellulose, oxidized starch, and oxidized traditional Chinese medicine polysaccharides. The amino-modified polysaccharide is a water-soluble chitosan derivative. Optionally, the water-soluble chitosan derivative is carboxymethyl chitosan, chitosan quaternary ammonium salt, or other water-soluble chitosan derivatives.

[0031] In the above embodiments, the aldehyde and amino groups of the aldehyde-modified polysaccharide and the amino-modified polysaccharide can be cross-linked via Schiff base reaction to form a hydrogel with a dynamic Schiff base cross-linked network. This hydrogel, through dynamic Schiff base cross-linking, exhibits a reversible network and thus better flexibility and deformation capability.

[0032] In one specific embodiment, the hydrogel is an oxidized hyaluronic acid-carboxymethyl chitosan hydrogel. In this embodiment, the oxidized hyaluronic acid-carboxymethyl chitosan hydrogel has the following advantages: (1) Biocompatibility: The precursors of oxidized hyaluronic acid-carboxymethyl chitosan hydrogel are hyaluronic acid and carboxymethyl chitosan, both of which are derived from biomacromolecules that are widely present in organisms and are mild and non-irritating. (2) Adhesion and water retention: Its hydrogel network has abundant hydroxyl and amino groups, which can adhere well to the skin surface; its porous network structure can lock in a large amount of water, which has the functions of moisturizing and absorbing wound exudate, and also provides enough space for the hydrogel to deform. (3) Enzymatic sustained release: The oxidized hyaluronic acid and carboxymethyl chitosan that make up the hydrogel are both natural polysaccharides that can be catalyzed and degraded by enzymes secreted by cells at the wound site, and continuously and effectively release gold-platinum nanoenzymes to act on the wound.

[0033] It should be noted that the hydrogel mainly provides a carrier and sustained-release function. The oxidized hyaluronic acid-carboxymethyl chitosan hydrogel in this embodiment is only a preferred Schiff base hydrogel system of the present invention. Other Schiff base hydrogel systems in the prior art that have good biocompatibility and degradability, and can enable the uniform dispersion of nanozymes in the hydrogel to give the system stable sustained-release performance and achieve long-term effects can also be applied to the present invention.

[0034] In one specific embodiment, the gold-platinum nanozyme is prepared by the following steps: S1: Prepare chloroauric acid solution, potassium chloroplatinate solution and tannic acid solution respectively; S2: Mix the chloroauric acid solution and the potassium chloroplatinate solution, then add the tannic acid solution under stirring, and stir the reaction at room temperature for 4-8 h; S3: Wash, centrifuge, and sonicate the product to obtain the gold-platinum nanozyme.

[0035] In the above embodiments, the present invention uses tannic acid as a reducing agent and stabilizer, and can rapidly and greenly prepare porous dendritic gold-platinum alloy nanoparticles with polyphenol surface modification in a one-step method. It does not require additional toxic reducing agents or surface modifiers, and the preparation process is simple, mild, and has good biosafety and reproducibility.

[0036] In a specific embodiment, in step S1, the concentration of the chloroauric acid solution is 4-6 mM, the concentration of the potassium chloroplatinate solution is 4-6 mM, and the concentration of the tannic acid solution is 15-30 mg / mL; in step S2, the volume ratio of the chloroauric acid solution to the potassium chloroplatinate solution is 1.8-2.2:1, and the volume of the tannic acid solution is the sum of the volumes of the chloroauric acid solution and the potassium chloroplatinate solution.

[0037] In the above embodiments, the volume ratio was determined by the absorbance of ox-TMB catalyzed by gold-platinum nanozymes. Specifically, gold-platinum nanozymes with different ratios were prepared by using chloroauric acid in proportions of 100% to 0% of the total amount of chloroauric acid and potassium chloroplatinate. Then, the prepared gold-platinum nanozymes with different amounts of chloroauric acid and potassium chloroplatinate were added to a 3,3',5,5'-tetramethylbenzidine (TMB) solution and reacted for 3 min. The absorbance of the supernatant after the reaction was measured at a wavelength of 652 nm. Wherein, when n 金 :n 铂 When the ratio is 2:1, the absorbance of ox-TMB catalyzed by this gold-platinum nanozyme is 0.73; increasing the chloroauric acid feed ratio, when n 金 :n 铂 When the ratio is 2.2:1, the absorbance of ox-TMB catalyzed by this gold-platinum nanozyme is 0.63; reducing the chloroauric acid feed ratio, when n 金 :n 铂 At a ratio of 1.8:1, the absorbance of ox-TMB catalyzed by this gold-platinum nanozyme was 0.67. That is, as the amount of chloroauric acid increased, the absorbance first increased and then decreased; this ratio was ultimately selected based on the absorbance.

[0038] On the other hand, the present invention also provides a method for preparing a composite hydrogel with pH-responsive dual enzyme activity switching characteristics as described in any one of the above claims, comprising the following steps: dispersing gold-platinum nanozymes in a hydrogel, mixing and allowing to stand to obtain the composite hydrogel.

[0039] In one specific embodiment, when the hydrogel is an oxidized hyaluronic acid-carboxymethyl chitosan hydrogel, the preparation method includes the following steps: S1': Prepare oxidized hyaluronic acid solution and carboxymethyl chitosan solution respectively; S2': Disperse the gold-platinum nanozyme in the oxidized hyaluronic acid solution, so that the gold-platinum nanozyme is uniformly suspended in the oxidized hyaluronic acid solution, and obtain an oxidized hyaluronic acid solution coated with gold-platinum nanozyme. S3': The oxidized hyaluronic acid solution coated with gold-platinum nanozyme is mixed with the carboxymethyl chitosan solution, and after mixing, it is allowed to stand to obtain the composite hydrogel.

[0040] In the above embodiments, oxidized hyaluronic acid and carboxymethyl chitosan can construct a dynamic cross-linked hydrogel network through Schiff base reaction, so that the gold-platinum nanozyme is uniformly dispersed in the hydrogel network to obtain the composite hydrogel.

[0041] In a specific embodiment, in step S1', when preparing the oxidized hyaluronic acid solution, the oxidized hyaluronic acid is prepared by the following steps: oxidizing hyaluronic acid with sodium periodate, then adding ethylene glycol and stirring to terminate the reaction, dialyzing the reaction solution in deionized water, and finally freeze-drying to obtain the oxidized hyaluronic acid.

[0042] It should be noted that the preparation method of oxidized hyaluronic acid is existing technology, and sodium periodate is the most commonly used oxidant in chemical oxidation methods. The above embodiments are only preferred methods for preparing oxidized hyaluronic acid in this invention, and other methods for obtaining oxidized hyaluronic acid in the prior art can also be applied to this invention.

[0043] In one specific embodiment, the hyaluronic acid used has a molecular weight of 800,000, the sodium periodate used has a concentration of 5 mMol / L, the carboxymethyl chitosan used has a degree of substitution of 98%, the concentrations of the oxidized hyaluronic acid solution and the carboxymethyl chitosan solution are both 5 mg / mL, and the final concentration of the gold-platinum nanozyme contained in the composite hydrogel is 500 μg / mL.

[0044] It should be noted that when the concentrations of both the oxidized hyaluronic acid solution and the carboxymethyl chitosan solution were 2.5%, they failed to gel even after prolonged standing, or even if they did gel, they could not form a solid structure. Furthermore, when the concentration of carboxymethyl chitosan was too high (greater than 7.5%), the viscosity of the solution was extremely high, making it difficult to mix the two precursor solutions evenly, resulting in uneven gel formation. The hydrogel formation time was 5-6 minutes when the concentrations of oxidized hyaluronic acid and carboxymethyl chitosan were 5% and 2.5%, respectively, and exceeded 10 minutes when the concentrations were 2.5% and 5%, respectively. Therefore, a concentration of 5% for both precursor solutions was chosen for preparing the hydrogel, as this concentration resulted in suitable gel formation time and state. 5% is only a preferred ratio in this embodiment; other ratios, such as 4% or 6%, can be selected according to the requirements of gel formation time and state when using this invention.

[0045] In another aspect, the present invention also provides the application of the composite hydrogel with pH-responsive dual enzyme activity switching properties described in any one of the above-mentioned claims in the preparation of a chronic wound healing agent for diabetes.

[0046] In this invention, a diabetic chronic wound healing agent prepared using the composite hydrogel with pH-responsive dual-enzyme activity switching properties can efficiently generate reactive oxygen species (ROS) in an acidic environment, exhibiting excellent bactericidal effects against drug-resistant bacteria. Simultaneously, it can promptly remove excess ROS in a neutral environment, reducing oxidative damage to normal tissue cells. This improves biocompatibility while maintaining antibacterial efficacy, surpassing existing antibacterial strategies that rely solely on strong oxidizing agents. Furthermore, by removing excess ROS and generating oxygen, it effectively alleviates oxidative stress and local hypoxia in the wound, promotes angiogenesis and cell migration, thereby significantly accelerating the wound healing process.

[0047] Example 1 A composite hydrogel with pH-responsive dual-enzyme activity switching properties is prepared by the following steps: (1) Prepare 5 mM chloroauric acid (HAuCl4) solution, 5 mM potassium chloroplatinate (K2PtCl4) solution and 20 mg / mL tannic acid (TA) solution respectively. Mix HAuCl4 solution and K2PtCl4 solution at a volume ratio of 2:1. Add an equal volume of TA solution under magnetic stirring and stir continuously at room temperature for 6 h. After the reaction is completed, sonicate the reaction solution, centrifuge at 5000 rpm for 5 min, discard the supernatant, wash with reverse osmosis purified water (RO water) under the above conditions, centrifuge and sonicate three times, and finally disperse and adjust the volume to 5 mg / mL with RO water for later use. The product is denoted as AuPt NPs (abbreviated as AuPt). (2) Add 5 mL of 5 mmol sodium periodate (NaIO4) solution to 100 mL of 1% hyaluronic acid (HA) solution and stir overnight at room temperature in the dark; then add 2 mL of ethylene glycol and stir for 2 h to terminate the reaction. Dialyze the reaction solution in deionized water for 3 days (molecular weight cutoff MWCO = 10 kDa), and finally freeze dry to obtain oxidized hyaluronic acid (OHA). (3) Prepare a 5% OHA solution and a 5% carboxymethyl chitosan (CMC) solution respectively. Add 50 μL of 500 μg / mL AuPt to 225 μL of CMC solution, then take 225 μL of OHA solution and mix well. Let stand for 3 min to obtain the composite hydrogel, which is denoted as AuPt / Gel.

[0048] Example 2 Unlike Example 1, the concentrations of AuPt added in step (3) of this example are 300, 700, and 900 μg / mL.

[0049] Example 3 Unlike Example 1, sodium alginate is added in step (2) of this example, that is, the hydrogel used in this example is sodium oxidized alginate-carboxymethyl chitosan hydrogel.

[0050] Comparative Example 1 Unlike Example 1, in step (1) of this comparative example, instead of tannic acid, pyrogallol, tea polyphenols and sulfonated lignin were added at the same mass concentration.

[0051] Comparative Example 2 Unlike Example 1, in step (1) of this comparative example, instead of potassium chloroplatinate, copper chloride and sodium tetrachloropalladium with the same molar concentration were added, and the products were denoted as AuCu NPs (abbreviated as AuCu) and AuPd NPs (abbreviated as AuPd), respectively.

[0052] Comparative Example 3 Unlike Example 1, this comparative example does not include step (1), and AuPt in step (3) is replaced with deionized water. The product obtained is oxidized hyaluronic acid-carboxymethyl chitosan hydrogel, denoted as Gel.

[0053] Comparative Example 4 Unlike Example 1, this comparative example does not include step (2). The hydrogel in step (3) is a methacryloyl hyaluronic acid (HAMA) photocrosslinked hydrogel. Step (3) specifically includes: Prepare a 5% w / v solution of methacrylamide hyaluronic acid (HAMA, degree of substitution 60%-70%) and add a photoinitiator (ammonium persulfate, 0.25% w / v). Add 50 μL of 500 μg / mL AuPt dropwise to 450 μL of HAMA solution and incubate under 365 nm UV light (intensity approximately 5 mW / cm²). 2 Irradiation for 30-60 seconds under the influence of the irradiation medium crosslinks to form a composite hydrogel.

[0054] Comparative Example 5 Unlike Example 1, this comparative example does not include step (2). The hydrogel in step (3) is a calcium alginate (Ca-Alg) ion-crosslinked hydrogel. Step (3) is specifically as follows: Prepare a 5% w / v sodium alginate solution and a 0.1 M calcium chloride (CaCl2) solution. Add 50 μL of 500 μg / mL AuPt dropwise to 450 μL of the sodium alginate solution, then immerse the solution in the CaCl2 solution for 1 h to crosslink and form a composite hydrogel.

[0055] Test Example 1 The products from step (1) of Example 1, Comparative Example 1, and Comparative Example 2 were added to TMB solution, and the absorbance changes within 180 s after the addition of each nanozyme were measured at a wavelength of 652 nm. The test results are as follows. Figures 1-2 As shown.

[0056] from Figure 1 It can be seen that among the nanozymes prepared using tannic acid, pyrogallol, tea polyphenols and sulfonated lignin, the nanozyme prepared using tannic acid has the most outstanding effect and the highest catalytic activity. Other substances with the same phenolic structure cannot achieve the catalytic activity that this invention can achieve.

[0057] from Figure 2 It can be seen that among the bimetallic nanozymes obtained by reducing gold and palladium, copper and platinum with tannic acid, the gold-copper nanozyme has very low catalytic activity, and although the gold-palladium nanozyme has higher catalytic activity, it is far less than that of the gold-platinum nanozyme of this invention.

[0058] Test Example 2 Adhesion tests were conducted on the hydrogels of Example 1 and Comparative Example 3, and the test results are as follows: Figure 3 As shown, where Figure 3 In the figure, A represents the test result of Comparative Example 3. Figure 3 B in the figure represents the test results of Example 1.

[0059] from Figure 3 As can be seen, the oxidized hyaluronic acid-carboxymethyl chitosan hydrogel in Comparative Example 1 exhibited detachment from the fingers after being squeezed and then relaxed. This phenomenon was also observed in subsequent tests on adhesion to small glass slides and 1.5 mL centrifuge tubes, where the hydrogel quickly fell off upon lifting. In contrast, the composite hydrogel of this invention, after the addition of gold-platinum nanozymes, showed significantly enhanced adhesion. It adhered to the fingers and could be stretched considerably without detaching from the skin surface. It also exhibited good adhesion to small glass slides and centrifuge tubes, allowing them to adhere to the fingers and move freely. This indicates that the addition of polyphenol-functionalized gold-platinum nanozymes enhanced the adhesiveness of the oxidized hyaluronic acid-carboxymethyl chitosan hydrogel.

[0060] Mechanical properties and adhesion were tested on the composite hydrogels of Example 1, Comparative Example 4, and Comparative Example 5. The test results showed that both methacryloyl hyaluronic acid and calcium alginate hydrogels exhibited a hard and brittle texture. Furthermore, methacryloyl hyaluronic acid required ultraviolet light irradiation and was equipment-dependent, while calcium alginate hydrogel was even more fragile; both also showed very weak adhesion. In contrast, the composite hydrogel of this invention, through dynamic Schiff base crosslinking, possesses a reversible network and exhibits excellent flexibility and deformation capability. It can maintain rapid gelation while possessing excellent tissue adhesion and suitable mechanical flexibility, overcoming the limitations of conventional hydrogels (such as HAMA and calcium alginate) with their single function.

[0061] Test Example 3 Rheological tests were performed on the hydrogels of Example 1 and Comparative Example 3 to characterize the viscoelastic properties of Gel and AuPt / Gel. The storage modulus (G') and loss modulus (G'') were used to reflect the elastic and viscous behavior of the hydrogels, respectively. The test results are as follows: Figure 4 As shown.

[0062] from Figure 4 It can be seen that in the shear strain scanning test results of the linear viscoelastic range of the two hydrogels, Gel and AuPt / Gel exhibited gel points at strains of 263% and 304%, respectively, indicating that the gel structure was disrupted and transformed into a fluid state after exceeding this threshold. Within the linear viscoelastic range, G' remained consistently higher than G'' for both hydrogels, and the linear viscoelastic range of AuPt / Gel was greater than that of Gel. This indicates that the polyphenol-functionalized oxidized hyaluronic acid-carboxymethyl chitosan hydrogel of this invention has higher mechanical strength than the oxidized hyaluronic acid-carboxymethyl chitosan hydrogel. The addition of polyphenol-functionalized gold-platinum nanozyme enhanced the mechanical properties of the oxidized hyaluronic acid-carboxymethyl chitosan hydrogel and extended its linear viscoelastic range.

[0063] Test Example 4 The morphology of the gold-platinum nanozyme prepared in step (1) of Example 1 was observed using transmission electron microscopy. The results are as follows: Figure 5 As shown. From Figure 5 It can be seen that the AuPt NPs of this invention are dendritic clusters composed of many smaller nanozymes. This structure increases the specific surface area of ​​the AuPt NPs and enhances their catalytic activity. Furthermore, statistical analysis of their particle size distribution yields the following results: Figure 6 As shown. From Figure 6 It can be seen that AuPt NPs are mainly distributed in the range of 100~1000 nm, with a median of 300 nm.

[0064] Example 1: The actual composite hydrogel is shown below. Figure 7 As shown, its microstructure was observed using a scanning electron microscope, and the results are as follows. Figure 8 As shown. From Figure 8 It can be seen that the pore size of the composite hydrogel of the present invention is 10~20 μm, and AuPt is uniformly distributed in it.

[0065] Test Example 5 The performance of the composite hydrogel in Example 1 in generating ROS was tested. Specifically, TMB was used as the substrate for catalytic oxidation. The absorbance change of the ox-TMB solution after the reaction at 652 nm was measured by UV-Vis spectrophotometer to evaluate the oxidase-like and peroxidase-like activities of AuPt.

[0066] First, the effect of pH on the activities of oxidase-like enzymes and peroxidase-like enzymes was determined by controlling the pH of the reaction solution. The results are as follows: Figure 9 As shown in A in the diagram. From Figure 9 As can be seen from A, the oxidase-like and peroxidase-like activities of AuPt NPs are highest at pH 4, and still have 38.95% and 80.36% catalytic activity at pH 5, respectively.

[0067] Subsequently, the effect of temperature on the activities of oxidase-like enzymes and peroxidase-like enzymes was determined by controlling the temperature of the reaction solution. The results are as follows: Figure 9 As shown in B in the diagram. From Figure 9 As can be seen from B, the oxidase-like and peroxidase-like activities of AuPt NPs are highest at a temperature of 45℃, and still have more than 88.30% and 94.42% of the catalytic activity at a temperature of 35℃, respectively.

[0068] The above test results show that the composite hydrogel of the present invention can generate ROS for sterilization in the acidic, high H2O2 microenvironment of bacterial-infected chronic wounds by means of its enhanced oxidase-like and peroxidase activities.

[0069] Test Example 6 The enzymatic kinetics of oxidase-like and peroxidase-like enzymes in gold-platinum nanozymes of Example 1 were determined. Specifically, the maximum reaction rates vo of oxidase-like and peroxidase-like enzymes in AuPt NPs were determined using TMB as the catalytic substrate. max and the Mi constant K m K m K is the substrate concentration at which the reaction rate reaches its maximum in an enzyme-catalyzed reaction. m The smaller the value, the stronger the affinity of the enzyme for the substrate. Figure 10 In Figures A and B, the reaction rates v of the oxidase-like enzyme and peroxidase of the gold-platinum nanozyme in Example 1 are plotted as the reciprocals of the TMB concentration c. The graph shows that the gold-platinum nanozyme of this invention exhibits a good linear relationship between 1 / v and 1 / c. The maximum reaction rate v of the oxidase-like enzyme activity of the gold-platinum nanozyme was calculated. max and the Mi constant K m They are 1.99 × 10 - 7 M -1 ·s -1 3.12 × 10 -4 M, the maximum reaction rate of peroxidase activity v max and the Mi constant K m They are 2.33 × 10 -7 M -1 ·s -1 5.68 × 10-4 M. Subsequently, the maximum reaction rate v of the two enzyme activities of the gold-platinum nanozyme was measured. max and the Mi constant K m A comparison was made with some nanozymes published in the literature at this stage, and the results are shown in Table 1: Table 1 Comparison of the activities of different enzymes

[0070] Note: In Table 1, NPs represent nanoparticles, NCs represent nanoclusters, and HRP represents horseradish oxidase.

[0071] As can be seen from Table 1, the maximum reaction rate of the gold-platinum nanozyme of the present invention is higher than that of other nanozymes, and the Michaelis constant is also generally lower than that of other nanozymes, indicating that the gold-platinum nanozyme of the present invention has high catalytic oxidation activity and good substrate affinity.

[0072] Test Example 7 The performance of the composite hydrogel in Example 1 for scavenging ROS was tested. Specifically, H2O2 was used as a catalytic substrate to evaluate the catalase-like activity of AuPtNPs. The results are as follows: Figure 11 As shown in A in the diagram. From Figure 11 As can be seen from A in the figure, the dissolved oxygen content gradually increases with the increase of reaction time, and the higher the H2O2 concentration, the greater the dissolved oxygen growth rate, indicating that the rate of dissolved oxygen production is faster.

[0073] The superoxide dismutase (SOD)-like activity of AuPt NPs was then investigated using the nitrotetrazole blue (NBT) colorimetric method, and the results are as follows: Figure 11 As shown in B in the diagram. From Figure 11 As can be seen from B in the figure, with the increase of AuPt NPs concentration, AuPt NPs have an effect on O• The disproportionation of 2 gradually increased, and the absorbance of the solution decreased sequentially after NBT was reduced, indicating that the SOD-like enzyme activity of AuPt NPs is positively correlated with its concentration. Calculations showed that at an AuPt NPs concentration of 30 μg / mL, the SOD-like enzyme activity of AuPt NPs on O• The clearance rate of 2 can reach over 75%.

[0074] The above two experimental results demonstrate that the AuPt NPs of this invention can decompose H2O2 and O• through their catalase-like and superoxide dismutase-like activities. 2. These two reactive oxygen species, AuPt / Gel, can scavenge ROS during the inflammatory phase of chronic wounds, reduce ROS damage to tissue cells, and promote wound healing.

[0075] Test Example 8 The antibacterial properties of the composite hydrogels in Examples 1 and 2 were tested. Specifically, the antibacterial properties of AuPt / Gel (for Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and Gram-negative Escherichia coli (E. coli)) were studied using the dilution-spreading plate method. 490 μL of PBS, 490 μL of PBS containing H2O2 (final concentration 100 μM), 490 μL of PBS + AuPt / Gel hydrogel discs, and 490 μL of PBS containing H2O2 (final concentration 100 μM) and AuPt / Gel hydrogel discs were added to four wells of a 48-well plate. The bacteria were diluted to 1 × 10⁻⁶ with PBS at pH 5.5. 7 CFU / mL, then add 10 μL of bacterial suspension to each group. Incubate the wells in a shaker at 37°C and 180 rpm for 3 h, then suspend the bacterial suspension in each well with PBS to a final volume of 1 mL. Take 50 μL of the incubated suspension and add it to a solid culture plate, spread it evenly with a spreader, and incubate in a 37°C oven for 24 h. Afterwards, take pictures and count the colonies to calculate the survival rate. The test results of Example 1 are as follows. Figure 12 As shown.

[0076] from Figure 12 It can be seen that after 3 hours of incubation, the AuPt / Gel + H2O2 group exhibited the best antibacterial activity (with a bactericidal rate of over 99% against both E. coli and MRSA), and neither single component could completely kill the bacteria. The acidic environment enhanced the oxidase-like (OXD) and peroxidase-like (POD) activities of AuPtNPs, allowing their OXD-like activity to coordinate with their POD-like activity for effective bactericidal action.

[0077] In Example 2, the sterilization rate reached over 95% when the AuPt loading was 300 μg / mL, and the sterilization rate reached over 99% when the AuPt loading was 700 and 900 mg / mL.

[0078] Test Example 9 The cell compatibility of the composite hydrogel in Example 1 was tested. Specifically, the cytotoxic effect of AuPt / Gel on cells was first quantitatively determined using the CCK-8 assay, and the results are as follows: Figure 13 As shown in A in the diagram. From Figure 13 As can be seen from A, even after AuPt / Gel was co-incubated with cells for 12 h and 24 h respectively, both types of cells still had more than 90% activity, indicating that the composite hydrogel of the present invention has little impact on cell activity.

[0079] AuPt / Gel was co-incubated with cells for 24 h, and the effect of AuPt / Gel on cells was further evaluated using a live / dead cell fluorescence staining method. The results are as follows: Figure 13 As shown in B in the diagram. From Figure 13 As shown in Figure B, under a fluorescence microscope, live cells stained with Calcein-AM exhibit green fluorescence, while dead cells stained with propidium iodide (PI) show red fluorescence. With increasing AuPt NPs concentration, the green fluorescence of the experimental group cells did not change significantly compared to the control group, further demonstrating that the AuPt NPs of this invention have good cell compatibility.

[0080] Test Case 10 The anti-inflammatory properties of the composite hydrogel in Example 1 were tested, specifically by evaluating the anti-inflammatory (intracellular ROS scavenging) performance of AuPt / Gel using ROS fluorescent dyes. A suspension of human umbilical vein endothelial cells (HUVECs) to be used was diluted to 5 × 10⁻⁶. 4 Cells were seeded at a concentration of 1 mL / well in 24-well plates and incubated at 37°C with 5% CO2 for 24 h. Then, 20 μM DCFH-DA fluorescent probe was added and the plates were incubated at 37°C in the dark for 30 min. Cells without H2O2 treatment served as a blank control, cells co-incubated with 300 μM H2O2 served as a positive control, and cells co-incubated with 300 μM H2O2 and AuPt / Gel served as the experimental group. Cell nuclei were then stained with Hoechst fluorescent dye. Results are shown below. Figure 14 As shown.

[0081] Figure 14 In the image, blue fluorescence represents the cell nucleus, and green fluorescence represents the binding of intracellular DCFH-DA to ROS. From... Figure 14 As can be seen, compared with the blank control group, the positive control group showed strong green fluorescence, while the green fluorescence intensity representing the intracellular ROS level in the experimental group with added AuPt / Gel was comparable to that in the blank control group, indicating that AuPt / Gel has good anti-inflammatory ability.

[0082] Test Example 11 The cell migration-promoting properties of the composite hydrogel in Example 1 were tested. Specifically, the cell migration-promoting properties of AuPt / Gel were evaluated using a cell scratch assay on HUVEC cells. Cells were cultured in an incubator for 24 h until they filled the bottom of the wells. The bottom cells were then vertically scratched along the diameter of the wells using a 200 μL pipette tip, washed three times with PBS, and then added with complete culture medium. The Transwell chambers were divided into two groups: one group was added to 50 μL of PBS, and the other to AuPt / Gel + 50 μL of PBS containing H2O2 (both with a final H2O2 concentration of 300 μM). The wells were placed in an anaerobic sealed bag and cultured in an incubator. The cells were periodically removed and photographed under a microscope. Finally, ImageJ was used to statistically analyze the migration area. The results are shown below. Figure 15 As shown.

[0083] from Figure 15 It can be seen that the AuPt / Gel + H2O2 group exhibited a higher migration rate. Compared with the control group's 25.90%, the migration rate of the AuPt / Gel + H2O2 group increased to 81.33%, indicating that the CAT-like activity of AuPt NPs decomposes H2O2 to generate O2, which promotes cell migration.

[0084] Test Example 12 To test the wound-healing effect of the composite hydrogel in Example 1, specifically: a bacterial diabetic mouse model was established. BALB / c mice were selected for constructing the diabetic model and fed a high-sugar, high-fat diet for two weeks. Before modeling, mice were fasted but allowed free water for 12 hours. Streptozotocin (STZ) was dissolved in fresh sodium citrate buffer (pH = 4.5) and administered at a dose of 50 mg / kg for 7 consecutive days. During this period, a high-sugar, high-fat diet was maintained. Mice with a blood glucose concentration ≥16.7 mM were selected as successfully modeled diabetic mice and continued to be fed a high-sugar, high-fat diet thereafter.

[0085] After successfully modeling the disease, mice were anesthetized, their back hair was shaved, and a 10 mm diameter full-thickness skin wound was made using a sterile biopsy punch after disinfection with 75% alcohol. 20 μL of Staphylococcus aureus (1 × 10⁻⁶) was inoculated into the wound. 8 Mice were fed with sterile, waterproof, and breathable dressings for two days to create a chronic bacterial wound in diabetic mice. On days 1, 3, 5, and 7, mice were treated with PBS, antibiotics, and AuPt / Gel, respectively. Wound area was recorded and tissue analysis was performed. The results of mouse wound healing rates are shown below. Figure 16 As shown, H&E-stained images of longitudinal sections of the wound skin 14 days after treatment are presented. Figure 17 As shown, a Masson-stained image of a longitudinal section of the wound skin 14 days after treatment is displayed. Figure 18 As shown.

[0086] Figure 16 In the diagram, A represents the changes in wound healing in diabetic mice promoted by AuPt / Gel, and B is a schematic diagram of the wound healing rate in diabetic mice. From... Figure 16It can be seen that after 14 days of treatment, the AuPt / Gel group had the highest wound healing rate, reaching 91.37%, which was significantly different from the 56.83% in the Control group. Furthermore, the AuPt / Gel group exhibited the fastest wound healing speed. The results indicate that the control group, lacking both bactericidal and cell-growth-promoting effects, showed weak healing efficacy; while AuPt / Gel, with its multiple functions, can catalyze bactericidal activity in the early stages and promote cell growth in the later stages, thereby accelerating wound healing. The experiment demonstrates that AuPt / Gel has a superior effect on promoting the healing of chronic diabetic wounds compared to antibiotics.

[0087] from Figure 17 It can be seen that the wound tissue in the control group had not yet healed, while the control group showed a large number of inflammatory cell infiltrations, indicating that the wounds of the mice in this group were still in the inflammatory stage. The AuPt / Gel group showed milder inflammatory cell infiltration and increased fibrous tissue proliferation, along with a large amount of granulation tissue, forming normal skin that was close to epithelium and had a relatively intact structure.

[0088] Collagen secreted by fibroblasts is also an important indicator for assessing wound healing; Masson staining was used to further evaluate collagen deposition at the wound site. Figure 18 As can be seen, the collagen fibers (blue) in the Control group were relatively sparse and thin, and inflammatory cell infiltration was present. Collagen deposition in the antibiotic group increased compared to the Control group, but not significantly. In contrast, the AuPt / Gel group showed abundant collagen fiber deposition, fewer inflammatory cells, and a more orderly arrangement of collagen fibers, indicating a tendency for wound healing. These results collectively and clearly demonstrate that AuPt / Gel can effectively promote the healing of chronic diabetic wounds.

[0089] In summary, the composite hydrogel of this invention uses a gold-platinum nanozyme as its core and a hydrogel as its carrier. It achieves bactericidal and ROS scavenging effects through the enzyme-like activity of the gold-platinum nanozyme under different conditions. In experiments, this composite hydrogel exhibited excellent antibacterial, anti-inflammatory, and wound-healing capabilities. By dynamically regulating ROS through abundant enzyme-like activity, the composite hydrogel intelligently generates or removes reactive oxygen species, achieving bactericidal and anti-inflammatory effects on chronic wounds. This demonstrates the application potential of bimetallic nanozyme composite hydrogels as biomedical materials, providing a novel biomaterial solution for the treatment of bacterial-infected diabetic chronic wounds. This solution allows for intelligent bactericidal or ROS scavenging through dynamic activity regulation to achieve anti-inflammatory effects, thereby promoting cell growth and wound healing.

[0090] It should be noted that the above embodiments are only test results of some embodiments of the present invention. Other embodiments of the present invention have similar performance. All of them can enable the composite hydrogel of the present invention to have excellent antibacterial, anti-inflammatory and wound healing abilities through the reversible enzyme activity of gold-platinum nanozymes under different pH conditions.

[0091] The above description is merely a representative embodiment of the present invention and is not intended to limit the present invention in any way. Any embodiment made by those skilled in the art without departing from the scope of the present invention and utilizing the disclosed technical content is an equivalent embodiment of the present invention. Any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A composite hydrogel with pH-responsive dual-enzyme activity switching property, characterized in that, The invention includes a nanozyme and a hydrogel supporting the nanozyme. The nanozyme is a gold-platinum nanozyme, which is porous and dendritic, and exhibits different enzyme-like activities under different pH conditions. The different enzyme-like activities can be reversibly switched under different pH conditions. The gold-platinum nanozyme exhibits oxidase-like and peroxidase-like activities in an acidic environment, and superoxide dismutase-like and catalase-like activities in a neutral environment. The hydrogel is a hydrogel with a dynamic Schiff base crosslinking network.

2. The composite hydrogel with pH-responsive dual-enzyme activity switchable property according to claim 1, wherein, The gold-platinum nanozyme has a particle size of 100~1000 nm, and the hydrogel has a pore size of 10~20 μm.

3. The composite hydrogel with pH-responsive dual-enzyme activity switchable property according to claim 1, wherein, The loading capacity of the gold-platinum nanozyme is greater than or equal to 300 μg / mL.

4. The composite hydrogel with pH-responsive dual-enzyme activity switchable property according to claim 1, wherein, The hydrogel is a hydrogel with a dynamic Schiff base cross-linking network constructed from aldehyde-modified polysaccharides and amino-modified polysaccharides. The aldehyde-modified polysaccharides are any one or more of oxidized hyaluronic acid, oxidized sodium alginate, oxidized cellulose, oxidized starch, and oxidized traditional Chinese medicine polysaccharides. The amino-modified polysaccharides are water-soluble chitosan derivatives.

5. The composite hydrogel with pH-responsive dual-enzyme activity switchable property according to any one of claims 1-4, wherein, The gold-platinum nanozyme was prepared by the following steps: S1: Prepare chloroauric acid solution, potassium chloroplatinate solution and tannic acid solution respectively; S2: Mix the chloroauric acid solution and the potassium chloroplatinate solution, then add the tannic acid solution under stirring, and stir the reaction at room temperature for 4-8 h; S3: Wash, centrifuge, and sonicate the product to obtain the gold-platinum nanozyme.

6. The composite hydrogel with pH-responsive dual-enzyme activity switchable property according to claim 5, wherein, In step S1, the concentration of the chloroauric acid solution is 4-6 mM, the concentration of the potassium chloroplatinate solution is 4-6 mM, and the concentration of the tannic acid solution is 15-30 mg / mL; in step S2, the volume ratio of the chloroauric acid solution to the potassium chloroplatinate solution is 1.8-2.2:1, and the volume of the tannic acid solution is the sum of the volumes of the chloroauric acid solution and the potassium chloroplatinate solution.

7. The method for preparing the composite hydrogel with pH-responsive dual-enzyme activity switchable characteristics according to any one of claims 1-6, characterized in that, Includes the following steps: The gold-platinum nanozyme was dispersed in the hydrogel, mixed well, and allowed to stand to obtain the composite hydrogel.

8. The method for preparing the composite hydrogel with pH-responsive dual-enzyme activity switching property according to claim 7, characterized in that, When the hydrogel is an oxidized hyaluronic acid-carboxymethyl chitosan hydrogel, the preparation method includes the following steps: S1': Prepare oxidized hyaluronic acid solution and carboxymethyl chitosan solution respectively; S2': Disperse the gold-platinum nanozyme in the oxidized hyaluronic acid solution, so that the gold-platinum nanozyme is uniformly suspended in the oxidized hyaluronic acid solution, and obtain an oxidized hyaluronic acid solution coated with gold-platinum nanozyme. S3': The oxidized hyaluronic acid solution coated with gold-platinum nanozyme is mixed with the carboxymethyl chitosan solution, and after mixing, it is allowed to stand to obtain the composite hydrogel.

9. The method of claim 8, wherein the method comprises the steps of: (a) mixing the first and second hydrogels to form a mixture; (b) adding the crosslinking agent to the mixture to form the composite hydrogel. In step S1', the oxidized hyaluronic acid solution is prepared by the following steps: hyaluronic acid is oxidized with sodium periodate, then ethylene glycol is added and stirred to terminate the reaction, the reaction solution is dialyzed in deionized water, and finally lyophilized to obtain the oxidized hyaluronic acid.

10. The application of the composite hydrogel with pH-responsive dual-enzyme activity switching properties as described in any one of claims 1-6 in the preparation of a reagent for chronic wound healing in diabetes.

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