Hydrogel, preparation method thereof and product containing hydrogel

Abstract

The invention relates to the field of medical materials, in particular to hydrogel and a preparation method thereof and a product containing the hydrogel, a sodium alginate solution and a gelatin solution are mixed and then cross-linked with a polylysine solution by adopting a calcium salt solution as a cross-linking agent, the flexibility of the prepared hydrogel is obviously improved, the hydrogel is not prone to degradation, and the hydrogel has the advantages that the hydrogel is not prone to disintegration, and the service life of the hydrogel is prolonged. The hydrogel prepared by the preparation method disclosed by the invention has the advantages that the material can be attached to the wound for a long time to play a better curative effect, in addition, the hydrogel also shows obviously improved antibacterial property and bacteriostasis, has good biocompatibility, and can promote cell proliferation and cell migration, enhance tissue repair and promote subcutaneous angiogenesis of the infected wound, so that the healing of the infectious wound is promoted. Epsilon-PLL (at) SA / Gel promotes infected wounds induced by staphylococcus aureus and E.coil by adjusting polarization of macrophages and skin microbiota. The invention provides a new research approach for prevention and treatment of infectious wounds.

Description

Technical Field

[0001] The present invention relates to the field of medical materials, and in particular to a hydrogel, a preparation method thereof, and a product containing the hydrogel. Background Art

[0002] Wound infections caused by bacteria such as Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) pose a significant challenge to clinical wound healing. Currently, the main treatment for infected wounds is systemic antibiotics combined with local debridement, which is accompanied by problems such as antibiotic resistance, difficulty in achieving effective antibacterial concentrations locally, and antibiotic ototoxicity and nephrotoxicity. Hydrogel wound dressings have become a promising material for treating such infected wounds, with the ability to release antimicrobial substances to combat infection while supporting tissue repair.

[0003] Sodium alginate (SA) is a natural biodegradable polysaccharide biomaterial extracted from algae. It is composed of α-l-glucuronic acid (G unit) and β-d-manuronic acid (M unit) polymerized through 1-4 glycosidic bonds, similar to biomimetic extracellular matrix components. The wound healing performance of SA dressing lies in its strong exudate absorption capacity, and its water absorption capacity is 5-7 times that of traditional dressings (15). SA quickly forms a gel after contact with the wound surface, inducing cell migration, proliferation and regeneration, and releasing bioactive substances to activate platelets to stop bleeding. Gelatin (Gel) is formed by the hydrolysis and denaturation of collagen and has been recognized by the US Food and Drug Administration (FDA) as a non-toxic and safe material. One of the most important properties of gelatin is its thermal reversibility: it is soluble in water at temperatures above 35°C and can form a transparent solution, which solidifies after cooling. This is due to the presence of a large number of active hydrophilic groups in the gelatin molecular chain, such as -NH2, -OH and -COOH, which allow the formation of gels through grafting modification.

[0004] Existing hydrogels made with sodium alginate and gelatin as film-forming substrates and calcium ions as cross-linking agents have problems with poor softness and easy degradation, making it difficult for the material to adhere to the wound for a long time, thereby increasing the number of medication applications and costs. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the defects of the sodium alginate / gelatin gel in the prior art, which has poor softness and is easy to degrade, resulting in the material being difficult to adhere to the wound for a long time, thereby providing a hydrogel and a preparation method thereof and a product containing the hydrogel.

[0006] To this end, the present application provides a method for preparing a hydrogel, comprising the following steps:

[0007] Step S1: mixing the sodium alginate solution and the gelatin solution to prepare a first mixed solution;

[0008] Step S2: mixing the first mixed solution with the polylysine solution to prepare a second mixed solution;

[0009] Step S3: mixing the second mixed solution with the calcium salt solution, cross-linking, and preparing a hydrogel.

[0010] Furthermore, in the first mixed solution, the mass ratio of sodium alginate to gelatin is 1:1-30, preferably 1:5-15.

[0011] Furthermore, the concentration of the sodium alginate solution is 1.5% to 2.5% m / v; and / or the concentration of the gelatin solution is 15% to 25% m / v.

[0012] Furthermore, the concentration of the sodium alginate solution is 1.5% to 2.5% m / v; and / or the concentration of the gelatin solution is 15% to 25% m / v.

[0013] In this application, % m / v refers to the amount of solute in 100 mL of solvent.

[0014] Furthermore, the polylysine is ε-polylysine.

[0015] Furthermore, the preparation method of the sodium alginate solution includes dissolving sodium alginate in an aqueous solution;

[0016] Optionally, the dissolution temperature is 25°C to 55°C.

[0017] Optionally, the dissolution time is 1h-2h.

[0018] Furthermore, the preparation method of the gelatin solution includes dissolving gelatin in an aqueous solution;

[0019] Optionally, the dissolution temperature is 35°C to 65°C.

[0020] Optionally, the dissolution time is 30 min-1 h.

[0021] The aqueous solution is a conventional aqueous solution in the art, which can be sterile water, deionized water, pure water, glucose aqueous solution or phosphate aqueous solution, etc.

[0022] Furthermore, in the second mixed solution, the mass percentage of polylysine to the total mass of sodium alginate and gelatin is 1% to 30%.

[0023] Furthermore, the calcium salt includes one or more of calcium chloride, calcium nitrate, and calcium sulfate; and / or the concentration of the calcium salt solution is 1.5% to 2.5% w / v; and / or the volume ratio of the second mixed solution to the calcium salt is 1:0.8 to 1.2; and / or the concentration of the polylysine solution is 5% to 25% w / v.

[0024] The present application also provides a hydrogel prepared according to any of the above-mentioned preparation methods.

[0025] The present application also provides a hydrogel dressing, comprising a hydrogel prepared by any of the above-mentioned preparation methods.

[0026] The present application also provides a hydrogel prepared by any of the above preparation methods having one or more of the following AH:

[0027] A. Use in preparing a product for promoting wound healing; preferably, the wound comprises an infected wound;

[0028] B. Use in the preparation of products promoting angiogenesis;

[0029] C. Use in the preparation of antibacterial and / or bacteriostatic products;

[0030] D. Use in the preparation of anti-inflammatory products;

[0031] E. Use in the preparation of products that promote cell proliferation and cell migration;

[0032] F. Use in the preparation of products that promote tissue repair;

[0033] G. Use in the preparation of products that stabilize skin flora;

[0034] H. Use in preparing a product that promotes polarization of skin macrophages M1 to M2.

[0035] The present application also provides a product comprising a hydrogel prepared by any of the above-mentioned preparation methods; optionally, the product includes a hydrogel dressing and / or an injection.

[0036] 1. The present invention provides a method for preparing a hydrogel, comprising the following steps: Step S1: mixing a sodium alginate solution with a gelatin solution to produce a first mixed solution; Step S2: mixing the first mixed solution with a polylysine solution to produce a second mixed solution; and Step S3: mixing the second mixed solution with a calcium salt solution and cross-linking to produce a hydrogel. The hydrogel prepared by mixing a sodium alginate solution with a gelatin solution, then with a polylysine solution, and then cross-linking using a calcium salt solution as a cross-linking agent exhibits significantly improved softness and is resistant to degradation, facilitating long-term adhesion of the material to wounds and achieving better therapeutic efficacy. Furthermore, the hydrogel exhibits significantly enhanced antibacterial and antimicrobial properties, possesses good biocompatibility, and can promote cell proliferation and migration, enhance tissue repair, and promote subcutaneous angiogenesis in infected wounds, thereby promoting the healing of infected wounds. ε-PLL@SA / Gel promotes the healing of infected wounds induced by Staphylococcus aureus and E.coil by regulating macrophage polarization and the skin microbiome. This invention provides a new research avenue for the prevention and treatment of infected wounds.

[0037] 2. The hydrogel preparation method provided by the present invention controls the mass ratio of sodium alginate to gelatin to be between 1:1 and 30. By controlling the mass ratio of sodium alginate to gelatin within this range, not only can the cross-linking of the materials be promoted to obtain a product with better performance, but it also facilitates preparation and improves processing efficiency.

[0038] 2. The hydrogel preparation method provided by the present invention controls the dissolution temperature of sodium alginate during preparation to 25°C to 55°C, allowing sodium alginate to dissolve rapidly in an aqueous solution, forming a stable and uniform sodium alginate aqueous solution. Controlling the dissolution temperature of gelatin solution during preparation to 35°C to 65°C allows gelatin to dissolve rapidly in an aqueous solution, forming a stable and uniform gelatin aqueous solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0040] Figure 1 Figures 1 and 2 show the test results of the preparation process changes, morphology and microstructure, injectability, skin compliance, and self-healing behavior of the hydrogel in Experimental Example 1, wherein A is a diagram showing the changes in the preparation process, B is a diagram showing the changes in the morphology of the hydrogel, C is a diagram showing the hydrogel following the movement of the finger joint, and D is a diagram showing the self-healing behavior of the hydrogel; E is the general morphology of the hydrogel; F is a diagram showing the microstructure of the hydrogel under an electron microscope; and G is the average pore size of the hydrogel.

[0041] Figure 2 These are the EDS analysis results and Fourier transform infrared spectra of the hydrogel in Experimental Example 2; A is the EDS analysis, and B is the Fourier transform infrared spectra; Transmittance is translated as transmittance; Wavenumber is translated as wavelength;

[0042] Figure 3 is the rheological property test result of the hydrogel in Experimental Example 3;

[0043] Figure 4 The cytotoxicity, cell fluorescence staining and viability, and hemolysis results of the hydrogels in Experimental Examples 4 and 5 are shown; A is the cell viability result of human fibroblasts (HSF cells); B is the cell viability result of human keratinocytes (HaCaT cells); C is the fluorescence staining image of human fibroblasts; D is the fluorescence staining image of human keratinocytes; E is the scratch test result of human fibroblasts; F is the scratch test result of human keratinocytes; G is the hemolysis result; where Cellviability is translated as cell viability, Fluorescence intensity is translated as fluorescence intensity, Absorance is translated as absorbance, and Control is translated as control group;

[0044] Figure 5 are the antibacterial test results of the hydrogel in Experimental Example 6, where A is the bacterial absorbance test result before incubation; B is the bacterial absorbance test result after incubation; C is the Escherichia coli inhibition zone diameter test result; D is the Staphylococcus aureus inhibition zone diameter test result; E is an image of the Escherichia coli inhibition zone; F is an image of the Staphylococcus aureus inhibition zone; G is the bacterial morphology under a scanning electron microscope; where "Inhibitory zone diameter" is translated as "inhibitory zone diameter"; "After contact" is translated as "after contact"; and "Before contact" is translated as "before contact";

[0045] Figure 6 The effect of the hydrogel on the back infection of mice in Experimental Example 7, wherein A is the observation of wound healing; B is the change in the healing area of ​​the wound on the back of the mouse; C is the wound closure rate of the wound on the back of the mouse; D is the healing of the infected wound observed by HE staining; E is the result of the number of new blood vessels; F is the result of the number of new hair follicles; G is the result of the collagen volume fraction under the skin;

[0046] Figure 7The results of staining the back of mice infected with the hydrogel in Experimental Example 7 are shown in Figure 7; A is an immunofluorescence staining of α-SMA; B is an immunofluorescence staining of CD31; C is an immunofluorescence staining of CD206; D is an immunofluorescence staining of CD80; E is the immunofluorescence level of CD206; F is the immunofluorescence level of CD80;

[0047] Figure 8 Figure 5 is the result of the changes in the skin microbiome during wound healing, where A and B are the changes in skin microbiome diversity over time; C and D are the results of phylum-level analysis of the skin microbiome; E and F are the results of linear discriminant analysis; G and H are the results of time-bacterial abundance change analysis. DETAILED DESCRIPTION

[0048] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0049] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0050] SA (Product No.: W201502), Gel (CAS No.: 9000-70-8) and calcium chloride (CaCl2, Product No.: C3881) were purchased from Sigma-Aldrich (US).

[0051] ε-Poly-L-lysine (ε-PLL), molecular formula: (C6H 12 N2O)n; molecular weight: 70,000-150,000, obtained from Shanghai Myrel Biochemical Technology Co., Ltd. (#M60714, Shanghai, China).

[0052] Example 1

[0053] This embodiment provides a method for preparing a hydrogel, comprising the following steps:

[0054] (1) 1.50 g of ε-PLL powder was dissolved in 10 mL of deionized water to prepare a 15.0% w / v ε-PLL solution.

[0055] 4 g of SA was dissolved in 200 mL of PBS and heated at 37°C for 1.5 h to prepare a 2% m / v SA solution. The solution was allowed to stand for 4 h to remove air bubbles.

[0056] 40 g of Gel was dissolved in 200 mL of PBS and heated at 50°C for 45 min to prepare a 20% m / v Gel solution. The solution was allowed to stand for 4 hours to remove air bubbles.

[0057] Calcium chloride was dissolved in deionized water to prepare a 2% m / v CaCl2 solution.

[0058] (2) SA solution and Gel solution were mixed at a volume ratio of 1:1 at 37°C and stirred thoroughly using a magnetic stirrer to form a uniform SA / Gel solution (referred to as the first mixed solution).

[0059] (3) The ε-PLL solution was added to the first mixed solution at a volume ratio of 1:50 (v / v) and stirred at room temperature to obtain an ε-PLL@SA / Gel precursor solution (referred to as the second mixed solution).

[0060] (4) The second mixed solution was mixed with an equal volume of 2% m / v CaCl2 solution. After standing at room temperature for 1 hour, a hydrogel was formed, which was recorded as PSG15.

[0061] See Figure 1 As shown, the SA solution and Gel solution exhibited stable fluid states at 37°C and 50°C, respectively. After thorough mixing, a transparent SA / Gel precursor solution was formed at 37°C. After cross-linking with CaCl2 solution, the mixture exhibited a gel-like state and remained stable when inverted, indicating its structural integrity.

[0062] Example 2

[0063] This embodiment provides a method for preparing a hydrogel, which is basically the same as that of Example 1, except that the concentration of the ε-PLL solution prepared in step (1) is adjusted from 15.0% w / v to 10.0% w / v. The remaining steps and process conditions are the same as those of Example 1, and a hydrogel is obtained, which is recorded as PSG10.

[0064] Example 3

[0065] This embodiment provides a method for preparing a hydrogel, which is basically the same as that of Example 1, except that the concentration of the ε-PLL solution prepared in step (1) is adjusted from 15.0% w / v to 5.0% w / v. The remaining steps and process conditions are the same as those of Example 1, and a hydrogel is obtained, which is recorded as PSG5.

[0066] Example 4

[0067] This embodiment provides a method for preparing a hydrogel, which is basically the same as that of Example 1, except that the concentration of the ε-PLL solution prepared in step (1) is adjusted from 15.0% w / v to 20.0% w / v. The remaining steps and process conditions are the same as those of Example 1, and a hydrogel is obtained, which is recorded as PSG20.

[0068] Example 5

[0069] This embodiment provides a method for preparing a hydrogel, which is basically the same as that of Example 1, except that the volume ratio of the SA solution to the Gel solution is adjusted from 1:1 to 10:1. The remaining steps and process conditions are the same as those of Example 1 to prepare the hydrogel.

[0070] Example 6

[0071] This embodiment provides a method for preparing a hydrogel, which is basically the same as that of Example 1, except that the volume ratio of the SA solution to the Gel solution is adjusted from 1:1 to 2:1. The remaining steps and process conditions are the same as those of Example 1 to prepare the hydrogel.

[0072] Example 7

[0073] This embodiment provides a method for preparing a hydrogel, which is basically the same as that of Example 1, except that the volume ratio of the SA solution to the Gel solution is adjusted from 1:1 to 1:3. The remaining steps and process conditions are the same as those of Example 1 to prepare the hydrogel.

[0074] Example 8

[0075] This embodiment provides a method for preparing a hydrogel, which is basically the same as that of Example 1, except that the volume ratio of the SA solution to the Gel solution is adjusted from 1:1 to 1:1.5. The remaining steps and process conditions are the same as those of Example 1 to prepare the hydrogel.

[0076] Example 9

[0077] This embodiment provides a method for preparing a hydrogel, comprising the following steps:

[0078] (1) Dissolve 1.5 g of ε-PLL powder in 10 mL of deionized water to prepare a 15.0% w / v ε-PLL solution.

[0079] 5 g of SA was dissolved in 200 mL of deionized water and heated at 30°C for 1 h to prepare a 2.5% m / v SA solution. The solution was allowed to stand for 4 h to remove air bubbles.

[0080] Dissolve 50 g of Gel in 200 mL of deionized water and heat at 40°C for 30 min to prepare a 25% m / v Gel solution. Let the solution stand for 4 hours to remove air bubbles.

[0081] Calcium nitrate was dissolved in deionized water to prepare a 2.5% m / v calcium nitrate solution.

[0082] (2) SA and Gel solution were mixed at a volume ratio of 1:1 at 40°C and stirred thoroughly using a magnetic stirrer to form a uniform SA / Gel solution (referred to as the first mixed solution).

[0083] (3) The ε-PLL solution was added to the first mixed solution at a volume ratio of 1:50 (v / v) and stirred at room temperature to obtain an ε-PLL@SA / Gel precursor solution (referred to as the second mixed solution).

[0084] (4) The second mixed solution was mixed with an equal volume of 2.5% m / v calcium nitrate solution and allowed to stand at room temperature for 1 hour to form a hydrogel.

[0085] Comparative Example 1

[0086] This comparative example provides a method for preparing a hydrogel, comprising the following steps:

[0087] (1) Dissolve 4 g of SA in 200 mL of PBS and heat at 37°C for 1.5 h to prepare a 2% m / v SA solution. Let the solution stand for 4 h to remove air bubbles.

[0088] 40 g of Gel was dissolved in 200 mL of PBS and heated at 50°C for 45 min to prepare a 20% m / v Gel solution. The solution was allowed to stand for 4 hours to remove air bubbles.

[0089] Calcium chloride was dissolved in deionized water to prepare a 2% m / v CaCl2 solution.

[0090] (2) SA and Gel solution were mixed at a volume ratio of 1:1 at 37°C and stirred thoroughly using a magnetic stirrer to form a uniform SA / Gel solution (referred to as the first mixed solution).

[0091] (3) The first mixed solution was mixed with an equal volume of 2% m / v CaCl2 solution to induce ionic crosslinking. After standing at room temperature for 1 hour, a hydrogel was formed, which was recorded as PSG0.

[0092] Comparative Example 2

[0093] This comparative example provides a method for preparing a hydrogel, comprising the following steps:

[0094] 1.50 g of ε-PLL powder, 4 g of SA and 40 g of Gel were mixed and dissolved in 410 ml of PBS. The mixture was heated at 37°C for 1.5 h, but it did not dissolve and contained a large number of bubbles. The temperature was adjusted to 50°C and the mixture was heated for another 1.5 h, but it still did not dissolve.

[0095] Comparative Example 3

[0096] This comparative example provides a method for preparing a hydrogel, comprising the following steps:

[0097] (1) 4 g SA and 40 g Gel were mixed and dissolved in 410 ml PBS, and heated at 50°C for 1.5 h to obtain a first mixed solution; the solution was allowed to stand for 4 h to remove bubbles.

[0098] (2) adding 1.50 g of ε-PLL powder to the mixed solution to obtain a second mixed solution;

[0099] (3) The second mixed solution was mixed with an equal volume of 2% m / v CaCl2 solution and allowed to stand at room temperature for 1 hour to form a hydrogel.

[0100] Comparative Example 4

[0101] This comparative example provides a method for preparing a hydrogel, comprising the following steps:

[0102] (1) Prepare ε-PLL solution, SA solution, Gel solution and CaCl2 solution according to the method of step (1) of Example 1.

[0103] (2) The ε-PLL solution was added to the SA solution at a volume ratio of 1:25 (v / v) to obtain a first mixed solution. Then, an equal volume of Gel solution to the SA solution was added to the first mixed solution and the mixture was stirred using a magnetic stirrer to form a uniform second mixed solution.

[0104] (3) The second mixed solution was mixed with an equal volume of 2% m / v CaCl2 solution and allowed to stand at room temperature for 1 hour to form a hydrogel.

[0105] Experimental Example 1

[0106] (1) Changes in preparation process and morphology and microstructure

[0107] PSG hydrogel was prepared according to the methods of Examples 1-3 and Comparative Example 1.

[0108] SA solution and GL solution were in a stable fluid state at 37°C and 50°C, respectively. After thorough mixing, a transparent SA / Gel precursor solution was formed at 37°C. After cross-linking with CaCl2 solution, the mixture was in a gel-like state and remained stable when inverted, indicating that its structure was intact (see Figure 1 (as shown in A in the figure).

[0109] All hydrogels appeared as transparent jelly, and ε-PLL was evenly distributed throughout the hydrogel (see Figure 1 E in the figure).

[0110] The surface morphology of the hydrogels was observed using a scanning electron microscope (ApreoC, ThermoFisher). Scanning electron microscope (SEM) images showed that all hydrogels exhibited a three-dimensional interconnected network structure (see Figure 1 The average pore size of PSG0 hydrogel is in the range of 130-150 μm (see Figure 1 The decrease in pore size may be due to the incorporation of more ε-PLL into the hydrogel, forming a denser structure.

[0111] (2) Injectability and skin compliance

[0112] Injectability is crucial for hydrogel dressings because it allows them to fill irregular wounds and promote in situ regeneration. The hydrogel also exhibits skin conformability, maintaining adhesion to the wound even during joint movement. Its self-healing properties enable the hydrogel to recover and extend its lifespan. The injectability and shape adaptability of ε-PLL@SA / Gel were tested by loading it into a syringe and extruding it through a 1 mL syringe.

[0113] Skin compliance: The hydrogel was placed on a finger joint to observe its skin compliance. After applying slight external pressure, the joint was bent to observe the gel's ability to adapt to the joint movement.

[0114] The results showed that the hydrogel could be continuously extruded through the needle to write the letters "ABCD", confirming the injectability of the hydrogel and its adaptability to the shape of the wound (see Figure 1 Furthermore, the hydrogel adhered to the finger joints and withstood bending and stretching forces without falling off or breaking, indicating good skin compliance (see Figure 1 (as shown in C in the figure).

[0115] (3) Self-healing behavior

[0116] Self-healing behavior of the hydrogel: The hydrogel disc of Example 1 was split into two halves: one half was stained with rhodamine B, and the other half remained transparent. The two halves were left in contact for 30 minutes, and the gel was lifted with tweezers to observe the healing process.

[0117] The results showed that when the two halves of the pink and transparent circular hydrogel disks were cut symmetrically and placed together without external force, the PSG hydrogels prepared in Examples 1-3 of the present invention exhibited self-healing behavior. After 30 minutes, the ruptured hydrogels healed again, regained their integrity and were able to bear their own weight (see Figure 1 (as shown in D in the figure).

[0118] Experimental Example 2 Characterization

[0119] 1. Energy Dispersive X-ray Spectroscopy (EDS) Analysis: 2 ml of PSG hydrogel prepared according to Examples 1-3 was prepared in a 20 mm diameter mold and freeze-dried. The freeze-dried hydrogel sample was sliced, and a thin layer of gold was sputtered onto the cross section for scanning electron microscopy. The elemental composition and content of the sample were analyzed using an energy dispersive spectrometer at an accelerating voltage of 20 kV.

[0120] See the results Figure 2 As shown in part A, the main elements of the PSG hydrogel are C, O, and Cl, indicating that some sodium ions in sodium alginate have successfully replaced calcium ions, confirming successful ionic cross-linking.

[0121] 2. Chemical structure analysis: Fourier transform infrared spectroscopy (FTIR, Nicolet 6700, ThermoNicolet Corporation) was used to analyze the chemical structures of the PSG hydrogel samples, SA, Gel, ε-PLL, and CaCl2 of Examples 1-3 and Comparative Example 1.

[0122] See the results Figure 2 As shown in Part B, the infrared spectra of the raw materials and the PSG hydrogels prepared in Examples 1-3 of the present invention show three different characteristic peaks: the OH stretching vibration peak at 3435 cm -1 , the stretching vibration peak of the symmetrical C=O bond is 1636cm -1 , and the stretching vibration peak of the out-of-plane carbon-hydrogen bond at 563 cm -1 With the increase of ε-PLL concentration, the intensity of these peaks, especially the OH peak, changed significantly. This indicates that with the increase of ε-PLL concentration, the carboxyl, amine and Ca groups in the hydrogel 2+ Cross-linking reaction occurs between ions, resulting in changes in peak intensity. In addition, at 2929 cm -1 A C—H stretching vibration peak was detected at , which was attributed to the interaction between ε-PLL and SA. These results confirmed the successful incorporation of ε-PLL into the hydrogel.

[0123] Experimental Example 3 Mechanical Properties

[0124] The hydrogels prepared in the examples and comparative examples were subjected to the following performance tests:

[0125] 1. Test Method

[0126] 1. Rheological properties

[0127] The rheological properties of the hydrogels were measured using a rheometer (MCR92, Anton Paar) at 25°C. Disc-shaped hydrogel samples with a diameter of 20 mm and a thickness of 5 mm were prepared, and their storage modulus (G') and loss modulus (G') were recorded in the frequency range of 1 to 100 Hz.

[0128] 2. Mechanical properties

[0129] The compressive mechanical properties of the hydrogels were tested using a universal testing machine (CMT6103, MTS). Cylindrical hydrogel samples (25 mm diameter x 5 mm height) were prepared for compression testing. A compressive load of 100 N was applied at a compression rate of 0.5 mm / s. Loading was stopped when the compressive strain reached 60%. The stress-strain curve was plotted, and the strain (%) at 10 kPa was recorded.

[0130] 3. Swelling rate and degradation rate

[0131] (1) Swelling rate measurement: The hydrogel was placed in a freeze dryer for 24 h to obtain freeze-dried PSG hydrogel. The freeze-dried PSG hydrogel of the same shape and volume was weighed (W0) and immersed in 10 mL of PBS (pH 7.4) at 37°C. Within a predetermined time, the hydrogel was removed, excess surface moisture was absorbed with filter paper, and the hydrogel was weighed again (Wt) until the weight stabilized, indicating equilibrium expansion. The swelling rate was calculated as (Wt-W0) / W0×100%. Each hydrogel sample was tested three times.

[0132] (2) Degradation rate measurement: Hydrogels of identical shape and volume were weighed (V0) and immersed in 10 mL of PBS (pH 7.4) at 37°C while shaking (100 rad / s). Within a predetermined time, the hydrogels were removed, washed, dried at 37°C for 48 hours, and weighed again (Vt). Degradation rate = (1-Vt / V0) × 100%. Each hydrogel sample was tested three times.

[0133] 2. Test Results

[0134] 1. Rheological properties test results

[0135] Rheological tests showed that at 37°C, the storage modulus (G') of the PSG hydrogels of all embodiments of the present invention was higher than the loss modulus (G"), indicating the formation of a hydrogel network with both solid and fluid properties, which is crucial for handling the dynamic forces at the wound site during the healing process. The hydrogel has both fluid and solid properties and good viscosity, which helps to alleviate the external forces on the wound (see Figure 3 ).

[0136] Table 1 Rheological properties test results at 100ω (rad / s)

[0137] project G' G" Example 1-PSG15 3319.6 532.6 Example 2 - PSG10 3674.7 582.6 Example 3-PSG5 4058.2 684.1 Example 4 - PSG20 2627.6 462.7 Example 5 2765.6 372.4 Example 6 3348.2 417.5 Example 7 2832.8 367.0 Example 8 3763.9 563.7 Example 9 3628.5 547.2

[0138] 2. Mechanical properties test results

[0139] Compression tests showed that the hydrogels prepared in Examples 1-6 of the present invention exhibited significantly improved softness compared to the hydrogels prepared in Comparative Examples 2-4, and the hydrogels became softer with increasing ε-PLL concentration. The hydrogels prepared in each example were able to withstand a maximum stress exceeding 10 kPa, demonstrating their ability to withstand external compressive forces and protect wounds while maintaining their softness.

[0140] Table 2 Mechanical properties test results

[0141] project Strain at 10 kPa (%) Example 1-PSG15 54.8 Example 2 - PSG10 51.2 Example 3-PSG5 47.0 Example 4 - PSG20 69.3 Example 5 64.6 Example 6 56.3 Comparative Example 2 42.8 Comparative Example 3 42.1 Comparative Example 4 36.7

[0142] 3. Swelling rate test results

[0143] The swelling rate is closely related to the hydrogel's ability to continuously absorb exudate. At pH 7.4, all hydrogels reached swelling equilibrium within 36 hours. Compared to Comparative Examples 1-4, the swelling rates of the hydrogels prepared in Examples 1-3 and 6-7 of the present invention were significantly improved. In particular, the swelling rates of the hydrogels in Examples 1-3 were all greater than 400%.

[0144] Table 3 Swelling rate test results after immersion for 36 h

[0145] project Swelling rate (%) Example 1-PSG15 453.25 Example 2 - PSG10 449.38 Example 3-PSG5 422.61 Example 6 416.24 Example 7 379.76 Comparative Example 1-PSG0 268.75 Comparative Example 2 368.54 Comparative Example 3 275.75 Comparative Example 4 364.63

[0146] 4. Degradation rate test results

[0147] The degradation rate is related to the durability and moisture retention of the hydrogel. At pH 7.4, all hydrogels showed significant degradation after 36 hours. Compared with the hydrogel prepared in Comparative Example 1, the degradation rate of the hydrogels prepared in Examples 1-9 was significantly lower.

[0148] Table 4 Degradation rate test results after immersion for 36 h

[0149]

[0150]

[0151] Experimental Example 4 Biocompatibility

[0152] 1. Experimental Purpose

[0153] The cytotoxicity and biocompatibility of hydrogel dressings are crucial for wound healing because they are in direct contact with damaged skin. In this study, the cytotoxicity of PSG hydrogel was evaluated using the CCK-8 assay.

[0154] 2. Experimental methods

[0155] (1) PSG0, PSG5, PSG10, and PSG15 hydrogels (1 mL each) were immersed in 10 mL DMEM containing 10% fetal bovine serum (FBS) and incubated at 37°C for 24 hours. The obtained extracts were sterilized by passing through a 0.22 μm filter. HSF cells and HaCaT cells were seeded into 96-well plates (5000 cells / well) and incubated overnight to allow the cells to adhere. The cells were cultured overnight for 24 hours to allow the cells to grow on the wall. Sample wells, blank wells, and control wells were set up. The old culture medium was discarded with a pipette. The extracts of the four hydrogels were added to the sample wells, 100 μl per well, and 100 μl of fresh complete culture medium was added to the control wells. Blank wells were set up in parallel without cells or hydrogels, and only fresh complete culture medium was added. After incubation for 1, 2, and 3 days, the culture medium was discarded, and 100 μL of fresh culture medium containing 10 wt% CCK-8 reagent was added to each well. After incubation at 37°C for 3 hours, measure absorbance at 450 nm using a microplate reader. Cell viability is calculated using the formula: [(OD sample - OD blank) / (OD control - OD blank)] × 100%. OD sample is the OD value of each sample well, OD blank is the OD value of the blank well, and OD control is the OD value of the control well.

[0156] (2)Live / Dead staining

[0157] PSG0, PSG5, PSG10, and PSG15 hydrogels (1 mL each) were immersed in 10 mL of DMEM containing 10% fetal bovine serum (FBS) and incubated at 37°C for 24 hours. The resulting extracts were sterilized by passing through a 0.22 μm filter. Cell viability was assessed using a live / dead cell staining kit (#C542, DOJINDO, Japan). HSF cells and HaCaT cells were seeded in 12-well plates (70,000 cells / well) and incubated overnight. Sample wells and control wells were set up, and the culture medium was replaced with 2 mL of hydrogel extract or fresh complete culture medium, respectively. After incubation for 1, 2, and 3 days, the cells were washed with PBS and stained with a working solution containing calcein-AM (2 μM) and propidium iodide (PI, 4.5 μM) and incubated at 37°C for 30 minutes. Calcein-AM (green) marks live cells, and propidium iodide (red) marks dead cells. Live cells fluoresce green, and dead cells fluoresce red. Images were captured using an inverted fluorescence microscope (Olympus, Tokyo, Japan).

[0158] (3) Blood compatibility evaluation

[0159] Fresh mouse blood was collected and centrifuged at 2000 rpm for 10 minutes. Red blood cell suspension (2 wt%) was obtained by resuspending with PBS. The samples were randomly divided into positive control group, negative control group and sample group. The sample group incubated each group of hydrogels with 200 μL of red blood cell suspension at 37 ° C for 4 hours, the negative control group incubated 0.9% NaCl with 200 μL of red blood cell suspension at 37 ° C for 4 hours, and the positive control group incubated 1% w / v TritonX100 with 200 μL of red blood cell suspension at 37 ° C for 4 hours. After centrifugation, the absorbance of the supernatant at 540 nm was measured. The hemolysis rate calculation formula is: hemolysis rate (%) = [(OD sample - OD negative control) / (OD positive control - OD negative control)] × 100%. Wherein OD sample is the OD value of each sample group, OD negative control is the OD value of the negative control group, and OD positive control is the OD value of the positive control group. Each sample was measured 3 times, and the average value of the measurement was taken.

[0160] 3. Experimental results

[0161] After incubation with different PSG hydrogel extracts for 3 days, the cell viability remained at about 100%, which is in line with international standards (>80%, ISO10993-5), indicating that the PSG hydrogel prepared by the present invention is non-toxic ( Figure 4 AB). Notably, on day 3, HSF cell viability was significantly increased, indicating that the hydrogel promoted the proliferation of HSFs.

[0162] In addition, live / dead staining (Calcein-AM / PI) was used to evaluate the viability of HaCaT and HSF cells after 1, 2, and 3 days of incubation. After 72 h, a large number of live cells (green fluorescence) and almost no dead cells (red fluorescence) were observed, indicating that the hydrogel had no obvious cytotoxic effect ( Figure 4 CD). The fluorescence intensity of fibroblasts increased significantly on days 2 and 3, further confirming the proliferative effect of the hydrogel on fibroblasts.

[0163] When the hydrogel dressing was in direct contact with an open wound, human red blood cells (RBCs) were used to assess blood compatibility in a hemolysis test. The supernatants of the different PSG hydrogels and the negative control (PBS) remained clear and transparent, with no signs of hemolysis (red). In contrast, the positive control (1% w / v TritonX100) caused hemolysis, turning the supernatant red. Further measurement of absorbance at 540 nm revealed that the absorbance values ​​of the PSG hydrogel and the negative control were both below 0.1, while the absorbance value of the positive control was above 0.6 ( Figure 4 G). This indicates that the hydrogel does not induce hemolysis and has good blood compatibility.

[0164] Experimental Example 5 Cell Healing Ability

[0165] 1. Experimental methods

[0166] PSG0, PSG5, PSG10 and PSG15 hydrogels (1 mL each) were immersed in 10 mL DMEM containing 10% fetal bovine serum (FBS) and incubated at 37°C for 24 hours. The obtained extract was sterilized by passing through a 0.22 μm filter. The effect of hydrogel extract on cell migration was evaluated by the scratch method. HSF and HaCaT were respectively seeded in 12-well plates (100,000 cells / well) and incubated overnight until the confluence reached 80%. A sterile pipette tip was used to make scratches, the initial wound width was recorded, and the cells were washed twice with PBS. The wells were then filled with hydrogel extracts or fresh complete culture medium, incubated for 24 hours, and the final wound width after 12 hours and 24 hours of incubation was recorded. The migration rate was calculated as follows: migration rate (%) = [(initial wound width - final wound width) / initial wound width] × 100%.

[0167] 2. Experimental results

[0168] The migration ability of HaCaT and HSF was evaluated by the scratch method. Compared with the control group and PSG0, the scratch distance was significantly shortened after 3 days of incubation with PSG5, PSG10, and PSG15 hydrogel extracts, indicating that the migration of both cells toward the center of the wound increased over time. This suggests that the hydrogel promotes wound healing by enhancing the migration of HaCaT and HSF. Figure 4 EF). PSG15 hydrogel increased the migration rate of human fibroblasts by >40% and the migration rate of human keratinocytes by >20% compared with the control group.

[0169] Experimental Example 6 Antibacterial properties of hydrogel

[0170] 1. Experimental methods

[0171] (1) Bacterial absorbance detection

[0172] The antibacterial properties of hydrogel dressings are crucial for treating infected wounds. Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) are common pathogens in infected wounds. The antibacterial effect of PSG15 hydrogel against E. coli and S. aureus was evaluated by measuring absorbance at 600 nm using a microplate reader. E. coli and S. aureus (5 × 10 CFU / mL) were inoculated in LB liquid medium. Subsequently, 1 mL of PSG15 hydrogel was dissolved in 5 mL of LB medium, and 100 μL of the bacterial suspension (5 × 10 CFU / mL) was added. The cells were incubated at 37°C on a shaker at 150 rpm for 24 hours. The bacterial absorbance at the start point (before incubation) and the end point (after 24 hours of incubation) was measured using a microplate reader at 600 nm. This experiment was repeated three times.

[0173] (2) Inhibition zone test

[0174] The inhibition zone experiment was used to investigate the antibacterial effects of PSG0, PSG5, PSG10 and PSG15 hydrogels against Escherichia coli and Staphylococcus aureus.

[0175] 200 μL of Escherichia coli and 200 μL of Staphylococcus aureus (1×10 26 CFU / mL) suspension was prepared and the inhibition zone was determined on LB agar plates.

[0176] Four hydrogels with a diameter of 1 cm were placed in the center of a culture dish. After 24 hours of incubation, the diameter of the inhibition zone was measured to determine the antibacterial activity. This experiment was repeated three times.

[0177] (3)Live / Dead staining

[0178] The antibacterial activity of PSG15 hydrogel against Escherichia coli and Staphylococcus aureus was further evaluated by live / dead staining. PSG15 hydrogel (1 mL) was immersed in 1 mL of Escherichia coli and 1 mL of Staphylococcus aureus suspension (both at a concentration of 1×10 8 CFU / mL) and incubated overnight. As a control, 1 ml of PBS was added to the suspension. After incubation, the PSG15 hydrogel was removed and the remaining bacterial suspension was centrifuged at 3000 rpm for 5 minutes.

[0179] Wash the bacterial microspheres three times with PBS. Stain with SYTO-9 (5 μM) and PI (1 μg / mL) at 37°C in the dark for 15 minutes. After staining, centrifuge (5000 rpm, 5 minutes) to remove the supernatant, and wash the bacteria three times with saline. Observe the bacterial suspension under a laser confocal scanning microscope.

[0180] (4) Bacterial electron microscopy

[0181] The morphology of Escherichia coli and Staphylococcus aureus cultured on PSG15 hydrogel was observed by scanning electron microscopy. 8 CFU / mL) and incubated overnight at 37°C. The bacterial suspension was centrifuged at 3000 rpm for 10 minutes, fixed with 4% glutaraldehyde for 5 hours, and then fixed with 2.5% glutaraldehyde for 1 hour at 4°C. Bacteria were dehydrated in a graded ethanol series (20%, 50%, 80%, and 100% w / v), with each step lasting 10 minutes. After dehydration, the bacteria were sputter-coated with a thin layer of gold and observed under a scanning electron microscope (S-4800, 5 kV, Hitachi, Tokyo, Japan).

[0182] 2. Experimental results

[0183] (1) Bacterial absorbance detection

[0184] The antibacterial activity of PSG was evaluated by absorbance measurement. The antibacterial activity of PSG15 hydrogel against Escherichia coli (E.coil) and Staphylococcus aureus (S.aureus) was 89.53% and 92.21%, respectively, showing excellent antibacterial performance (see Figure 5 AB in the figure).

[0185] (2) Inhibition zone test

[0186] The results showed that compared with PSG0 prepared in comparative example 1, PSG5, PSG10 and PSG15 hydrogels could significantly improve the antibacterial effect, and with the increase of polylysine concentration, the diameter of the inhibition zone increased and the antibacterial efficiency was higher (see Figure 5 (shown in CE).

[0187] (3)Live / Dead staining

[0188] SYTO-9 and PI stain live and dead bacteria respectively. Compared with the PBS control group, a large amount of red fluorescence was shown, and the number of dead bacteria was significantly increased (see Figure 5 The PSG hydrogels prepared in Examples 1-3 of the present invention exhibit excellent antibacterial activity, making them a promising option for preventing and treating wound infections.

[0189] (4) Bacterial electron microscopy

[0190] Scanning electron microscopy images showed that untreated Escherichia coli and Staphylococcus aureus maintained normal morphology, with smooth and intact surfaces. After 12 hours of exposure to PSG hydrogel, the bacteria showed severe deformation, wall rupture, and cytoplasm leakage, indicating that the antibacterial activity of the PSG hydrogels prepared in Examples 1-3 of the present invention is achieved by destroying the integrity of the bacterial wall and membrane (see Figure 5 (shown as G in the figure).

[0191] Experimental Example 7 Animal Experiment

[0192] 1. Experimental methods

[0193] (1) Animals and group administration

[0194] C57BL / 6 mice (6–8 weeks, male, 20–22 g) were purchased from Jiangsu GemPharmatech Co., Ltd., China. All animal experiments were performed in accordance with the guidelines for the care and use of laboratory animals.

[0195] After one week of adaptive feeding, mice were anesthetized with an intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg). The anesthetized mice were placed in the prone position, and the hair on their backs was shaved with electric clippers and then completely removed with a depilatory cream. The area was then disinfected with 75% ethanol. A full-thickness skin wound (8 mm in diameter) was created on the back of each mouse using a skin biopsy punch. Mice were randomly assigned to seven groups of eight using a random number table. These groups included a control group (E-Model), a model group, a hydrogel-treated group, and an Urgotul silver / silver dressing group. The model group consisted of groups E (E-Model) and S (S-Model), the hydrogel-treated group included groups E (E-PSG15) and S (S-PSG15), and the Urgotul silver / silver dressing group included groups E (E-Ag) and S (E-Ag). Except for the control group, wounds in groups E and S were infected with Escherichia coli and Staphylococcus aureus suspensions (1 × 10 CFU / mL), respectively, for 48 hours. Starting on day 3, the wounds were treated every three days. Mice in the control and model groups were gently cleansed with sterile saline. After wound cleansing in the experimental groups, PSG15 hydrogel (1 cm diameter, 5 mm height cylinder) and Urgotul Ag / Silver (Urgotul Ag / Silver, purchased from Urgotul Medical, France) commercial dressings were applied to the hydrogel-treated group and Urgotul Ag / Silver dressing group, respectively. The wounds were covered with sterile medical gauze and secured with surgical tape. The mice were housed individually and provided with adequate food and water. A 1 cm diameter circular template was placed around the wound to ensure consistency during photography.

[0196] The calculation formula for wound healing percentage (%) is: wound closure rate (%) = [(S0-Sn) / S0] × 100%, where S0 is the initial wound area on day 0, and Sn is the wound area on the day of dressing change.

[0197] (2) Hematoxylin-eosin (HE) and Masson's trichrome staining: On day 15, all mice were sacrificed, wound tissues were excised, and fixed with 4% (w / v) paraformaldehyde. Tissue specimens were embedded in paraffin and sectioned.

[0198] For HE staining, paraffin sections were immersed in xylene twice for 10 minutes each. Sections were rehydrated through a graded ethanol series (100%, 95%, 80%, 70%) and then rinsed in distilled water for 5 minutes. Sections were stained with hematoxylin solution for 5 minutes and then rinsed in tap water for 10 minutes. Sections were immersed in 1% acid alcohol (1% hydrochloric acid, 70% ethanol) for 3 seconds and then rinsed in tap water for 5 minutes. Sections were stained with eosin solution for 1 minute. Sections were dehydrated through a series of graded ethanol solutions (70%, 80%, 90%, 100%) for 1 minute each. Sections were cleared in xylene twice for 5 minutes each, mounted with a xylene-based mounting medium, and allowed to dry. Masson's trichrome staining was first performed by dewaxing and rehydrating. The tissue was then stained with Weigert's iron hematoxylin (5 minutes) and Biebrich's red acid fuchsin solution (10 minutes). After differentiation with phosphotungstic-phosphomolybdic acid solution for 10 minutes, the sections were stained with aniline blue for 5 minutes. After acid differentiation and dehydration, the tissues were washed twice in xylene for 5 minutes each. Finally, the tissues were mounted on coverslips using a xylene-based mounting medium.

[0199] (3) Immunohistochemical staining: After dewaxing and rehydration, the tissue sections were placed in antigen retrieval buffer (EDTA, pH 9.0) and microwaved for 8 minutes for antigen retrieval. The slides were then cooled naturally and washed three times with PBS (pH 7.4) for 5 minutes each. The primary antibody diluted in PBS was applied and the sections were incubated in a wet chamber at 4°C overnight. After washing three times with PBS (pH 7.4) on a shaker (5 minutes each), the secondary antibody was applied and incubated in the dark at room temperature for 50 minutes. The cell nuclei were stained with DAPI (4′, 6-diamino-2-phenylindole) in the dark for 10 minutes and then washed three times with PBS. The slides were then mounted with anti-fluorescence quenching mounting medium. The sections were observed and imaged under a fluorescence microscope.

[0200] (4) 16S rRNA gene sequencing: CD31 was used to confirm the presence of vascular endothelial cells, and α-SMA (α-smooth muscle actin) was used as a specific marker for vascular rings, which is associated with tissue fibrosis and wound contraction. 16S rRNA gene sequencing was performed according to the manufacturer's instructions. DNA was extracted from the samples using the CTAB method. The V3-V4 region of the 16S ribosomal RNA (rRNA) gene was amplified using barcodes using specific primers 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-ggactacnngggtatcatat-3′). PCR amplification was performed using High-Fidelity PCR Master Mix (New England Biolabs, USA). The PCR program included an initial denaturation at 98°C for 1 minute, followed by 30 cycles of 98°C for 10 seconds, 50°C for 30 seconds, and 72°C for 30 seconds, and a final extension at 72°C for 5 minutes. PCR products were purified using a DNA Gel Extraction Kit (Qiagen, Germany). Sequencing libraries were used. Ultra TM IIDNA library preparation kit (New England Biolabs, USA) was used according to the manufacturer's recommendations. The library was sequenced on the Illumina NovaSeq platform (Illumina, USA). Raw tags were merged and split according to barcode and primer sequences to obtain clean tags. Data quality control was performed using FASTP software to obtain high-quality clean tags. Chimeric sequences were removed by alignment with the Silva database to obtain valid tags.

[0201] (5) Statistical analysis: All data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 9 software. Visualization was performed using RStudio (version 4.1.1). One-way analysis of variance (ANOVA) and Tukey's post hoc test were used to determine statistically significant differences between groups (P < 0.05 or P < 0.01).

[0202] 2. Experimental results

[0203] After inflicting trauma to the back of mice, the experimental groups were infected with Staphylococcus aureus and Escherichia coli suspensions for 48 hours. On the third day, purulent exudate appeared in the infected wound, confirming the successful establishment of a full-thickness infection wound model.

[0204] Macroscopic observation of wound healing showed that compared with the model group, PSG15 hydrogel significantly promoted the healing of wounds infected with Escherichia coli and Staphylococcus aureus (p less than 0.005). The healing efficiency of the PSG group was similar to that of the commercial group (see Figure 6 (shown in Figures AC).

[0205] Histopathological analysis further demonstrated that the healing effect of PSG15 hydrogel was superior to that of the other groups. H&E staining on day 15 showed that new blood vessels and follicles were formed in the PSG group and the Urgotul Ag / Silver group. Masson trichrome staining showed that the PSG group had more collagen deposition than the model group, indicating enhanced tissue repair (see Figure 6 (DG in the middle). PSG15 hydrogel can maintain a moist wound environment, which is conducive to tissue regeneration.

[0206] The CD31 and α-SMA staining were significantly increased in the hydrogel-treated group and the Urgotul silver / silver dressing group compared with the other groups (see Figure 7 This finding suggests that PSG15 hydrogel treatment promoted subcutaneous angiogenesis in infected wounds, which is crucial for tissue repair and regeneration. CD80 and CD206 serve as markers for macrophage M1 and M2 subtypes, respectively. M1 macrophages are known to trigger immune responses around wounds, leading to inflammation and tissue damage. In contrast, M2 macrophages help reduce local inflammation, improve tissue microenvironment, and promote wound repair. Compared with the model group, CD206 expression was increased and CD80 expression was decreased in the hydrogel group and Urgotul silver / silver dressing group (see Figure 7 This suggests that PSG15 hydrogel promotes the transformation of M1 macrophages to M2 macrophages in the late stage of wound healing, contributing to its anti-inflammatory and pro-repair effects.

[0207] Experimental Example 8 Changes in skin microbiota during wound healing

[0208] To investigate whether PSG15 hydrogel treatment could regulate changes in the skin microbiota during wound healing, skin samples were collected from all groups at three different time points: 3, 7, and 14 days after infection in Experiment 7. Skin microbiome analysis, including diversity analysis (Shannon index) and differential microbial analysis (Lefse analysis, LDA>2), was performed using MicrobiomeAnalyst 2.0.

[0209] On day 3 after infection, the skin microbiome diversity of the E. coli and S. aureus model groups decreased significantly, while the other groups showed no significant changes compared with the control group. By day 7, the microbiome diversity of the E. coli and S. aureus model groups gradually recovered, and by day 14, there was no significant difference in diversity between the groups (see Figure 8 AB in the figure).

[0210] At the phylum level, the skin microbiome was mainly composed of Firmicutes and Bacteroidetes. On days 3 and 7, the Proteobacteria group in both the E. coli and S. aureus model groups increased significantly, with the increase in the E. coli group being more significant (see Figure 8(Figures 8E-F). In contrast, the commercial dressing group and the PSG-treated group significantly suppressed this increase. Using linear discriminant analysis (LDA), with an effect size of |LDA|>2 and p<0.05, we found that Escherichia species in the Escherichia coli family increased significantly on day 3 in the E. coli model group, closely associated with E. coli infection. Similarly, Staphylococci increased significantly in the S. aureus model group, suggesting S. aureus infection. These results confirmed the successful establishment of the infection model (see Figures 8E-F). Skin microbial diversity is important for preventing chronic infection and promoting wound healing. We further used a heat map to analyze the relative abundance of the top 10 bacterial genera that showed significant differences on day 3. The heat map showed that bacterial abundance decreased during the healing process of wounds infected with E. coli and S. aureus. Based on these results, we found that infection with E. coli and S. aureus significantly reduced the diversity of the skin microbiome in the wound area, leading to an increase in the relative abundance of harmful bacteria (see Figures 8G-H). Application of PSG15 hydrogel effectively inhibited the proliferation of Escherichia coli and Staphylococcus aureus and maintained the stability of the wound skin microbiome, significantly alleviating this effect. This suggests that PSG15 hydrogel has a beneficial effect not only in inhibiting pathogenic bacteria but also in maintaining the balance of the wound microbiome, contributing to an overall positive impact on wound healing. These findings highlight the dual role of PSG15 hydrogel in infection control and microbiome stabilization, underscoring its potential to promote wound healing, particularly in infected wounds.

[0211] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for preparing a hydrogel, characterized in that: The steps include: Step S1: mixing the sodium alginate solution and the gelatin solution to prepare a first mixed solution; Step S2, mixing the first mixed solution with the polylysine solution to prepare a second mixed solution; In step S3, the second mixed solution is mixed with the calcium salt solution, and cross-linked to obtain a hydrogel.

2. The method for preparing the hydrogel according to claim 1, wherein: In the first mixed solution, the mass ratio of sodium alginate to gelatin is 1:1-30.

3. The method for preparing the hydrogel according to claim 1 or 2, characterized in that: The concentration of the sodium alginate solution is 1.5% to 2.5% m / v; and / or the concentration of the gelatin solution is 15% to 25% m / v; and / or the concentration of the polylysine solution is 5% to 25% w / v.

4. The method for preparing the hydrogel according to any one of claims 1 to 3, characterized in that: The preparation method of the sodium alginate solution comprises dissolving sodium alginate in an aqueous solution; Optionally, the dissolution temperature is 25°C to 55°C.

5. The method for preparing the hydrogel according to any one of claims 1 to 3, characterized in that: The preparation method of the gelatin solution comprises dissolving gelatin in an aqueous solution; Optionally, the dissolution temperature is 35°C to 65°C.

6. The method for preparing the hydrogel according to any one of claims 1 to 5, characterized in that: The polylysine is ε-polylysine.

7. The method for preparing the hydrogel according to any one of claims 1 to 6, characterized in that: The calcium salt includes one or more of calcium chloride, calcium nitrate, and calcium sulfate; and / or the concentration of the calcium salt solution is 1.5% to 2.5% w / v; and / or the volume ratio of the second mixed solution to the calcium salt is 1:0.8 to 1.

2.

8. The hydrogel prepared according to the preparation method according to any one of claims 1 to 7.

9. The hydrogel prepared by the method according to any one of claims 1 to 7 has one or more of the following AH: A. Use in preparing a product for promoting wound healing; preferably, the wound comprises an infected wound; B. Use in the preparation of products that promote angiogenesis, increase the number of new hair follicles and / or increase the volume of subcutaneous collagen; C. Use in the preparation of antibacterial and / or bacteriostatic products; D. Use in the preparation of anti-inflammatory products; E. Use in the preparation of products that promote cell proliferation and cell migration; F. Use in the preparation of products that promote tissue repair; G. Use in the preparation of products that stabilize skin flora; H. Use in preparing a product that promotes polarization of skin macrophages M1 to M2.

10. A product, characterized in that A hydrogel prepared by the preparation method according to any one of claims 1 to 7; optionally, the product includes a hydrogel dressing and / or an injection.

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