Staged sequential coating hydrogel dressing for diabetic foot ulcer as well as preparation method and application thereof

Abstract

The invention discloses a staged sequential coating hydrogel dressing for diabetic foot ulcer as well as a preparation method and application thereof, and relates to the technical field of biomedical materials. The dressing comprises a first hydrogel and a second hydrogel which can be sequentially applied to a wound surface, the first hydrogel comprises gelatin, a cross-linking agent and a natural polyphenol compound; the second hydrogel is prepared from gelatin, a cross-linking agent and vitepofen. The method comprises the following steps: firstly, coating a cleaned wound part with first hydrogel, and controlling infection and relieving inflammatory response by utilizing the antibacterial and anti-inflammatory functions of the first hydrogel; and then, 3-5 days after the first time of coating, coating the second hydrogel on the same part to inhibit scar formation and promote tissue regeneration. The first hydrogel and the second hydrogel both have skin adhesion and self-healing ability. Through a staged sequential coating strategy, different biological stages of diabetic foot ulcer wound healing are intervened, and scar-free wound repair is promoted synergistically.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, in particular to a staged sequential coating hydrogel dressing for diabetic foot ulcers and a preparation method and application thereof. BACKGROUND

[0002] Chronic wounds have become a major clinical problem worldwide, causing heavy social and economic burden. For example, the annual prevalence of diabetic foot ulcers worldwide is about 18.6 million, and the 5-year mortality rate is as high as 50-70%. Normal wound healing follows four ordered stages of coagulation, inflammation, cell proliferation and migration, and remodeling. However, diabetic foot ulcers are long-term stagnant in the inflammation stage, leading to excessive inflammatory response and bacterial infection, hindering the healing process.

[0003] To regulate the microenvironment of diabetic foot ulcer inflammation imbalance, bacterial infection and active oxygen accumulation, natural polyphenol compounds are widely studied due to their antibacterial, antioxidant and anti-inflammatory activities. Among them, gallic acid can also inhibit bacterial colonization by reducing the pH value of the wound (alkaline to neutral / acidic). Traditional dressings (such as gauze, film) are easy to damage the newly formed tissue when removed, and alginate dressings need secondary fixation. In contrast, hydrogel dressings, with their excellent bioadhesion and biodegradability, can controllably release drugs, making them ideal carriers for delivering polyphenols. However, existing research on polyphenol hydrogels focuses on "accelerating" wound closure, but ignores the negative impact of scar formation on the quality of life of patients.

[0004] In view of this, the present application is proposed. SUMMARY

[0005] The present application aims to provide a staged sequential coating hydrogel dressing for diabetic foot ulcers and a preparation method and application thereof. To address the problems of prolonged inflammation period and excessive scar formation during the healing process of diabetic foot ulcers, the present application simulates the normal wound healing process by sequentially coating the first and second hydrogels, and synergistically promotes scarless repair.

[0006] The present application is implemented as follows: In a first aspect, the present application provides a staged sequential coating hydrogel dressing for diabetic foot ulcers, which comprises a first hydrogel and a second hydrogel that can be sequentially applied to the wound surface. The first hydrogel comprises gelatin, a crosslinking agent and a natural polyphenol compound. The second hydrogel comprises gelatin, a crosslinking agent and verteporfin.

[0007] In some preferred embodiments, the first hydrogel comprises gelatin 2%-20% w / v, a crosslinking agent 1-5 mg / ml and a natural polyphenol compound 5-20 mg / ml.

[0008] wherein the natural polyphenol compound is selected from at least one of gallic acid, epigallocatechin gallate, rosmarinic acid, salvianolic acid B, anthocyanin, catechin, resveratrol, curcumin, ellagic acid.

[0009] In some preferred embodiments, the second hydrogel comprises: gelatin 2% -20% w / v, crosslinking agent 1-5mg / ml and verteporfin 0.5-5mg / ml.

[0010] In some preferred embodiments, the crosslinking agent is selected from at least one of transglutaminase, tyrosinase, peroxidase, glutaraldehyde, genipin.

[0011] In the second aspect, the application provides a preparation method of a staged sequential coating hydrogel dressing for diabetic foot ulcers, the preparation method of the first hydrogel comprising the following steps: adding a crosslinking agent and a natural polyphenol compound into a gelatin solution, uniformly mixing, and then incubating at 30-40℃ with oscillation until the first hydrogel is formed. The preparation method of the second hydrogel comprises the following steps: adding a crosslinking agent and verteporfin into a gelatin solution, uniformly mixing, and then incubating at 30-40℃ with oscillation until the second hydrogel is formed.

[0012] In some preferred embodiments, the solvent of the verteporfin is dimethyl sulfoxide, and the concentration of dimethyl sulfoxide in the second hydrogel is ≤0.1%.

[0013] In some preferred embodiments, the oscillation rate of the first hydrogel is 800-1200rpm, and the oscillation time is 8-12h; the oscillation rate of the second hydrogel is 800-1200rpm, and the oscillation time is 0.5-1h.

[0014] In the third aspect, the application provides a use of a hydrogel dressing in the preparation of a medicine for scarless repair of diabetic foot ulcers.

[0015] In the third aspect, the application provides a use of a hydrogel dressing in the preparation of a medicine for scarless repair of diabetic foot ulcers.

[0016] The application has the following beneficial effects: The first and second hydrogel dressings provided by the application both have excellent tissue adhesion and self-healing properties, and the components are highly biocompatible. The first hydrogel can shorten the inflammation period through antibacterial and anti-inflammatory effects, and the second hydrogel can inhibit scar formation in the proliferation period, simulate the natural healing process of normal wounds, and promote scarless repair of diabetic foot ulcers. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some of the embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0018] Figure 1 A schematic diagram of the use process of the hydrogel dressing provided by the present application; Figure 2 A scanning electron microscope image, a relative nucleic acid leakage and a protein release amount statistical diagram of Test Example 1 of the present application; Figure 3 An immunoblot test result diagram and a flow cytometry analysis result diagram of Test Example 2 of the present application; Figure 4 An immunoblot test result diagram of Test Example 3 of the present application; Figure 5 A mouse wound antibacterial and anti-inflammatory result diagram of Test Example 4 of the present application; Figure 6 A mouse wound healing effect diagram of Test Example 5 of the present application; Figure 7 A mouse regenerated skin tissue staining result diagram of Test Example 6 of the present application; Figure 8 A fluorescence staining result diagram of Test Example 7 of the present application. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be purchased on the market.

[0020] Studies have shown that the underlying mechanism of scar formation is closely related to fibroblasts and their derived myofibroblasts, which are key factors leading to fibrosis and abnormal collagen deposition. For example, Engrailed-1 (En1) lineage positive fibroblasts promote scar formation, while En1 lineage negative subtypes promote skin regeneration. By using the Yes-related protein inhibitor verteporfin to block the activation of En1 in fibroblasts, scar formation can be effectively reduced and skin regeneration repair can be induced.

[0021] Therefore, the inventors found that by sequentially coating the polyphenol hydrogel and the verteporfin hydrogel in stages, the normal and orderly wound healing cascade process can be better simulated, which is expected to drive the transition from the inflammatory phase to the proliferation phase and inhibit scar formation, and synergistically promote scar-free repair of diabetic foot ulcers.

[0022] In a first aspect, the present application provides a staged sequential hydrogel dressing for diabetic foot ulcer, the hydrogel dressing comprising a first hydrogel and a second hydrogel which can be sequentially applied to a wound surface; The first hydrogel comprises gelatin, a cross-linking agent and a natural polyphenol compound, having antibacterial and anti-inflammatory functions. The second hydrogel comprises gelatin, a cross-linking agent and verteporfin, for promoting scarless repair.

[0023] The first hydrogel comprises: Gelatin 2% -20% w / v, for example 2%, 4%, 6%, 8%, 10% and 20%, etc. any value between 2-20% and two range values between any values; Cross-linking agent 1-5mg / ml, for example 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL and 5 mg / mL, etc. any value between 1-5mg / mL and two range values between any values. The cross-linking agent is selected from at least one of transglutaminase, tyrosinase, peroxidase, glutaraldehyde, genipin; Natural polyphenol compound 5-20 mg / mL, for example 5 mg / mL, 10 mg / mL, 15 mg / mL and 20 mg / mL, etc. any value between 5-20 mg / mL and two range values between any values. The natural polyphenol compound is selected from at least one of gallic acid, epigallocatechin gallate, rosmarinic acid, salvianolic acid B, anthocyanin, catechin, resveratrol, curcumin, ellagic acid.

[0024] The second hydrogel comprises: Gelatin 2% -20% w / v, for example 2%, 4%, 6%, 8%, 10% and 20%, etc. any value between 2-20% and two range values between any values; Cross-linking agent 1-5mg / ml, for example 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL and 5 mg / mL, etc. any value between 1-5mg / mL and two range values between any values. The cross-linking agent is selected from at least one of transglutaminase, tyrosinase, peroxidase, glutaraldehyde, genipin; Verteporfin 0.5-5 mg / mL, for example 0.5 mg / mL, 1 mg / mL, 2 mg / mL and 5 mg / mL, etc. any value between 0.5-5mg / mL and two range values between any values. The solvent of the verteporfin is dimethyl sulfoxide, and the concentration of dimethyl sulfoxide in the second hydrogel is ≤0.1%.

[0025] In an optional embodiment, the first and second hydrogels both have skin adhesion and self-healing ability, and the first hydrogel has antibacterial and anti-inflammatory functions, and the second hydrogel can promote scarless repair of diabetic foot ulcers.

[0026] In a second aspect, the application provides a preparation method of a staged sequential coating hydrogel dressing for diabetic foot ulcers, the preparation method of the first hydrogel comprising the following steps: adding a crosslinking agent and a natural polyphenol compound to a gelatin solution, and uniformly mixing and then incubating at 30-40℃ with shaking until the first hydrogel is formed; The preparation method of the second hydrogel comprises the following steps: adding a crosslinking agent and verteporfin to a gelatin solution, and uniformly mixing and then incubating at 30-40℃ with shaking until the second hydrogel is formed.

[0027] In some preferred embodiments, the first hydrogel is incubated at a shaking rate of 800-1200 rpm for 8-12 h, and the second hydrogel is incubated at a shaking rate of 800-1200 rpm for 0.5-1 h.

[0028] In a third aspect, the application provides a use of a hydrogel dressing in the preparation of a medicine for scarless repair of diabetic foot ulcers.

[0029] In the above method, the staged sequential coating of the hydrogel dressing comprises the following steps, as shown in the following table: Figure 1 First, the first hydrogel is coated on the site of a diabetic foot ulcer, and after 3-5 days of the first coating, the second hydrogel is coated on the same site.

[0030] The first and second hydrogel dressings of the application are coated on the site of a diabetic foot ulcer in a staged sequential manner, which simulates the normal healing process and synergistically promotes scarless repair. Specifically, the first and second hydrogels are coated in a staged manner by gelatin-natural polyphenol (first hydrogel) and gelatin-verteporfin (second hydrogel): the first hydrogel has high antibacterial activity by destroying bacterial cell membranes, and regulates the Nrf2-NF-κB signaling pathway to reduce inflammation and accelerate the transition from the inflammatory phase to the proliferative phase; the second hydrogel inhibits the En1 signaling pathway in fibroblasts to down-regulate the expression of YAP and α-SMA to reduce scar formation. The two hydrogels synergistically promote angiogenesis and epithelialization, achieving scarless repair of diabetic foot ulcers.

[0031] The features and properties of the application are further described in detail below in conjunction with the examples.

[0032] Example 1 This example provides a preparation method of a gelatin hydrogel, comprising the following steps: Gelatin stock solution (20%, w / v): Accurately weigh 2.0 g of gelatin powder and add it to 10 mL of ultrapure water to achieve a final concentration of 20%. Place the mixture in a 55°C oil bath and stir magnetically until the gelatin powder is completely dissolved to obtain a clear solution.

[0033] Crosslinking agent: Preparation of TG enzyme stock solution (50 mg / mL): Accurately weigh 10 mg of TG enzyme powder, dissolve it in 200 µL of ultrapure water, place the solution in a 37°C constant temperature mixer, and gently shake until completely dissolved.

[0034] Take 500 µL of preheated 20% gelatin stock solution into a 2 mL centrifuge tube and place it in a constant-temperature mixer (37°C, 1000 rpm). Add 500 µL of ultrapure water. Then, quickly add 20 µL of TG enzyme stock solution (corresponding to 1.0 mg TG enzyme) to the gelatin solution and mix thoroughly to ensure uniform enzyme distribution. The gelation process usually completes within several hours. The formation of a gel can be determined by the inverted container method: when the solution loses its fluidity and forms a self-supporting, non-flowing solid gel, cross-linking is complete.

[0035] Example 2 This embodiment provides a method for preparing a staged sequential application of hydrogel dressing for diabetic foot ulcers, including the following steps: S1. Preparation of the stock solution: Gelatin stock solution (20%, w / v): Accurately weigh 2.0 g of gelatin powder and add it to 10 mL of ultrapure water to achieve a final concentration of 20%. Place the mixture in a 55°C oil bath and stir magnetically until the gelatin powder is completely dissolved to obtain a clear solution.

[0036] TG enzyme stock solution (50 mg / mL): Accurately weigh 10 mg of TG enzyme powder and dissolve it in 200 µL of ultrapure water. Place the solution in a 37°C constant temperature mixer and gently shake until completely dissolved.

[0037] S2. Preparation of the first hydrogel: Prepare gallic acid (GGA) stock solution (20 mg / mL) in advance, and add 500 µL of gallic acid stock solution to 500 µL of preheated 20% gelatin stock solution. Take 60 µL of TG enzyme stock solution (corresponding to 3.0 mg TG enzyme) and quickly add it to the above mixture. Place it in a constant temperature mixer until cross-linking is completed to obtain the first hydrogel.

[0038] S3. Preparation of the second hydrogel: Prepare verteporfen stock solution (100 mg / mL DMSO solution) in advance. Take 1 µL of verteporfen stock solution, add 100 µL of 10% gelatin aqueous solution, and then add 10 µL of TG enzyme stock solution. Place the mixture in a constant temperature mixer until cross-linking is completed to obtain the second hydrogel.

[0039] Test Example 1: In vitro antibacterial performance test of gallic acid hydrogel (GGA) Gram-positive methicillin-resistant Staphylococcus aureus (MRSA, ATCC 43300) was selected as the experimental strain. 100 μL of GGA hydrogel was mixed with an equal volume of MRSA bacterial suspension (10 7 CFU / mL), and co-cultured at 37°C for 4 hours. The bacterial solution was collected, gradient diluted, and plated on LB agar plates to count colony-forming units (CFU) to evaluate the in vitro antibacterial effect. In addition, the mixed bacterial solution was centrifuged at 3000 rpm, fixed with 2.5% glutaraldehyde at 4°C overnight, dehydrated with gradient ethanol (30%-100%), dried, and then observed under a scanning electron microscope to observe the bacterial morphology. The cell membrane permeability was evaluated by detecting the release of extracellular nucleic acids (OD260) and proteins (BCA method). The specific grouping is as follows: The results are shown in Figure 2 According to a in Figure 2 , the CFU experiment results show that GGA hydrogel treatment significantly reduces the survival rate of MRSA, and the antibacterial effect is better than that of commercial 3M hydrogel. According to b in Figure 2 , the scanning electron microscopy results show that the cell membrane of MRSA in the GGA group is severely deformed and ruptured, while the bacteria in the simple gelatin (G) group are intact. According to c-d in Figure 2 , the release of extracellular nucleic acids and proteins in the GGA treatment group is significantly higher than that in the G group, which proves that gallic acid achieves antibacterial effect by destroying the cell membrane structure.

[0040] Test Example 2: In vitro anti-inflammatory activity evaluation of GGA hydrogel The macrophage polarization model was used to evaluate the in vitro anti-inflammatory effect of GGA hydrogel. RAW 264.7 cells were co-cultured with different hydrogels for 24 hours, and then the cell supernatant and cells were collected. The levels of anti-inflammatory factor TGF-β1 and pro-inflammatory factor IL-12 in the supernatant were detected by enzyme-linked immunosorbent assay. At the same time, RAW 264.7 cells were labeled with anti-F4 / 80, anti-CD80, and anti-CD206 antibodies, and the macrophage phenotype (M1 type: CD80+F4 / 80+; M2 type: CD206+F4 / 80+) was analyzed by flow cytometry. In addition, Western blotting was used to detect the expression of Nrf2 and NF-κB proteins in RAW 264.7 cells to clarify the anti-inflammatory mechanism of GGA hydrogel.

[0041] The results are shown in Figure 3 According to a in Figure 3 , the flow cytometry analysis results show that GGA hydrogel significantly promotes the polarization of macrophages to M2 phenotype. According to Figure 3From Fig. 3b-c, ELISA results further confirmed that GGA hydrogel group significantly increased the secretion of anti-inflammatory factor TGF-β1 and reduced the level of pro-inflammatory factor IL-12. Figure 3 From Fig. 3d, Western blot results showed that GGA hydrogel could significantly increase the expression of Nrf2 and inhibit the activation of NF-κB signaling pathway, indicating that it could reduce inflammatory response by regulating Nrf2 / NF-κB signaling pathway.

[0042] Test Example 3: Evaluation of GVert hydrogel in vitro skin regeneration potential To evaluate the regulatory effect of GVert hydrogel on key proteins of skin regeneration, in vitro experiments were conducted using NIH / 3T3 fibroblasts. NIH / 3T3 cells were seeded in a six-well plate and cultured, and three groups were set up for comparison: blank control group (no drug added in the culture medium), free verteporfin group (final concentration of verteporfin in the culture medium was 10 µg / mL), GVert hydrogel group (GVert hydrogel extract containing 10 µg / mL verteporfin was added in the culture medium). After 24 hours of continuous culture, the cell lysate of the three groups was collected, and the expression levels of Yes-associated protein (YAP) and α-smooth muscle actin (α-SMA) were detected by Western blot.

[0043] Results are shown in Figure 4 Compared with the blank control group, the expression of YAP and α-SMA proteins in the GVert hydrogel group was significantly reduced, and the inhibitory effect was comparable to that of the free verteporfin group.

[0044] Test Example 4: Study on the antibacterial and anti-inflammatory effects of hydrogel on MRSA-infected diabetic mouse wounds A full-thickness skin defect model (wound diameter 6 mm) of MRSA-infected diabetic mice was established. The successfully modeled mice were randomly divided into 5 groups (n=5): control group (no treatment), G hydrogel group, GGA hydrogel group, and 3M commercial gel group. The hydrogels of each group were directly applied to the infected wounds, and the wound tissues were collected on the 3rd day. ELISA was used to detect the concentrations of inflammatory factors (IL-6, IL-10), and rapid bacterial detection test paper was used to analyze the bacterial residues on the wound.

[0045] Results are shown in Figure 5 From Fig. 4a, after 3 days of GGA hydrogel application, the bacterial detection test paper only showed the quality control line, indicating that the bacteria in the wound were completely eliminated. From Fig. 4b-c, after 3 days of GGA hydrogel application, the IL-10 concentration in the wound site was the highest, and the IL-6 concentration was the lowest, confirming its high anti-inflammatory performance. Figure 5 Figure 5

[0046] Test Example 5: Evaluation of the wound healing effect of hydrogel on MRSA-infected diabetic mice​​ After the success of the MRSA infected diabetic mouse model, fresh hydrogel was applied to the wound site. The specific grouping is as follows: Control group (no treatment), G hydrogel group, GGA hydrogel group, GGA+GVert hydrogel group (after 3 days of GGA hydrogel application, GVert hydrogel is applied), 3M commercial gel group. The wound healing was monitored for 14 consecutive days, and the wound images were recorded on days 0, 3, 7, and 14, and the relative wound area was calculated.

[0047] The results are shown in Figure 6 , the Control group and the G hydrogel group were delayed in healing due to MRSA infection; the GA+GVert group had a significant reduction in wound area on day 7, attributed to the strong antibacterial and anti-inflammatory effects of the GGA hydrogel; by day 14, the wounds in this group had almost no scar residue, indicating that the verteprofin in the GVert hydrogel effectively promoted skin regeneration. In contrast, the commercial 3M hydrogel group had poor wound healing results.

[0048] Test Example 6: Study of the Effect of Hydrogel on Wound Repair in MRSA Infected Diabetic Mice On days 7 and 14, the mice were sacrificed, and the regenerated skin tissue at the wound site was fixed with 4% paraformaldehyde, dehydrated, and embedded in paraffin, then sliced into 5 µm sections using a microtome. Histological analysis was performed using hematoxylin & eosin staining, Masson's trichrome staining, and Sirius red staining, respectively, to evaluate skin regeneration.

[0049] The results are shown in Figure 7 , according to a in Figure 7 , on day 14, the epidermal layer in the GGA+GVert group was regenerated completely and the epidermal thickness was close to that of normal skin; according to b in Figure 7 , Masson's trichrome staining showed complete epidermal-dermal structure and dense and orderly collagen fiber arrangement, accompanied by hair follicle regeneration; according to c in Figure 7 , Sirius red staining showed the highest ratio of type I / III collagen.

[0050] Test Example 7: Exploration of the Mechanism of Hydrogel Promoting Scarless Wound Healing To explore the mechanism of hydrogel promoting scarless wound healing, immunofluorescence staining was performed on the tissue sections at the wound site for cytokeratin 14 (CK14), platelet endothelial cell adhesion molecule (CD31), YAP, and alpha-SMA. After deparaffination and dehydration, the sections were placed in citric acid-EDTA antigen retrieval solution and heated at 95°C for 20 minutes for antigen retrieval; 2% BSA was added and incubated at room temperature for 1.5 hours, then incubated with the primary antibody overnight at 4°C in a humidified box, followed by reaction with the fluorescently labeled secondary antibody; finally, the nuclei were labeled with DAPI, and the fluorescence microscope was used for imaging.

[0051] Results are shown in Figure 8 , according to Figure 8 a, CK14 staining results showed that GGA+GVert hydrogel group formed mature stratum corneum, and the hair follicle structure was significantly regenerated; according to Figure 8 b, CD31 staining results showed that the vascular density of this group was significantly higher than that of the control group, and the hair follicle cells of this group also showed CD31 positive, suggesting that Nestin + cells participated in angiogenesis during hair follicle morphogenesis; according to Figure 8 c-d, YAP and alpha-SMA staining results showed that this group could significantly inhibit YAP nuclear translocation, block the mechanical transduction pathway, and reduce the activation of myofibroblasts, achieving scar-free skin regeneration.

[0052] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A staged sequential application hydrogel dressing for diabetic foot ulcers, characterized in that, The hydrogel dressing includes a first hydrogel and a second hydrogel that can be sequentially applied to the wound. The first hydrogel comprises gelatin, a cross-linking agent, and a natural polyphenol compound; The second hydrogel comprises gelatin, a crosslinking agent, and verteporfen.

2. The staged sequential application of hydrogel dressing for diabetic foot ulcers according to claim 1, characterized in that, The first hydrogel comprises: 2%-20% gelatin w / v, 1-5 mg / ml crosslinking agent and 5-20 mg / ml natural polyphenol compound.

3. A staged sequential application hydrogel dressing for diabetic foot ulcers according to claim 1 or 2, characterized in that, The natural polyphenolic compound is selected from at least one of gallic acid, epigallocatechin gallate, rosmarinic acid, salvianolic acid B, anthocyanins, catechins, resveratrol, curcumin, and ellagic acid.

4. The staged sequential application of hydrogel dressing for diabetic foot ulcers according to claim 1, characterized in that, The second hydrogel comprises: 2%-20% gelatin w / v, 1-5 mg / ml crosslinking agent and 0.5-5 mg / ml verteporfen.

5. A staged sequential hydrogel dressing for diabetic foot ulcers according to claim 1, characterized in that, The cross-linking agent is selected from at least one of transglutaminase, tyrosinase, peroxidase, glutaraldehyde, and genipin.

6. A method for preparing a staged sequentially applied hydrogel dressing for diabetic foot ulcers as described in any one of claims 1-5, characterized in that, The preparation method of the first hydrogel includes the following steps: adding a cross-linking agent and a natural polyphenol compound to a gelatin solution, mixing evenly, and then incubating with shaking at 30-40°C until the first hydrogel is formed; The preparation method of the second hydrogel includes the following steps: adding a crosslinking agent and vertiporfin to a gelatin solution, mixing evenly, and then incubating with shaking at 30-40°C until the second hydrogel is formed.

7. The method for preparing a staged sequential hydrogel dressing for diabetic foot ulcers according to claim 6, characterized in that, The solvent for verteporfen is dimethyl sulfoxide, and the concentration of dimethyl sulfoxide in the second hydrogel is ≤0.1%.

8. The method for preparing a staged sequentially applied hydrogel dressing for diabetic foot ulcers according to claim 6, characterized in that, The first hydrogel has an oscillation rate of 800-1200 rpm and an oscillation time of 8-12 h; the second hydrogel has an oscillation rate of 800-1200 rpm and an oscillation time of 0.5-1 h.

9. The use of a hydrogel dressing as described in any one of claims 1-5 in the preparation of a medicament for scarless repair of diabetic foot ulcers.

10. The application according to claim 9, characterized in that, The staged sequential application of the hydrogel dressing includes the following steps: first, the first hydrogel is applied to the diabetic foot ulcer site, and then the second hydrogel is applied to the same site 3-5 days after the first application.

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