Antioxidant and antibacterial injectable hydrogel responding to hydrogen peroxide and glucose as well as preparation method and application of injectable hydrogel

Abstract

The invention relates to antibacterial hydrogel, in particular to injectable hydrogel which is antioxidant, responds to hydrogen peroxide and glucose and is antibacterial, a preparation method of the injectable hydrogel and application of the injectable hydrogel to preparation of antibacterial hydrogel dressing for repairing diabetic infectious wounds. The injectable hydrogel capable of resisting oxidation, responding to hydrogen peroxide and glucose and resisting bacteria has a porous three-dimensional network structure and mainly comprises gelatin modified by 4-carboxyphenylboronic acid, tannic acid, zinc chloride and water. The porous three-dimensional network structure is obtained by chemical crosslinking of gelatin modified by 4-carboxyphenylboronic acid and tannic acid, and zinc chloride is loaded in the hydrogel by complexing with the tannic acid. The hydrogel disclosed by the invention has good self-healing ability, injectable ability, oxidation resistance, hydrogen peroxide and glucose response property, good biocompatibility and excellent antibacterial property.

Description

Technical Field

[0001] This invention relates to an antibacterial hydrogel, specifically to an injectable hydrogel that is antioxidant, responsive to hydrogen peroxide and glucose, and antibacterial, as well as its preparation method and its application in the preparation of antibacterial hydrogel dressings for the repair of diabetic infected wounds. Background Technology

[0002] Diabetes is a prevalent chronic disease with an increasing incidence rate. 19-34% of patients develop chronic diabetic wounds such as diabetic foot ulcers, a common, complex, and costly complication of diabetes. The care and treatment of diabetic ulcers is one of the most challenging problems in clinical skin repair. Inflammatory responses, high levels of matrix metalloproteinase expression, reactive oxygen species, excessively high glucose levels, and recurrent bacterial infections are all key factors contributing to the development of chronic wounds. Dressings are a common treatment for chronic wounds. Traditional dry wound dressings, such as gauze, bandages, and absorbent cotton, while protecting the wound and absorbing some exudate, still have many drawbacks that hinder wound healing, such as easy adhesion to the wound, secondary damage during dressing changes, and inability to prevent infection. Therefore, there is an urgent need to develop a dressing specifically for diabetic chronic wounds to fill the gap in the chronic wound dressing market.

[0003] Ideal dressings should be absorbent, easy to remove and replace, safe, antibacterial, non-polluting, and promote wound healing. Compared to traditional dry dressings, hydrogel dressings provide a moist healing environment for the wound, prevent secondary damage, avoid secondary bacterial infection, are less likely to adhere to newly formed tissue, and are easy to functionalize. Furthermore, during wound healing, hydrogel dressings can improve the wound microenvironment, reduce reactive oxygen species levels, inhibit inflammation, and promote cell migration, collagen deposition, and angiogenesis, thereby accelerating wound healing.

[0004] Gelatin is a natural collagen molecule with good biocompatibility, which is beneficial for promoting wound healing. However, gelatin has poor mechanical properties and lacks antibacterial properties. Tannic acid, as a polyphenol, has excellent antioxidant, antibacterial, and anti-inflammatory properties. It can form borate ester bonds with phenylboronic acid, acting as an adhesive. The borate ester bond is a reversible covalent bond, which gives the hydrogel injectability and self-healing properties. Phenylboronic acid can react with and consume H2O2 and glucose, making the hydrogel responsive. Traditional antibacterial ingredients such as antibiotics have the problem of drug resistance, while zinc itself has good antibacterial properties and does not produce drug-resistant bacteria. Appropriate concentrations of zinc can also promote cell migration and have anti-inflammatory effects. As a bioactive ion, zinc also has the advantages of abundant sources, low cost, high stability, and biosafety. Summary of the Invention

[0005] In view of the above-mentioned problems in the prior art, the purpose of this invention is to provide a hydrogel with antioxidant, hydrogen peroxide- and glucose-responsive properties, and good antibacterial properties for the repair of diabetic infected wounds, as well as its preparation method and application. The hydrogel of this invention has good self-healing ability, injectability, antioxidant, hydrogen peroxide- and glucose-responsive properties, and good biocompatibility and excellent antibacterial properties.

[0006] In a first aspect, the present invention provides an injectable hydrogel that is antioxidant, responsive to hydrogen peroxide and glucose, and antibacterial. The injectable hydrogel, which is antioxidant, responsive to hydrogen peroxide and glucose, and antibacterial, has a porous three-dimensional network structure. Its main components are 4-carboxyphenylboronic acid-modified gelatin, tannic acid, zinc chloride, and water. The porous three-dimensional network structure is obtained by chemical cross-linking of the 4-carboxyphenylboronic acid-modified gelatin and tannic acid, and zinc chloride is loaded in the hydrogel through complexation with tannic acid.

[0007] Preferably, the raw material composition of the injectable hydrogel with antioxidant, hydrogen peroxide-responsive, glucose- and antibacterial properties includes: 9-16.5 wt% 4-carboxyphenylboronic acid modified gelatin, 0.45-0.83 wt% tannic acid, 0.08-0.27 wt% zinc chloride, and 83-90.5 wt% water, for a total of 100 wt%.

[0008] Preferably, the self-healing time of the injectable hydrogel, which is antioxidant, responsive to hydrogen peroxide and glucose, and antibacterial, is 30s to 2min, and more preferably 30 to 60s.

[0009] Secondly, the present invention provides a method for preparing an injectable hydrogel that is antioxidant, responsive to hydrogen peroxide and glucose, and antibacterial. The preparation method includes: Step (A) At room temperature, 4-carboxyphenylboronic acid and an activator are dissolved in an organic solvent and stirred for a certain time to obtain a 4-carboxyphenylboronic acid solution. In step (B), gelatin is dissolved in water at a temperature range of 25–50°C to obtain a gelatin solution; 4-carboxyphenylboronic acid solution is added to the gelatin solution and stirred for a certain time to obtain a first 4-carboxyphenylboronic acid modified gelatin solution; subsequently, the first 4-carboxyphenylboronic acid modified gelatin solution is dialyzed and freeze-dried to obtain 4-carboxyphenylboronic acid modified gelatin solid. In step (C), tannic acid and zinc chloride are dissolved in water at room temperature to obtain aqueous solutions of tannic acid and zinc chloride, respectively. Then, the aqueous solutions of tannic acid and zinc chloride are stirred evenly to obtain a tannic acid-zinc chloride complex solution. In step (D), at room temperature, 4-carboxyphenylboronic acid modified gelatin solid is dissolved in water to obtain a second 4-carboxyphenylboronic acid modified gelatin solution, which is then mixed with a tannic acid-zinc chloride complex solution and stirred for a certain period of time to obtain an injectable hydrogel with antioxidant, responsive to hydrogen peroxide and glucose, and antibacterial properties.

[0010] Preferably, in step (A), the activator is N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the organic solvent is dimethyl sulfoxide; and the reaction time is 2-4 h.

[0011] Preferably, in step (A), the amounts of 4-carboxyphenylboronic acid, N-hydroxysuccinimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide are 1-2 wt%, 4-5 wt%, and 6-7 wt% of dimethyl sulfoxide, respectively.

[0012] Preferably, in step (B), the amount of gelatin in the gelatin solution is 3.5 to 4.5 wt% of water; the volume ratio of gelatin solution to 4-carboxyphenylboronic acid solution is 0.8 to 1.2:1; and the reaction time is 24 to 36 h.

[0013] Preferably, in step (C), the amount of tannic acid in the tannic acid aqueous solution is 3-4 wt% of water, and the amount of zinc chloride in the zinc chloride aqueous solution is 0.6-2.4 wt% of water; the volume ratio of the tannic acid aqueous solution to the zinc chloride aqueous solution is 1:1-1.2; and the stirring time is 1-2 h.

[0014] Preferably, in step (D), the concentration of the second 4-carboxyphenylboronic acid modified gelatin solution is 15-20 wt%; the volume ratio of the second 4-carboxyphenylboronic acid modified gelatin solution to the tannic acid-zinc chloride complex solution is 1-3:1; and the stirring time is 30-60 s.

[0015] Thirdly, the present invention provides the use of the antioxidant, hydrogen peroxide- and glucose-responsive, antibacterial injectable hydrogel described in any of the above claims in the preparation of antibacterial hydrogel dressings for the prevention and / or treatment of diabetic infectious wounds.

[0016] Beneficial effects:

[0017] In this invention, the injectable hydrogel is prepared through a physicochemical reaction and possesses strong DPPH scavenging ability, self-healing properties, and injectability. It can respond to accelerated degradation by H2O2 and glucose, exhibits good biocompatibility, and possesses strong antibacterial properties (e.g., for a concentration of 1×10⁻⁶). 7 The kill rates of Staphylococcus aureus and Escherichia coli at CFU / mL were 99.9% and 99.7%, respectively. Attached Figure Description

[0018] Figure 1 The image shown is a sol-gel optical image of the GPT@Zn2 hydrogel in the example. Figure 2 The rheological test results of the GPT@Zn2 hydrogel in the examples are shown below; Figure 3 The results of the self-healing and injectability tests of the GPT@Zn2 hydrogel in the examples are shown. Figure 4 The results of degradation tests of GPT@Zn2 hydrogel in PBS, H2O2 and glucose environments are shown in the examples. Figure 5 The DPPH scavenging rates of GPT, GPT@Zn1, GPT@Zn2, and GPT@Zn3 hydrogels in the examples are shown. Figure 6 The antibacterial coating results of GPT, GPT@Zn1, GPT@Zn2, and GPT@Zn3 hydrogels in the examples are shown. Figure 7 The figures show the cell viability results of the 24-hour cytotoxicity test of GPT, GPT@Zn1, GPT@Zn2, and GPT@Zn3 hydrogels in the examples; the two groups of bars on the left represent the relative cell viability of L929 cells after 24 hours of co-culturing with the hydrogel and the relative cell viability of L929 cells after 48 hours of co-culturing with the hydrogel; the two groups of bars on the right represent the relative cell viability of HUVECs cells after 24 hours of co-culturing with the hydrogel and the relative cell viability of HUVECs cells after 48 hours of co-culturing with the hydrogel; the bars from left to right are the Control group, GPT hydrogel group, GPT@Zn1 hydrogel group, GPT@Zn2 hydrogel group, and GPT@Zn3 hydrogel group. Figure 8 The images shown are fluorescence micrographs of the cells living and dead cells of the hydrogels prepared by GPT, GPT@Zn1, GPT@Zn2, and GPT@Zn3 in the examples. Figure 9 The results of in vivo antibacterial experiments on GPT and GPT@Zn2 hydrogels in the examples are shown. Figure 10 The results of the healing rate of GPT and GPT@Zn2 hydrogels used in the examples to treat infected wounds in diabetic rats are shown. Detailed Implementation

[0019] The present invention is further illustrated by the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0020] The injectable hydrogel of this invention, possessing antioxidant, hydrogen peroxide- and glucose-responsive, and antibacterial properties, exhibits a three-dimensional porous structure (a porous three-dimensional network structure). Its main components are 4-carboxyphenylboronic acid-modified gelatin, tannic acid, zinc chloride, and water. The chemical cross-linking of tannic acid with the phenylboronic acid in the 4-carboxyphenylboronic acid-modified gelatin to form borate ester bonds is the primary chemical bond forming the hydrogel. A single tannic acid molecule can form borate ester bonds with multiple 4-carboxyphenylboronic acids, thus enabling tannic acid to cross-link the gelatin and form a three-dimensional hydrogel structure. Simultaneously, tannic acid and gelatin form numerous hydrogen bonds, and tannic acid complexes with zinc ions, forming secondary cross-linking, ultimately resulting in the overall three-dimensional cross-linked network of the hydrogel.

[0021] The injectable hydrogel comprises the following raw materials: 8.5–16.5 wt% 4-carboxyphenylboronic acid modified gelatin, 0.4–0.9 wt% tannic acid, 0.08–0.3 wt% zinc chloride, and 83–95 wt% water, totaling 100 wt%. The combination of these components and the aforementioned proportions were determined through extensive experimentation. This combination and proportion range allows the hydrogel of the present invention to possess an excellent balance of antioxidant properties, responsiveness to hydrogen peroxide and glucose, antibacterial properties, and biocompatibility. If the mass ratio of 4-carboxyphenylboronic acid modified gelatin to tannic acid exceeds the aforementioned range, gelation will fail. Preferably, the injectable hydrogel with antioxidant, hydrogen peroxide and glucose responsiveness, and antibacterial properties comprises: 9–16.5 wt% 4-carboxyphenylboronic acid modified gelatin, 0.45–0.83 wt% tannic acid, 0.08–0.27 wt% zinc chloride, and 83–90.5 wt% water, totaling 100 wt%.

[0022] Gelatin, a natural collagen molecule, possesses excellent biocompatibility and promotes wound healing. Gelatin is modified with 4-carboxyphenylboronic acid, enabling it to chemically cross-link with tannic acid. Furthermore, phenylboronic acid reacts with and consumes H₂O₂ and glucose, giving the hydrogel responsiveness. Tannic acid, a polyphenol, exhibits excellent antioxidant, antibacterial, and anti-inflammatory properties. It can form borate ester bonds with phenylboronic acid, acting as an adhesive. The borate ester bond is a reversible covalent bond, granting the hydrogel injectability and self-healing properties. Zinc, a bioactive ion, is abundant, low-cost, highly stable, and biosafe. Zinc itself possesses good antibacterial properties without inducing drug-resistant bacteria, and appropriate concentrations can promote cell migration and anti-inflammatory effects. Zinc chloride is incorporated into the hydrogel through complexation with tannic acid, allowing for a stable and continuous release of zinc.

[0023] The hydrogel described in this invention can still recover into a whole after its overall structure is destroyed, and has self-healing ability and injectability.

[0024] The antioxidant, hydrogen peroxide- and glucose-responsive, and antibacterial injectable hydrogel of this invention exhibits the following hydrogen peroxide and glucose responsiveness: the degradation rate in a hydrogen peroxide environment for 24 hours is increased from 35-45% to 80-90% compared to a PBS environment; the degradation rate in a glucose environment for 24 hours is increased from 35-45% to 55-65% compared to a PBS environment; and the degradation rate in an environment containing both hydrogen peroxide and glucose for 24 hours is increased from 35-45% to 95-100% compared to a PBS environment.

[0025] The present invention also provides a method for preparing an injectable hydrogel that is antioxidant, responsive to hydrogen peroxide and glucose, and antibacterial.

[0026] Step (A) involves dissolving 4-carboxyphenylboronic acid and an activator in an organic solvent at room temperature and stirring for a certain time to obtain a 4-carboxyphenylboronic acid solution. The organic solvent can be dimethyl sulfoxide. The activator can be N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide. Preferably, the amounts of 4-carboxyphenylboronic acid, N-hydroxysuccinimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide are 1–2 wt%, 4–5 wt%, and 6–7 wt% of dimethyl sulfoxide, respectively. The stirring time can be 2–4 hours. The stirring reaction activates the carboxyl group of 4-carboxyphenylboronic acid.

[0027] In step (B), gelatin is dissolved in water at a temperature range of 25–50°C to obtain a gelatin solution; 4-carboxyphenylboronic acid solution is added to the gelatin solution and stirred for a certain time to obtain a 4-carboxyphenylboronic acid modified gelatin solution; subsequently, the 4-carboxyphenylboronic acid modified gelatin solution is dialyzed and then freeze-dried to obtain a 4-carboxyphenylboronic acid modified gelatin solid.

[0028] In step (B), gelatin is completely dissolved in ultrapure water to obtain a gelatin solution. The amount of gelatin in the gelatin solution can be 3.5–4.5 wt% of ultrapure water. A 4-carboxyphenylboronic acid solution is added dropwise to the gelatin solution and the mixture is stirred for 24–36 hours. The volume ratio of the gelatin solution to the 4-carboxyphenylboronic acid solution can be 0.8–1.2:1 (e.g., 0.8:1). The obtained (first) 4-carboxyphenylboronic acid modified gelatin solution is dialyzed in a dialysis bag for 72–120 hours to remove impurities, and then freeze-dried to obtain 4-carboxyphenylboronic acid modified gelatin solid.

[0029] In step (C), tannic acid and zinc chloride are completely dissolved in ultrapure water at room temperature to obtain tannic acid solution and zinc chloride solution, respectively. The tannic acid solution and zinc chloride solution are then mixed under stirring to obtain a tannic acid-zinc chloride complex solution.

[0030] In step (C), the amount of tannic acid in the tannic acid solution is 3-4 wt% of water, and the amount of zinc chloride in the zinc chloride solution is 0.6-2.4 wt% of water. The tannic acid solution and the zinc chloride solution are then mixed and stirred at a volume ratio of 1:1-1.2 (e.g., 1:1) for 1-2 hours (e.g., 2 hours) to obtain a tannic acid-zinc chloride complex solution.

[0031] In step (D), 4-carboxyphenylboronic acid modified gelatin solid is dissolved in ultrapure water at room temperature to obtain a (second) 4-carboxyphenylboronic acid modified gelatin solution. Subsequently, the (second) 4-carboxyphenylboronic acid modified gelatin solution and the tannic acid-zinc chloride complex solution are mixed in a certain proportion and stirred for a certain period of time to obtain an injectable hydrogel with antioxidant, responsive to hydrogen peroxide and glucose, and antibacterial properties.

[0032] In step (D), the concentration of the (second) 4-carboxyphenylboronic acid modified gelatin solution is 15–20 wt%. The volume ratio of the (second) 4-carboxyphenylboronic acid modified gelatin solution to the tannic acid zinc chloride complex solution is 1–3:1 (e.g., 2:1). The stirring time can be 30–60 s (e.g., 30 s).

[0033] The present invention also provides the application of the above-mentioned injectable hydrogel material with antioxidant, hydrogen peroxide- and glucose-responsive properties, and antibacterial properties in the preparation of dressings for diabetic infected wounds.

[0034] In the injectable hydrogel of this invention, a borate ester bond is formed between 4-carboxyphenylboronic acid-modified gelatin and tannic acid, allowing for gelation without the introduction of an additional crosslinking agent. Furthermore, the borate ester bond endows the hydrogel with hydrogen peroxide and glucose responsiveness. In addition, the hydrogel of this invention also exhibits good injectability and self-healing properties, making it more suitable for wound dressing applications.

[0035] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0036] Example 1

[0037] Methods for preparing injectable hydrogels with antioxidant, hydrogen peroxide and glucose responsiveness, and antibacterial properties include:

[0038] Step (A) At room temperature, 1.5 g of 4-carboxyphenylboronic acid, 4 g of N-hydroxysuccinimide and 6 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide were dissolved in 100 mL of dimethyl sulfoxide and stirred for 2 hours to obtain a carboxyl-activated 4-carboxyphenylboronic acid solution.

[0039] In step (B), 4g of gelatin was completely dissolved in water at 35°C to obtain a gelatin solution, with the amount of gelatin being 4wt% of the water. Then, the activated 4-carboxyphenylboronic acid solution from the previous step was completely added to the gelatin solution, with a volume ratio of 0.8:1 between the gelatin solution and the 4-carboxyphenylboronic acid solution. The mixture was stirred for 36 hours to obtain a 4-carboxyphenylboronic acid-modified gelatin solution. This solution was then dialyzed for 3 days and frozen for 36 hours to obtain a 4-carboxyphenylboronic acid-modified gelatin solid.

[0040] In step (C), 0.3 g of tannic acid and 0.06 g of zinc chloride were completely dissolved in 10 mL of ultrapure water at room temperature. Then, the tannic acid solution and the zinc chloride solution were mixed and stirred at a volume ratio of 1:1 for 1.5 h to obtain 20 mL of tannic acid-zinc chloride complex solution.

[0041] In step (D), 30 mg of 4-carboxyphenylboronic acid modified gelatin solid was dissolved in 200 μL of ultrapure water at room temperature. Then, the 4-carboxyphenylboronic acid modified gelatin solution was mixed with 100 μL of tannic acid-zinc chloride complex solution and stirred for 30 s to obtain an injectable hydrogel, labeled GPT@Zn1, with a final zinc chloride content of 0.09%.

[0042] Example 2

[0043] The method in Example 1 was repeated according to the above proportions, except that: the amount of zinc chloride used was 0.12g, the sample was labeled as GPT@Zn2, and the final zinc chloride content of the hydrogel was 0.18%.

[0044] Example 3

[0045] The method in Example 1 was repeated according to the above proportions, except that: the amount of chlorination was 0.18g, the sample was labeled as GPT@Zn3, and the final zinc chloride content of the hydrogel was 0.27%.

[0046] Comparative Example 1

[0047] Step (A) At room temperature, 1.5 g of 4-carboxyphenylboronic acid, 4 g of N-hydroxysuccinimide and 6 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide were dissolved in 100 mL of dimethyl sulfoxide and stirred for 2 hours to obtain a carboxyl-activated 4-carboxyphenylboronic acid solution.

[0048] In step (B), 4g of gelatin was completely dissolved in water at 35°C to obtain a gelatin solution, with the amount of gelatin being 4wt% of the water. Then, the activated 4-carboxyphenylboronic acid solution from the previous step was completely added to the gelatin solution, with a volume ratio of 0.8:1 between the gelatin solution and the 4-carboxyphenylboronic acid solution. The mixture was stirred for 36 hours to obtain a 4-carboxyphenylboronic acid-modified gelatin solution. This solution was then dialyzed for 3 days and frozen for 36 hours to obtain a 4-carboxyphenylboronic acid-modified gelatin solid.

[0049] In step (C), at room temperature, 30 mg of 4-carboxyphenylboronic acid modified gelatin solid was dissolved in 200 μL of ultrapure water, and 1.5 mg of tannic acid was dissolved in 100 μL of ultrapure water. Then, the 4-carboxyphenylboronic acid modified gelatin solution and the tannic acid solution were mixed and stirred for 30 s to finally obtain an injectable hydrogel, labeled as GPT.

[0050] like Figure 1 The sol-gel optical image of GPT@Zn2 hydrogel shown demonstrates that a uniform and stable composite gel can be successfully prepared.

[0051] Figure 2 The rheological test results of GPT@Zn2 hydrogel are presented, in which the storage modulus (G') of the hydrogel is significantly higher than the gel loss modulus (G”), further confirming the successful and stable preparation of the hydrogel.

[0052] Figure 3 The self-healing and injectability properties of GPT@Zn2 hydrogel were characterized. After cutting and staining a single piece of hydrogel, a complete gel could be obtained by splicing the sections together, and it could be picked up with tweezers, demonstrating the excellent self-healing properties of the hydrogel. The hydrogel could be uniformly extruded into a 1mL syringe, demonstrating its injectability.

[0053] Figure 4 The degradation results of GPT@Zn2 hydrogel in PBS, H2O2, and glucose environments are presented. GPT@Zn2 hydrogel was placed in PBS solution, 0.6 mM H2O2 solution, 4 g / L glucose solution, and 0.6 mM H2O2 + 4 g / L glucose solution, respectively. The hydrogel was lyophilized at different time points, and the mass of the lyophilized samples was measured and the percentage of remaining mass was calculated. The results show that the hydrogel degrades faster in H2O2 and glucose environments, indicating that the hydrogel is responsive to hydrogen peroxide and glucose.

[0054] Figure 5The DPPH scavenging rates of GPT, GPT@Zn1, GPT@Zn2, and GPT@Zn3 hydrogels are presented. The DPPH scavenging rate was tested as follows: a 0.1 mM DPPH solution was prepared using ethanol. 100 μL of hydrogel sample was incubated with 1 mL of DPPH ethanol solution at 37°C for 10 min on a shaker. 200 μL of the supernatant was then measured at 517 nm using a microplate reader. The DPPH scavenging rate was calculated from the absorbance. The formula for DPPH scavenging rate is: DPPH scavenging rate (%) = (Ao - An) / (Ap - An) × 100%, where Ao, An, and Ap are the absorbances of the sample group, negative control, and positive control, respectively. The positive control was the group without sample (200 μL DPPH ethanol solution); the negative control was anhydrous ethanol (200 μL ethanol). The DPPH scavenging rate of the hydrogels was approximately 90%, demonstrating the excellent antioxidant properties of the hydrogels.

[0055] Figure 6 The antibacterial coating results for GPT, GPT@Zn1, GPT@Zn2, and GPT@Zn3 hydrogels are shown. 1 mL of 1×10⁻⁶ g / mL solution was used. 7 Staphylococcus aureus and Escherichia coli (CFU / mL) were co-cultured with 50 μL of gel for 8 h before being plated. The bacterial plate results showed that the hydrogels exhibited excellent antibacterial effects, with GPT@Zn3 showing the strongest antibacterial activity.

[0056] Figure 7 24-hour cytotoxicity assays were performed on GPT, GPT@Zn1, GPT@Zn2, and GPT@Zn3 hydrogels. L929 cell culture medium consisted of 89% α-MEM basal medium, 10% FBS (bovine serum albumin), and 1% penicillin-dip antibiotics; HUVEC cell culture medium consisted of 89% DMEM basal medium, 10% FBS, and 1% penicillin-dip antibiotics. The hydrogels were placed in the culture medium (50 μL of hydrogel per 1 mL of medium; no hydrogel was added to the control group). After 24 hours, the supernatant was collected to obtain the hydrogel extract. The L929 and HUVEC cell suspensions were then subjected to a 5 × 10⁻⁶ ppm hydrochloric acid treatment. 3 Cells were added to 96-well plates at a specific density and cultured for 24 hours. The culture medium was then aspirated, 100 μL of extraction buffer was added, and the plates were cultured for another 24 hours. Cells were then washed three times with PBS, followed by staining with Alma Blue assay for 2 hours. Finally, fluorescence intensity was measured using a microplate reader. Figure 5 The relative cell viability of the hydrogel groups was all above 90%, indicating that the hydrogels had no obvious cytotoxicity to L929 cells and HUEVC cells and had good biocompatibility.

[0057] Figure 8These are fluorescence micrographs of cell viability and mortality of hydrogels prepared with GPT, GPT@Zn1, GPT@Zn2, and GPT@Zn3 in the examples. L929 cell suspension was subjected to a 3×10⁻⁶ ppm concentration. 4 Cells were added to 24-well plates at a density of 24 μL and cultured in a cell culture incubator for 24 h. The culture medium was aspirated, and 100 μL of medium containing H2O2 (600 μM) and 50 μL of GPT or GPT@Zn2 hydrogel were added (the control group only added medium without H2O2 and hydrogel, and the H2O2 group did not add hydrogel). The cells were cultured in the incubator for another 6 h, then washed three times with PBS, stained with calcein / propidium iodide staining agent for 15 min, and finally photographed using a laser confocal fluorescence microscope.

[0058] according to Figure 8 In the control group, the vast majority of L929 cells were viable, with few dead cells, indicating a high survival rate and good condition. However, after stimulation with H2O2, most L929 cells died. The addition of hydrogel increased the number of viable cells and decreased the number of dead cells. This is because the hydrogel removes harmful H2O2, thus protecting the cells.

[0059] Figure 9 This is a plate-spreading result of the in vivo antibacterial experiment of GPT and GPT@Zn2 hydrogels in animals. First, male rats of uniform age were selected and intraperitoneally injected with streptozotocin citrate buffer at a dose of 65 mg / kg to establish a diabetic rat model. Then, two 1 cm diameter circular wounds were created on the back of each rat. 20 μL of a 1×10⁻⁶ solution was added to each wound. 7 The wound was treated with a CFU / ml Staphylococcus aureus bacterial suspension, with 50 μL of hydrogel added to the hydrogel group. A transparent dressing was then applied to the wound and secured with tape. On the first postoperative day, one mouse from each group was harvested, and the wound, surrounding 1–2 mm of skin, and subcutaneous fascia were removed. The tissue was then placed in 4 mL of physiological saline for a bacterial smear test. The results confirmed that the GPT@Zn2 hydrogel maintained significant antibacterial activity in animals.

[0060] Figure 10 This study illustrates the wound healing effects of GPT and GPT@Zn2 in diabetic animals with infectious wounds in the examples. The rats in the aforementioned model were continuously observed, and the remaining wound area was recorded. A comparison of the remaining wound areas in each group revealed that, compared to the control group and GPT hydrogel, the GPT@Zn2 hydrogel group had the smallest remaining wound area at days 4, 7, and 14, indicating that GPT@Zn2 hydrogel has a significant wound-healing promoting effect.

[0061] Comparative Example 2

[0062] 30 mg of solid gelatin was dissolved in 200 μL of ultrapure water, and 1.5 mg of tannic acid was dissolved in 100 μL of ultrapure water. The gelatin solution and the tannic acid solution were then mixed and stirred for 30 seconds. The results showed that Comparative Example 2 failed to form a gel. This is because the pure gelatin and tannic acid, when stirred for a short time, only formed hydrogen bonds, not borate ester bonds. Hydrogen bonds are weak and unstable, therefore they cannot form a hydrogel.

[0063] Comparative Example 3

[0064] Step (A) At room temperature, 1.5 g of 4-carboxyphenylboronic acid, 4 g of N-hydroxysuccinimide and 6 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide were dissolved in 100 mL of dimethyl sulfoxide and stirred for 2 hours to obtain a carboxyl-activated 4-carboxyphenylboronic acid solution.

[0065] In step (B), 4g of gelatin was completely dissolved in water at 35°C to obtain a gelatin solution, with the amount of gelatin being 4wt% of the water. Then, the activated 4-carboxyphenylboronic acid solution from the previous step was completely added to the gelatin solution, with a volume ratio of 0.8:1 between the gelatin solution and the 4-carboxyphenylboronic acid solution. The mixture was stirred for 36 hours to obtain a 4-carboxyphenylboronic acid-modified gelatin solution. This solution was then dialyzed for 3 days and frozen for 36 hours to obtain a 4-carboxyphenylboronic acid-modified gelatin solid.

[0066] In step (C), at room temperature, 30 mg of 4-carboxyphenylboronic acid modified gelatin solid was dissolved in 200 μL of ultrapure water, and 5 mg of tannic acid was dissolved in 100 μL of ultrapure water. Then, the 4-carboxyphenylboronic acid modified gelatin solution and the tannic acid solution were mixed and stirred for 30 s.

[0067] Excessive tannic acid leads to over-polymerization with 4-carboxylated modified gelatin, preventing the formation of a stable three-dimensional hydrogel network.

[0068] Comparative Example 4

[0069] Step (A) At room temperature, 1.5 g of 4-carboxyphenylboronic acid, 4 g of N-hydroxysuccinimide and 6 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide were dissolved in 100 mL of dimethyl sulfoxide and stirred for 2 hours to obtain a carboxyl-activated 4-carboxyphenylboronic acid solution.

[0070] In step (B), 4g of gelatin was completely dissolved in water at 35°C to obtain a gelatin solution, with the amount of gelatin being 4wt% of the water. Then, the activated 4-carboxyphenylboronic acid solution from the previous step was completely added to the gelatin solution, with a volume ratio of 0.8:1 between the gelatin solution and the 4-carboxyphenylboronic acid solution. The mixture was stirred for 36 hours to obtain a 4-carboxyphenylboronic acid-modified gelatin solution. This solution was then dialyzed for 3 days and frozen for 36 hours to obtain a 4-carboxyphenylboronic acid-modified gelatin solid.

[0071] In step (C), 0.3 g of tannic acid and 0.24 g of zinc chloride were completely dissolved in 10 mL of ultrapure water at room temperature. Then, the tannic acid solution and the zinc chloride solution were mixed and stirred at a volume ratio of 1:1 for 1.5 h to obtain 20 mL of tannic acid-zinc chloride complex solution.

[0072] In step (D), 30 mg of 4-carboxyphenylboronic acid modified gelatin solid was dissolved in 200 μL of ultrapure water at room temperature. Then, the 4-carboxyphenylboronic acid modified gelatin solution was mixed with 100 μL of tannic acid-zinc chloride complex solution and stirred for 30 s to obtain an injectable hydrogel labeled GPT@Zn4. The final zinc chloride content of the hydrogel was 0.36%.

[0073] Due to the high zinc concentration, the hydrogel exhibits high cytotoxicity, and GPT@Zn4 significantly reduces the relative cell activity in cell compatibility experiments.

Claims

1. An injectable hydrogel that is antioxidant, responsive to hydrogen peroxide and glucose, and antibacterial, characterized in that, The injectable hydrogel, which is antioxidant, responsive to hydrogen peroxide and glucose, and antibacterial, has a porous three-dimensional network structure. Its main components are 4-carboxyphenylboronic acid-modified gelatin, tannic acid, zinc chloride, and water. The porous three-dimensional network structure is obtained by chemical cross-linking of 4-carboxyphenylboronic acid-modified gelatin and tannic acid, and zinc chloride is loaded in the hydrogel by complexing with tannic acid.

2. The injectable hydrogel with antioxidant, hydrogen peroxide- and glucose-responsive, and antibacterial properties according to claim 1, characterized in that, The injectable hydrogel with antioxidant, hydrogen peroxide-responsive, glucose- and antibacterial properties comprises the following raw materials: 9-16.5 wt% 4-carboxyphenylboronic acid modified gelatin, 0.45-0.83 wt% tannic acid, 0.08-0.27 wt% zinc chloride, and 83-90.5 wt% water, for a total of 100 wt%.

3. The injectable hydrogel with antioxidant, hydrogen peroxide- and glucose-responsive, and antibacterial properties according to claim 1 or 2, characterized in that, The self-healing time of the injectable hydrogel, which is antioxidant, responsive to hydrogen peroxide and glucose, and antibacterial, is 30s to 2min, preferably 30 to 60s.

4. A method for preparing an injectable hydrogel with antioxidant, hydrogen peroxide- and glucose-responsive, and antibacterial properties as described in any one of claims 1-3, characterized in that, include: Step (A) At room temperature, 4-carboxyphenylboronic acid and an activator are dissolved in an organic solvent and stirred for a certain time to obtain a 4-carboxyphenylboronic acid solution. In step (B), gelatin is dissolved in water at a temperature range of 25–50°C to obtain a gelatin solution; 4-carboxyphenylboronic acid solution is added to the gelatin solution and stirred for a certain time to obtain a first 4-carboxyphenylboronic acid modified gelatin solution; subsequently, the first 4-carboxyphenylboronic acid modified gelatin solution is dialyzed and freeze-dried to obtain 4-carboxyphenylboronic acid modified gelatin solid. In step (C), tannic acid and zinc chloride are dissolved in water at room temperature to obtain aqueous solutions of tannic acid and zinc chloride, respectively. Then, the aqueous solutions of tannic acid and zinc chloride are stirred evenly to obtain a tannic acid-zinc chloride complex solution. In step (D), at room temperature, 4-carboxyphenylboronic acid modified gelatin solid is dissolved in water to obtain a second 4-carboxyphenylboronic acid modified gelatin solution, which is then mixed with a tannic acid-zinc chloride complex solution and stirred for a certain period of time to obtain an injectable hydrogel with antioxidant, responsive to hydrogen peroxide and glucose, and antibacterial properties.

5. The preparation method according to claim 4, characterized in that, In step (A), the activator is N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the organic solvent is dimethyl sulfoxide; and the reaction time is 2-4 h.

6. The preparation method according to claim 5, characterized in that, In step (A), the amounts of 4-carboxyphenylboronic acid, N-hydroxysuccinimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide are 1-2 wt%, 4-5 wt%, and 6-7 wt% of dimethyl sulfoxide, respectively.

7. The preparation method according to any one of claims 4 to 6, characterized in that, In step (B), the amount of gelatin in the gelatin solution is 3.5 to 4.5 wt% of water; the volume ratio of gelatin solution to 4-carboxyphenylboronic acid solution is 0.8 to 1.2:1; and the reaction time is 24 to 36 h.

8. The preparation method according to any one of claims 4 to 7, characterized in that, In step (C), the amount of tannic acid in the tannic acid aqueous solution is 3-4 wt% of water, and the amount of zinc chloride in the zinc chloride aqueous solution is 0.6-2.4 wt% of water; the volume ratio of the tannic acid aqueous solution to the zinc chloride aqueous solution is 1:1-1.2; and the stirring time is 1-2 h.

9. The preparation method according to any one of claims 4 to 8, characterized in that, In step (D), the concentration of the second 4-carboxyphenylboronic acid modified gelatin solution is 15-20 wt%; the volume ratio of the second 4-carboxyphenylboronic acid modified gelatin solution to the tannic acid-zinc chloride complex solution is 1-3:1; and the stirring time is 30-60 s.

10. The use of any one of the antioxidant, hydrogen peroxide- and glucose-responsive, and antibacterial injectable hydrogels according to claims 1-3 in the preparation of antibacterial hydrogel dressings for the prevention and / or treatment of diabetic infected wounds.

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