Hydrogel dressing for promoting healing of diabetic infected wounds and method of making same

Abstract

The application aims to provide a hydrogel dressing for promoting healing of diabetic infected wounds and a preparation method thereof, wherein polydopamine is copolymerized with a cationic polymer such as polyethyleneimine, the PEI-PDA copolymer is introduced into a methacrylated gelatin system, and a multifunctional hydrogel with good mechanical properties, tissue adhesion and self-healing ability is prepared by using dynamic crosslinking generated by PEI-PDA and GelMA. The hydrogel can promote healing of diabetic infected wounds by adsorbing inflammatory mediators, removing bacteria and excessive ROS in the wound.

Description

Technical Field

[0001] This invention belongs to the field of diabetic wound repair, and relates to a hydrogel dressing for promoting the healing of diabetic wounds and its preparation method. Background Technology

[0002] Diabetic foot ulcer (DFU), one of the most common complications of diabetes, is a leading cause of non-traumatic lower limb amputations worldwide. Studies show that early prevention and treatment of skin ulcers in diabetic patients can prevent up to 85% of amputations. The pathogenesis of DFU is complex, involving the interaction of multiple factors that cause it to progress through the normal healing stages. Chronic inflammation in the wound microenvironment caused by local circulatory disturbances, oxidative stress, and increased expression of pro-inflammatory cytokines is considered a major cause of poor wound healing in diabetic patients. Furthermore, infection can exacerbate the inflammatory response and local tissue damage, even spreading to deeper tissues, increasing the risk of amputation and potentially leading to sepsis. However, for the patient population, conventional treatments for diabetic foot ulcers have drawbacks such as limited therapeutic efficacy, long treatment cycles, and high costs; antibiotic resistance caused by long-term or frequent use makes treating infections more difficult; and poor patient compliance poses a challenge to the treatment of diabetic foot ulcers. Therefore, seeking an economical, simple, and comfortable treatment method for early intervention in diabetic foot ulcers is an effective treatment strategy.

[0003] In recent years, the intersection of materials science, nanotechnology, and bioengineering with medicine has led to the rapid development of regenerative medicine. Under normal circumstances, wound healing proceeds through three main overlapping and continuous phases: the hemostasis and inflammatory response phase, the proliferation phase, and the tissue remodeling phase. In the complex and dynamic process of wound healing, any dysregulation of the wound microenvironment or alteration of the components involved in healing can lead to healing failure. Therefore, future wound treatment products must possess multiple functions to meet the needs of each stage of wound healing.

[0004] Hydrogel dressings are hydrophilic three-dimensional network polymer materials obtained by polymer cross-linking. Due to their high water content, they can provide a suitable moist healing environment for wound healing, reduce wound temperature, and alleviate discomfort. The porous network structure facilitates the exchange of moisture and gas, absorbing wound exudate. Furthermore, hydrogel dressings possess certain mechanical strength and tissue adhesion, enabling them to adhere to the wound surface, isolate the external environment, inhibit bacterial growth, and provide a suitable delivery system for loading cells and other biomolecules that promote wound healing. Dopamine (DA) can undergo oxidative self-polymerization in alkaline aqueous solutions, forming stable functional coatings on various material surfaces. Polydopamine (PDA) has excellent adhesive properties; its phenolic hydroxyl and quinone groups can capture oxygen free radicals, thus exerting an antioxidant effect. The antibacterial effect of traditional hydrogel dressings mainly relies on antibiotics or other antibacterial substances contained in the hydrogel matrix. Adding other antibacterial substances not only increases the complexity and cost of hydrogel synthesis but may also lead to uneven release of antibacterial substances, resulting in inconsistent antibacterial effects. In comparison, hydrogels carrying inherent antibacterial properties are more advantageous. Polyethyleneimine (PEI) is a polymer synthesized from ethyleneimine monomers. Its molecular structure contains a large number of positively charged amino groups (NH2), giving it significant cationic properties. PEI interacts with negatively charged components in microbial cell membranes (such as lipopolysaccharides and proteins), disrupting the cell membrane and leading to cell death. Currently, the use of PDA-PEI to construct coatings for carrier modification to achieve antibacterial properties in medical devices has been reported. However, PDA-PEI coatings suffer from drawbacks such as slow deposition rates, insufficient coating stability, poor bioavailability, and rapid metabolism, limiting their potential for medical applications. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a hydrogel dressing for promoting the healing of diabetic infected wounds. The method involves copolymerizing polydopamine with a cationic polymer such as polyethyleneimine, introducing a PEI-PDA copolymer into a methacrylic anhydride-modified gelatin (GelMA) system, and utilizing the dynamic crosslinking generated between PEI-PDA and GelMA to prepare a multifunctional hydrogel with good mechanical properties, tissue adhesion, and self-healing ability. This hydrogel can promote the healing of diabetic infected wounds by adsorbing inflammatory mediators and clearing bacteria and excess ROS from the wound.

[0006] The above objectives are achieved through the following technical solutions:

[0007] A method for preparing a hydrogel dressing for promoting healing of diabetic infected wounds includes the following steps:

[0008] Add GelMA to the buffer solution, heat and stir until completely dissolved, then cool to room temperature;

[0009] First, add the PEI-PDA copolymer coating to the dissolved GelMA, then add the UV curing agent, mix thoroughly, and then cure under UV light to obtain GelMA@PEI-PDA.

[0010] As a preferred technical solution of the present invention, the mass-volume ratio (mg / mL) of GelMA and buffer solution is 1:9-3:9.

[0011] As a more preferred technical solution of the present invention, the mass-to-volume ratio (mg / mL) of the GelMA and PEI-PDA copolymer coating is 2:1-5:1.

[0012] As a more preferred technical solution of the present invention, the volume ratio of the dissolved GelMA and PEI-PDA copolymer coating is 1:100-5:100.

[0013] As a preferred technical solution of the present invention, 2g GelMA is added to 9 mL tris buffer solution, heated and stirred at 60°C until completely dissolved, and when cooled to room temperature, 1 mL PEI-PDA copolymer coating and 10 μL of light curing agent are added and shaken at high speed until fully mixed, and cured under ultraviolet light for 1 hour.

[0014] As a preferred technical solution of the present invention, the PEI-PDA preparation method is as follows:

[0015] Dopamine hydrochloride and polyethyleneimine were added to a Tris buffer solution with a pH of 8.5 and reacted at 25°C with slight vibration for 6 h to obtain a PEI-PDA copolymer coating.

[0016] As a preferred embodiment of the present invention, the 250 mg dopamine hydrochloride and 243 μL polyethyleneimine are added to 557 μL Tris buffer solution.

[0017] As a preferred technical solution of the present invention, the method for preparing GelMA is as follows:

[0018] Gelatin was added to PBS buffer solution, heated and stirred until dissolved, and then cooled to 50°C to obtain a gelatin solution. Methacrylic anhydride solution was slowly added dropwise to the gelatin solution, and the reaction was continued at 50°C for 4 hours. After cooling to room temperature, it was placed in a dialysis bag with a molecular weight of 1000 and dialyzed with deionized water at 4°C for 4 days with stirring. After dialysis, GelMA was frozen at 80°C until completely frozen and then placed in a freeze dryer for lyophilization to obtain GelMA. The final product was stored at 4°C until use.

[0019] As a preferred technical solution of the present invention, the photocuring agent is 1173 photoinitiator (2-hydroxy-2-methyl-1-phenyl-1-propanone).

[0020] The beneficial effects are:

[0021] This invention innovatively copolymerizes polydopamine (PDA) with cationic polymers such as polyethyleneimine (PEI) and introduces PEI-PDA into a methacrylic anhydride-modified gelatin (GelMA) system to prepare a multifunctional hydrogel with excellent mechanical properties and various biological properties. This hydrogel can promote wound healing by adsorbing inflammatory mediators and clearing bacteria and excess ROS from wounds. The structure and microstructure of this system were characterized using NMR, UV, and electron microscopy; its mechanical properties were evaluated in detail using tensile and adhesion tests; the biocompatibility of the hydrogel dressing was tested using hemolysis, CCK-8 assays, and live / dead cell staining; the antibacterial and antioxidant properties of the hydrogel were assessed using antibacterial and ROS scavenging tests; finally, a full-thickness skin defect model of MRSA infection in diabetic mice was constructed, and the therapeutic effect of the hydrogel was evaluated through a series of experiments including histopathological staining and immunofluorescence staining. Attached Figure Description

[0022] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0023] Figure 1 (A) A schematic diagram of the synthesis of GelMA@PEI-PDA hydrogel; (B) Hydrogel promotes the healing of diabetic infected wounds;

[0024] Figure 2 (A) 1H NMR images of GelMA and gelatin; (B) PEI-PDA UV spectra changes at different deposition times;

[0025] Figure 3 The internal morphology of the hydrogel under SEM;

[0026] Figure 4 (A) Gel-sol changes of GelMA@PEI-PDA hydrogel; (B) Effect of DA and PEI content on the adhesive strength of GelMA@PEI-PDA hydrogel; (C) GelMA@PEI-PDA hydrogel adhered to a finger.

[0027] Figure 5 The tensile stress-strain curve of the hydrogel;

[0028] Figure 6 The self-healing ability of hydrogels;

[0029] Figure 7Biocompatibility of hydrogels: (A) Live and dead cell staining of HaCaT cells after different hydrogel treatments; (B) Viability of HUVEC cells after different hydrogel treatments; (C) Viability of L929 cells after different hydrogel treatments; (D) Viability of HaCaT cells after different hydrogel treatments; (E) Hemolysis rate of different hydrogel dressings; (F) Schematic diagram of hemolysis pattern.

[0030] Figure 8 To assess the antioxidant capacity of hydrogels: (A) Effects of different hydrogels on HaCaT migration under simulated oxidative stress conditions (H2O2); (B) DPPH scavenging rate of different hydrogels; (C) Quantitative graph of scratch assay results; (D) DPPH scavenging effect of different hydrogels; (E) Relative ROS content in HaCaT cells after treatment with different hydrogels; (F) Flow cytometry images of cells stained with DCFH-DA after H2O2 stimulation and treatment with different hydrogels. *P<0.05, **P<0.01;

[0031] Figure 9 The antibacterial properties of the hydrogel are as follows: (A) Antibacterial effects of different concentrations of GelMA@PEI-PDA on E. coli, S. aureus, and MRSA; (B) Antibacterial rate of GelMA@PEI-PDA on E. coli; (C) Antibacterial rate of GelMA@PEI-PDA on S. aureus; (D) Antibacterial rate of GelMA@PEI-PDA on MRSA; (E) SEM image of MASR after treatment with GelMA@PEI-PDA hydrogel.

[0032] Figure 10 (A) Flowchart of animal experiments; (B) Representative images of wounds in each group during the healing process and a schematic diagram of the wound healing process;

[0033] Figure 11 Representative H&E staining images of wound samples from different groups on days 7 and 14;

[0034] Figure 12 To promote inflammation reduction and tissue regeneration with hydrogels: (A) Representative images of CD206 (M2a) and CD163 (M2c) immunostaining in wound samples on days 7 and 14; (B) Statistical data of CD206-positive cells in the wound; (C) Statistical data of CD163-positive cells in the wound. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001;

[0035] Figure 13To promote wound angiogenesis with hydrogels: (A) Representative images of CD31 and α-SMA immunostaining; (B) Statistical data of CD31-positive cells in the wound; (C) Statistical data of α-SMA-positive cells in the wound; *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001;

[0036] Figure 14 Representative Masson staining images of wounds from different groups on days 7 and 14. Detailed Implementation

[0037] The present invention will be further described in detail below through specific embodiments. However, those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of protection of the present invention. In addition, where specific technical operation steps or conditions are not specified in the embodiments, they are all carried out in accordance with the general techniques or conditions described in the literature in the field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially. Mouse skin fibroblast cell line L929 was purchased from Wuhan Pronosai Life Science Technology Co., Ltd.; human umbilical vein endothelial cells HUVEC and human immortalized keratinocytes HacaT were purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0038] Reagent Name Manufacturer DMEM high glucose medium Gibco, USA FBS Clark, USA Penicillin-Streptomycin Bilateral Antibody Thermo, USA 0.25% trypsin + EDTA Hyclone, USA CCK8 reagent kit MCE, USA Live / Dead Cell Imaging Kit Beyotime Biotechnology, China Reactive oxygen species detection kit Beyotime Biotechnology, China DPPH MCE, USA Yeast extract Aladdin Biochemistry, China trypsin Aladdin Biochemistry, China Agar powder Aladdin Biochemistry, China Tris-HCl, pH 8.5 Beyotime Biotechnology, China Dopamine hydrochloride (98% purity) McLean Biochemicals, China Polyethyleneimine Sigma, USA streptozotocin MCE, USA Sodium citrate Aladdin Biochemistry, China Citric acid Aladdin Biochemistry, China

[0039] Example 1

[0040] (1) Preparation of GelMA:

[0041] 20 g of gelatin was weighed and added to 300 mL of PBS buffer solution, and heated and stirred until dissolved in 1×PBS. After the gelatin dissolved, it was cooled to 50°C, and 4.83 mL of methacrylic anhydride solution was slowly added dropwise to the above solution using a graduated cylinder. The reaction was continued at 50°C for 4 h. After the solution cooled to room temperature, it was placed in a dialysis bag with a molecular weight of 1000 and dialyzed with deionized water at 4°C for 4 days with stirring. After dialysis, GelMA was frozen at 80°C until completely frozen and then placed in a freeze dryer for lyophilization to obtain GelMA. The final product was stored at 4°C until use.

[0042] (2) Preparation of PEI-PDA copolymer coating and PDA:

[0043] Add 250 mg of dopamine hydrochloride and 243 μL of polyethyleneimine to 557 μL of Tris buffer solution (pH = 8.5), place in a glass weighing bottle, and react for 6 h at 25°C with gentle vibration to obtain the PEI-PDA copolymer coating.

[0044] (3) Preparation of hydrogels:

[0045] 2g of GelMA was added to 9 mL of tris buffer solution, heated and stirred at 60°C until completely dissolved, and cooled to room temperature. 1 mL of PEI-PDA copolymer coating and 10 μL of 1173 photoinducer were added and shaken at high speed until fully mixed. GelMA@PEI-PDA was obtained by curing under UV light for 1 h.

[0046] Example 2

[0047] The difference between this embodiment and Embodiment 1 is that:

[0048] 2g of GelMA was added to 9 mL of tris buffer solution, heated and stirred at 60°C until completely dissolved, cooled to room temperature, 1 mL of PDA and 10 μL of 1173 photoinducer were added and shaken at high speed until fully mixed, and cured under ultraviolet light for 1 h to obtain GelMA@PDA.

[0049] 250 mg of dopamine hydrochloride was added to 1 mL of buffer solution (pH = 8.5) and reacted at 25°C with gentle vibration for 6 h to obtain PDA.

[0050] Example 3

[0051] The difference between this embodiment and Embodiment 1 is that:

[0052] Add 2 g of GelMA to 10 mL of tris buffer solution, heat and stir at 60 °C until completely dissolved, cool to room temperature, add 10 μL of 1173 photoinitiator and 243 μL of PEI, mix thoroughly, and cure under UV light for 1 h to obtain GelMA@PEI.

[0053] Structural characterization of GelMA: ¹H NMR was used to analyze methacrylic anhydride (MA), gelatin (Gel), and methacrylic anhydride-modified gelatin (GelMA) to confirm the degree of substitution (DS) of MA in gel.

[0054] Example 4

[0055] The difference between this embodiment and Example 1 is that the mass-volume ratio (mg / mL) of GelMA and buffer solution is 1:9, resulting in GelMA@PEI-PDA-1:9.

[0056] Example 5

[0057] The difference between this embodiment and Example 1 is that the mass-volume ratio (mg / mL) of GelMA and buffer solution is 3:9, resulting in GelMA@PEI-PDA-3:9.

[0058] Example 6

[0059] The difference between this embodiment and Example 1 is that the mass-to-volume ratio (mg / mL) of the GelMA and PEI-PDA copolymer coating is 1:1, resulting in GelMA@PEI-PDA-1:1.

[0060] Example 7

[0061] The difference between this embodiment and Example 1 is that the mass-to-volume ratio (mg / mL) of the GelMA and PEI-PDA copolymer coating is 5:1, resulting in GelMA@PEI-PDA-5:1.

[0062] The results obtained in Examples 1 to 7 were verified.

[0063] The structure of PEI-PDA was characterized by tracing the polymerization process using UV-Vis spectroscopy on a UV-1800 spectrometer, with a light source wavelength of 340 nm and a scanning range of 200 nm–600 nm. The microstructure of the hydrogel was also determined by tracing the polymerization process using UV-Vis spectroscopy on a UV-1800 spectrometer, with a light source wavelength of 340 nm and a scanning range of 200 nm–600 nm.

[0064] This invention innovatively copolymerizes polydopamine (PDA) with cationic polymers such as polyethyleneimine (PEI) and introduces PEI-PDA into a methacrylic anhydride-modified gelatin (GelMA) system to prepare a multifunctional hydrogel with both excellent mechanical properties and various biological properties. This hydrogel can promote wound healing by adsorbing inflammatory mediators, clearing bacteria and excess ROS from wounds, and so on. Figure 1 As shown.

[0065] Methacrylate anhydride gelatin (GelMA) was prepared by grafting methacrylate (MA) groups onto gelatin via esterification. The 1H NMR spectra of GelMA and gelatin samples are shown below. Figure 2 As shown in (A), the signal peaks at 5.61 ppm and 5.3 ppm in the 1H NMR spectrum of GelMA belong to the C=C bond vibrations after the acidification of the methacrylic acid functional group. This proves that MA successfully modifies gelatin. By integrating the peak areas, the degree of substitution of MA in gelatin is calculated to be 20% according to the formula.

[0066] Under aerobic and weakly alkaline conditions, dopamine (DA) can undergo oxidative self-polymerization to form polydopamine (PDA). However, the self-polymerization of pure DA is time-consuming and unstable. Adding polymers containing primary amines, such as polyacrylamide (PAM) and polyethyleneimine (PEI), can promote DA polymerization and further improve surface hydrophilicity. The Schiff base reaction and / or Michael addition between PEI and DA, as well as the oxidative self-polymerization of DA, can be detected by UV-Vis spectroscopy, such as... Figure 2 In (B), the shoulder peaks at 280 nm (catechol group) and 300 nm (dehydrodopamine) gradually increase in intensity over time, indicating the presence of various intermediates in the self-polymerization of DA and its copolymerization with PEI. The PEI-PDA solution exhibits strong absorption peaks at approximately 400 nm and 365 nm (C=C--C=N and C=CC=O), indicating a cross-linking reaction between DA and PEI molecules.

[0067] The properties of the hydrogels obtained in Examples 1 to 3 are compared as follows:

[0068] Tensile property testing: Dumbbell-shaped gel strips of each component were placed in the fixture of a universal testing machine. Tensile testing was performed at a speed of 10 mm / min, with at least three tests per group. Hydrogel dressings are hydrophilic three-dimensional network polymer materials obtained by polymer cross-linking. Their porous network structure facilitates the exchange of moisture and gas, and absorbs wound exudate. For example... Figure 3 The microstructure of the GelMA hydrogel shown is an intricate fibrous network. With the incorporation of PEI-PDA, the pore size of the hydrogel gradually decreases. Figure 4 (A) shows that the GelMA@PEI-PDA hydrogel exhibits a sol-gel change under UV curing. The adhesive strength of the GelMA@PEI-PDA hydrogel is determined by the PEI and PDA content in the hydrogel. Figure 4 As shown in (B), the hydrogel exhibits the strongest adhesion when the PEI / DA ratio is 5%. Furthermore, the GelMA@PEI-PDA hydrogel can adhere to the surface of human skin. Figure 4(C) A 2 g sample of GelMA@PEI-PDA hydrogel adhered to a finger and left no significant residue upon peeling. The GelMA@PEI-PDA hydrogel exhibited excellent mechanical properties, attributed to the interaction between the GelMA, PDA, and PEI-PDA networks. Tensile tests were conducted on four hydrogel samples (GelMA, GelMA@PDA, GelMA@PEI, and GelMA@PEI-PDA) to systematically evaluate their mechanical properties. Test results showed that with the incorporation of PEI-PDA, the elastic modulus of the hydrogel increased accordingly, while the elongation at break decreased significantly. GelMA@PEI-PDA exhibited an elongation at break of 220%, meeting the requirements for high toughness and low modulus in gel materials. Figure 5 As shown.

[0069] The hydrogel contains various non-covalent interactions, including π-π stacking between PDA-PDA groups and hydrogen bonding between hydroxyl, amino, and catechol groups. These interactions endow the GelMA@PEI-PDA hydrogel with excellent self-healing properties, enabling it to self-heal after damage. Figure 6 As shown, the mechanical properties of the hydrogel were evaluated. The hydrogel has good extensibility and can adapt to the movement of the joint. The hydrogel was cut to separate it, and then the broken ends were brought into contact with each other. The hydrogel self-healed and still has good extensibility to adapt to the movement of the finger joint.

[0070] Hemolysis assay using hydrogel: Red blood cells were separated from mouse whole blood by centrifugation (1000 rpm). The obtained red blood cells were washed three times with PBS buffer and then diluted with PBS to a final concentration of 5% (v / v). The resulting red blood cell dilution was incubated with hydrogel (10 mg / mL) at 37 °C and oscillated at 1000 rpm for 1 h. 0.1% Triton X-100 was used as a positive control, and PBS buffer was used as a negative control. The contents of the centrifuge tubes were centrifuged for 5 min (1000 rpm), and 100 µL of the supernatant was transferred to a 96-well plate. The absorbance of the solution was read at 540 nm using a microplate reader (FLUO star Omega). The percentage of hemolysis was calculated using the following formula: Hemolysis (%) = [(Ap–Ab) / (At–Ab)] × 100%, where Ap is the absorbance value of the copolymer solution at a given concentration. At is the absorbance value of the Tritonx-100 positive control, and Ab is the absorbance value of PBS.

[0071] Discard the culture medium in the 96-well plate, add hydrogel soaking solution, and incubate for 24 h, 48 h, and 72 h. Then, add 10 μL of CCK-8 solution to each well, and record the change in absorbance at 450 nm after 2 h. Cell viability = [(As-Ab) / (Ac-Ab)] × 100%, where As is the absorbance of the experimental wells, Ac is the absorbance of the control group, and Ab is the absorbance of the blank wells.

[0072] DPPH scavenging assay: The hydrogel sample (10 mg / mL) was dispersed in 100 µM DPPH, stirred, and incubated in the dark for half an hour. The wavelength at 517 nm was calculated. DPPH degradation was calculated using the following formula: DPPH scavenging% = (Ab - Ah) / Ab × 100%, where Ab and Ah are the absorbance of the blank (DPPH) and the absorbance of the hydrogel (DPPH + hydrogel), respectively.

[0073] ROS clearance assay: L929 cells were seeded at a density of 1 × 10⁶ cells / well in 6-well plates and incubated for 24 h. Then, 100 μM H₂O₂ was added for 1 h of stimulation. The old culture medium was removed, and soaking solutions of different hydrogels (10 mg / mL) were added. Fresh culture medium was used as a positive control. After 2 hours, cells were collected and resuspended in serum-free DCFH-DA (10 μM / L). After incubation at 37°C for 20 min, cells were washed three times with PBS to thoroughly remove any uninfiltrated DCFH-DA. Intracellular reactive oxygen species levels were detected using a BD™ C6 flow cytometer.

[0074] Establishment of a diabetic mouse model with infected wounds: A diabetic mouse model was induced by intraperitoneal injection of STZ at a dose of 50 mg / kg into 6-week-old male C57BL / 6 mice. Successful modeling was defined as a non-fasting blood glucose level >16.7 mM two weeks later, allowing for subsequent experiments. Before surgery, mice were anesthetized with 1.5% isoflurane. Two full-thickness skin defects (1.0 cm in diameter) were created on both sides of the mouse's back using a skin biopsy punch. 20 μL of MARA (1×10⁸ CFU / mL) was instilled into each wound, which was then covered with a 3M transparent dressing and secured with sterile gauze. Starting on the first day post-surgery, the medication was applied every other day, and the wound condition was observed. Wound condition was recorded using a digital camera on days 0, 3, 7, 10, 14, and 18 after the first application of medication, and the wound area was measured using ImageJ. On days 7 and 14 post-treatment, mice were sacrificed, and the tissue surrounding the wound was removed, cleaned with physiological saline, and then immersed in 4% paraformaldehyde fixative for subsequent experiments.

[0075] Eosin (H&E) staining: After washing the fixed skin, the tissue was dehydrated in a gradient of ethanol solutions and then embedded in paraffin. The paraffin blocks were cut into 10 μm sections using a microtome. The paraffin sections were mounted on glass slides, dried, and then dewaxed and rehydrated. The obtained sections were stained with hematoxylin for 10-30 min, rinsed with running water to remove excess dye, and then destained in 1% hydrochloric acid ethanol solution before staining with 0.5% eosin ethanol solution for 2-5 min. After staining, the sections were mounted with resin and photographed under an inverted microscope.

[0076] Masson staining: After washing the fixed skin, it was dehydrated in a gradient of ethanol solutions and then embedded in paraffin. The paraffin block was cut into 10 μm sections using a microtome. The paraffin sections were mounted on glass slides, dried, and then dewaxed and rehydrated. The obtained sections were stained with R1 nuclear stain for 1 min, rinsed with running water to remove excess dye, stained with R2 sulphur stain for 45 s, rinsed with running water to remove excess dye, separated with R3 separating solution for 6 min, discarded, stained with R4 blue counterstain for 5 min, rapidly dehydrated in ethanol solution, and mounted with neutral resin. The sections were then photographed under an inverted microscope.

[0077] Statistical analysis of data: GraphPad Prism software was used for quantitative analysis and graphing. One-way ANOVA was used for all data. Data are presented in the form of mean ± standard deviation (SD). Student t test was used for pairwise comparisons between groups. P < 0.05 was considered statistically significant.

[0078] Live and dead cell staining: HaCaT cells were seeded at 5 × 10⁵ cells per well in 6-well plates, with 3 replicates per group. The next day, the culture medium in the 96-well plates was discarded, and different hydrogel soaking solutions were added for further culture. After 24 h, 48 h, and 72 h, the culture medium was removed, and the cells were washed once with PBS. An appropriate amount of 1 mL of Calcein AM / PI detection working solution was added, and the cells were incubated at 37°C in the dark for 30 min. After incubation, the staining effect was observed under a fluorescence microscope (Calcein AM showed green fluorescence, Ex / Em = 494 / 517 nm; PI showed red fluorescence, Ex / Em = 535 / 617 nm).

[0079] Ideal dressings require excellent biocompatibility. To confirm the biocompatibility of hydrogel dressings, L929 cells, HaCaT cells, and HUVEC cells were used to evaluate the in vitro cytotoxicity of the hydrogel. Figure 7As shown in Figure BD, after culturing in different hydrogel soaking media for 24, 48, and 72 hours, the viability of L929 cells was consistently above 80%. Similar results were also observed in experiments with HaCaT and HUVEC cells. Cell morphology changes after hydrogel treatment were then observed using a live / dead staining method, with live and dead cells stained with green and red fluorescent dyes, respectively. The results showed that most HaCaT cells exhibited normal morphology, and cells treated with various hydrogels showed a similar proliferation trend to the control group. Figure 7 As shown in Figure A, the hemocompatibility of the hydrogel was assessed by in vitro hemolytic activity testing. Figure 7 As shown. GelMA@PDA, GelMA@PEI, and GelMA@PEI-PDA were co-incubated with red blood cells at 37 °C for 1 hour. The supernatant of all samples showed similar transparency to the negative control group (PBS). Figure 7 As shown in F, the hemolysis rates of GelMA@PDA, GelMA@PEI, and GelMA@PEI-PDA are all <5%. Figure 7 As shown in E, this indicates that the hydrogel has good blood compatibility.

[0080] Cell viability assay: The hydrogel was soaked in PBS until it swelled to equilibrium. The treated hydrogel was then immersed in cell culture medium and incubated overnight in a cell culture incubator to obtain the hydrogel soaking solution (10 mg / mL). The cytotoxicity of the hydrogel was evaluated by the CCK-8 assay. L929 cells, HaCaT cells, and HUVEC cells were seeded at 4.0 × 10³ cells per well in 96-well plates, with four replicate wells per group.

[0081] Dopamine (PDA) possesses strong reducing power due to its phenolic hydroxyl group, quinone group, and catechol in its structure, enabling it to capture oxygen free radicals. This study evaluated the antioxidant activity of GelMA@PEI-PDA. The reducing power of GelMA@PEI-PDA was determined using the DPPH assay. Figure 8 As shown in Figure D, after incubation with GelMA@PEI-PDA and GelMA@PEI-PDA, the color of DPPH gradually changed from purple to yellow, and 85.99% and 81.14% of DPPH were eliminated, respectively. Figure 8 As shown in Figure B. Furthermore, a 100 μM H2O2 environment was used to simulate a ROS environment to induce cellular oxidative stress damage, in order to evaluate the ability of GelMA@PEI-PDA to control intracellular reactive oxygen species levels. L929 cells were incubated with 100 μM H2O2 for 2 h, then treated with different hydrogels for 2 h. Intracellular ROS levels were then measured using the standard DCFH-DA method, and quantitative analysis was performed by flow cytometry. Figure 8 As shown in E and F.

[0082] Scratch assay: HaCaT cells were cultured in 6-well plates (1 × 10⁶ cells / well) for 24 hours. A 200 μL pipette tip was used to linearly scratch the cell monolayer, and the cells were washed with PBS to remove cell debris. The cells were then co-incubated with FBS-free hydrogel soaking medium containing 100 μM H₂O₂. Images of the scratched HaCaT cells were captured at 0 and 12 h and analyzed using ImageJ software. Cell migration rate was calculated as follows: Cell migration rate (%) = [(A0 - At) / A0] × 100%. A0 is the scratched area at 0 h, and At is the scratched area without cell migration at 12 h.

[0083] After treatment with GelMA@PDA and GelMA@PEI-PDA, the relative percentage of ROS in L929 cells decreased from 46.16% to 37.79% and 35.11%, respectively. Keratinocytes play an important role in tissue repair; the effect of hydrogels on the migration of keratinocytes under oxidative stress was investigated using a scratch assay. Figure 8 As shown in Figure A, compared with the Control group, cell migration was significantly increased in both the GelMA@PDA and GelMA@PEI-PDA groups, with the GelMA@PDA group exhibiting the highest cell migration rate.

[0084] Antibacterial assay: The hydrogel (10 mg / mL) was mixed with suspensions of Staphylococcus aureus, Escherichia coli, and MRSA (1×10⁶ CFU / mL). The samples were incubated at 37°C with shaking at 220 rpm for 4 h. After serial dilution of the bacterial suspension, 10 µL was evenly spread onto LB agar. The samples were incubated at 37°C for 24 h, then removed and photographed. After bacterial treatment with the samples, the bacteria were washed three times with PBS, centrifuged to precipitate, and fixed with 1 mL of glutaraldehyde (2.5%). Subsequently, the fixed bacteria were dehydrated three times with ethanol solutions of increasingly higher concentrations. Finally, the bacterial morphology was observed using SEM.

[0085] The antibacterial properties of different concentrations of GelMA@PEI-PDA hydrogel were investigated using *Escherichia coli*, *Staphylococcus aureus*, and methicillin-resistant *Staphylococcus aureus* (MRSA) as experimental subjects. First, bacteria were co-cultured with different concentrations of GelMA@PEI-PDA hydrogel at 37 °C for 6 hours. Then, the treated bacteria were plated on agar plates and incubated for 18 hours. The corresponding colony-forming units (CFU) on the culture dishes were photographed to evaluate the antibacterial activity of the GelMA@PEI-PDA hydrogel. Figure 9A) After 18 h, bacterial colonies were widely formed in the control group culture dishes. Compared with the control group, the inhibition rates of *E. coli* with GelMA@PEI-PDA hydrogels containing 2, 4, 6, 8, and 10 mg of GelMA@PEI-PDA were 57.01%±7.20%, 81.22%±3.63%, and 96.10%±0.48%, 98.15%±0.37%, and 99.52%±0.24%, respectively. The inhibition rates of *Staphylococcus aureus* were 68.95%±3.51%, 78.83%±2.88%, and 93.95%±0.80%, 98.70%±0.46%, and 99.99%, respectively. The inhibition rates of methicillin-resistant Staphylococcus aureus were 51.05%±1.80%, 97.43%±0.81%, and 99.88%±0.10%, 99.99%, 99.99%, respectively. Figure 9 These results indicate that the GelMA@PEI-PDA hydrogel exhibits good antibacterial activity against Staphylococcus aureus, MRSA, and Escherichia coli.

[0086] Using methicillin-resistant Staphylococcus aureus (MRSA) as the experimental subject, the effect of GelMA@PEI-PDA hydrogel on its morphology at different time points was observed using scanning electron microscopy (SEM) to reveal the antibacterial process of the hydrogel. In the control group, the bacterial surface was smooth, morphologically intact, and regularly shaped after 2 h and 6 h of treatment. However, in the GelMA@PEI-PDA hydrogel treatment group, wrinkles appeared on the bacterial surface after 2 h; after 6 h, the bacterial cell wall and cell membrane were significantly damaged, and intracellular contents flowed out. Figure 9 E). Scanning electron microscopy results also showed that the GelMA@PEI-PDA hydrogel can disrupt the bacterial outer membrane and kill bacteria.

[0087] The above in vitro experimental results indicate that GelMA@PEI-PDA hydrogel possesses antibacterial and antioxidant properties, demonstrating good potential for wound repair as a strong dressing for chronic diabetic wounds. The therapeutic effect of GelMA@PEI-PDA hydrogel on a full-thickness MRSA-infected mouse model of diabetic wounds was evaluated. Figure 10 As shown, Figure 10 Figure A illustrates the entire process of GelMA@PEI-PDA treatment in wounds. A diabetic mouse model was established by intraperitoneal injection of STZ into C57BL / 6 mice for 5 consecutive days. Then, MRSA was inoculated into a 1 cm diameter circular wound on the back of the diabetic mice and sealed for 1 day until abscess formation. Subsequently, the wounds were treated with PBS (Control group), GelMA@PEI, GelMA@PDA, and GelMA@PEI-PDA hydrogel dressings, respectively. Figure 10Figure B shows representative images of wounds from different groups at different time points. The healing efficiency of GelMA@PEI-PDA hydrogel was significantly higher than that of the untreated group. On day 3, the control group still had a large amount of exudate on the wound surface, while the infection in the GelMA@PEI-PDA group was basically controlled. On day 7, the inflammation in the control group began to subside, while the wounds in the hydrogel-treated group had begun to shrink. On day 14, the GelMA@PEI-PDA wounds were basically closed. On day 18, the control group mice still had scabs on the wound surface, while the GelMA@PEI-PDA wounds only had scar tissue remaining.

[0088] Mouse skin tissue was collected and stained with H&E. The histopathological structure of the damaged skin was observed to assess the wound healing and regeneration process. Figure 11 As shown, on day 7, a large number of inflammatory cells were found in the untreated wound due to bacterial infection. In the hydrogel treatment group, the inflammation at the wound site had largely subsided, and the epidermal cells regenerated regularly. On day 14, the wound in the GelMA@PEI-PDA hydrogel group closed, and the squamous epithelial arrangement and collagen fibers became more regular. In the control group, however, an inflammatory response was still observed under the scab. Although the wounds in the GelMA@PDA and GelMA@PEI groups did not completely close, the local inflammation was basically controlled. The healed skin tissue also regenerated regularly. During the experiment, no hydrogel rejection reaction occurred in any group at any of the above stages. These results indicate that hydrogels promote the healing of diabetic infected wounds in vivo.

[0089] To investigate wound inflammation resolution and tissue regeneration, a possible phenotypic transition from M2a macrophages to M2c macrophages was observed in skin tissue on days 7 and 14. Figure 12 Immunofluorescence results showed that the number of CD206-labeled M2a macrophages was statistically highest in the GelMA@PEI-PDA group on day 7, followed by GelMA@PEI, GelMA@PDA, and the control group. However, a completely opposite trend was observed on day 14: GDFE showed the lowest level, while the control group showed the highest. Regarding CD163-labeled M2c macrophages, the number of positive cells was relatively low in all groups on day 7. Strong positive staining was observed in the GelMA@PEI-PDA group on day 14, followed by GelMA@PDA, GelMA@PEI, and the control group. Compared to the other groups, the GelMA@PEI-PDA group had higher CD206 levels on day 7 and more CD163-stained cells on day 14, indicating that GDFE hydrogel has the ability to induce macrophage polarization towards M2c. In summary, these results indicate that GDFE hydrogel can promote macrophage polarization from the anti-inflammatory M2a phenotype to the pro-regenerative M2c phenotype, and facilitate the transition from the inflammatory phase to the proliferative phase in diabetic wound infections.

[0090] Immunofluorescence staining: After dewaxing, the antigen was repaired with EDTA. The slides were placed in a protective humidifier, completely covered with 5% blank goat serum, and incubated at 37°C for 30 min. Following the manufacturer's instructions, the antibody was diluted in antibody dilution buffer, the blocking solution was removed, and the diluted primary antibody was added. The sample was incubated overnight at 4°C. The sample was then placed at room temperature and warmed for 15 min. The working antibody solution was removed, and the sample was washed with TBST buffer. Following the manufacturer's instructions, the antibody was diluted in antibody dilution buffer, and the secondary antibody was incubated in the dark for 1 h. The working secondary antibody solution was removed, and the sample was washed with TBST buffer. DAPI working solution was added to the sample, and the sample was incubated at room temperature in the dark for 10 min. The working DAPI solution was removed, and the sample was washed with TBST buffer. After adding an anti-fluorescence attenuation mounting medium, the sample was observed and images were acquired under a fluorescence microscope. CD31 and α-SMA were used as markers for neovascularization and mature angiogenesis, respectively, and immunofluorescence staining was used to evaluate the wound. Figure 13 The CD31 positivity rate in the GelMA@PEI-PDA group was statistically highest on day 7, followed by GelMA@PEI, GelMA@PDA, and the control group. On day 14, the CD31 positivity rate followed the same trend as on day 7, but the overall number decreased slightly. The α-SMA positivity rate in the GelMA@PEI-PDA group was statistically highest on day 7, followed by GelMA@PEI, GelMA@PDA, and the control group. On day 14, the α-SMA positivity rate followed the same trend as on day 7, but the overall number increased slightly. Compared to the other three groups, CD31 expression and α-SMA levels were significantly increased in the hydrogel-treated wounds, indicating that angiogenesis occurred at the wound site in this group. Compared to day 7, the overall number of CD31 positivity rates decreased slightly and the α-SMA positivity rate increased slightly on day 14, as excess immature capillaries closed and the disordered vascular network gradually matured.

[0091] Proper collagen deposition and remodeling are crucial indicators of wound healing. Masson staining was used to observe collagen density in regenerated tissue on days 7 and 14. Figure 14 As shown, collagen density increased in all groups from day 7 to day 14, with the highest collagen density observed in the GelMA@PEI-PDA hydrogel group, followed by GelMA@PEI, GelMA@PDA, and the control group. On day 14, the wound treated with GelMA@PEI-PDA was almost completely closed, and the collagen distribution in the wound became more regular. This indicates that GelMA@PEI-PDA hydrogel can significantly promote collagen deposition and remodeling.

[0092] GelMa@PDA-PEI hydrogels were successfully prepared in Examples 1 and 4 to 7 of this application. The successful synthesis of hydrogels with a three-dimensional network structure was confirmed by NMR, UV, and SEM techniques. Adhesion, stretching, and self-healing experiments showed that GelMa@PDA-PEI possesses good mechanical properties, with the hydrogel prepared in Example 1 exhibiting the best performance. Cells treated with GelMa@PDA-PEI hydrogel showed normal morphology and exhibited a similar proliferation trend to the control group. Blood compatibility tests showed that the hemolysis rate in each group was <5%. GelMa@PDA-PEI hydrogel can reduce intracellular and extracellular ROS levels, reduce cellular oxidative stress damage, and promote cell migration. It has a highly effective broad-spectrum antibacterial effect, killing bacteria by disrupting the bacterial outer membrane. In a diabetic mouse MRSA infection model, GelMa@PDA-PEI hydrogel can alleviate local inflammatory responses and promote wound tissue regeneration.

[0093] This invention utilizes dynamic cross-linking generated by PEI-PDA and GelMA to provide a simple method with excellent mechanical properties, tissue adhesion, and self-healing capabilities to adapt to different wound surfaces. The hydrogel of this invention exhibits good biocompatibility, can kill bacteria, clear intracellular and extracellular ROS, and improve migration barriers caused by oxidative stress. In a diabetic mouse MRSA infection model, the hydrogel can clear bacteria from the wound surface, reduce wound inflammation, promote collagen and angiogenesis, and accelerate wound healing.

Claims

1. A method for preparing a hydrogel dressing for promoting the healing of diabetic infected wounds, characterized in that; Includes the following steps: Add GelMA to the buffer solution, heat at 60°C and stir until completely dissolved, then cool to room temperature; First, add the PEI-PDA copolymer coating to the dissolved GelMA, then add the UV curing agent, mix thoroughly, and then cure under UV light to obtain GelMA@PEI-PDA; The mass-to-volume ratio (mg / mL) of GelMA to buffer is 1:9-3:9; The mass-to-volume ratio (mg / mL) of the GelMA and PEI-PDA copolymer coating is 2:1-5:

1.

2. The method for preparing the hydrogel dressing for promoting the healing of diabetic infected wounds as described in claim 1, characterized in that; The PEI-PDA preparation method is as follows: Dopamine hydrochloride and polyethyleneimine were added to a Tris buffer solution with a pH of 8.5 and reacted at 25°C with slight vibration for 6 h to obtain a PEI-PDA copolymer coating.

3. The method for preparing the hydrogel dressing for promoting the healing of diabetic infected wounds as described in claim 1, characterized in that: The 250 mg dopamine hydrochloride and 243 μL polyethyleneimine were added to 557 μL of Tris buffer solution.

4. The method for preparing the hydrogel dressing for promoting the healing of diabetic infected wounds as described in claim 1, characterized in that; The method for preparing GelMA is as follows: Gelatin was added to PBS buffer solution, heated and stirred until dissolved, and then cooled to 50°C to obtain a gelatin solution. Methacrylic anhydride solution was slowly added dropwise to the gelatin solution, and the reaction was continued at 50°C for 4 hours. After cooling to room temperature, it was placed in a dialysis bag with a molecular weight of 1000 and dialyzed with deionized water at 4°C for 4 days with stirring. After dialysis, GelMA was frozen at 80°C until completely frozen and then placed in a freeze dryer for lyophilization to obtain GelMA. The final product was stored at 4°C until use.

5. The method for preparing the hydrogel dressing for promoting the healing of diabetic infected wounds as described in claim 1, characterized in that: The photocuring agent is 1173 photoinitiator.

6. A hydrogel dressing, characterized in that: The hydrogel dressing for promoting the healing of diabetic infected wounds was prepared by the method described in any one of claims 1 to 5.

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