A hydrogel for wound repair and a preparation method and application thereof

Abstract

This invention relates to hydrogels, specifically to a hydrogel for wound repair, its preparation method, and its applications. Through material design and optimization, this invention successfully prepared a multifunctional composite hydrogel system composed of N-isopropylacrylamide, gelatin, tannic acid, poly-L-lysine, and ferric chloride, and systematically investigated its physicochemical properties and biological functions. This hydrogel possesses a unique network structure, balancing mechanical properties and flexibility, and can dynamically adapt to the wound microenvironment and closely adhere to the wound surface. Tannic acid and Fe... 3+ Coordination and complexation enhance antioxidant properties, protecting against oxidative stress damage; poly-L-lysine and tannic acid synergistically endow it with highly effective broad-spectrum antibacterial properties, inhibiting the proliferation of Staphylococcus aureus and Escherichia coli; mouse experiments show that it can significantly accelerate the healing of full-thickness skin defects. This PGTPL composite hydrogel integrates multiple functions and has great potential for application in the treatment of infected wounds.

Description

Technical Field

[0001] This invention relates to hydrogels, specifically to a hydrogel for wound repair, its preparation method, and its applications. Background Technology

[0002] Traditional dressings have several limitations in treating infected wounds. First, their mechanical properties are incompatible with soft tissue; insufficient elasticity leads to edge curling, and poor adhesion results in frequent detachment. Second, their passive protective mode makes it difficult to effectively regulate the wound microenvironment and actively close the wound to promote healing. Third, traditional materials generally lack active antibacterial functions, providing only limited barrier effects through physical isolation, and their inhibitory effect on multidrug-resistant bacteria is poor. Furthermore, most traditional dressings lack antioxidant functions, making it difficult to alleviate the persistent oxidative stress in chronic wounds. Dressing changes can easily damage newly formed tissue and fail to provide an active microenvironment that promotes angiogenesis and cell migration. These shortcomings collectively lead to prolonged wound healing periods, high infection recurrence rates, and severely impact clinical treatment outcomes.

[0003] The PTPD hydrogel dressing disclosed in the inventor's patent application (2026105053633, April 16, 2026) overcomes the technical barriers of traditional wound dressings in clinical applications through innovative functional design. This dressing achieves seamless adhesion to complex wounds with excellent mechanical compliance and actively drives wound closure through its thermosensitive contraction properties, significantly improving wound healing efficiency. However, the wound healing process faces the dual challenges of oxidative stress and microbial infection: excessive accumulation of reactive oxygen species induced by inflammation not only leads to cell and biomolecular damage but also destroys growth factor activity; while bacterial proliferation in the wound further exacerbates the risk of infection and hinders the healing process. Limited by its antioxidant and antibacterial properties, the application efficacy of PTPD hydrogel in the treatment of infected wounds is significantly restricted. Therefore, it is urgent to systematically improve its comprehensive therapeutic performance through material molecular structure design optimization and multifunctional synergistic strategies to meet clinical application needs. Summary of the Invention

[0004] Purpose of the invention Based on previous research and the needs of wound repair, this invention constructs a composite hydrogel system, PGTPL, with multiple intelligent response characteristics through molecular engineering and material design strategies.

[0005] Technical solution A hydrogel for wound repair, characterized in that it comprises: N-isopropylacrylamide, gelatin, poly-L-lysine, tannic acid, and Fe. 3+ Solution, cross-linking agent, initiator.

[0006] The hydrogel for wound repair is characterized in that it is prepared from the following raw materials in the following mass / volume ratio, using 4 mL of deionized water as a solvent: N-isopropylacrylamide 0.60–0.90 g, gelatin 0.12–0.18 g, poly-L-lysine 0.04–0.06 g, tannic acid 0.016–0.024 g, and Fe at a concentration of 20 mg / mL. 3+ The solution contains 30.0–45.0 μL, crosslinking agent 4.8–7.2 mg, and initiator 16–24 mg. The hydrogel for wound repair is characterized by being composed of the following raw materials: N-isopropylacrylamide 0.75 g, gelatin 0.15 g, poly-L-lysine 0.05 g, tannic acid 20 mg, and Fe... 3+ The solution concentration was 20 mg / mL (37.5 µL), containing 6 mg of crosslinking agent, 20 mg of initiator, and 4 mL of water as solvent.

[0007] The hydrogel for wound repair is characterized in that the crosslinking agent is N,N'-methylenebisacrylamide, N,N'-dihydroxymethylbisacrylamide, N,N'-ethylidenebisacrylamide, or polyethylene glycol diacrylate. The initiator is any one or any combination of two or more of the following: polyethylene glycol dimethacrylate, 1,3-propylene glycol diacrylate, ethylene glycol dimethacrylate, and diallyl dimethyl ammonium chloride; the initiator is any one or any combination of two or more of the following: ammonium persulfate, potassium persulfate, azobisisobutyramidine hydrochloride, azobisisobutyramidine imidazoline hydrochloride, hydrogen peroxide, tert-butyl hydroperoxide, sodium bisulfite, sodium metabisulfite, and ascorbic acid.

[0008] The hydrogel for wound repair, characterized in that the Fe... 3+ The solution is an aqueous solution of ferric chloride, ferric nitrate, or ferric sulfate.

[0009] A method for preparing a hydrogel for wound repair, characterized by comprising the following steps: S1. Dissolve and mix the raw materials: N-isopropylacrylamide, gelatin and poly-L-lysine are placed in pure water, heated to 50 ℃ and stirred for 10 min to obtain a uniform mixture. S2. Metal ion coordination modification: Add ferric ion aqueous solution to the homogeneous mixture, stir until completely mixed, then add tannic acid solution, and continue stirring for 20 min to allow tannic acid to fully participate in the reaction. S3. Preparation of crosslinking initiation system: Add N,N'-methylenebisacrylamide crosslinking agent to the mixed system after reaction, stir evenly, then add ammonium persulfate initiator and stir quickly until the system is evenly mixed; S4. Static polymerization molding: Pour the well-mixed reaction solution into a mold and allow it to stand at room temperature for 30 minutes to polymerize and obtain the initial hydrogel. S5. Purification treatment: The initial hydrogel is soaked in pure water for 24 hours to remove unreacted monomers from the system, finally obtaining PGTPL hydrogel. Further: A method for preparing a hydrogel for wound repair, characterized in that... S1. Dissolve and mix the raw materials: Accurately weigh 0.75 g N-isopropylacrylamide, 0.15 g gelatin and 0.05 g poly-L-lysine, place the three raw materials in 4 mL of purified water, heat to 50 ℃ and stir for 10 min to obtain a uniform mixture; S2. Metal ion coordination modification: Add 37.5 µL of ferric ion aqueous solution with a concentration of 20 mg / mL to the homogeneous mixture, stir until completely mixed, then add 20 mg of tannic acid solution, and continue stirring for 20 min to allow tannic acid to fully participate in the reaction. S3. Preparation of crosslinking initiation system: Add 6 g of N,N'-methylenebisacrylamide crosslinking agent to the mixed system after reaction, stir evenly, add 20 mg of ammonium persulfate initiator, and stir quickly until the system is evenly mixed; S4. Static polymerization molding: Pour the well-mixed reaction solution into a mold and allow it to stand at room temperature for 30 minutes to polymerize and obtain the initial hydrogel. S5. Purification treatment: The initial hydrogel is soaked in pure water for 24 h to remove unreacted monomers from the system, and finally PGTPL hydrogel is obtained.

[0010] The application of the hydrogel in the preparation of wound repair dressings.

[0011] The wound can be any one of the following: burn wound, acute or chronic infected wound, skin trauma wound, postoperative wound that is difficult to heal, diabetic ulcer wound, pressure ulcer wound, skin abrasion wound, skin erosion wound, skin defect wound, or radiation-induced skin injury wound.

[0012] Invention Design Concept This system uses temperature-responsive poly(N-isopropylacrylamide) (PNIPAM) as the core intelligent response unit, and precisely constructs a three-dimensional cross-linked network framework using controlled free radical polymerization technology. Simultaneously, it introduces gelatin molecules with excellent biocompatibility, forming an interpenetrating network structure through physical cross-linking, effectively enhancing the material's bioactivity and tissue affinity. Furthermore, it innovatively integrates tannic acid-iron ions (TA-Fe). 3+The dynamic coordination system and polylysine (PLL) antibacterial functional component endow the material with self-healing and antioxidant properties through dynamic coordination bonds, and utilize the cationic antibacterial mechanism of PLL to achieve efficient antibacterial effect. Finally, a multi-component synergistic and multi-mechanism complementary composite functional system PGTPL is formed, providing a more comprehensive solution for wound repair.

[0013] Beneficial effects This invention, through material design and optimization, successfully prepared a multifunctional composite hydrogel system composed of N-isopropylacrylamide, gelatin, tannic acid, poly-L-lysine, and ferric chloride, and systematically investigated its physicochemical properties and biological functions. This hydrogel possesses a unique network structure, balancing mechanical properties and flexibility, and can dynamically adapt to the wound microenvironment and closely adhere to the wound surface. Tannic acid and Fe... 3+ Coordination and complexation enhance antioxidant properties, protecting against oxidative stress damage; poly-L-lysine and tannic acid synergistically endow it with highly effective broad-spectrum antibacterial properties, inhibiting the proliferation of Staphylococcus aureus and Escherichia coli; mouse experiments show that it can significantly accelerate the healing of full-thickness skin defects. This PGTPL composite hydrogel integrates multiple functions and has great potential for application in the treatment of infected wounds. Attached Figure Description

[0014] Figure 1 The preparation process of hydrogels; Figure 2 (A) SEM image and (B) EDS image of the hydrogel Figure 3 Swelling properties of hydrogels with different formulations. (A) Changes in swelling rate of hydrogels with different formulations after 24 h; (B) Equilibrium swelling rate of hydrogels with different formulations. Figure 4 Rheological properties of hydrogels. (A) Rheological properties of hydrogels with different formulations; (B) Rheological properties of NIPAM / Gel / TA-PL hydrogels at different temperatures.

[0015] Figure 5 1. Adhesion and mechanical compliance of hydrogels. (A) Hydrogel adheres to a finger; (B) The finger with hydrogel adhered to it bends; (C) Pigskin with hydrogel adhered to it bends; (D) Pigskin with hydrogel adhered to it twists. Figure 6 Antioxidant properties of hydrogels with different formulations (the top right figure shows the DPPH alcohol solution of different formulation hydrogels after soaking at 37°C for half an hour. From left to right, they are DPPH alcohol solutions of PTPD hydrogel, PGTPL hydrogel, PNIAPM / TA-PL hydrogel, PGPL hydrogel, PPL hydrogel and blank DPPH alcohol solution). Figure 71. Tensile properties of hydrogels. (A) Tensile-stress-strain curves of hydrogels; (B) Fracture strength, elastic modulus, toughness, and elongation at break of hydrogels; Figure 8 Shrinkage properties of hydrogels with different formulations; Figure 9 Cell viability of hydrogels with different formulations Figure 10 Antibacterial properties of different hydrogel materials. (A) Antibacterial zone experiment diagrams of different dressings; (B) Size of the antibacterial zone of different dressings; Figure 11 Evaluation of the in vivo wound healing performance of thermosensitive hydrogel. (A) Changes in mouse wounds treated with multifunctional hydrogel, sterile gauze, and commercially available dressings from 0 to 14 days; (B) Dynamic changes in mouse wounds under different treatment conditions; (C) Wound healing rate of mice under different treatment conditions. Detailed Implementation

[0016] Example 1 1.1 Experiment Content 1.1.1 Orthogonal experiments to optimize the hydrogel formulation Based on the preliminary screening results of single-factor experiments, this invention selects peak storage modulus, shrinkage area ratio, and DPPH free radical scavenging rate as key performance evaluation indicators. For the three key preparation parameters affecting hydrogel performance (gelatin content, tannic acid addition, and crosslinking agent dosage), a three-factor, three-level experimental design method is adopted, with specific parameter settings shown in Table 1. By constructing an L9(3^3) orthogonal experimental matrix, the influence of changes in the levels of each factor on the performance indicators of the composite hydrogel is systematically investigated, thereby establishing the structure-property relationship between preparation process parameters and material properties, and achieving optimized control of the preparation process.

[0017] Table 1. Factor-level table for orthogonal experiments

[0018] 1.1.2 Scanning Electron Microscopy (SEM) Testing The prepared PGTPL hydrogel was subjected to SEM testing. Specific procedures are as follows: The prepared hydrogel samples were quenched with liquid nitrogen and then dried in a freeze dryer for 24 hours. After removal, the gel was cut into thin slices and fixed on a stage with conductive tape. The conductivity was improved by vacuuming and gold sputtering. Finally, the microstructure of the samples was observed using an EVO18 scanning electron microscope at a test voltage of 3.00 kV.

[0019] 1.1.3 Swelling property test and rheological property test A. Swelling performance tests were conducted on the hydrogels with different formulations. Specific procedures included: First, hydrogel samples with a diameter of 20 mm and a thickness of 1.5 mm were prepared, ensuring uniform size. After freeze-drying to constant weight, the initial mass of the samples was measured, followed by immersion in deionized water at 25°C. The samples were removed after 0.5, 1, 2, 4, 8, 12, 24, and 36 hours, and weighed after surface drying. Each group of samples was tested three times, and the swelling rate was calculated using the average value according to the formula:

[0020] In the formula, Wd is the original mass in the dry state, in g; Wt is the mass after water absorption and swelling, in g.

[0021] B. Rheological property testing The rheological properties of different hydrogels were determined, including the rheological properties of PGTPL hydrogel at 20℃ and 37℃. Specifically, a rotational rheometer equipped with a 50 mm parallel plate was used to test the dynamic rheological properties of the composite hydrogel at 20℃. Hydrogel samples with a diameter of 25 mm and a thickness of 1.5 mm were fixed on the test stage, and the distance between the rotor and the sample was adjusted. A strain amplitude scanning mode was used, with a strain range of 0.1%-1000% and a frequency of 10 rad / s. Each sample was tested three times. Based on the test data, the storage modulus G' and loss modulus G'' curves of the samples at room temperature were plotted and analyzed.

[0022] 1.1.4 Antioxidant performance test The antioxidant properties of the dynamic hydrogel were evaluated by investigating its DPPH (2,2-diphenyl-1-trinitrohydrazide hydrate) free radical scavenging ability. Briefly, a 0.1 mM DPPH solution was prepared in ethanol, and a freshly prepared hydrogel (20 mm × 20 mm × 1 mm) was added to 10 mL of the DPPH solution. The solution was then placed in a shaker at 37 °C with a speed of 100 r / min. -1 Shake in the dark for 30 minutes. Measure the absorbance of the blank sample at 517 nm using a UV spectrometer. Each group has 3 replicates. The DPPH free radical scavenging effect is calculated using the following formula:

[0023] A control A is the absorbance of the DPPH solution without the addition of a hydrogel-like substance. sample The absorbance of the DPPH solution was measured after the addition of hydrogel.

[0024] 1.1.5 Tensile property test The mechanical properties of PGTPL hydrogels were determined using a universal testing machine. Different formulations of hydrogel prepolymers were injected into dumbbell-shaped molds to create dumbbell-shaped gel samples measuring 50 mm × 8.5 mm × 2.5 mm, followed by tensile testing. The tensile speed was set to 10 mm / min, resulting in the tensile stress-strain curves of the hydrogels.

[0025] 1.1.6 Temperature-sensitive shrinkage performance test The specific procedure for determining the thermosensitive shrinkage properties of hydrogels with different formulations is as follows: The hydrogel prepolymer solution was poured into six-well plates and polymerized at 4°C for 24 h. The volume change was measured by placing the sample in a 37°C water bath to evaluate the thermal response. The initial and final dimensions are denoted as L0 and L, respectively, and the shrinkage area ratio was calculated as follows:

[0026] 1.1.7 Cytotoxicity assay To determine the cell viability of hydrogels with different formulations, the specific procedures are as follows: The cytotoxicity of PGTPL, PTPL, PGPL, and PPL was evaluated using the MTT assay. The prepared hydrogel samples were placed in DMEM medium at a concentration of 60 mg mL⁻¹ and incubated at 37°C for 24 hours. The extract was then filtered through a 0.22 µm membrane.

[0027] Mouse fibroblasts (L929) were cultured to a concentration of 5 × 10⁻⁶. 4 Cells / mL, 100 μL was seeded into 96-well plates and incubated at 37℃ in a 5% CO2 incubator for 24 hours. After removing the original culture medium, the experimental group was added to DMEM medium containing sample extract, while the control group was incubated with PBS, and cultured for another 12 hours. 50 μL of MTT reagent was added to each well and incubated for 4 hours, and the absorbance at 490 nm was measured using a microplate reader.

[0028]

[0029] In the formula, ODsample is the absorbance of the sample, ODblank is the absorbance of the blank control group, and ODcontrol is the absorbance of the control group.

[0030] 1.1.8 Antibacterial test To evaluate the antibacterial properties of the hydrogel, two bacteria, *Escherichia coli* and *Staphylococcus aureus*, were used to test its antibacterial activity. The bactericidal performance of the hydrogel was verified by the inhibition zone test. 100 μL of bacterial suspension (10... 6 CFU·mL -1 Add the bacterial solution evenly to an LB agar plate. Divide the plate with the bacterial solution into three regions, and place a 5 mm diameter PGTPL hydrogel, PTPD hydrogel, and commercially available dressing in the center of each region. Incubate at 37 °C for 24 hours and record the size of the inhibition zone.

[0031] 1.1.9 In vivo wound healing experiment The healing-promoting effects of different dressings were evaluated by applying them to full-thickness wounds in mice. Specific procedures included: This invention is based on a randomized controlled trial design. Fifteen 8-week-old male BALB / c mice were selected as experimental subjects and randomly divided into a blank control group, a commercial dressing control group, and an experimental group, with a sample size of 5 mice in each group (n=5). The experimental animals were anesthetized by intraperitoneal injection of 1.25% tribromoethanol solution, with the injection dose strictly controlled at 0.2 mL / 10g body weight. In the preoperative preparation stage, the hair on the back of the mice was first initially removed using an electric shaver, followed by a second hair removal using depilatory cream to ensure complete removal of hair from the surgical area. Subsequently, the surgical area was strictly disinfected using povidone-iodine. Following aseptic techniques, a full-thickness skin defect wound was prepared on the back of the mice using a scalpel, reaching the myofascia layer. During postoperative care, the blank control group used sterile gauze to cover the wound, the commercial dressing control group used Kefu hydrocolloid dressing purchased from Taobao, and the experimental group used PGTPL hydrogel dressing. Throughout the entire wound healing cycle, continuous dynamic imaging of the wound healing process was recorded using intelligent digital devices, and the wound area was quantitatively measured and analyzed using ImageJ image analysis software, providing precise quantitative evidence for subsequent data statistics and results analysis. The animal experiments have been approved by the Ethics Committee of China Pharmaceutical University, with ethics approval number: YSL-202504045.

[0032] 1.2 Results and Discussion 1.2.1 Design Strategy for Hydrogels As shown in Figure 1, this invention successfully constructs a multifunctional hydrogel system based on a design strategy that combines multiple cross-linking mechanisms and synergistic functional groups. Using N-isopropylacrylamide (NIPAM) as a thermosensitive functional monomer, and leveraging its low critical solution temperature (LCST ≈ 32℃), NIPAM molecules spontaneously form a physical cross-linked network through hydrophobic interactions under physiological temperature conditions. This network structure not only endows the hydrogel with temperature-responsive properties and mechanical support, but also provides an ideal carrier platform for the intelligent controlled release of drugs.

[0033] Based on the following explanation of the reaction mechanism involving tannic acid molecules, in this hydrogel system, the abundant functional groups on the gelatin segments and the phenolic hydroxyl groups of tannic acid (TA) form physical cross-links through hydrogen bonds and ionic bonds; particularly crucially, some phenolic groups in the TA structure are... 3+ Under oxidation, it transforms into a quinone structure. This quinone structure can undergo Michael addition and Schiff base reactions with the amino groups in gelatin molecules, thereby forming a stable covalent cross-linked network. The introduction of this covalent cross-linking significantly improves the structural stability and mechanical strength of the gel network. Simultaneously, precise control of the cross-linking density optimizes the gel swelling behavior and degradation rate, providing a key performance foundation for the application of hydrogels in the biomedical field. Polylysine (PLL), as a cationic polymer, participates in the cross-linking process to strengthen the network structure and disrupts bacterial cell membranes through electrostatic adsorption, achieving highly efficient antibacterial function.

[0034] The synergistic effect of physical cross-linking (hydrogen bonds, ionic bonds) and chemical cross-linking (coordinate bonds, covalent bonds) endows the gel with both high toughness and self-healing properties. Furthermore, the excellent biocompatibility of gelatin, the antioxidant activity of TA, and the thermosensitive properties of NIPAM work together to successfully integrate multiple functions such as mechanical adaptation, tissue adhesion, and antibacterial and anti-inflammatory properties into this hydrogel system, effectively meeting the complex needs of repairing infected and exudative wounds.

[0035]

[0036] The reaction of tannic acid molecules 1.2.2 Formulation optimization of hydrogels Based on a pre-designed factor level table, this invention systematically prepared hydrogel samples with different component ratios. The maximum shear modulus of the hydrogels was precisely measured using a rheometer; this parameter served as a quantitative indicator to evaluate the stability of the hydrogel network structure. To investigate the dynamic mechanical behavior of the hydrogels under physiological conditions, the area shrinkage rate of the hydrogels was continuously monitored over 24 hours at a constant temperature of 37℃, serving as a key basis for evaluating their dynamic mechanical properties. Regarding the characterization of antioxidant properties, the scavenging efficiency of the hydrogels against DPPH free radicals within 30 minutes was measured at a reaction temperature of 37℃, thereby systematically evaluating their antioxidant activity. The data obtained from the above experiments were rigorously recorded and organized; detailed results are shown in Table 2.

[0037] This invention employs a critical comprehensive analysis method to systematically process experimental data. This method integrates the dynamic fluctuations of the data with the intrinsic correlations between variables by constructing a multivariate data integration model. Using a hierarchical weighting algorithm, it scientifically calculates the comprehensive evaluation index of each formulation. To further explore the influence of each factor on the experimental results, range analysis is used to statistically analyze the comprehensive scoring results. The results show that among all influencing factors, gelatin quality has the most significant impact on the physicochemical and mechanical properties of the hydrogel. Secondly, crosslinking agent quality has a significant impact on the crosslinking density and swelling properties of the hydrogel. In contrast, tannic acid quality has a relatively weak impact on the hydrogel properties. Based on the above quantitative analysis, the optimal formulation for the main influencing factors of the hydrogel was determined to be A3B2C3, with the specific parameters: 0.15 g gelatin, 20 mg tannic acid, and 6 mg crosslinking agent. The hydrogel prepared under this formulation exhibits the best comprehensive performance in terms of mechanical properties, swelling properties, and biocompatibility, providing important theoretical basis and practical guidance for subsequent related research.

[0038] Table 2. Orthogonal Experiment Table for Hydrogels

[0039] 1.2.3 Preparation of hydrogels Preparation of PGTPL hydrogel: Accurately weigh 0.75 g N-isopropylacrylamide, 0.15 g gelatin, and 0.05 g poly-L-lysine, dissolve them in 4 mL of purified water, heat to 50 °C, and stir for 10 minutes to mix thoroughly. Then, add 37.5 µL of 20 mg / mL hydrogel. -1 Fe 3+The solution was stirred until completely mixed, and 20 mg of tannic acid (TA) solution was added. Stirring continued for 20 minutes to allow the tannic acid to react fully. Then, 6 mg of the crosslinking agent N,N'-methylenebisacrylamide (MBA) was added to the solution and stirred until homogeneous. 20 mg of the initiator ammonium persulfate was added, and the mixture was stirred rapidly to ensure homogeneity. The mixture was poured into a mold and allowed to stand at room temperature for polymerization for half an hour. Finally, the resulting hydrogel was immersed in purified water for 24 hours to remove unreacted monomers, thus obtaining hydrogel PGTPL.

[0040] Preparation of PTPL hydrogel: Accurately weigh 0.75 g N-isopropylacrylamide and 0.05 g poly-L-lysine, dissolve them in 4 mL of purified water, heat to 50 °C, and stir for 10 minutes to mix thoroughly. Then, add 37.5 µL of 20 mg / mL hydrogel. -1 Fe 3+ The solution was stirred until completely mixed, and 20 mg of tannic acid (TA) solution was added. Stirring continued for 20 minutes to allow the tannic acid to react fully. Then, 6 mg of the crosslinking agent N,N'-methylenebisacrylamide (MBA) was added to the solution and stirred until homogeneous. 20 mg of the initiator ammonium persulfate was added, and the mixture was stirred rapidly to ensure homogeneity. The mixture was poured into a mold and allowed to stand at room temperature for polymerization for half an hour. Finally, the resulting hydrogel was immersed in purified water for 24 hours to remove unreacted monomers, thus obtaining the PTPL hydrogel.

[0041] Preparation of PGPL hydrogel: Accurately weigh 0.75 g N-isopropylacrylamide, 0.15 g gelatin, and 0.05 g poly-L-lysine, dissolve them in 4 mL of purified water, heat to 50 °C, and stir for 10 minutes to mix thoroughly. Then, add 6 mg of crosslinking agent N,N'-methylenebisacrylamide (MBA) to the solution and stir until homogeneous. Add 20 mg of initiator ammonium persulfate and stir rapidly to ensure homogeneity. Add 20 µL of accelerator tetramethylethylenediamine (TEMED) and stir rapidly until homogeneous. Pour the mixture into a mold and allow it to polymerize at room temperature for half an hour. Finally, soak the prepared hydrogel in purified water for 24 hours to remove unreacted monomers, thereby obtaining PGPL hydrogel.

[0042] Preparation of PPL hydrogel: Accurately weigh 0.75 g N-isopropylacrylamide and 0.05 g poly-L-lysine, dissolve them in 4 mL of purified water, heat to 50 °C, and stir for 10 minutes to mix thoroughly. Then, add 6 mg of crosslinking agent N,N'-methylenebisacrylamide (MBA) to the solution and stir until homogeneous. Add 20 mg of initiator ammonium persulfate and stir rapidly to ensure homogeneity. Add 20 µL of accelerator tetramethylethylenediamine (TEMED) and stir rapidly until homogeneous. Pour the mixture into a mold and allow it to polymerize at room temperature for half an hour. Finally, soak the prepared hydrogel in purified water for 24 hours to remove unreacted monomers, thereby obtaining PPL hydrogel.

[0043] Note: P: Poly-N-isopropylacrylamide (PNIPAM) G: Gelatin T: Tannic acid PL: Poly-L-lysine 1.2.4 SEM Analysis Figure 2 The presence of numerous pores of varying sizes in A is due to the formation of a thermosensitive poly(N-isopropylacrylamide) (PNIPAM) network through free radical polymerization, while gelatin and polylysine (PLL) form a secondary network through physical entanglement and hydrogen bonding. Furthermore, TA and Fe... 3+ Dynamic coordination bonds are formed, further stabilizing the network structure and fixing the pore morphology during solvent evaporation or freeze-drying. Different types of pore structures also exist in the network. Interconnected open pore structures allow water to flow, accelerating water entry and exit and causing the hydrogel to shrink faster when heated, while isolated closed pore structures are beneficial to the mechanical buffering properties of the gel network. Figure 2 The EDS plot of B shows a high C / N ratio in the hydrogel, which is consistent with the characteristics of organic polymer-based hydrogels. The presence of a certain amount of iron ions in the network indicates the presence of TA-Fe. 3+ Cross-linked network.

[0044] 1.2.5 Swelling Properties of Multifunctional Hydrogels The degree and rate of swelling are fundamental properties of hydrogels, and their swelling performance is closely related to the hydrophilic groups and the structure of the hydrogel. For example... Figure 3 As shown in Figure A, each formulation exhibits rapid swelling behavior within half an hour and reaches swelling equilibrium within 24 hours. Figure 3 As shown in Figure B, in the PGTPL multifunctional hydrogel, gelatin and tannic acid possess a large number of hydrophilic groups such as -OH / -NH2 / -COOH, and the electrostatic complex of tannic acid with poly-L-lysine (PL)+ -TA - It can form reversible physical crosslinks, which are more conducive to network expansion than chemical crosslinks. The moderately loose crosslinked network results in the highest swelling rate, with an equilibrium swelling rate of 9.20 g / g. The equilibrium swelling rates of PPL and PTPL are 7.85 g / g and 7.80 g / g, respectively, showing similar values. The former's network expansion is moderate due to the self-crosslinking (ε-amino condensation) of poly-L-lysine, but lacking the additional effect of tannins or gelatin. The latter's network expansion is moderate because the self-crosslinking of poly-L-lysine is nearing saturation; even with the addition of tannins, the ionic crosslinking network and crosslinking density increase, but the improvement is minimal. In PGPL hydrogel, the gel network is dominated by strong electrostatic crosslinking. Gelatin and poly-L-lysine form stable ionic crosslinks, significantly increasing the crosslinking density. The hydrophilic effect of gelatin is offset by the high crosslinking density, resulting in the lowest equilibrium swelling rate of 7.16 g / g.

[0045] 1.2.6 Rheological properties of hydrogels like Figure 4 A. By comparing the rheological property curves of four different hydrogel formulations, it was found that the storage modulus (G′) and loss modulus (G″) of the multifunctional PGTPL hydrogel were higher than those of other hydrogels within the strain range of 0.1% to 100%, indicating that the multifunctional hydrogel has higher structural stability. This phenomenon is closely related to the crosslinking density of the hydrogel network and the interaction of the internal network. The PGTPL hydrogel has the highest storage modulus of 4448 Pa, and its excellent mechanical properties are due to the synergistic effect of multiple crosslinked networks: N-isopropylacrylamide monomers form a stable network backbone through chemical crosslinking, while the gelatin triple helix structure enhances the network integrity through physical crosslinking, and TA-Fe... 3+ Metal coordination bonds form a stable chemical cross-linked network, enhancing network rigidity. This synergistic effect of "chemical cross-linking + physical cross-linking" endows the material with high strength and toughness. The significantly reduced modulus of PTPL hydrogel indicates insufficient mechanical support due to the lack of a gelatin-based physical cross-linked network. (TA-Fe) 3+ Crosslinking may be unevenly distributed due to the steric hindrance of polylysine, and purely chemical crosslinked networks lack energy dissipation mechanisms, making them prone to stress concentration. In contrast, the physical crosslinking of gelatin in PGPL hydrogels provides the main mechanical support, while the electrostatic interaction between polylysine and gelatin assists network formation. The lack of TA-Fe... 3+ The strong chemical crosslinking limits the maximum modulus. The PPL hydrogel network contains only poly(N-isopropylacrylamide) network, with low crosslinking density and weak interaction forces, resulting in a loose network structure and limited mechanical properties.

[0046] In addition, such as Figure 4As shown in Figure B, when the testing temperature of the PGTPL hydrogel was increased to 37℃, the storage modulus of the hydrogel increased from 4448 Pa to 9817 Pa, and the mechanical properties doubled. This is due to the interaction of multiple networks within the hydrogel's internal structure. When the temperature rises to 37℃ (above the low critical solution temperature of PNIPAM, LCST≈32℃), the PNIPAM molecular chains undergo a phase transition, the hydrophobic isopropyl groups dehydrate and aggregate, leading to polymer network shrinkage and enhanced intramolecular hydrophobic interactions, significantly improving the material's stiffness. Simultaneously, the increased temperature promotes TA-Fe... 3+ Dynamic recombination of coordination bonds increases coordination density, further strengthening the network structure. Gelatin maintains stable physical cross-linking at physiological temperatures, forming an interpenetrating reinforcement effect with the shrinking PNIPAM network. The cationic properties of PLL stabilize the network structure through electrostatic interactions during this process. This temperature-induced synergistic enhancement of multiple molecular interactions results in a storage modulus at 37°C that is twice that at 20°C, perfectly mimicking the mechanical requirements under human body temperature conditions and providing better structural support for wounds.

[0047] 1.2.7 Adhesion properties of multifunctional hydrogels To evaluate the adhesive properties of the PGTPL multifunctional hydrogel, experimental studies were conducted on its adhesion to biological tissues such as fingers and pig skin. Figure 5 As shown in A and B, the hydrogel exhibits excellent adhesion and compliance properties, adhering firmly to the surface of human skin. Even during joint flexion and extension movements, it maintains stable adhesion without detachment. Figure 5 Results C and D indicate that the PGTPL multifunctional hydrogel forms an adhesion interface with the pigskin surface, and withstands complex bending and twisting motions of the pigskin. From the perspective of its mechanism of action, the strong adhesion properties of PGTPL multifunctional hydrogel stem from multiple synergistic effects. The amphiphilic structure of PNIPAM facilitates the formation of hydrogen bonds with the surface of biological tissues. The abundant amino, carboxyl, and phenolic hydroxyl groups in gelatin, tannic acid, and polylysine molecules, through hydrogen bonding and electrostatic interactions, achieve effective binding with the tissue surface. Simultaneously, these three molecules also construct a stable hydrogel network structure through hydrogen bonding and electrostatic interactions. Furthermore, the chelating effect between iron ions and tannic acid acts as cross-linking sites, significantly enhancing the strength and toughness of the hydrogel and ensuring stable adhesion under stress and deformation. The hydrogel's inherent flexibility allows it to fill the microscopic unevenness of biological tissue surfaces, achieving thorough wetting. The interaction between hydrophilic groups and polar groups on the tissue surface forms a physical adsorption layer. Combined with the excellent biocompatibility conferred by its high water content, this results in a stable and durable interfacial adhesion effect.

[0048] 1.2.8 Antioxidant properties of hydrogels Inflammatory responses trigger the excessive production of free radicals, which attack intracellular biomolecules, damage cell membranes, proteins, and nucleic acids, leading to cell death. Simultaneously, they exacerbate inflammatory responses, inhibit the activity of angiogenesis-related factors, hinder angiogenesis, and severely impair wound healing; therefore, timely removal is essential. Antioxidant materials can be used to promote wound healing by scavenging excess reactive free radicals. This invention evaluated the antioxidant capacity of PGTPL hydrogel, PTPL hydrogel, PGPL hydrogel, and PPL hydrogel samples using DPPH scavenging rates. The lower the DPPH free radical content, the lower the absorbance measured by ultraviolet spectrophotometry.

[0049] like Figure 6 As shown, the PGTPL hydrogel with added tannic acid and gelatin exhibits the highest DPPH free radical scavenging efficiency, with a DPPH scavenging rate of 76.2%, demonstrating the strongest antioxidant properties. This is due to the abundance of phenolic hydroxyl groups (-OH) in tannic acid molecules, which can donate hydrogen atoms or electrons to directly react with DPPH free radicals, thus exhibiting free radical scavenging ability. Furthermore, the ε-amino group of poly-L-lysine forms a dynamic hydrogen bond network with the tannic acid hydroxyl groups, enhancing the orientation stability of tannic acid molecules and increasing the exposure rate of active sites. In addition, the hydrophilic network of gelatin promotes the diffusion and migration of tannic acid molecules within the hydrogel. Compared to the PGTPL hydrogel, the PTPL hydrogel shows a slightly lower DPPH scavenging efficiency, with a DPPH scavenging rate of 67.8%. This may be because the hydrogel network lacks gelatin buffering, leading to dense ionic cross-linking between tannic acid and poly-L-lysine, resulting in partial encapsulation of the tannic acid phenolic hydroxyl groups. Moreover, the rigid network also restricts the molecular movement of tannic acid, reducing the accessibility of active sites. The lack of core phenolic hydroxyl groups in tannins significantly reduces the DPPH scavenging efficiency of PGPL hydrogels to 45.9%. However, the formation of a hydrogen bond network between gelatin's -OH / -NH2 groups and poly-L-lysine slightly improves electron transfer efficiency. PNIPAM-PL hydrogels, on the other hand, only achieve 38.6% efficiency. This is because their hydrogel network relies solely on electron transfer from the ε-amino groups on the poly-L-lysine chain, lacking the auxiliary networks of tannins and gelatin. This single, weak mechanism leads to low efficiency. Furthermore, the DPPH scavenging rate of PTPD hydrogels discussed in the previous chapter is approximately 55.7%. This is primarily because the dopamine grafting rate of gelatin is typically <20%, resulting in a much lower number of available catechol groups compared to tannin systems. Dopamine may be partially oxidized to a quinone structure during polymerization, reducing the content of active hydroxyl groups. Additionally, polyvinyl alcohol may lack active functional groups, serving only as a mechanical reinforcing phase without contributing antioxidant capacity. Its dense crystalline regions may also hinder the exposure of dopamine groups, reducing the probability of contact with free radicals.

[0050] 1.2.9 Tensile properties of hydrogels A hydrogel of this size was prepared using a 5 × 2 × 0.2 cm mold, and a tensile test was performed on it at a speed of 10 mm / min. Figure 7 As shown, firstly, the elastic modulus of PGTPL hydrogel is approximately 24.7 kPa, close to the range of human soft tissue modulus (1-100 kPa), indicating its excellent mechanical adaptability. This ensures that the dressing can move synchronously with skin deformation, avoiding edge lifting or secondary damage caused by modulus mismatch, and maintaining good interfacial contact to promote healing. Furthermore, as... Figure 7 As shown in Figure B, its elongation at break is 162.5%, significantly exceeding the normal elongation of skin (approximately 50-70%), ensuring that the hydrogel dressing will not break due to daily activities when used on active areas such as joints. Furthermore, the hydrogel exhibits good tensile strength of 30.2 kPa, significantly higher than the skin tension at the wound site (typically <10 kPa), resisting daily friction and minor impacts, maintaining the integrity of the dressing structure, and avoiding healing interference caused by frequent changes. The hydrogel's toughness is 16.2 kPa, indicating that the material has good energy dissipation capabilities, absorbing energy through plastic deformation upon impact and preventing sudden breakage.

[0051] 1.2.10 Shrinkage area ratio of hydrogels like Figure 8 Analysis of the shrinkage area ratios of hydrogel systems with different components revealed a significant component-performance correlation. The shrinkage area ratios of PGTPL, PTPL, PGPL, and PPL hydrogels were 42.3%, 52.0%, 46.9%, and 67.2%, respectively. This variation is primarily influenced by the internal network structure of the hydrogels. The PPLL hydrogel system exhibits only electrostatic interactions (the -NH3 group of poly-L-lysine). + The -CONH- group of PNIPAM exhibits the weakest intermolecular forces, resulting in high chain segment freedom during phase transitions and thus the largest shrinkage rate. The PTPL hydrogel incorporates TA-Fe... 3+ After coordination bonding, dynamic metal coordination significantly enhances network stability and reduces the shrinkage area ratio. The addition of gelatin to PGPL hydrogel increases crosslinking points through physical entanglement and hydrogen bonding, limiting the shrinkage of poly(N-isopropylacrylamide) segments. In contrast, the physical crosslinking of gelatin in PGTPL hydrogel is related to TA-Fe... 3+ Chemical coordination produces a synergistic effect, maximizing the crosslinking density. The phenolic hydroxyl groups of tannic acid can react with Fe... 3+ It can coordinate with gelatin and form hydrogen bonds. The ε-amino group of poly-L-lysine participates in both electrostatic crosslinking and metal coordination. The thermosensitive α-helical structure of gelatin and the LCST behavior of PNIPAM produce an interlocking effect.

[0052] 1.2.11 Biocompatibility of Hydrogels In assessing cytotoxicity, this invention used the MTT assay and employed L929 mouse fibroblasts as the experimental cell line, using an extraction method for toxicity evaluation. Figure 9 As shown, after co-culturing cells with extracts from PGTPL hydrogel, PNIPAM / TA hydrogel, PGPL hydrogel, and PPL hydrogel samples, the cell viability of all four hydrogels exceeded 85%. This excellent performance stems from the inherent compatibility of the basic biomaterials: the N-isopropylacrylamide polymer chain exhibits hydrophilicity at physiological temperatures (<32℃), and its amide group (-CONH-) is similar to the phospholipid structure of cell membranes, reducing rejection reactions; gelatin, as a collagen hydrolysis product, retains the RGD (arginine-glycine-aspartic acid) cell adhesion sequence, promoting cell attachment and proliferation; the ε-amino group of poly-L-lysine carries a positive charge, mimicking the cationic properties of the extracellular matrix (ECM); and tannic acid and Fe... 3+ The coordination crosslinking is performed at physiological concentrations (≤ 0.1 mM) to avoid iron ion toxicity. The biocompatibility exhibited by PGTPL hydrogel indicates that it is a safe biomedical material that can be used as a wound dressing.

[0053] 1.2.12 Antibacterial properties of hydrogels like Figure 10 As shown in the inhibition zone test, PGTPL hydrogel exhibits significant inhibitory effects on both types of bacteria, particularly Staphylococcus aureus. The number of colonies around the hydrogel is significantly reduced, and the inhibition zone diameter reaches 1.82 cm. In the Escherichia coli environment, the inhibition zone is slightly smaller, with a diameter of 1.73 cm. The broad-spectrum antibacterial properties of PGTPL hydrogel are mainly due to the following mechanisms: First, the membrane disruption mechanism of polylysine. The positively charged ε-amino group electrostatically binds to the negatively charged phospholipid head group of the bacterial membrane, inserting into the bacterial cell membrane to form a pore (approximately 2 nm in diameter). This effect is effective against both Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli). Second, TA-Fe... 3+ Synergistic antibacterial effect. Tannic acid blocks bacterial iron metabolism by chelating iron ions, Fe... 3+ Catalyzing the production of reactive oxygen species (ROS) damages bacterial DNA. Finally, gelatin may also play a supporting role; gelatin degradation can generate antimicrobial peptide fragments, enhancing antimicrobial properties and improving the dispersibility of polylysine in the gel network, thus strengthening the effect of polylysine.

[0054] In patent application 2026105053633, the PTPD hydrogel showed limited inhibitory activity against two types of bacteria. The inhibition zone diameter for Staphylococcus aureus was 1.51 cm, while the inhibitory effect on Escherichia coli was significantly reduced, with an inhibition zone diameter of only 0.62 cm. This is because the catechol groups in the PTPD hydrogel are primarily effective against Gram-positive bacteria, inhibiting Staphylococcus aureus reproduction by interfering with the peptidoglycan cross-linking of Staphylococcus aureus. The poor effect on Escherichia coli is due to the outer membrane barrier of E. coli hindering catechol penetration. Furthermore, the polyvinyl alcohol in the PTPD hydrogel has certain negative effects; its hydrophilic surface promotes biofilm formation, and the crystalline regions hinder the exposure of dopamine active sites, thus affecting the antibacterial properties of the PTPD hydrogel. Commercially available dressings showed no inhibitory effect against either type of bacteria. Based on the antibacterial performance testing of the hydrogel, the PGTPL hydrogel system has significant clinical translational value. Its balance of broad-spectrum antibacterial activity and excellent biocompatibility makes it suitable for the diverse needs of modern wound management.

Claims

1. A hydrogel for wound repair, characterized in that, include: N-Isopropylacrylamide, gelatin, poly-L-lysine, tannic acid, Fe 3+ Solution, cross-linking agent, initiator.

2. The hydrogel for wound repair according to claim 1, prepared from the following raw materials, using 4 mL of deionized water as a solvent: 0.60–0.90 g of N-isopropylacrylamide, 0.12–0.18 g of gelatin, 0.04–0.06 g of poly-L-lysine, 0.016–0.024 g of tannic acid, and Fe at a concentration of 20 mg / mL. 3+ Solution 30.0–45.0 μL, crosslinking agent 4.8–7.2 mg, initiator 16–24 mg.

3. The hydrogel for wound repair according to claim 2, characterized in that, Composed of the following raw materials: N-isopropylacrylamide 0.75 g, gelatin 0.15 g, poly-L-lysine 0.05 g, tannic acid 20 mg, Fe... 3+ The solution concentration was 20 mg / mL (37.5 µL), containing 6 mg of crosslinking agent, 20 mg of initiator, and 4 mL of water as solvent.

4. A hydrogel for wound repair according to any one of claims 1-3, characterized in that, The crosslinking agent is any one or any combination of two or more of N,N'-methylenebisacrylamide, N,N'-dihydroxymethylbisacrylamide, N,N'-ethylidenebisacrylamide, polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, 1,3-propylene glycol diacrylate, and diallyl dimethylammonium chloride; the initiator is any one or any combination of two or more of ammonium persulfate, potassium persulfate, azobisisobutyramidine hydrochloride, azobisisobutyramidine imidazoline hydrochloride, hydrogen peroxide, tert-butyl hydroperoxide, sodium bisulfite, sodium metabisulfite, and ascorbic acid.

5. A hydrogel for wound repair according to claims 1-3, characterized in that... The Fe 3+ The solution is an aqueous solution of ferric chloride, ferric nitrate, or ferric sulfate.

6. A method for preparing a hydrogel for wound repair, characterized in that, To achieve this, follow these steps: S1. Dissolve and mix the raw materials: N-isopropylacrylamide, gelatin and poly-L-lysine are placed in pure water, heated to 50 ℃ and stirred for 10 min to obtain a uniform mixture. S2. Metal ion coordination modification: Add ferric ion aqueous solution to the homogeneous mixture, stir until completely mixed, then add tannic acid solution, and continue stirring for 20 min to allow tannic acid to fully participate in the reaction. S3. Preparation of crosslinking initiation system: Add N,N'-methylenebisacrylamide crosslinking agent to the mixed system after reaction, stir evenly, then add ammonium persulfate initiator and stir quickly until the system is evenly mixed; S4. Static polymerization molding: Pour the well-mixed reaction solution into a mold and allow it to stand at room temperature for 30 minutes to polymerize and obtain the initial hydrogel. S5. Purification treatment: The initial hydrogel is soaked in pure water for 24 h to remove unreacted monomers from the system, and finally PGTPL hydrogel is obtained.

7. The method according to claim 6, characterized in that, S1. Dissolve and mix the raw materials: Accurately weigh 0.75 g N-isopropylacrylamide, 0.15 g gelatin and 0.05 g poly-L-lysine, place the three raw materials in 4 mL of purified water, heat to 50 ℃ and stir for 10 min to obtain a uniform mixture; S2. Metal ion coordination modification: Add 37.5 µL of ferric ion aqueous solution with a concentration of 20 mg / mL to the homogeneous mixture, stir until completely mixed, then add 20 mg of tannic acid solution, and continue stirring for 20 min to allow tannic acid to fully participate in the reaction. S3. Preparation of crosslinking initiation system: Add 6 g of N,N'-methylenebisacrylamide crosslinking agent to the mixed system after reaction, stir evenly, add 20 mg of ammonium persulfate initiator, and stir quickly until the system is evenly mixed; S4. Static polymerization molding: Pour the well-mixed reaction solution into a mold and allow it to stand at room temperature for 30 minutes to polymerize and obtain the initial hydrogel. S5. Purification treatment: The initial hydrogel is soaked in pure water for 24 h to remove unreacted monomers from the system, and finally PGTPL hydrogel is obtained.

8. The application of the hydrogel according to any one of claims 1-5 in the preparation of wound repair dressings.

9. The application according to claim 7, characterized in that, The The wound can be any one of the following: burn wound, acute or chronic infected wound, skin trauma wound, postoperative wound that is difficult to heal, diabetic ulcer wound, pressure ulcer wound, skin abrasion wound, skin erosion wound, skin defect wound, or radiation-induced skin injury wound.

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