A photocrosslinked dual-network hydrogel that promotes skin wound healing and its preparation method

Abstract

This invention belongs to the field of biomedical materials technology, specifically relating to a photocrosslinked dual-network hydrogel that promotes skin wound healing and its preparation method. Glycidyl methacrylate is added dropwise to an aqueous solution of ε-polylysine to obtain methacrylated polylysine. An aqueous solution of hyaluronic acid and an aqueous solution of sodium periodate are mixed under light-protected conditions and stirred to obtain oxidized hyaluronic acid. The methacrylated polylysine solution and the oxidized hyaluronic acid solution are mixed and vortexed until homogeneous to obtain a mixed solution. This mixed solution is injected into a mold and cured under ultraviolet light to obtain the photocrosslinked dual-network hydrogel. The dual-network hydrogel of this invention not only effectively solves the contradiction between mechanical strength and biocompatibility that is difficult to achieve simultaneously in traditional single-network hydrogels, but also integrates multiple healing mechanisms through functional complementarity between components.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a photocrosslinked dual-network hydrogel that promotes skin wound healing and its preparation method. Background Technology

[0002] As the largest and most multifunctional organ in the human body, the skin not only acts as a physical barrier against microbial invasion and ultraviolet radiation, but also maintains internal environmental balance through mechanisms such as regulating water evaporation, temperature homeostasis, and immune responses. It is noteworthy that this tissue exposed to the body surface often suffers severe suppression of its self-repair capacity when faced with exogenous mechanical injuries (including sharp instrument wounds, thermal burns, and surgical incisions) and endogenous metabolic abnormalities (such as diabetic ulcers and vascular dysfunction). This suppression is often due to factors such as extracellular matrix (ECM) damage, chronic inflammatory responses, and pathogen colonization. Skin wound healing is a complex biological process. While commonly used gauze dressings provide basic protection, they can cause problems such as wound adhesion and hinder tissue regeneration. Furthermore, inappropriate dressing selection often leads to delayed wound healing.

[0003] Although hydrogel dressings can maintain a moist wound environment through a three-dimensional hydrophilic network, existing systems mostly employ a single-network cross-linking architecture. Limited by the lack of energy dissipation mechanisms in the network topology, they generally suffer from functional limitations such as insufficient fracture toughness and are prone to brittle fracture under complex stress conditions. Particularly noteworthy is that single-function designs struggle to meet the dynamic needs of multi-stage wound healing. For example, polylysine (PLL) possesses natural antibacterial properties but has poor mechanical properties; hyaluronic acid (HA), while promoting cell migration, is easily degraded.

[0004] Existing technologies lack composite systems that synergistically enhance both, and the key issues of combining photocrosslinking and dynamic networks remain unresolved. Therefore, there is a need to provide a hydrogel with good wound healing properties as a medical antibacterial dressing. Summary of the Invention

[0005] The purpose of this invention is to provide a photocrosslinked dual-network hydrogel that promotes skin wound healing and its preparation method, so as to solve the above-mentioned technical problems.

[0006] To achieve the above-mentioned technical objectives, the technical solution of the present invention is as follows:

[0007] A method for preparing a photocrosslinked dual-network hydrogel that promotes skin wound healing includes the following steps:

[0008] S1. Glycidyl methacrylate is added dropwise to 5-15 wt% of ε-polylysine aqueous solution and reacted at 60-90℃ for 4-15 h. After the reaction is completed, the solution is filtered through a 0.22 μm filter membrane, dialyzed with distilled water, and the dialysate is freeze-dried to obtain methacrylated polylysine, which is then sealed and stored at -80℃.

[0009] S2. Mix 0.5-6 wt% hyaluronic acid aqueous solution and 1-18 wt% sodium periodate aqueous solution under light-protected conditions, stir and react at 25-70°C for 3-8 hours, add ethylene glycol to quench the reaction, dialyze the resulting reaction solution at room temperature, freeze-dry to obtain oxidized hyaluronic acid, and store at 4°C in the dark.

[0010] S3. Dissolve oxidized hyaluronic acid in PBS aqueous solution to obtain oxidized hyaluronic acid solution;

[0011] A solution of methacrylated polylysine was obtained by mixing methacrylated polylysine with an aqueous photoinitiator LAP and vortexing until completely dissolved.

[0012] Equal volumes of methacrylated polylysine solution and oxidized hyaluronic acid solution were mixed and vortexed to obtain a mixture. The mixture was then injected into a mold and cured under ultraviolet light to obtain a photocrosslinked double-network hydrogel.

[0013] As a further improvement, in step S3, an aqueous solution of an anti-inflammatory drug is added to the mixture, wherein the concentration of the anti-inflammatory drug in the aqueous solution is 0.1~5.5wt%, and the volume ratio of the aqueous solution of the anti-inflammatory drug to the methacrylated polylysine solution is 1:5~15.

[0014] As a further improvement, the anti-inflammatory drug is one or more of curcumin, quercetin, rutin, catechin, soy isoflavones, luteolin, andrographolide, resveratrol, and artemisinin.

[0015] As a further improvement, in step S3, the concentration of the oxidized hyaluronic acid solution is 2~12wt%, and the pH of the PBS aqueous solution is 8.

[0016] As a further improvement, in step S3, the concentration of methacrylated polylysine in the methacrylated polylysine solution is 0.25 g / mL, and the concentration of the photoinitiator LAP aqueous solution is 0.1~0.25 wt%.

[0017] As a further improvement, in step S1, the volume ratio of the glycidyl methacrylate to the aqueous ε-polylysine solution is 4:100.

[0018] As a further improvement, in step S2, the volume ratio of the hyaluronic acid aqueous solution to the sodium periodate aqueous solution is 5:1.

[0019] As a further improvement, in step S3, the ultraviolet curing specifically uses 365 nm ultraviolet light at a concentration of 365~1000 mW / cm². 2 Under these conditions, the light exposure time is 5~1600s.

[0020] The present invention also provides a photocrosslinked dual-network hydrogel.

[0021] Due to the adoption of the above technical solution, the beneficial effects of the present invention are as follows:

[0022] This invention provides a photocrosslinked dual-network hydrogel that promotes skin wound healing and its preparation method. It not only has excellent in vitro antibacterial properties, good biosafety and strong biocompatibility, but also achieves multifunctional synergistic optimization through innovative molecular design strategies.

[0023] In this invention, a polylysine main network is first constructed by photo-initiated polymerization, and its cationic properties endow the material with inherent antibacterial activity. At the same time, the introduced oxidized hyaluronic acid forms a secondary network through dynamic Schiff base reaction. With the participation of anti-inflammatory drugs, the mechanical properties of the hydrogel network are significantly improved while retaining the cell migration-promoting properties of hyaluronic acid and the anti-inflammatory properties of anti-inflammatory drugs.

[0024] The dual-network hydrogel of this invention not only effectively solves the contradiction between mechanical strength and biocompatibility in traditional single-network hydrogels, but also integrates multiple healing mechanisms through functional complementarity between components. Attached Figure Description

[0025] Figure 1 These are the 1H NMR spectrum and infrared spectrum measured in Example 2, where A is the 1H NMR spectrum of methacrylated polylysine and ε-polylysine, and B is the infrared spectrum of methacrylated polylysine, ε-polylysine, hyaluronic acid, oxidized hyaluronic acid and double network hydrogel GE-OHA2.

[0026] Figure 2 This is a schematic diagram of the preparation process of the dual-network hydrogel;

[0027] Figure 3 These are comparison charts of gelation time for GE-OHA1, GE-OHA2, and GE mono-network hydrogels, and comparison charts of flow state before and after gelation for GE-OHA2 and GE mono-network hydrogels. In the charts, A is a comparison chart of gelation time for GE-OHA1, GE-OHA2, and GE mono-network hydrogels, and B is a comparison chart of flow state before and after gelation for GE-OHA2 and GE mono-network hydrogels.

[0028] Figure 4 The images show the mechanical properties of GE-OHA1, GE-OHA2, and GE single-network hydrogels, as well as the frequency scan of GE-OHA2 with a fixed strain of 1.0%. In the images, A is the stress-strain curve, B is the comparison of the compressive modulus of the hydrogels, and C is the frequency scan of the double-crosslinked hydrogel GE-OHA2.

[0029] Figure 5 The results are the swelling performance test results and hemolysis performance test results of GE-OHA1, GE-OHA2 and GE single network hydrogels. Among them, A is the swelling performance test result of the hydrogel and B is the hemolysis performance test result of the hydrogel.

[0030] Figure 6 The results show the stability of GE-OHA1, GE-OHA2, and GE mono-network hydrogels under different pH conditions. Among them, A represents the stability of GE-OHA2 under different pH conditions, B represents the stability of GE-OHA1 under different pH conditions, and C represents the stability of GE mono-component hydrogels under different pH conditions.

[0031] Figure 7 The results show the drug release capacity of GE-OHA1@Rut, GE-OHA2@Rut, and GE@Rut. Among them, A represents the drug release capacity of GE-OHA2@Rut, B represents the drug release capacity of GE-OHA1@Rut, and C represents the drug release capacity of GE@Rut single-component drug-loaded hydrogel.

[0032] Figure 8 The results show the antioxidant capacity of GE-OHA2@Rut and GE@Rut, where A represents the DPPH scavenging capacity of the two hydrogels and B represents the ABTS scavenging capacity of the two hydrogels.

[0033] Figure 9 These are the results of colony growth and colony count in the in vitro antimicrobial performance evaluation. Among them, A is the result of colony growth in the in vitro antimicrobial performance evaluation, and B is the result of colony count in the in vitro antimicrobial performance evaluation.

[0034] Figure 10 These are the results of cytotoxicity evaluation and biocompatibility assay, where A is the cytotoxicity evaluation result and B is the biocompatibility assay result.

[0035] Figure 11 This is a graph showing the effect of hydrogels on cell migration and growth.

[0036] Figure 12 These are the results of TNF-α immunofluorescence assay and IL-10 immunofluorescence assay, where A is the result of TNF-α immunofluorescence assay and B is the result of IL-10 immunofluorescence assay.

[0037] Figure 13 The results are the evaluation results of skin wound treatment, H&E staining results, and Masson staining results. Among them, A is the evaluation result of skin wound treatment, B is the H&E staining result, and C is the Masson staining result.

[0038] Figure 14These are the results of TNF-α immunofluorescence staining and IL-10 immunofluorescence staining after skin wound treatment. Among them, A is the result of TNF-α immunofluorescence staining and B is the result of IL-10 immunofluorescence staining. Detailed Implementation

[0039] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0040] Example 1: A method for preparing a photocrosslinked dual-network hydrogel that promotes skin wound healing, specifically including the following steps:

[0041] S1. Weigh 10g of ε-polylysine (EPL) and dissolve it in 100mL of deionized water to prepare a 10wt% ε-polylysine solution.

[0042] 4 mL of glycidyl methacrylate (GMA) was added dropwise to 100 mL of ε-polylysine solution. The mixture was stirred in an oil bath at 80 °C for 6 h. After passing through a 0.22 μm filter membrane, the solution was transferred to a dialysis bag with a molecular weight cutoff of 1 kDa and dialyzed with distilled water for 48 h. The dialysate was then freeze-dried at -50 °C for 48 h to obtain white methacrylated polylysine (GE) powder, which was then sealed and stored at -80 °C.

[0043] S2. Dissolve 2g of hyaluronic acid in 100mL of water to obtain a 2wt% hyaluronic acid aqueous solution;

[0044] Dissolve 1g of sodium periodate in 20mL of water to obtain a 5wt% sodium periodate aqueous solution;

[0045] Under light-protected conditions, 100 mL of hyaluronic acid aqueous solution and 20 mL of sodium periodate aqueous solution were mixed and reacted with magnetic stirring at 25 °C for 5 h. Then, 2 mL of ethylene glycol was added to quench the reaction. The reaction solution was transferred to a 3.5 kDa dialysis bag and dialyzed at room temperature for 72 h. After dialysis, the solution was freeze-dried at -50 °C for 48 h to obtain white oxidized hyaluronic acid (OHA) powder, which was stored at 4 °C in the dark.

[0046] S3. Dissolve oxidized hyaluronic acid in 1 mL of PBS aqueous solution (phosphate buffered saline solution) with pH 8 to obtain a 5 wt% oxidized hyaluronic acid solution;

[0047] Methacrylated polylysine was dissolved in a 0.1% (w / w) aqueous solution of photoinitiator LAP, and vortexed until completely dissolved to obtain a 0.25 g / mL methacrylated polylysine solution;

[0048] Equal volumes of oxidized hyaluronic acid solution and methacrylated polylysine solution were mixed and vortexed until homogeneous to obtain a mixture. The mixture was then injected into a mold at 365 nm and 800 mW / cm². 2 A double-network hydrogel GE-OHA1 was formed after 90 seconds of ultraviolet light irradiation.

[0049] Example 2: A method for preparing a photocrosslinked dual-network hydrogel that promotes skin wound healing. The specific preparation steps are the same as in Example 1, except that the concentration of the oxidized hyaluronic acid solution in this example is 10 wt%, and the resulting dual-network hydrogel is named GE-OHA2.

[0050] The methacrylated polylysine and ε-polylysine prepared in this embodiment were characterized by 1H NMR spectroscopy.

[0051] Infrared spectroscopy was used to characterize the methacrylated polylysine, ε-polylysine, hyaluronic acid, oxidized hyaluronic acid, and dual-network hydrogel GE-OHA2 in this embodiment.

[0052] The 1H NMR characterization and infrared spectrum obtained in this embodiment are as follows: Figure 1 As shown, A is the 1H NMR spectrum of methacrylated polylysine and ε-polylysine, and B is the infrared spectrum of methacrylated polylysine, ε-polylysine, hyaluronic acid, oxidized hyaluronic acid and double-network hydrogel GE-OHA2.

[0053] As can be seen from Figure A, 6.19 ppm and 5.72 ppm are generated by the terminal olefin H in the methacrylate group, indicating the successful preparation of methacrylated polylysine GE;

[0054] As shown in Figure B, the stretching vibration peak of CO-saturated aldehydes appears at 1728 cm⁻¹. -1 The presence of this group nearby indicates that the hydroxyl groups in hyaluronic acid have been irreversibly oxidized to aldehyde groups by sodium periodate, proving the successful preparation of oxidized hyaluronic acid; 1645 cm⁻¹ appears in GE-OHA2. -1 The imine bond peak indicates the occurrence of the Schiff base reaction, proving the successful preparation of GE-OHA2 hydrogel; the ethyl methacrylate group absorption peak appears at 815 cm⁻¹. -1 and 1105cm -1 The presence of light nearby indicates that the photocured methacrylated polylysine was successfully transformed into a hydrogel.

[0055] Example 3: A method for preparing a photocrosslinked dual-network hydrogel that promotes skin wound healing, specifically including the following steps:

[0056] S1. Weigh 5g of ε-polylysine (EPL) and dissolve it in 100mL of deionized water to prepare a 5wt% ε-polylysine solution.

[0057] 4 mL of glycidyl methacrylate (GMA) was added dropwise to 100 mL of ε-polylysine solution. The mixture was stirred in an oil bath at 60 °C for 4 h. After passing through a 0.22 μm filter membrane, the solution was transferred to a dialysis bag with a molecular weight cutoff of 0.5 kDa and dialyzed with distilled water for 48 h. The dialysate was then freeze-dried at 0 °C for 12 h to obtain white methacrylated polylysine (GE) powder, which was then sealed and stored at -80 °C.

[0058] S2. Dissolve 0.5g of hyaluronic acid in 100mL of water to obtain a 0.5wt% hyaluronic acid aqueous solution;

[0059] Dissolve 0.2g of sodium periodate in 20mL of water to obtain a 1wt% sodium periodate aqueous solution;

[0060] Under light-protected conditions, 100 mL of hyaluronic acid aqueous solution and 20 mL of sodium periodate aqueous solution were mixed and reacted with magnetic stirring at 70 °C for 5 h. Then, 2 mL of ethylene glycol was added to quench the reaction. The reaction solution was transferred to a 3.5 kDa dialysis bag and dialyzed at room temperature for 72 h. After dialysis, the solution was freeze-dried at -50 °C for 48 h to obtain white oxidized hyaluronic acid (OHA) powder, which was stored at 4 °C in the dark.

[0061] S3. Dissolve oxidized hyaluronic acid in 1 mL of PBS aqueous solution with pH 8 to obtain a 0.5 wt% oxidized hyaluronic acid solution;

[0062] Methacrylated polylysine was dissolved in a 0.25% (w / w) aqueous solution of photoinitiator LAP, and vortexed until completely dissolved to obtain a 0.25 g / mL methacrylated polylysine solution;

[0063] Equal volumes of oxidized hyaluronic acid solution and methacrylated polylysine solution were mixed and vortexed until homogeneous to obtain a mixture. This mixture was then injected into a mold at 365 nm and 1000 mW / cm². 2 A double-network hydrogel is formed after 5 seconds of ultraviolet light irradiation.

[0064] Example 4: A method for preparing a photocrosslinked dual-network hydrogel that promotes skin wound healing, specifically including the following steps:

[0065] S1. Weigh 15g of ε-polylysine (EPL) and dissolve it in 100mL of deionized water to prepare a 15wt% ε-polylysine solution.

[0066] 4 mL of glycidyl methacrylate (GMA) was added dropwise to 100 mL of ε-polylysine solution. The mixture was stirred in an oil bath at 90 °C for 15 h. After passing through a 0.22 μm filter membrane, the solution was transferred to a dialysis bag with a molecular weight cutoff of 5 kDa and dialyzed with distilled water for 48 h. The dialysate was then freeze-dried at -25 °C for 96 h to obtain white methacrylated polylysine (GE) powder, which was then sealed and stored at -80 °C.

[0067] S2. Dissolve 6g of hyaluronic acid in 100mL of water to obtain a 6wt% hyaluronic acid aqueous solution;

[0068] Dissolve 3.6g of sodium periodate in 20mL of water to obtain an 18wt% sodium periodate aqueous solution;

[0069] Under light-protected conditions, 100 mL of hyaluronic acid aqueous solution and 20 mL of sodium periodate aqueous solution were mixed and reacted with magnetic stirring at 50 °C for 5 h. Then, 2 mL of ethylene glycol was added to quench the reaction. The reaction solution was transferred to a 3.5 kDa dialysis bag and dialyzed at room temperature for 72 h. After dialysis, the solution was freeze-dried at -50 °C for 48 h to obtain white oxidized hyaluronic acid (OHA) powder, which was stored at 4 °C in the dark.

[0070] S3. Dissolve oxidized hyaluronic acid in 1 mL of PBS aqueous solution with pH 8 to obtain a 6 wt% oxidized hyaluronic acid solution;

[0071] Methacrylated polylysine was dissolved in a 0.15% (w / w) aqueous solution of photoinitiator LAP, and vortexed until completely dissolved to obtain a 0.25 g / mL methacrylated polylysine solution;

[0072] Equal volumes of oxidized hyaluronic acid solution and methacrylated polylysine solution were mixed and vortexed until homogeneous to obtain a mixture. This mixture was then injected into a mold at 365 nm and 365 mW / cm². 2 A double-network hydrogel is formed by irradiation with ultraviolet light for 1600 seconds.

[0073] Example 5: A method for preparing a photocrosslinked dual-network drug-loaded hydrogel that promotes skin wound healing. The specific steps are the same as in Example 1, except that in step S3, oxidized hyaluronic acid solution, anti-inflammatory drug aqueous solution, and methacrylated polylysine solution are mixed, vortexed to obtain a mixture, and then injected into a mold. Photocrosslinking is then performed to obtain the dual-network drug-loaded hydrogel GE-OHA1@Rut. The volume ratio of oxidized hyaluronic acid solution, anti-inflammatory drug aqueous solution, and methacrylated polylysine solution is 0.5:1:0.5.

[0074] In this embodiment, the method for preparing the anti-inflammatory drug aqueous solution is as follows: using rutin powder as the anti-inflammatory drug, 1 mg of rutin powder is dissolved in 1 mL of deionized water and stirred continuously until it is uniformly dissolved to obtain a 0.1 wt% rutin aqueous solution.

[0075] The procedure for preparing the photocrosslinked dual-network drug-loaded hydrogel in this embodiment is as follows: Figure 1 As shown.

[0076] Example 6: A method for preparing a photocrosslinked dual-network drug-loaded hydrogel that promotes skin wound healing. The specific steps are the same as in Example 2, except that in step S3, oxidized hyaluronic acid solution, anti-inflammatory drug aqueous solution, and methacrylated polylysine solution are mixed, vortexed to obtain a mixture, and then injected into a mold. Photocrosslinking is then performed to obtain the dual-network drug-loaded hydrogel GE-OHA2@Rut. The volume ratio of oxidized hyaluronic acid solution, anti-inflammatory drug aqueous solution, and methacrylated polylysine solution is 0.5:1:0.5.

[0077] In this embodiment, the method for preparing the anti-inflammatory drug aqueous solution is as follows: using rutin powder as the anti-inflammatory drug, 1 mg of rutin powder is dissolved in 1 mL of deionized water and stirred continuously until it is uniformly dissolved to obtain a 0.1 wt% rutin aqueous solution.

[0078] Example 7: A method for preparing a photocrosslinked dual-network drug-loaded hydrogel that promotes skin wound healing. The specific steps are the same as in Example 3, except that in step S3, oxidized hyaluronic acid solution, anti-inflammatory drug aqueous solution, and methacrylated polylysine solution are mixed, vortexed to obtain a mixture, injected into a mold, and then photocrosslinked to obtain a photocrosslinked dual-network drug-loaded hydrogel. The volume ratio of oxidized hyaluronic acid solution, anti-inflammatory drug aqueous solution, and methacrylated polylysine solution is 0.5:1:0.5.

[0079] In this embodiment, the method for preparing the anti-inflammatory drug aqueous solution is as follows: using rutin powder as the anti-inflammatory drug, 55 mg of rutin powder is dissolved in 1 mL of deionized water and stirred continuously until it is uniformly dissolved to obtain a 5.5 wt% rutin aqueous solution.

[0080] Example 8: A method for preparing a photocrosslinked dual-network drug-loaded hydrogel that promotes skin wound healing. The specific steps are the same as in Example 4, except that in step S3, oxidized hyaluronic acid solution, anti-inflammatory drug aqueous solution, and methacrylated polylysine solution are mixed, vortexed to obtain a mixture, injected into a mold, and then photocrosslinked to obtain a photocrosslinked dual-network drug-loaded hydrogel. The volume ratio of oxidized hyaluronic acid solution, anti-inflammatory drug aqueous solution, and methacrylated polylysine solution is 0.5:1:0.5.

[0081] In this embodiment, the method for preparing the anti-inflammatory drug aqueous solution is as follows: using rutin powder as the anti-inflammatory drug, 35 mg of rutin powder is dissolved in 1 mL of deionized water and stirred continuously until it is uniformly dissolved to obtain a 3.5 wt% rutin aqueous solution.

[0082] Example 9: A method for preparing a photocrosslinked dual-network drug-loaded hydrogel that promotes skin wound healing. The specific steps are the same as in Example 1, except that in step S3, oxidized hyaluronic acid solution, anti-inflammatory drug aqueous solution and methacrylated polylysine solution are mixed, vortexed to obtain a mixture, the mixture is injected into a mold, and then photocrosslinked to obtain a dual-network drug-loaded hydrogel. The volume ratio of oxidized hyaluronic acid solution, anti-inflammatory drug aqueous solution and methacrylated polylysine solution is 5:1:5.

[0083] In this embodiment, the method for preparing the anti-inflammatory drug aqueous solution is as follows: using rutin powder as the anti-inflammatory drug, 1 mg of rutin powder is dissolved in 1 mL of deionized water and stirred continuously until it is uniformly dissolved to obtain a 0.1 wt% rutin aqueous solution.

[0084] Example 10: A method for preparing a photocrosslinked dual-network drug-loaded hydrogel that promotes skin wound healing. The specific steps are the same as in Example 1, except that in step S3, oxidized hyaluronic acid solution, anti-inflammatory drug aqueous solution and methacrylated polylysine solution are mixed, vortexed to obtain a mixture, the mixture is injected into a mold, and then photocrosslinked to obtain a dual-network drug-loaded hydrogel. The volume ratio of oxidized hyaluronic acid solution, anti-inflammatory drug aqueous solution and methacrylated polylysine solution is 1:1:1.

[0085] In this embodiment, the method for preparing the anti-inflammatory drug aqueous solution is as follows: using rutin powder as the anti-inflammatory drug, 1 mg of rutin powder is dissolved in 1 mL of deionized water and stirred continuously until it is uniformly dissolved to obtain a 0.1 wt% rutin aqueous solution.

[0086] Examples 11-18 provide a method for preparing a photocrosslinked dual-network drug-loaded hydrogel that promotes skin wound healing. The specific steps are the same as in Example 5, except that different anti-inflammatory drugs are used, as shown in Table 1.

[0087] Table 1. Anti-inflammatory drugs used in the dual-network drug-loaded hydrogels of Examples 11-18

[0088]

[0089] Comparative Example 1: A single-network hydrogel, prepared by the following method:

[0090] S1. Weigh 10g of ε-polylysine (EPL) and dissolve it in 100mL of deionized water to prepare a 10wt% ε-polylysine solution.

[0091] 4 mL of glycidyl methacrylate (GMA) was added dropwise to 100 mL of ε-polylysine solution. The mixture was stirred in an oil bath at 80 °C for 6 h. After passing through a 0.22 μm filter membrane, the solution was transferred to a dialysis bag with a molecular weight cutoff of 1 kDa and dialyzed with distilled water for 48 h. The dialysate was then freeze-dried at -50 °C for 48 h to obtain white methacrylated polylysine (GE) powder, which was then sealed and stored at -80 °C.

[0092] S2. Weigh 0.25g of methacrylated polylysine and dissolve it in 1mL of 0.1% (w / w) photoinitiator LAP aqueous solution. Vortex until completely dissolved to obtain a 0.25g / mL methacrylated polylysine solution.

[0093] A methacrylated polylysine solution was injected into a mold at 365 nm with a strength of 10 mW / cm. 2 A single-network hydrogel is formed by irradiation with ultraviolet light for 60 seconds.

[0094] Comparative Example 2: A single-network drug-loaded hydrogel, prepared by the following method:

[0095] S1. Weigh 10g of ε-polylysine (EPL) and dissolve it in 100mL of deionized water to prepare a 10wt% ε-polylysine solution.

[0096] 4 mL of glycidyl methacrylate (GMA) was added dropwise to 100 mL of ε-polylysine solution. The mixture was stirred in an oil bath at 80 °C for 6 h. After passing through a 0.22 μm filter membrane, the solution was transferred to a dialysis bag with a molecular weight cutoff of 1 kDa and dialyzed with distilled water for 48 h. The dialysate was then freeze-dried at -50 °C for 48 h to obtain white methacrylated polylysine (GE) powder, which was then sealed and stored at -80 °C.

[0097] S2. Weigh 0.25g of methacrylated polylysine and dissolve it in 1mL of 0.1% (w / w) photoinitiator LAP aqueous solution. Vortex until completely dissolved to obtain a 0.25g / mL methacrylated polylysine solution.

[0098] After uniformly mixing the methacrylated polylysine solution and the anti-inflammatory drug aqueous solution, the mixture was injected into the mold. (365nm, strength 10mW / cm) 2 A single-network drug-loaded hydrogel GE@Rut was formed by 60 seconds of ultraviolet light irradiation;

[0099] In this embodiment, the preparation method of the anti-inflammatory drug aqueous solution is the same as in Example 5.

[0100] The gelation time of the hydrogels of Comparative Example 1 and Examples 1 and 2 was compared. Then, the flow state of the hydrogels prepared in Example 1 and Comparative Example 1 before and after photocrosslinking and curing was compared using the 45°C tilting method.

[0101] like Figure 3 The figures show a comparison of the gelation times of GE-OHA1, GE-OHA2, and GE single-network hydrogels, as well as a comparison of the flow states of GE-OHA2 and GE single-network hydrogels before and after gelation. Figure A shows the gelation time comparison of GE-OHA1, GE-OHA2, and GE single-network hydrogels, while Figure B shows the flow state comparison of GE-OHA2 and GE single-network hydrogels before and after gelation. Figure A shows that GE-OHA2 has the shortest gelation time, gelling within 60 seconds. Figure B shows that all hydrogels exhibited a flow state before gelation. After gelation, both the single-network and double-network hydrogels did not flow when tilted at 45°C, and the liquid level did not change, indicating that gelation had occurred.

[0102] The hydrogels prepared in Examples 1, 2 and Comparative Example 1 were characterized for mechanical properties to obtain stress-strain curves and compression modulus results. Then, the strain was fixed at 1.0% to obtain the frequency scan diagram of the double crosslinked hydrogel GE-OHA2 in Example 2.

[0103] like Figure 4 As shown, the mechanical properties of GE-OHA1, GE-OHA2 and GE single-network hydrogels are characterized, and the frequency scan of GE-OHA2 is shown when the strain is fixed at 1.0%. Among them, A is the stress-strain curve, B is the comparison of the compressive modulus of the hydrogels, and C is the frequency scan of the double crosslinked hydrogel GE-OHA2.

[0104] As can be seen from A and B, compared with GE single-network hydrogel, the compressive modulus of GE-OHA composite hydrogel gradually increases, the mechanical properties increase, and the mechanical properties of the composite hydrogel GE-OHA2 with high oxidized hyaluronic acid content are even better.

[0105] As shown in Figure C, the storage modulus (G′) curve of GE-OHA2 gel exhibits a higher value than the loss modulus (G″). The mechanical properties of this thermosensitive gel remain relatively stable after the phase transition, indicating consistent mechanical behavior after the phase transition. The G′ curve of the gel consistently exceeds G″, maintaining a relatively stable trend. These results demonstrate that the gel retains its mechanical properties and exhibits a certain degree of elasticity after the phase transition, which meets the management requirements of wound dressings and prevents wound dressing detachment during application.

[0106] The hydrogels prepared in Examples 1, 2, and 1 (Comparative Example 1) were subjected to swelling and hemolysis tests. The results were obtained by... Figure 5 As shown, A represents the swelling performance test result of the hydrogel, and B represents the hemolysis performance test result of the hydrogel.

[0107] As shown in A, the equilibrium swelling rates of GE, GE-OHA1, and GE-OHA2 are 1080±25%, 810±24%, and 640±27%, respectively. With the increase of OHA substitution, the degree of photocrosslinking of the gel under ultraviolet irradiation increases, forming a denser three-dimensional network structure, which restricts the diffusion of water molecules and leads to a decrease in swelling rate. Although the swelling rate of GE-OHA2 is relatively low, it still has a swelling capacity of 640%, which is sufficient to absorb wound exudate and maintain a moderately moist environment. At the same time, the denser network structure is beneficial to improving the mechanical strength and structural stability of the gel, making it more suitable for wound dressing applications.

[0108] As shown in B, the hemolysis rate of all three hydrogel materials is less than 5%, indicating that the hydrogels do not cause in vitro hemolysis and have good blood compatibility.

[0109] The hydrogels prepared in Examples 1, 2, and 1 were subjected to stability tests under different pH conditions. The results were obtained by... Figure 6 As shown in the figure, A represents the stability of GE-OHA2 under different pH conditions, B represents the stability of GE-OHA1 under different pH conditions, and C represents the stability of GE single-component hydrogel under different pH conditions. All three gels showed a slower degradation rate at physiological conditions (pH=7.4) compared to acidic conditions, indicating that the hydrogels are more stable under physiological conditions, which is beneficial for skin application. Comparative analysis of the three figures shows that the degradation rate of the three gels is GE > GE-OHA1 > GE-OHA2, indicating that GE-OHA2 gel is more stable and less prone to degradation.

[0110] The drug release capacity of the drug-loaded hydrogels prepared in Examples 5, 6, and Comparative Example 2 was determined, and the results are as follows: Figure 7 As shown, A represents the drug release capacity of GE-OHA2@Rut, B represents the drug release capacity of GE-OHA1@Rut, and C represents the drug release capacity of the single-component GE@Rut drug-loaded hydrogel. The drug release rate of GE@Rut did not change significantly at different pH levels. For both the dual-network drug-loaded hydrogels GE-OHA1@Rut and GE-OHA2@Rut, the drug release rate was fastest from 0 to 30 min, slowed down from 30 to 80 min, and then flattened out from 80 to 180 min. The maximum release rates for the two groups were 75% and 70%, respectively, indicating that the dual-network drug-loaded hydrogel had a better sustained-release effect.

[0111] The antioxidant capacity of the drug-loaded hydrogels prepared in Example 6 and Comparative Example 2 was determined, and the results were obtained by... Figure 8As shown, A represents the scavenging ability of the two hydrogels on DPPH, and B represents their scavenging ability on ABTS. Free radicals in wound healing affect healing; therefore, hydrogels should possess the ability to scavenge free radicals. When GE-OHA2@Rut and GE@Rut were added to the DPPH solution, the intensity of the characteristic absorption peak of DPPH at 517 nm was significantly reduced; when GE-OHA2@Rut and GE@Rut were added to the ABTS solution, the characteristic absorption peak of ABTS at 734 nm was reduced. This indicates that the hydrogels GE-OHA2@Rut and GE@Rut can effectively scavenge free radicals and have strong antioxidant capacity, thus promoting skin wound regeneration.

[0112] The in vitro antibacterial properties of the hydrogels from Examples 2, 6, Comparative Example 1, and Comparative Example 2 were evaluated. Specifically, frozen bacterial strains (Escherichia coli and Staphylococcus aureus) were inoculated onto MHA medium to prepare a concentration of approximately 1.5 × 10⁻⁶. 8 CFU / mL bacterial suspension. Using a sterile cotton swab, streak the bacterial suspension onto the entire agar surface. Cut hydrogel into approximately equal-sized circles, moisten with a small amount of distilled water, place in the center of the culture medium, and incubate at 37°C for 24 hours. Observe colony growth.

[0113] The bacterial concentration was 1×10 6 Staphylococcus aureus and Escherichia coli (CFU / mL) were divided into 5 groups: Control group, Rutin group, GE-OHA2 group, GE group, GE-OHA2@Rut group, and GE@Rut group. Hydrogels were immersed in a mixture of bacterial suspension and MHB medium. For the Rutin group, the bacterial suspension was mixed with 0.1 wt% rutin solution, incubated at 37°C and 160 rpm for 4 hours, then diluted 1000 times and plated. After incubation at 37°C for 24 hours, colony counting was performed.

[0114] like Figure 9 As shown, the results of colony growth and colony count in the in vitro antimicrobial performance evaluation are shown. Among them, A is the result of colony growth in the in vitro antimicrobial performance evaluation, and B is the result of colony count in the in vitro antimicrobial performance evaluation.

[0115] The results showed that GE-OHA2, GE, GE-OHA2@Rut, and GE@Rut all had good anti-Staphylococcus aureus effects, while the four synthesized hydrogels, although having weak anti-Escherichia coli activity, also had some ability to resist Gram-negative bacilli, indicating that the dual-network hydrogel of the present invention has excellent in vitro antibacterial activity.

[0116] Cytotoxicity evaluation was performed on the hydrogels of Examples 2, 6, Comparative Example 1, and Comparative Example 2. The specific method was as follows: the hydrogel samples were sterilized and a 0.1 g / mL hydrogel extract was prepared. The hydrogel extract was diluted to 30 mg / mL and stored at 4°C for later use. L929 cells were cultured at a rate of 1×10⁻⁶. 4 Cells were seeded per well into 96-well plates and cultured for 24 hours. Then, hydrogel extract was added and the cells were incubated in a CO2 cell culture incubator for 24 hours, 48 ​​hours, and 72 hours. After incubation, 10% CCK8 solution was added and incubated for 1 hour. Cell viability was then measured using a microplate reader.

[0117] Biocompatibility of the hydrogels of Examples 2, 6, Comparative Example 1 and Comparative Example 2 was determined by live-dead staining imaging. Specifically, the prepared hydrogel extract was incubated with L929 cells for 72 hours, and the cell growth status was observed by inverted fluorescence microscope.

[0118] like Figure 10 As shown, the results are cytotoxicity evaluation and biocompatibility assay, where A represents the cytotoxicity evaluation result and B represents the biocompatibility assay result.

[0119] As shown in A, the cell survival rate of the hydrogel extract treatment group can reach over 75%. After 2 and 3 days of incubation, the cells in each group proliferated normally. The above results indicate that the prepared hydrogel does not affect fibroblast proliferation and has good biocompatibility.

[0120] As shown in B, cells in all experimental groups exhibited a spindle-shaped morphology and strong green fluorescence, indicating good cell growth. This result is consistent with the cytotoxicity experiment, demonstrating that all four hydrogels prepared possess good biocompatibility and can meet the survival requirements of cells.

[0121] The effects of the hydrogels of Examples 2, 6, Comparative Example 1 and Comparative Example 2 on cell migration and growth ability were evaluated by Transwell assay. The specific method was as follows: L929 cells were incubated with hydrogel extract for 24 h, and the growth status of the cells was observed and the migration was recorded by taking pictures using an inverted microscope.

[0122] like Figure 11 As shown, the results of the effect of hydrogel on cell migration and growth are as follows. It can be seen that the GE and GE-OHA2@Rut groups do not inhibit the migration of L929 cells. The number of L929 cells that migrated in the GE-OHA2 and GE@Rut extract treatment groups was significantly increased compared with the Control group. The migration in the GE@Rut treatment group was more than 1.5 times that of the Control group, indicating that the prepared hydrogel has the ability to promote migration.

[0123] The anti-inflammatory ability of the hydrogels from Examples 2, 6, Comparative Example 1, and Comparative Example 2 was evaluated by immunofluorescence assay of TNF-α in cells. The specific method was as follows: RAW264.7 cells were pretreated with the prepared hydrogel extract for 16 h, and then stimulated with 2 μg / mL LPS for 6 h. After the treatment, the cells were stained with immunofluorescence of the anti-inflammatory factor TNF-α, and the results were recorded by Leica inverted fluorescence microscope.

[0124] The anti-inflammatory ability of the hydrogels of Examples 2, 6, Comparative Example 1 and Comparative Example 2 was evaluated by cellular IL-10 immunofluorescence assay, and the results were recorded by Leica inverted fluorescence microscope.

[0125] like Figure 12 The results shown are the immunofluorescence assay results for TNF-α and IL-10, where A represents the immunofluorescence assay result for TNF-α and B represents the immunofluorescence assay result for IL-10.

[0126] As shown in Figure A, the LPS-treated group exhibited significant green fluorescence compared to the Control group, indicating that LPS successfully induced a cellular inflammation model. The green fluorescence of the hydrogel GE, GE@Rut, and GE-OHA2@Rut treatment groups was significantly weaker than that of the LPS-treated group, especially the GE-OHA2@Rut group, which showed almost no green fluorescence. This result indicates that the GE-OHA2@Rut treatment group significantly reduced the content of the pro-inflammatory factor TNF-α induced by LPS, demonstrating excellent anti-inflammatory capabilities.

[0127] As shown in B, the hydrogels GE, GE@Rut, and GE-OHA2@Rut all exhibited significant green fluorescence, especially the GE-OHA2@Rut group, which showed strong green fluorescence of IL-10. This result indicates that the GE-OHA2@Rut treatment group enhanced the content of the anti-inflammatory factor IL-10 and has excellent anti-inflammatory ability.

[0128] The hydrogels prepared in Examples 2 and 6 were used to evaluate their effect on wound healing in skin wound treatment. Specifically, KM mice were used, and circular full-thickness skin wounds of the same size (9 mm wide) were constructed on their backs. All mice were randomly divided into 4 groups: Control, GE-OHA2, Rutin, and GE-OHA2@Rut. The wound healing status of each group of mice was recorded at different time points (day 4, day 7, day 11, and day 14).

[0129] The hydrogels prepared in Examples 2 and 6 were used to treat skin wounds. The effect of the hydrogels on wound healing was further investigated by histological analysis of the regenerated skin tissue and H&E staining results.

[0130] The hydrogels prepared in Example 2 and Example 6 were used for skin wound treatment. Through histological analysis of the regenerated skin tissue, the effect of the hydrogel on wound healing was further studied by the results of Masson staining.

[0131] As Figure 13 shown, they are the evaluation results of skin wound treatment, the results of H&E staining, and the results of Masson staining. Among them, A is the evaluation result of skin wound treatment, B is the result of H&E staining, and C is the result of Masson staining;

[0132] As can be seen from A, starting from the fourth day, it was observed that the wound surface of the GE-OHA2@Rut group began to significantly decrease, and the wound surface at 14 days had basically healed. The healing rate of the experimental group (95%) was significantly better than that of the control group (70%), indicating that the GE-OHA2@Rut treatment group had a faster re-epithelialization process, and the hydrogel could promote the healing of the wound surface;

[0133] As can be seen from B, the epidermis layer could be clearly seen in the skin of the hydrogel treatment group. In terms of the epidermal thickness, hair follicles, and the degree of sebaceous gland formation, the double-network drug-loaded hydrogel GE-OHA2@Rut had the best effect and stronger ability to promote skin regeneration.

[0134] As can be seen from C, after 14 days of treatment, the stratum corneum and regenerated epidermis appeared in the experimental groups of GE-OHA2, Rutin, and GE-OHA2@Rut. Both the epidermis and the stratum corneum covered the dermis, but no new epidermis was formed in the control group.

[0135] The hydrogels prepared in Example 2 and Example 6 were used for skin wound treatment, and immunofluorescence staining of inflammatory factors TNF-α and IL-10 was performed to further study the effect of the hydrogel on wound healing.

[0136] As Figure 14 shown, they are the results of immunofluorescence staining of TNF-α and the results of immunofluorescence staining of IL-10 after skin wound treatment. Among them, A is the result of immunofluorescence staining of TNF-α, and B is the result of immunofluorescence staining of IL-10. It can be seen from the figure that the expression levels of TNF-α expressed by M1 macrophages are in the order of: GE-OHA2@Rut < Rutin < Control < GE-OHA2, and the expression level of inflammatory factors of GE-OHA2@Rut is the lowest; the expression levels of IL-10 expressed by M2 macrophages are in the order of: GE-OHA2@Rut > Rutin > GE-OHA2 > Control, and the expression level of anti-inflammatory factors of GE-OHA2@Rut is the highest.

[0137] These results indicate that the gel GE-OHA2@Rut can release rutin, inhibit the synthesis and release of inflammatory factors, produce the anti-inflammatory factor IL-10 to reduce the inflammatory response, further reduce the inflammatory time of infected wounds and accelerate the healing of skin wounds.

[0138] In summary, this study innovatively constructed a dual-network drug-loaded hydrogel system based on oxidized hyaluronic acid, photocrosslinked polylysine, and anti-inflammatory drugs.

[0139] Using a molecular design strategy, a polylysine main network is first constructed by photo-initiated polymerization, whose cationic properties endow the material with inherent antibacterial activity. Then, oxidized hyaluronic acid is introduced to form a secondary network through dynamic Schiff base reaction, and the anti-inflammatory drug rutin is introduced. This improves mechanical properties while retaining the cell migration-promoting properties of hyaluronic acid and the anti-inflammatory properties of rutin.

[0140] This dual-network synergistic mechanism not only resolves the contradiction between mechanical strength and biocompatibility in traditional single-network hydrogels, but also integrates multiple healing mechanisms through functional complementarity between components: the amino group of ε-polylysine provides reaction sites for photocrosslinking, ensuring the spatial controllability of network formation; the aldehyde modification of oxidized hyaluronic acid not only enhances the crosslinking density, but also continuously regulates the wound microenvironment through the slow release of degradation products (cell survival rate >90%).

[0141] Compared to existing single-function dressings, the dual-network drug delivery system proposed in this study achieves synergistic optimization of antibacterial activity, mechanical toughness, and regeneration-promoting function for the first time, providing an innovative solution for the development of a new generation of smart wound dressings.

[0142] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a photocrosslinked dual-network hydrogel that promotes skin wound healing, characterized in that, Includes the following steps: S1. Glycidyl methacrylate is added dropwise to 5-15 wt% of ε-polylysine aqueous solution and reacted at 60-90℃ for 4-15 h. After the reaction is completed, the solution is filtered through a 0.22 μm filter membrane, dialyzed with distilled water, and the dialysate is freeze-dried to obtain methacrylated polylysine, which is then sealed and stored at -80℃. The volume ratio of the glycidyl methacrylate to the aqueous ε-polylysine solution is 4:

100. S2. Mix 0.5-6 wt% hyaluronic acid aqueous solution and 1-18 wt% sodium periodate aqueous solution under light-protected conditions, stir and react at 25-70°C for 3-8 hours, add ethylene glycol to quench the reaction, dialyze the resulting reaction solution at room temperature, freeze-dry to obtain oxidized hyaluronic acid, and store at 4°C in the dark. S3. Dissolve oxidized hyaluronic acid in PBS aqueous solution to obtain oxidized hyaluronic acid solution; The concentration of the oxidized hyaluronic acid solution is 10 wt%, and the pH of the PBS aqueous solution is 8; A solution of methacrylated polylysine was obtained by mixing methacrylated polylysine with an aqueous photoinitiator LAP and vortexing until completely dissolved. In the methacrylated polylysine solution, the concentration of methacrylated polylysine is 0.25 g / mL, and the concentration of the photoinitiator LAP aqueous solution is 0.1~0.25 wt%. Equal volumes of methacrylated polylysine solution and oxidized hyaluronic acid solution were mixed and vortexed to obtain a mixture. The mixture was then injected into a mold and cured under ultraviolet light to obtain a photocrosslinked double network hydrogel. An aqueous solution of an anti-inflammatory drug is also added to the mixture. The concentration of the anti-inflammatory drug in the aqueous solution is 0.1-5.5 wt%, and the volume ratio of the aqueous solution of the anti-inflammatory drug to the methacrylated polylysine solution is 1:0.5-5.

2. The method for preparing the photocrosslinked dual-network hydrogel for promoting skin wound healing according to claim 1, characterized in that, The anti-inflammatory drugs are one or more of the following: curcumin, quercetin, rutin, catechin, soy isoflavones, luteolin, andrographolide, resveratrol, and artemisinin.

3. The method for preparing the photocrosslinked dual-network hydrogel for promoting skin wound healing according to claim 1, characterized in that, In step S2, the volume ratio of the hyaluronic acid aqueous solution to the sodium periodate aqueous solution is 5:

1.

4. The method for preparing the photocrosslinked dual-network hydrogel for promoting skin wound healing according to claim 1, characterized in that, In step S3, the ultraviolet curing specifically involves using 365 nm ultraviolet light at a concentration of 365~1000 mW / cm². 2 Under these conditions, the light exposure time is 5~1600s.

5. The photocrosslinked dual-network hydrogel prepared by the method for preparing the photocrosslinked dual-network hydrogel for promoting skin wound healing as described in claim 1.

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