Preparation method and application of antibacterial hydrogel capable of being rapidly cured

Abstract

The invention belongs to the field of materials, and particularly relates to a preparation method and application of antibacterial hydrogel capable of being rapidly cured. The preparation method comprises the following steps: uniformly mixing dopamine-modified hyaluronic acid and ferric chloride, carrying out a metal coordination reaction to prepare a first layer of hydrogel network, uniformly mixing norbornene-modified collagen and azine-modified four-arm polyethylene glycol, and carrying out a click chemical reaction to prepare a second layer of hydrogel network; the rapidly-cured antibacterial hydrogel is obtained through interpenetrating and crosslinking of two layers of networks and is applied to skin repair. The antibacterial hydrogel capable of being rapidly cured, provided by the invention, has the advantages of more uniform system, no particle property, simple preparation method, good biocompatibility and excellent antibacterial property, can effectively resist various pathogenic microorganisms, and shows a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of materials, and in particular relates to a preparation method and application of a rapidly curable antibacterial hydrogel based on click chemistry. Background Art

[0002] The skin is the largest organ in the human body, accounting for approximately 10% of body weight. Due to its direct contact with the external environment, it is highly susceptible to damage. Different wounds heal in varying amounts, influenced by the depth, area, and location of the wound. Prolonged wound healing can lead to infection and serious complications, resulting in significant medical costs and prolonged pain and emotional distress for the patient. A wide variety of wound dressings have been developed to manage skin wounds. However, commonly used wound dressings, such as gauze, bandages, and Band-Aids, are passive dressings. These traditional wound dressings offer advantages such as simplicity, cost-effectiveness, and widespread availability. However, they generally lack active wound healing functions, such as controlling infection, promoting cell growth, or absorbing excess exudate. With advances in medical technology, modern wound dressings have gradually replaced traditional wound dressings. Modern wound dressings are an innovative class of wound care products that not only provide basic protection and absorption functions but also possess properties that promote wound healing. They primarily include active dressings, hydrogel dressings, foam dressings, alginate dressings, and negative pressure wound therapy (NPWT).

[0003] Hydrogel dressings are a type of modern wound care product, and their unique physical and chemical properties play an important role in the field of wound healing. Hydrogels are composed of a three-dimensional network structure that can absorb and retain large amounts of water without dissolving. First, the high water content of hydrogels not only keeps the wound in a relatively moist environment, reducing the risk of scarring, but also has a cooling sensation and low tissue adhesion, thereby alleviating the patient's pain and discomfort. In addition, hydrogel dressings soften necrotic tissue by maintaining a moist environment, which helps with autolytic debridement. Secondly, their good swelling properties give hydrogels good absorption capacity, allowing them to absorb large amounts of exudate, reducing the frequency of dressing changes and lowering care costs. Based on the above properties, hydrogels have been widely studied as a potential material in the field of wound healing.

[0004] Collagen is a fundamental protein in the extracellular matrix (ECM). It is widely distributed in mammalian tissues, such as skin, cartilage, tendons, and ligaments, and accounts for approximately 25-33% of the total protein content in the human body. Its widespread application in biomaterials stems from its significant biological functions, including promoting cell adhesion and proliferation, as well as its biocompatibility, low immunogenicity, rapid hemostasis, and biodegradability. Hyaluronic acid (HA), also known as hyaluronic acid, is a naturally occurring polysaccharide that was first isolated from the bovine vitreous humor in 1934. This polysaccharide is a key component of the extracellular matrix (ECM), with a molecular structure composed of linearly arranged repeating disaccharide units. HA exhibits physiological properties including structural support, filling, lubrication, and water retention. HA is a biocompatible polymer with diverse biological functions, such as moisturizing and anti-wrinkle effects, and it promotes the natural healing process. Furthermore, numerous in vitro and in vivo studies have highlighted the role of HA in wound healing by promoting the migration and differentiation of mesenchymal and epithelial cells, as well as promoting angiogenesis and collagen deposition. Furthermore, HA provides a favorable microenvironment for wound healing and shows great potential in scar treatment and overall wound recovery.

[0005] Skin wound healing is a complex process involving multiple stages, and inappropriate treatments may negatively impact this recovery process. Consequently, there is growing interest in improving methods for treating skin wounds. Hydrogel dressings, due to their unique physical and chemical properties, have shown great potential in treating minor surface injuries. Hydrogels facilitate the regeneration of vascular tissue and skin appendages. Furthermore, as a skin tissue engineering material, hydrogels show broad application prospects in replacing skin grafts to treat severe, deep skin injuries. Although numerous studies have explored the application of hydrogels in healing skin wounds, the effective treatment of chronic and deep wounds remains an ongoing challenge. Future research will focus on reducing the cost of hydrogels while improving their performance, and addressing the treatment of chronic wounds will continue to be a key focus of future research. Summary of the Invention

[0006] In view of the above shortcomings of the existing technology, the present invention provides a preparation method and application of a rapidly curable antibacterial hydrogel based on click chemistry. Dopamine-modified hyaluronic acid and ferric chloride are evenly mixed and subjected to a metal coordination reaction to prepare a first layer of hydrogel network. Norbornene-modified collagen and azine-modified four-arm polyethylene glycol are evenly mixed and subjected to a click chemistry reaction to prepare a second layer of hydrogel network. The two layers of network are interpenetrating and cross-linked to obtain a rapidly curable antibacterial hydrogel, which is applied to skin repair.

[0007] To achieve the above-mentioned purpose, the present invention adopts the following technical solution: a method for preparing a rapidly curable antibacterial hydrogel, characterized in that the method for preparing the rapidly curing antibacterial hydrogel comprises the following steps:

[0008] (1) mixing hyaluronic acid and dopamine hydrochloride to react to obtain a dopamine-modified hyaluronic acid polymer;

[0009] (2) subjecting a uniformly mixed system comprising a dopamine-modified hyaluronic acid polymer and ferric chloride to a metal coordination reaction to form a first hydrogel network;

[0010] (3) mixing collagen with succinic anhydride to react, wherein the succinic anhydride is dissolved in an acetone solution to obtain a succinic anhydride-modified collagen polymer;

[0011] (4) mixing the succinic anhydride-modified collagen polymer with 5-norbornene-2-methylamine to obtain a norbornene-modified collagen polymer;

[0012] (5) mixing and reacting four-arm polyethylene glycol carboxylic acid with tetrazine hydrochloride to obtain tetrazine-modified four-arm polyethylene glycol polymer;

[0013] (6) subjecting the homogeneous mixture of the norbornene-modified collagen polymer and the oxazine-modified four-arm polyethylene glycol polymer to a click chemical reaction to form a second hydrogel network;

[0014] (7) Dopamine-modified hyaluronic acid polymer and norbornene-modified collagen polymer are dissolved in a phosphate buffer solution at a mass ratio of 1:1 to form a uniform precursor solution; ferric chloride and a oxazine-modified four-arm polyethylene glycol polymer mixed solution are added to the precursor solution; and the mixture is rapidly mixed and cross-linked at room temperature to obtain a rapidly curable antibacterial hydrogel.

[0015] In the step (1), the reaction molar ratio of the carboxyl group on the hyaluronic acid to EDC, NHS and dopamine hydrochloride is 1:2:2:1.5.

[0016] In the step (2), the concentration of the dopamine-modified hyaluronic acid polymer is 5-10 w / v%, and the concentration of ferric chloride is 2.5-5 w / v%.

[0017] The mass ratio of collagen to succinic anhydride in step (3) is 1:15.

[0018] The mass ratio of succinic anhydride to acetone in step (3) is 1:7.5.

[0019] In the step (4), the reaction molar ratio of the succinic anhydride-modified collagen polymer to 5-norbornene-2-methylamine is 1:1.5.

[0020] The reaction molar ratio of the four-arm polyethylene glycol carboxylic acid to the tetrazine hydrochloride in the step (5) is 1:(4-6).

[0021] In the step (6), the concentration of the norbornene-modified collagen polymer is 5-10 w / v%, and the concentration of the oxazine-modified four-arm polyethylene glycol polymer is 5-10 w / v%.

[0022] An antibacterial hydrogel wound dressing is prepared by using the antibacterial hydrogel prepared by the above method.

[0023] Further preferred on the basis of the above scheme: application of the antibacterial hydrogel wound dressing in skin repair.

[0024] The rapidly curable antibacterial hydrogel provided by the present invention has a more uniform system and is free of particles. Other advantages include:

[0025] (1) Simple preparation method. The rapidly curable antibacterial hydrogel provided by the present invention combines the two modes of action of metal coordination chemical reaction and click chemical reaction. Its advantage is that the rapid characteristics of metal coordination reaction and click chemical reaction enable the hydrogel system to cure rapidly. At the same time, the preparation method is simple and can be prepared in large quantities.

[0026] (2) Good biocompatibility. The rapidly curing antibacterial hydrogel of the present invention functionalizes the commonly used synthetic material hyaluronic acid and the natural material collagen to obtain dopamine-modified hyaluronic acid polymer and norbornene-modified collagen polymer. These two materials are non-toxic to cells and do not restrict skin healing.

[0027] (3) Excellent antibacterial properties. The fast-curing antibacterial hydrogel proposed in the present invention cleverly combines the photothermal antibacterial properties of dopamine and the unique efficacy of the antibacterial agent iron ions, thereby achieving an excellent synergistic antibacterial effect. Dopamine, as a bioactive substance with photothermal conversion capabilities, can rapidly heat up under light conditions, thereby effectively killing pathogenic microorganisms. At the same time, it has good biocompatibility and will not cause damage to surrounding normal tissues. Iron ions, as a common antibacterial agent, can further enhance the antibacterial properties of the hydrogel by interfering with the metabolic process of microorganisms and inhibiting their growth and reproduction. When these two antibacterial mechanisms work synergistically in the double-network hydrogel, not only the antibacterial efficiency is improved, but also the antibacterial spectrum is broadened, enabling it to effectively fight against a variety of pathogenic microorganisms, showing broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a diagram of the preparation mechanism of the present invention.

[0029] FIG2 is a hydrogen nuclear magnetic spectrum of the relevant materials detected in Example 2.

[0030] Figure 3 These are the appearance and microstructure of the hydrogel tested in Example 3.

[0031] Figure 4 This is a diagram showing the self-healing performance of the hydrogel tested in Example 4.

[0032] FIG5 is a graph showing the adhesion performance of the hydrogel tested in Example 5.

[0033] Figure 6 This is a diagram of the swelling performance of the hydrogel tested in Example 6.

[0034] Figure 7 This is a graph showing the degradation performance of the hydrogel tested in Example 7.

[0035] FIG8 is an infrared thermal image and a temperature change curve of the hydrogel tested in Example 8.

[0036] FIG9 is a physical picture of the inhibition zones of Escherichia coli and Staphylococcus aureus detected in the hydrogel in Example 9.

[0037] Figure 10 This is a diagram showing the repair of rat skin defects in a skin repair experiment using the hydrogel tested in Example 10.

[0038] Figure 11 This is an H&E staining image of rat skin in a skin repair experiment using the hydrogel tested in Example 10. DETAILED DESCRIPTION

[0039] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0040] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0041] Example 1:

[0042] A method for preparing a rapidly curing antibacterial hydrogel, wherein the method comprises the following steps:

[0043] (1) mixing hyaluronic acid and dopamine hydrochloride to react to obtain a dopamine-modified dopamine polymer;

[0044] Specifically, 1g of sodium hyaluronate was dissolved in water. EDC and NHS were added at room temperature under a nitrogen atmosphere, with the reaction molar ratio of carboxyl groups on the hyaluronic acid, EDC, and NHS being 1:2:2 for activation for 20 minutes. Dopamine hydrochloride was then added at a reaction molar ratio of 1:1.5 between the carboxyl groups on the hyaluronic acid and dopamine hydrochloride. The pH was maintained at 5-6, and the reaction was continued at room temperature for 12 hours. The product was dialyzed for 3 days to a Mw of 7000 Da, and then lyophilized.

[0045] (2) subjecting a uniformly mixed system comprising a dopamine-modified hyaluronic acid polymer and ferric chloride to a metal coordination reaction to form a first hydrogel network;

[0046] Specifically: weigh a certain amount of HA-PDA and FeCl3, dissolve them in PBS, mix them evenly according to the proportion in the table below, and let them stand to form a gel.

[0047] Table 1 Mixing ratio of HA-PDA and FeCl3

[0048]

[0049] (3) mixing collagen with succinic anhydride to react and obtain succinic anhydride-modified collagen polymer;

[0050] Specifically, 500mg of collagen was weighed and stirred overnight in 75mL of 1% acetic acid solution to dissolve as much as possible. The solution was then centrifuged at 8000rpm for 15min, and the supernatant, i.e., the Col solution, was collected. Under ice-bath conditions, the pH of the Col solution was adjusted to approximately 10 using a 4M NaOH solution. Succinic anhydride, i.e., SAH, was dissolved in an appropriate amount of acetone solution and slowly added dropwise to the Col solution. The solution was mechanically stirred overnight, and the pH of the reaction solution was maintained at approximately 9 throughout the entire process. After the reaction was completed, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 14,000Da and dialyzed in deionized water at 4°C for 3d. The filtrate was then filtered, collected, and freeze-dried to obtain the succinic anhydride-modified collagen Col-SAH.

[0051] (4) mixing the succinic anhydride-modified collagen polymer with 5-norbornene-2-methylamine to obtain a norbornene-modified collagen polymer;

[0052] Specifically, 200 mg of Col-SAH was weighed and thoroughly dissolved in deionized water. 234.6 mg of EDC and 140.9 mg of NHS were added and stirred for 30 minutes to activate the carboxyl groups. Then, 56.7 mg of Nb was added and allowed to react overnight. Afterward, the mixture was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in deionized water at 4°C for three days. The mixture was then freeze-dried to obtain norbornene-modified collagen Col-Nb.

[0053] (5) mixing and reacting four-arm polyethylene glycol carboxylic acid with tetrazine hydrochloride to obtain tetrazine-modified four-arm polyethylene glycol polymer;

[0054] Specifically, 200 mg of 4armPEG-COOH was dissolved in dichloromethane (DCM), followed by the addition of 52 mg of pyBOP and 20 μL of DIPEA. The mixture was activated on ice for 30 minutes, followed by the addition of 10.8 mg of Tz. The reaction was allowed to react overnight in the dark. The reaction was terminated, and the solution was reprecipitated in ether. The precipitate was collected, redissolved in deionized water, and placed in a dialysis bag with a molecular weight cutoff of 3500 Da. The mixture was dialyzed in deionized water at 4°C for three days and freeze-dried to obtain tetrazine-modified 4armPEG-COOH, also known as PEG-Tz.

[0055] (6) subjecting a homogeneous mixture system comprising a norbornene-modified collagen polymer and an oxazine-modified four-arm polyethylene glycol polymer to a click chemistry reaction to form a second hydrogel network;

[0056] Specifically, appropriate amounts of Col-Nb and PEG-Tz were fully dissolved in PBS, and the two were evenly mixed according to the proportions in the table below, and allowed to stand to form a gel.

[0057] Table 2 Mixing ratio of Col-Nb and PEG-Tz

[0058]

[0059] (7) mixing and cross-linking the first and second hydrogel networks prepared above to obtain a double-network hydrogel;

[0060] Specifically, referring to the above method, the materials were fully dissolved in PBS. HA-PDA (final concentration of 5 wt%) was then mixed with Col-Nb (final concentration of 5 wt%), FeCl3 (final concentration of 2.5 wt%), and PEG-Tz (final concentration of 5 wt%). Finally, the two mixed solutions were mixed and allowed to stand to gel. The gelation time of the hydrogels was determined by the test tube inversion method.

[0061] Example 2:

[0062] Nuclear magnetic resonance spectroscopy detection:

[0063] The hydrogen spectra of HA-PDA, Col-Nb and PEG-Tz were measured by a 400M nuclear magnetic resonance spectrometer, with a corresponding deuterated reagent as the solvent and TMS as the internal standard.

[0064] The NMR results of HA-PDA are as follows Figure 2aAs shown in the figure, δ1.92 ppm is attributed to the N-acetyl protons of hyaluronic acid, while δ6.65-6.90 ppm corresponds to the aromatic protons of the catechol ring. The chemical shift shifts at δ6.65-6.90 ppm indicate successful dopamine modification onto hyaluronic acid. Quantitative integration of the integrated area ratio revealed a grafting yield of 31.5%.

[0065] The NMR results of Col-Nb are as follows Figure 2b As shown in the figure, δ7.2 ppm is attributed to the amide bond protons of succinic anhydride, indicating that succinic anhydride was successfully modified onto collagen. δ5.5-6.5 ppm corresponds to the olefin protons of the norbornene double bond. The chemical shift changes at δ5.5-6.5 ppm indicate that norbornene was successfully modified onto collagen. Due to the complex structure of collagen, its grafting rate could not be calculated.

[0066] The NMR results of PEG-Tz are as follows Figure 2c In the figure, δ7.5-8.5 ppm belongs to the characteristic peak of the benzene ring in the tetrazine group, indicating that the tetrazine group has been successfully modified onto the four-arm polyethylene glycol.

[0067] Example 3:

[0068] Morphology characterization and SEM images:

[0069] The hydrogel prepared in Example 1 was placed in liquid nitrogen for freezing, and then its cross section was fixed on the conductive glue, sprayed with gold at 0.2 mA for 3 minutes, and then placed on a field ring scanning electron microscope to observe the microstructure of the hydrogel. Figure 3 As shown in FIG, the double-network hydrogel microstructure is porous with a pore size of about 100 to 200 μm.

[0070] Example 4:

[0071] Self-healing performance characterization:

[0072] Prepare hydrogel samples with a certain shape, such as rectangle, circle or dumbbell; then cut the prepared hydrogel sample into two halves, ensuring that the cut surface is flat, and allow the two cut parts of the hydrogel to naturally fit together in the air. No other external force or external stimulation is applied during the healing process. After a period of healing, manually stretch the hydrogel to observe whether it can regain its shape and conduct a qualitative evaluation of the self-healing performance.

[0073] Its self-healing properties are Figure 4 As shown in the figure, the hydrogel exhibits significant self-healing properties. Without any external force or external stimulation, the hydrogel can quickly and automatically complete the healing process. The healing time of the hydrogel is only 15 minutes. This rapid healing ability indicates that it has an efficient self-repair mechanism and can restore its structure and function in a short time.

[0074] Example 5

[0075] Adhesion performance characterization:

[0076] The hydrogel samples were directly adhered to the surfaces of different materials, such as glass, metal, pigskin, etc., and vertically pulled or peeled off manually or mechanically to observe the adhesion of the hydrogel to the substrate.

[0077] Its adhesion properties such as Figure 5a As shown in the figure, the hydrogel can achieve strong adhesion on the surface of a variety of substrates of different materials, including but not limited to glass, metal, plastic and biological tissue, and shows good adhesion effect under various test conditions. In order to further quantitatively evaluate the adhesion ability of the hydrogel, the present invention uses weights of different weights for testing. The hydrogel sample is fixed on the surface of the target substrate, and then the weight of the weight is gradually increased below it to test the maximum load that the hydrogel can withstand. Figure 5b Experimental results show that the hydrogel can successfully adhere to and stably support a maximum weight of 50g, demonstrating extremely high adhesion strength.

[0078] Example 6:

[0079] Swelling performance characterization:

[0080] The double network hydrogel was placed in a PBS buffer system at 37°C and pH = 7.4 to study the swelling of the double network hydrogel. Double network hydrogels were prepared with n = 3, 8 mm in diameter and 4 mm in height, weighed to obtain W0, immersed in 2 mL of PBS buffer solution, placed in a shaker at 37°C, and taken out at 1 h, 3 h, 5 h, 12 h, 24 h, 48 h and 72 h, and the surface moisture was absorbed with filter paper and weighed to obtain W0. t The swelling ratio calculation formula is: Swelling ratio S t (%)=[(W t -W0) / W0]×100%.

[0081] Its swelling performance diagram is as follows Figure 6 As shown, the hydrogel's swelling rate increased rapidly within a short period of time, reaching a maximum of 227.29% at 6 hours. With further immersion time, the swelling rate decreased slightly, ultimately maintaining a swelling rate of around 175%. This swelling behavior demonstrates that the prepared double-network hydrogel can rapidly absorb large amounts of water in a short period of time. Furthermore, the ability to maintain a high swelling rate of 175% also demonstrates the hydrogel's excellent water retention capacity, maintaining its structural integrity after absorbing water and preventing network rupture due to excessive swelling.

[0082] Example 7:

[0083] Degradation performance characterization:

[0084] The prepared double network hydrogel, n=3, was freeze-dried and weighed to obtain W1, immersed in PBS buffer solution, placed in a 37°C shaker, and samples were taken out at 12h, 24h, 2d, 3d, 5d, 7d, 10d, and 14d, rinsed three times with PBS buffer solution, and freeze-dried and weighed to obtain W2. The residual mass ratio is calculated as follows: k (%) = W2 / W1×100%.

[0085] Its degradation properties such as Figure 7 As shown in the figure, in the initial stage of degradation, i.e. the first 24 hours, the degradation rate of the hydrogel was relatively fast. As the degradation progressed, the degradation rate of the hydrogel gradually slowed down. Finally, the degradation equilibrium was reached after 14 days, and the maximum mass loss ratio of the hydrogel system was 59.95%.

[0086] Example 8:

[0087] Photothermal effect assessment:

[0088] In the experiment, 808nm laser was used to test the photothermal effect of hydrogel samples. 2 Under the conditions of irradiation, a cylindrical hydrogel sample with a diameter of 8 mm and a height of 4 mm was continuously irradiated for 10 minutes. The surface temperature dynamics of the sample was monitored and recorded in real time using a thermal infrared imaging camera with a sampling frequency of once per minute.

[0089] Infrared thermal imaging data such as Figure 8a As shown in Figure 3, after 10 minutes of laser irradiation, the surface temperature of the hydrogel increased significantly and eventually reached an equilibrium temperature of 51.8°C. Figure 8b This is the temperature change curve of the hydrogel under 808nm laser irradiation.

[0090] Example 9:

[0091] Characterization of antibacterial performance:

[0092] The present invention verifies the antibacterial ability of the double network hydrogel by the antibacterial zone experiment. The bacteria used in the present invention are Escherichia coli ATCC25922 and Staphylococcus aureus CMCC(B)26003. 6The CFUs / mL bacterial solution was evenly spread on the surface of the agar medium, and a 6mm diameter hydrogel was placed on the surface of the agar medium. It was first placed in a 4°C refrigerator overnight, and then cultured in a 37°C shaker for 24 hours. The diameter of the sample's turnover inhibition zone was then measured three times in different directions, and the average value was taken. In addition, in order to explore whether the photothermal effect can further enhance the antibacterial properties of the hydrogel, a photothermal group was also set up in the experiment. In the photothermal group, an 808nm laser was used to irradiate the hydrogel, and the photothermal conversion effect of the laser was used to increase the local temperature of the hydrogel, thereby examining its synergistic antibacterial effect under photothermal stimulation.

[0093] Its antibacterial performance is shown in Figure 9. After the hydrogel with a diameter of 6 mm spreads on the culture medium, the covered area is almost sterile and can effectively inhibit the growth of surrounding bacteria. This phenomenon shows that the hydrogel can inhibit bacteria in a short time. Figure 9a As shown, after 12 hours, the diameter of the inhibition zone of the E. coli group was about 9.853 mm, and the diameter of the inhibition zone of the photothermal group was about 11.722 mm. Figure 9b As shown in the figure, after 12 hours, the diameter of the inhibition zone of the Staphylococcus aureus group was approximately 11.925 mm, and the diameter of the inhibition zone of the photothermal group was approximately 12.632 mm. Comparing the two data sets, it can be found that the diameter of the inhibition zone of the photothermal group was significantly larger than that of the hydrogel group, indicating that the antibacterial ability of the hydrogel was further enhanced under the synergistic effect of the photothermal effect.

[0094] Example 10:

[0095] Rat skin full-thickness defect repair experiment:

[0096] The present invention uses 27 4-5 week old Sprague-Dawley rats as experimental animal models. The rats are anesthetized with 20 ml / kg of 1.25% avertin. After anesthesia, the hair on the backs of the rats is partially shaved to expose the experimental skin area. The exposed skin area is then disinfected with iodine. After disinfection, a full-thickness skin defect model with a diameter of 8 mm is created on the rats' backs using ophthalmic scissors.

[0097] SD rats were randomly divided into 3 groups:

[0098] Rats in group A were housed in single cages without any wound treatment.

[0099] Group B rats: On day 0, skin defect wounds in Group B rats were treated and evenly covered with a double-network hydrogel to promote wound healing. Subsequently, rats were housed individually to prevent cross-contamination and infection. Wounds were photographed on days 1, 7, and 14 postoperatively to visually compare wound healing status and trends. Additionally, tissue samples were collected from selected rats on days 7 and 14 postoperatively. The procedure was as follows: Ophthalmic scissors were used to carefully excise the skin and subcutaneous tissue within a 1 cm radius around the wound, ensuring that the sampling area encompassed the wound and surrounding normal tissue. Tissue samples were immediately fixed in 4% paraformaldehyde solution for subsequent tissue embedding and sectioning. Following sectioning, sections were stained with hematoxylin and eosin (H&E). Microscopic observation of histological changes in wound healing, including key indicators such as inflammation, granulation tissue formation, collagen deposition, and epidermal regeneration, was performed to comprehensively evaluate the efficacy of the double-network hydrogel on skin defect repair.

[0100] Rats in group C: First, the skin defect wounds of rats in group C were treated on day 0, and the double-network hydrogel was used to evenly cover the wound surface. Then, the wound was irradiated with an 808nm laser to activate the photothermal effect of the hydrogel, thereby promoting wound healing. Subsequently, the rats were housed in single cages to avoid mutual interference and infection. The wounds were photographed and recorded on the 1st, 7th, and 14th days after surgery to visually compare the healing status and change trends of the wounds. In addition, tissue sampling was performed on some rats on the 7th and 14th days after surgery. The specific operation was as follows: Use ophthalmic scissors to carefully cut the skin and subcutaneous tissue within 1 cm around the wound to ensure that the sampling area includes the wound and the normal tissue around it. The collected tissue samples were immediately placed in 4% paraformaldehyde solution for fixation for subsequent tissue embedding and sectioning. After slicing, we performed hematoxylin-eosin (H&E) staining on the tissue sections and observed the histological changes of wound healing under a microscope, including key indicators such as inflammatory response, granulation tissue formation, collagen deposition, and epidermal regeneration, so as to comprehensively evaluate the effect of the double-network hydrogel on skin defect repair.

[0101] Depend on Figure 10It can be observed from the wound repair diagram that on the 7th day, the wounds in the control group showed a relatively dry state and the healing process was relatively slow. In contrast, the wounds treated with hydrogel showed a more moist environment, which shows that the hydrogel can effectively retain the moisture of the wound and provide a good moist environment for tissue repair. In addition, the skin edge of the wound in the hydrogel-treated group began to shrink, and the wound was oval as a whole, and its size was significantly smaller than that of the control group, indicating that the hydrogel can promote early contraction and healing of the wound. On the 14th day, the wounds in all experimental groups were further reduced, and the wound diameter was about 2.7mm. Among them, the photothermal group combined with 808nm laser irradiation had the most significant wound healing effect and the smallest wound size.

[0102] The H&E staining diagram is as follows Figure 11 As shown, on day 7, the wounds in all experimental groups were still in the early stages of wound healing, known as the inflammatory phase. Numerous pale pink inflammatory cells infiltrated the wound area. By day 14, the wounds had entered the final stage of healing, known as the maturation phase. At this point, the inflammatory cells gradually decreased, replaced by a large number of proliferating fibroblasts and newly formed collagen fibers. The collagen fibers were pink and orderly arranged, demonstrating the stability and maturity of the tissue structure.

Claims

1. A method for preparing a rapidly curable antibacterial hydrogel, characterized in that: The preparation method of the fast-curing antibacterial hydrogel comprises the following steps: (1) mixing hyaluronic acid and dopamine hydrochloride to react to obtain a dopamine-modified hyaluronic acid polymer; (2) subjecting a uniformly mixed system comprising a dopamine-modified hyaluronic acid polymer and ferric chloride to a metal coordination reaction to form a first hydrogel network; (3) mixing collagen with succinic anhydride to react, wherein the succinic anhydride is dissolved in an acetone solution to obtain a succinic anhydride-modified collagen polymer; (4) mixing the succinic anhydride-modified collagen polymer with 5-norbornene-2-methylamine to obtain a norbornene-modified collagen polymer; (5) mixing and reacting four-arm polyethylene glycol carboxylic acid with tetrazine hydrochloride to obtain tetrazine-modified four-arm polyethylene glycol polymer; (6) subjecting the homogeneous mixture of the norbornene-modified collagen polymer and the oxazine-modified four-arm polyethylene glycol polymer to a click chemical reaction to form a second hydrogel network; (7) Dopamine-modified hyaluronic acid polymer and norbornene-modified collagen polymer are dissolved in a phosphate buffer solution at a mass ratio of 1:1 to form a uniform precursor solution; ferric chloride and a oxazine-modified four-arm polyethylene glycol polymer mixed solution are added to the precursor solution; and the mixture is rapidly mixed and cross-linked at room temperature to obtain a rapidly curable antibacterial hydrogel.

2. The method for preparing the rapidly curable antibacterial hydrogel according to claim 1, wherein: In the step (1), the reaction molar ratio of the carboxyl group on the hyaluronic acid to EDC, NHS and dopamine hydrochloride is 1:2:2:1.

5.

3. The method for preparing the rapidly curable antibacterial hydrogel according to claim 1, characterized in that: In the step (2), the concentration of the dopamine-modified hyaluronic acid polymer is 5-10 w / v%, and the concentration of ferric chloride is 2.5-5 w / v%.

4. The method for preparing the rapidly curable antibacterial hydrogel according to claim 1, wherein: The mass ratio of collagen to succinic anhydride in step (3) is 1:

15.

5. The method for preparing the rapidly curable antibacterial hydrogel according to claim 1, wherein: The mass ratio of succinic anhydride to acetone in step (3) is 1:7.

5.

6. The method for preparing the rapidly curable antibacterial hydrogel according to claim 1, wherein: In the step (4), the reaction molar ratio of the succinic anhydride-modified collagen polymer to 5-norbornene-2-methylamine is 1:1.

5.

7. The method for preparing the rapidly curable antibacterial hydrogel according to claim 1, wherein: The reaction molar ratio of the four-arm polyethylene glycol carboxylic acid to the tetrazine hydrochloride in the step (5) is 1:(4-6).

8. The method for preparing the rapidly curable antibacterial hydrogel according to claim 1, wherein: In the step (6), the concentration of the norbornene-modified collagen polymer is 5-10 w / v%, and the concentration of the oxazine-modified four-arm polyethylene glycol polymer is 5-10 w / v%.

9. An antibacterial hydrogel wound dressing, characterized in that: An antibacterial hydrogel wound dressing is prepared using the antibacterial hydrogel prepared by the method according to any one of claims 1 to 8.

10. The antibacterial hydrogel wound dressing according to claim 9, characterized in that: Application of the antibacterial hydrogel wound dressing in skin repair.

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