A composite hydrogel for promoting wound healing and a preparation method thereof

Abstract

The present application belongs to the technical field of skin dressings, and particularly relates to a composite hydrogel for promoting wound healing and a preparation method thereof; the composite hydrogel for promoting wound healing comprises the following raw materials in parts by weight: 60-80 parts of a composite core, 3-8 parts of type I collagen, 8-15 parts of gelatin, and 8-15 parts of an inclusion compound; the present application uses a natural biological substrate as a core carrier, improves the stability and activity of functional components through inclusion compound technology, relies on the oxidation-sensitive characteristics of the outer layer to accurately remove inflammatory stress and provide antibacterial protection, and through the active peptide of the middle layer, endogenous stem cells are directionally recruited to activate the repair potential, the composite core guides the ordered deposition of collagen in a biomimetic structure to realize scar repair, and the quality of wound healing is significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of skin dressing technology, specifically relating to a composite hydrogel that promotes wound healing and its preparation method. Background Technology

[0002] Wound repair is a key research area in biomedicine, especially the repair of chronic and infected wounds. It presents a clinical challenge due to multiple factors, including inflammatory imbalance, infection risk, and insufficient infiltration of repair cells. Ideal wound repair materials must simultaneously meet multiple requirements, such as biosafety, antibacterial protection, inflammation regulation, cell recruitment, and anti-scarring properties, to adapt to the dynamic healing process of wounds from the inflammatory phase, proliferative phase, to the remodeling phase.

[0003] Current wound repair materials often suffer from limited functionality and fragmented repair mechanisms: some materials focus solely on antibacterial effects, relying on the continuous release of exogenous antibacterial agents, which easily leads to bacterial resistance and struggles to maintain cell compatibility; others rely on exogenous growth factors to activate the repair process, which not only suffers from easily inactivated biological activity and a short half-life, but also poses safety risks such as abnormal proliferation due to excessive factor intake. Therefore, developing a repair material that can promote wound healing and activate repair potential is crucial to overcoming current technological bottlenecks. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention proposes a composite hydrogel for promoting wound healing and its preparation method. The composite hydrogel uses a natural bio-based substrate as its core carrier, employing inclusion complex technology to enhance the stability and activity of functional components. The outer layer leverages its oxidation-sensitive properties to precisely eliminate inflammatory stress and provide antibacterial protection. The middle layer uses active peptides to directionally recruit endogenous stem cells, activating their repair potential. The core layer uses a biomimetic structure to guide orderly collagen deposition, achieving anti-scar repair. The functions of each layer are dynamically adapted to the wound healing sequence, simultaneously resolving multiple contradictions related to infection control, inflammation regulation, and tissue remodeling while ensuring excellent biocompatibility. This addresses the technical problems of traditional wound repair materials, such as limited functionality, susceptibility to infection, imbalanced inflammation regulation, and poor anti-scarring effects, significantly improving the quality of wound healing.

[0005] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0006] This invention provides a composite hydrogel for promoting wound healing, comprising the following raw materials in parts by weight: 60-80 parts composite core, 3-8 parts type I collagen, 8-15 parts gelatin, 0.1-0.3 parts SDF-1α mimic peptide (stromal cell-derived factor-1α mimic peptide), 25-35 parts hyaluronic acid, and 8-15 parts inclusion complex.

[0007] The composite core comprises raw materials in the following mass ratio: silkworm silk: asiaticoside = 1: 0.0001-0.0002;

[0008] The method for preparing the composite core includes the following steps:

[0009] (1) Boil the silkworm silk in a sodium carbonate solution with a mass-volume concentration of 0.5% for 30 min, repeat twice to completely remove the sericin, wash with deionized water, dry to form silk fibroin fiber, dissolve the silk fibroin fiber in a 9.3 M lithium bromide solution with a mass-volume ratio of 1 g: 10 mL, stir in a 60 ℃ water bath until completely dissolved, then perform dialysis to completely remove lithium bromide, centrifuge to remove impurities, and obtain a clear silk fibroin protein aqueous solution with a mass-volume concentration of 6-8%;

[0010] (2) Dissolve asiaticoside in DMSO (dimethyl sulfoxide) to prepare a stock solution with a concentration of 1-2 mg / mL. Slowly add the stock solution to the silk fibroin aqueous solution. The final concentration of asiaticoside is 10-20 μg / mL. Stir magnetically for 2 hours to mix it evenly and form a mixed solution.

[0011] (3) The mixed solution is loaded into the syringe of the electrospinning equipment, and electrospinning is performed. The process parameters are set as follows: injection speed 0.5-1 mL / h, voltage 15-20 kV, receiving distance 12-20 cm, and ambient humidity controlled at 40-50%. After obtaining the nanofiber membrane on the receiving plate, it is placed in 75% ethanol vapor for 30 min to obtain the composite core.

[0012] The inclusion complex comprises the following raw materials in the following mass ratio: hydroxypropyl-β-cyclodextrin:curcumin = 3-10:1;

[0013] The method for preparing the inclusion complex includes the following steps:

[0014] (a) Dissolve hydroxypropyl-β-cyclodextrin in deionized water at a ratio of 1 g to 100-150 mL to form a hydroxypropyl-β-cyclodextrin solution.

[0015] (b) Dissolve curcumin in anhydrous ethanol at a ratio of 1 g to 10-20 mL. Then, add the curcumin dropwise to the hydroxypropyl-β-cyclodextrin solution while stirring vigorously. Continue stirring in the dark for 24 h. Then filter and freeze dry to obtain the inclusion complex.

[0016] This invention also provides a method for preparing a composite hydrogel that promotes wound healing, specifically including the following steps:

[0017] S1. Dissolve type I collagen in a 0.1% (v / v) aqueous acetic acid solution and stir overnight to prepare a collagen solution. Dissolve gelatin in PBS (phosphate buffer, pH 7.4) and stir to dissolve in a 37°C water bath to prepare a gelatin solution.

[0018] S2, mix collagen solution and gelatin solution at a volume ratio of 1:1, keep the temperature constant at 37 ℃ and stir gently to avoid generating bubbles, to form mixture i;

[0019] S3, SDF-1α mimic peptide was dissolved in PBS, and EDC (carbodiimide) and NHS (N-hydroxysuccinimide) were added as cross-linking agents. The mixture was activated at 4 °C for 30 min to form activated SDF-1α mimic peptide. The ratio of SDF-1α mimic peptide, EDC, NHS and PBS was 5 mg: 5-10 mg: 5 mg: 1-2 mL. The activated SDF-1α mimic peptide was added to mixture i and reacted slowly at 4 °C for 12 h to allow the mimic peptide to be covalently grafted onto the amino groups of gelatin and collagen through amide bonds, thus obtaining mixture ii.

[0020] S4, the composite core was immersed in the mixture ii, and then transferred to a constant temperature environment of 37 ℃ and allowed to stand for 1 h to perform thermally induced cross-linking, thereby forming a strong stem cell recruitment middle layer outside the core. The surface was gently rinsed with PBS to obtain the complex.

[0021] S5. Hyaluronic acid was dissolved in PBS to prepare a 1% (w / v) hyaluronic acid solution. 3-(acrylamido)phenylboronic acid, EDC, and NHS were added to the hyaluronic acid solution, wherein the ratio of 3-(acrylamido)phenylboronic acid, EDC, NHS, and hyaluronic acid solution was 0.1-0.15 g:0.5 g:0.3 g:100 mL. The reaction was carried out at room temperature in the dark for 24 h, purified by dialyzing, and freeze-dried to obtain oxidized hyaluronic acid.

[0022] S6. Dissolve oxidized hyaluronic acid and its inclusion complex in PBS to form a pregel solution. Immerse the complex prepared in step S4 completely in the pregel solution. After removing it, irradiate it under 365 nm ultraviolet light for 30-90 s to carry out an ultraviolet cross-linking reaction, causing the acrylamide groups on the oxidized hyaluronic acid chain to undergo covalent cross-linking, forming a dense antioxidant outer layer on the outside of the complex. After rinsing with PBS, sterilize to obtain a composite hydrogel that promotes wound healing.

[0023] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0024] The composite hydrogel for promoting wound healing prepared in this invention is composed of a composite core and inclusion complexes. Its antioxidant outer layer constructs an initial protective barrier for the wound due to its oxidation-sensitive properties. During the inflammatory phase, under the high reactive oxygen species (ROS) environment, it preferentially releases curcumin, which can both remove excess ROS to inhibit the spread of inflammatory factors and ensure the preservation of the activity of the middle layer's mimic peptides by reducing the risk of damage to stem cells caused by inflammatory stress. After the inflammation subsides, the antioxidant outer layer gradually degrades as the ROS level decreases, and begins to continuously release SDF-1α mimic peptides, actively recruiting endogenous stem cells to the wound and avoiding cell infiltration barriers caused by structural barriers. Its collagen-gelatin network provides an attachment scaffold for stem cells that migrate to the wound after the outer layer degrades, and precisely anchors stem cell surface receptors through covalently grafted mimic peptides. During the slow degradation process in the wound proliferation phase, it provides a nutrient transport channel for the core scaffold, ensuring that the active ingredients in the core can be gradually released with the repair process. At the same time, the cytokines secreted by the recruited endogenous stem cells can enhance the regulatory effect of the core active ingredients on fibroblasts. The composite core silk fibroin scaffold combines the structural advantages of biomimetic extracellular matrix. Its porous nature perfectly matches the wound microenvironment exposed after the middle layer degrades, providing a continuous growth space for recruited stem cells. The core-loaded asiaticoside precisely targets the critical node transitioning from the proliferation phase to the remodeling phase, inhibiting excessive fibroblast proliferation to reduce disordered collagen deposition, and complementing the orderly collagen synthesis guided by stem cells. This avoids the drawback of existing anti-scarring ingredients inhibiting the repair process when used alone. Through interlayer synergy, the active ingredients are precisely released spatiotemporally, avoiding the safety risks of exogenous factors being easily inactivated and potentially causing abnormal proliferation. It also makes the repair process more in line with human physiological mechanisms, enabling the wound to complete a high-quality healing process entirely through its own repair mechanisms without external intervention. Attached Figure Description

[0025] Figure 1 The diagram shows the anti-inflammatory properties of the composite hydrogel for promoting wound healing prepared in this invention.

[0026] Figure 2 The graph shows the free radical scavenging rate of the composite hydrogel for promoting wound healing prepared in this invention.

[0027] Figure 3 This is a staining image of the composite hydrogel for promoting wound healing prepared in this invention on wound healing. Detailed Implementation

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0029] Unless otherwise specified, all materials used in the following examples are new materials purchased from the market. Among them, the SDF-1α mimic peptide is: SDF-1 mimic peptide-4 (SMP;LSYKCGCKFGGGFRCPCRYSL-NH2).

[0030] Example 1: This example provides a composite hydrogel that promotes wound healing. The composite hydrogel that promotes wound healing comprises the following raw materials in parts by weight: 60 parts composite core, 3 parts type I collagen, 8 parts gelatin, 0.1 parts SDF-1α mimic peptide, 25 parts hyaluronic acid, and 8 parts inclusion complex.

[0031] The composite core comprises raw materials in the following mass ratio: silkworm silk: asiaticoside = 1: 0.0001;

[0032] The method for preparing the composite core includes the following steps:

[0033] (1) Boil the silkworm silk in a sodium carbonate solution with a mass-volume concentration of 0.5% for 30 min, repeat twice to completely remove the sericin, wash with deionized water, dry to form silk fibroin, dissolve the silk fibroin in a 9.3M lithium bromide solution with a mass-volume ratio of 1g:10mL, stir in a 60℃ water bath until completely dissolved, put the solution into a dialysis bag, cut off the molecular weight of 8 kDa, dialyze in deionized water for 3 days, change the water every 4 hours to completely remove lithium bromide, finally, centrifuge the dialyzed silk fibroin solution at 9000 rpm for 20 min to remove impurities, and obtain a clear silk fibroin aqueous solution with a mass-volume concentration of 6%;

[0034] (2) Dissolve asiaticoside in DMSO to prepare a stock solution with a concentration of 1 mg / mL. Slowly add the stock solution to the silk fibroin aqueous solution. The final concentration of asiaticoside is 10 μg / mL. Stir magnetically for 2 h to mix it evenly and form a mixed solution.

[0035] (3) The mixed solution was loaded into the syringe of the electrospinning equipment and electrospinned. The process parameters were set as follows: injection speed 0.5 mL / h, voltage 15 kV, receiving distance 12 cm, and ambient humidity controlled at 40%. After obtaining the nanofiber membrane on the receiving plate, it was placed in 75% ethanol vapor for 30 min to obtain the composite core.

[0036] The inclusion complex comprises the following raw materials in the following mass ratio: hydroxypropyl-β-cyclodextrin:curcumin = 3:1;

[0037] The method for preparing the inclusion complex includes the following steps:

[0038] (a) Hydroxypropyl-β-cyclodextrin was dissolved in deionized water at a ratio of 1 g to 100 mL to form a hydroxypropyl-β-cyclodextrin solution.

[0039] (b) Dissolve curcumin in anhydrous ethanol at a ratio of 1 g to 10 mL. Then, add it dropwise to a hydroxypropyl-β-cyclodextrin solution while stirring vigorously. Continue stirring in the dark for 24 h. Then filter and freeze dry to obtain the inclusion complex.

[0040] This embodiment also provides a method for preparing a composite hydrogel that promotes wound healing, specifically including the following steps:

[0041] S1. Dissolve type I collagen in a 0.1% acetic acid aqueous solution and stir overnight to prepare a 3 mg / mL collagen solution. Dissolve gelatin in PBS and stir in a 37 ℃ water bath to prepare an 8 mg / mL gelatin solution.

[0042] S2, mix collagen solution and gelatin solution at a volume ratio of 1:1, keep the temperature constant at 37 ℃ and stir gently to avoid generating bubbles, to form mixture i;

[0043] S3, SDF-1α mimic peptide was dissolved in PBS, and EDC and NHS were added as cross-linking agents. The mixture was cross-linked and activated at 4 °C for 30 min to form activated SDF-1α mimic peptide. The ratio of SDF-1α mimic peptide, EDC, NHS and PBS was 5 mg: 5 mg: 5 mg: 1 mL. The activated SDF-1α mimic peptide was added to mixture i and reacted slowly at 4 °C for 12 h to allow the mimic peptide to be covalently grafted onto the amino groups of gelatin and collagen through amide bonds, thus obtaining mixture ii.

[0044] S4. Immerse the composite core in the mixture ii to ensure complete coverage, then transfer it to a constant temperature environment of 37 °C and let it stand for 1 h to induce cross-linking. At this temperature, collagen will spontaneously cross-link to form a stable network, and gelatin will also play an auxiliary role in gelation, thereby forming a strong stem cell recruitment middle layer outside the core. Gently rinse the surface with PBS to obtain the complex.

[0045] S5. Hyaluronic acid was dissolved in PBS to prepare a 1% (w / v) hyaluronic acid solution. 3-(acrylamido)phenylboronic acid, EDC, and NHS were added to the hyaluronic acid solution, wherein the ratio of 3-(acrylamido)phenylboronic acid, EDC, NHS, and hyaluronic acid solution was 0.1 g:0.5 g:0.3 g:100 mL. The reaction was carried out at room temperature in the dark for 24 h to graft the phenylboronic acid group onto the HA chain. After dialysis purification and freeze-drying, oxidized hyaluronic acid was obtained.

[0046] S6, Dissolve oxidized hyaluronic acid and its inclusion complex in PBS to form a pregel solution. Completely immerse the composite prepared in step S4 into the pregel solution, ensuring that the surface is fully and uniformly covered. After removal, irradiate it under 365 nm ultraviolet light for 30 s to carry out an ultraviolet cross-linking reaction, causing the acrylamide groups on the oxidized hyaluronic acid chain to undergo covalent cross-linking, forming a dense antioxidant outer layer on the outside of the composite structure. Rinse with PBS and then sterilize to obtain a composite hydrogel that promotes wound healing.

[0047] Example 2: This example provides a composite hydrogel that promotes wound healing. The composite hydrogel that promotes wound healing comprises the following raw materials in parts by weight: 70 parts composite core, 5 parts type I collagen, 10 parts gelatin, 0.2 parts SDF-1α mimic peptide, 30 parts hyaluronic acid, and 10 parts inclusion complex.

[0048] The composite core comprises raw materials in the following mass ratio: silkworm silk: asiaticoside = 1: 0.00015;

[0049] The method for preparing the composite core includes the following steps:

[0050] (1) Boil the silkworm silk in a sodium carbonate solution with a mass-volume concentration of 0.5% for 30 min, repeat twice to completely remove the sericin, wash with deionized water, dry to form silk fibroin, dissolve the silk fibroin in a 9.3M lithium bromide solution with a mass-volume ratio of 1g:10mL, stir in a 60℃ water bath until completely dissolved, put the solution into a dialysis bag, retain the molecular weight cutoff of 10 kDa, dialyze in deionized water for 3 days, change the water every 6 hours to completely remove lithium bromide, finally, centrifuge the dialyzed silk fibroin solution at 9000 rpm for 20 min to remove impurities, and obtain a clear silk fibroin aqueous solution with a mass-volume concentration of 7%;

[0051] (2) Dissolve asiaticoside in DMSO to prepare a stock solution with a concentration of 1.5 mg / mL. Slowly add the stock solution to the silk fibroin aqueous solution. The final concentration of asiaticoside is 15 μg / mL. Stir magnetically for 2 hours to mix it evenly and form a mixed solution.

[0052] (3) The mixed solution was loaded into the syringe of the electrospinning equipment and electrospinned. The process parameters were set as follows: injection speed 0.8 mL / h, voltage 18 kV, receiving distance 15 cm, and ambient humidity controlled at 50%. After obtaining the nanofiber membrane on the receiving plate, it was placed in 75% ethanol vapor for 30 min to obtain the composite core.

[0053] The inclusion complex comprises the following raw materials in the following mass ratio: hydroxypropyl-β-cyclodextrin:curcumin = 7:1;

[0054] The method for preparing the inclusion complex includes the following steps:

[0055] (a) Hydroxypropyl-β-cyclodextrin was dissolved in deionized water at a ratio of 1 g to 120 mL to form a hydroxypropyl-β-cyclodextrin solution.

[0056] (b) Dissolve curcumin in anhydrous ethanol at a ratio of 1 g to 15 mL. Then, add the curcumin to the hydroxypropyl-β-cyclodextrin solution dropwise while stirring vigorously. Continue stirring in the dark for 24 h. Then filter and freeze dry to obtain the inclusion complex.

[0057] This embodiment also provides a method for preparing a composite hydrogel that promotes wound healing, specifically including the following steps:

[0058] S1. Dissolve type I collagen in a 0.1% acetic acid aqueous solution and stir overnight to prepare a 5 mg / mL collagen solution. Dissolve gelatin in PBS and stir in a 37 ℃ water bath to prepare a 10 mg / mL gelatin solution.

[0059] S2, mix collagen solution and gelatin solution at a volume ratio of 1:1, keep the temperature constant at 37 ℃ and stir gently to avoid generating bubbles, to form mixture i;

[0060] S3, SDF-1α mimic peptide was dissolved in PBS, and EDC and NHS were added as cross-linking agents. The mixture was cross-linked and activated at 4 °C for 30 min to form activated SDF-1α mimic peptide. The ratio of SDF-1α mimic peptide, EDC, NHS and PBS was 5 mg:7 mg:5 mg:1-2 mL. The activated SDF-1α mimic peptide was added to mixture i and reacted slowly at 4 °C for 12 h to allow the mimic peptide to be covalently grafted onto the amino groups of gelatin and collagen through amide bonds, thus obtaining mixture ii.

[0061] S4. Immerse the composite core in the mixture ii to ensure complete coverage, then transfer it to a constant temperature environment of 37 °C and let it stand for 1 h to induce cross-linking. At this temperature, collagen will spontaneously cross-link to form a stable network, and gelatin will also play an auxiliary role in gelation, thereby forming a strong stem cell recruitment middle layer outside the core. Gently rinse the surface with PBS to obtain the complex.

[0062] S5. Hyaluronic acid was dissolved in PBS to prepare a 1% (w / v) hyaluronic acid solution. 3-(acrylamido)phenylboronic acid, EDC, and NHS were added to the hyaluronic acid solution, wherein the volume ratio of 3-(acrylamido)phenylboronic acid, EDC, NHS, and hyaluronic acid solution was 0.12 g:0.5 g:0.3 g:100 mL. The reaction was carried out at room temperature in the dark for 24 h to graft the phenylboronic acid group onto the HA chain. After dialysis purification and freeze-drying, oxidized hyaluronic acid was obtained.

[0063] S6, Dissolve oxidized hyaluronic acid and its inclusion complex in PBS to form a pregel solution. Completely immerse the composite prepared in step S4 into the pregel solution, ensuring that the surface is fully and uniformly covered. After removal, irradiate it under 365 nm ultraviolet light for 60 s to carry out an ultraviolet cross-linking reaction, causing the acrylamide groups on the oxidized hyaluronic acid chain to undergo covalent cross-linking, forming a dense antioxidant outer layer on the outside of the composite structure. Rinse with PBS and then sterilize to obtain a composite hydrogel that promotes wound healing.

[0064] Example 3: This example provides a composite hydrogel that promotes wound healing. The composite hydrogel that promotes wound healing comprises the following raw materials in parts by weight: 80 parts composite core, 8 parts type I collagen, 15 parts gelatin, 0.3 parts SDF-1α mimic peptide, 35 parts hyaluronic acid, and 15 parts inclusion complex.

[0065] The composite core comprises raw materials in the following mass ratio: silkworm silk: asiaticoside = 1: 0.0002;

[0066] The method for preparing the composite core includes the following steps:

[0067] (1) Boil the silkworm silk in a sodium carbonate solution with a mass-volume concentration of 0.5% for 30 min, repeat twice to completely remove the sericin, wash with deionized water, dry to form silk fibroin, dissolve the silk fibroin in a 9.3M lithium bromide solution with a mass-volume ratio of 1g:10mL, stir in a 60℃ water bath until completely dissolved, put the solution into a dialysis bag, retain the molecular weight cutoff of 14 kDa, dialyze in deionized water for 3 days, change the water every few hours to completely remove lithium bromide, finally, centrifuge the dialyzed silk fibroin solution at 9000 rpm for 20 min to remove impurities, and obtain a clear silk fibroin aqueous solution with a mass-volume concentration of 8%;

[0068] (2) Dissolve asiaticoside in DMSO to prepare a stock solution with a concentration of 2 mg / mL. Slowly add the stock solution to the silk fibroin aqueous solution. The final concentration of asiaticoside is 20 μg / mL. Stir magnetically for 2 hours to mix it evenly and form a mixed solution.

[0069] (3) The mixed solution is loaded into the syringe of the electrospinning equipment, and electrospinning is performed. The process parameters are set as follows: injection speed 0.1 mL / h, voltage 20 kV, receiving distance 20 cm, and ambient humidity controlled at 50%. After obtaining the nanofiber membrane on the receiving plate, it is placed in 75% ethanol vapor for 30 min to obtain the composite core.

[0070] The inclusion complex comprises the following raw materials in the following mass ratio: hydroxypropyl-β-cyclodextrin:curcumin = 10:1;

[0071] The method for preparing the inclusion complex includes the following steps:

[0072] (a) Hydroxypropyl-β-cyclodextrin was dissolved in deionized water at a ratio of 1 g to 150 mL to form a hydroxypropyl-β-cyclodextrin solution.

[0073] (b) Dissolve curcumin in anhydrous ethanol at a ratio of 1 g to 20 mL. Then, add it dropwise to a hydroxypropyl-β-cyclodextrin solution while stirring vigorously. Continue stirring in the dark for 24 h. Then filter and freeze dry to obtain the inclusion complex.

[0074] This embodiment also provides a method for preparing a composite hydrogel that promotes wound healing, specifically including the following steps:

[0075] S1. Dissolve type I collagen in a 0.1% acetic acid aqueous solution and stir overnight to prepare an 8 mg / mL collagen solution. Dissolve gelatin in PBS and stir in a 37 ℃ water bath to prepare a 15 mg / mL gelatin solution.

[0076] S2, mix collagen solution and gelatin solution at a volume ratio of 1:1, keep the temperature constant at 37 ℃ and stir gently to avoid generating bubbles, to form mixture i;

[0077] S3, SDF-1α mimic peptide was dissolved in PBS, and EDC and NHS were added as cross-linking agents. The mixture was cross-linked and activated at 4 °C for 30 min to form activated SDF-1α mimic peptide. The ratio of SDF-1α mimic peptide, EDC, NHS and PBS was 5 mg:10 mg:5 mg:2 mL. The activated SDF-1α mimic peptide was added to mixture i and reacted slowly at 4 °C for 12 h to allow the mimic peptide to be covalently grafted onto the amino groups of gelatin and collagen through amide bonds, thus obtaining mixture ii.

[0078] S4. Immerse the composite core in the mixture ii to ensure complete coverage, then transfer it to a constant temperature environment of 37 °C and let it stand for 1 h to induce cross-linking. At this temperature, collagen will spontaneously cross-link to form a stable network, and gelatin will also play an auxiliary role in gelation, thereby forming a strong stem cell recruitment middle layer outside the core. Gently rinse the surface with PBS to obtain the complex.

[0079] S5. Hyaluronic acid was dissolved in PBS to prepare a 1% (w / v) hyaluronic acid solution. 3-(acrylamido)phenylboronic acid, EDC, and NHS were added to the hyaluronic acid solution, wherein the volume ratio of 3-(acrylamido)phenylboronic acid, EDC, NHS, and hyaluronic acid solution was 0.15 g:0.5 g:0.3 g:100 mL. The reaction was carried out at room temperature in the dark for 24 h to graft the phenylboronic acid group onto the HA chain. After dialysis purification and freeze-drying, oxidized hyaluronic acid was obtained.

[0080] S6, Dissolve oxidized hyaluronic acid and its inclusion complex in PBS to form a pregel solution. Completely immerse the composite prepared in step S4 into the pregel solution, ensuring that the surface is fully and uniformly covered. After removal, irradiate it under 365 nm ultraviolet light for 90 s to carry out an ultraviolet cross-linking reaction, causing the acrylamide groups on the oxidized hyaluronic acid chain to undergo covalent cross-linking, forming a dense antioxidant outer layer on the outside of the composite structure. Rinse with PBS and then sterilize to obtain a composite hydrogel that promotes wound healing.

[0081] The difference between Comparative Example 1 and Example 2 is that the composite kernel was removed; the rest is exactly the same as Example 2.

[0082] The difference between Comparative Example 2 and Example 2 is that the inclusion complex was omitted; otherwise, they are exactly the same as Example 2.

[0083] The difference between Comparative Example 3 and Example 2 is that the addition of the SDF-1α mimic peptide was omitted; otherwise, they are exactly the same as Example 2.

[0084] Experimental example:

[0085] 1. Cytotoxicity

[0086] HUVECs were seeded in 96-well plates and incubated with complete culture medium for 24 hours to allow for cell adhesion. Cells were then co-cultured with the composite hydrogels for promoting wound healing prepared in Examples 1-3 and Comparative Examples 1-3 of this invention in a CO2 cell culture incubator for 24 hours, followed by washing three times with sterile buffer. Then, 450 μL of fresh culture medium and 50 μL of Cell Counting Reagent-8 (CCK-8) reaction solution were added to each well, and the cells were incubated again in a CO2 cell culture incubator for approximately 2 hours. The absorbance at 450 nm was measured using a microplate reader, and the data were recorded and cell viability was calculated. The results are shown in Table 1.

[0087] 2. Representative Gram-positive and Gram-negative bacteria, Staphylococcus aureus and Escherichia coli, were selected to evaluate antibacterial performance. The composite hydrogels for promoting wound healing prepared in Examples 1-3 and Comparative Examples 1-3 of this invention were added to 5.00 mL of bacterial solution (bacterial concentration of 1.00 × 10⁷ CFUs / mL) and incubated at 37.0℃ in a shaker for 6 hours, with shaking at 150 rpm. Subsequently, each group of bacterial solutions was diluted 1.00 × 10⁵ times and further evenly spread on Luria Broth agar plates, incubated for 24 hours, and the colony count was recorded. The antibacterial rate of each group was calculated according to the following formula: R = [(N₀ - N) / N₀ × 100%]. The results are recorded in Table 1.

[0088] 3. Wound healing rate

[0089] Six-week-old female BALB / c mice were randomly divided into Example 1-3 and Comparative Example 1-3 groups. First, an acute full-thickness skin wound of approximately 8 mm was created on the backs of all mice after hair removal. Then, a pre-cooled composite hydrogel solution was uniformly sprayed onto the wound site. The medication was administered immediately after injury and again on the first day post-injury. The wound was observed and measured at specific time points, and the wound area was calculated using ImageJ software. The wound healing rate was calculated using the following formula: Wound healing rate = (Soriginal wound - Sremaining wound) / Soriginal wound × 100%. The results are recorded in Table 1. Skin tissue samples were collected from the mice.

[0090] 4. In vitro anti-inflammatory

[0091] Using the composite hydrogels for promoting wound healing prepared in Examples 1-3 and Comparative Examples 1-3 of this invention as samples, RAW264.7 cells in logarithmic growth phase were adjusted to a cell suspension with a density of 5 × 10⁴ cells / mL. The suspension was then added to 12-well cell culture plates, 1 mL per well, and cultured in an incubator. After cell adhesion, the original culture medium was discarded, and 1 mL of the corresponding fresh culture medium and sample extract (2 mg / mL) were added. The plates were incubated for 2 h, followed by the addition of LPS solution to a final concentration of 1 µg / mL and further cultured for 24 h. After culture, the cell supernatant was collected for inflammatory factor detection. Standards were prepared, samples were incubated and resuspended, and then analyzed by flow cytometry. Cytokine standards were transferred to 2 mL centrifuge tubes and serially diluted with Assay Diluent to a total volume of 300 µL. Centrifuge tubes containing only 300 µL of Assay Diluent were used as zero wells. The capture microspheres were pre-mixed thoroughly. Then, the microspheres, sample, and PE-labeled cytokine detection antibody were mixed at a 1:1:1 ratio and incubated at room temperature in the dark for 2 h. For detection, 100 µL of wash buffer was added to each centrifuge tube, and the mixture was thoroughly vortexed. The system was then used in a flow cytometer for analysis. Results are shown below. Figure 1 As shown.

[0092] Table 1: Performance Test Results of Composite Hydrogels Promoting Wound Healing

[0093]

[0094] As shown in Table 1, the cell survival rate of all groups was higher than 95%, indicating that the entire preparation system had good biocompatibility and that the addition of each component did not introduce cytotoxicity. The antibacterial rate of Comparative Example 2 was significantly lower than that of the other groups, proving that the addition of the inclusion complex enhanced the antibacterial properties of the composite hydrogel that promotes wound healing. The wound healing rate of Examples 1-3 reached 99.4%, indicating that the composite core, inclusion complex and other substances added in this invention provided a good environment for promoting wound healing.

[0095] Figure 1 The figure shows the results of the positive expression quantitative analysis of TNF-α, IL-6 and IL-10. As can be seen from the figure, the expression of TNF-α and IL-6 inflammatory factors in the Example 2 group was much lower than that in the untreated blank group. At the same time, the expression of IL-10 in the Example 2 group was much higher than that in the blank group. This indicates that the composite hydrogel for promoting wound healing prepared in this invention has good anti-inflammatory ability and provides favorable conditions for wound healing. Figure 2 The free radical scavenging rate of Comparative Example 2 was much lower than that of the other groups, indicating that the addition of the inclusion complex enhanced the antioxidant capacity of the composite hydrogel that promotes wound healing. Figure 3The images show HE and Masson staining of mouse wound tissue sections on day 7. The HE staining image reveals that on day 7, the Example 2 group exhibited dense granulation tissue and well-developed epithelial cells, while the control group did not show this phenomenon. In the Masson staining image, the Example 2 group showed significant collagen deposition and differentiation, while the control group still had residual blood clots. This indicates that the composite hydrogel prepared in this invention promotes wound healing, thereby facilitating collagen deposition in mouse wounds and accelerating wound healing.

[0096] In summary, the composite hydrogel preparation process for promoting wound healing in this invention uses natural bio-based materials as its core. The entire process, from raw material pretreatment to gradient molding, ensures both efficient retention of component activity and biocompatibility, providing a safe and reliable foundation for wound repair. The outer layer utilizes its oxidation-sensitive properties to achieve precise protection and release of active ingredients during the inflammatory phase, clearing obstacles for the repair process. The middle layer relies on the targeted recruitment of active peptides to activate endogenous repair potential, providing a cellular basis for tissue regeneration. The core layer uses a biomimetic structure to guide orderly collagen deposition, achieving highly efficient anti-scar repair. This design not only resolves the multiple contradictions in wound repair—infection control, inflammation regulation, and tissue remodeling—but also avoids the safety risks that exogenous components may bring, providing a new direction for the research and development of wound repair materials that combines theoretical value and application prospects.

[0097] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for preparing a composite hydrogel that promotes wound healing, characterized in that, The composite hydrogel that promotes wound healing comprises the following raw materials in parts by weight: 60-80 parts composite core, 3-8 parts type I collagen, 8-15 parts gelatin, 0.1-0.3 parts SDF-1α mimic peptide, 25-35 parts hyaluronic acid, and 8-15 parts inclusion complex. The composite core comprises raw materials in the following mass ratio: silkworm silk: asiaticoside = 1: 0.0001-0.0002; The method for preparing the composite core includes the following steps: (1) Degumming and purifying the silk of domestic silkworms to form silk fibroin fibers, dissolving the silk fibroin fibers in lithium bromide solution and stirring to obtain an aqueous solution of silk fibroin protein; (2) Dissolve asiaticoside in DMSO to prepare a stock solution. Slowly add the stock solution dropwise to the silk fibroin aqueous solution and stir magnetically to form a mixed solution. (3) The mixed solution was electrospun to form a nanofiber membrane, and then treated with ethanol vapor to obtain a composite core; The inclusion complex comprises the following raw materials in the following mass ratio: hydroxypropyl-β-cyclodextrin:curcumin = 3-10:1; The method for preparing the inclusion complex includes the following steps: (a) Hydroxypropyl-β-cyclodextrin is dissolved in deionized water to form a hydroxypropyl-β-cyclodextrin solution; (b) Dissolve curcumin in anhydrous ethanol, add it to a hydroxypropyl-β-cyclodextrin solution under vigorous stirring, stir in the dark, filter, freeze dry to obtain inclusion complex; The preparation method of the composite hydrogel that promotes wound healing specifically includes the following steps: S1, prepare a collagen solution from type I collagen and prepare a gelatin solution from gelatin; S2, mix and stir the collagen solution and gelatin solution at a volume ratio of 1:1 to form mixture i; S3, dissolve the SDF-1α mimic peptide in PBS, add EDC and NHS for cross-linking activation to form an activated SDF-1α mimic peptide, add the activated SDF-1α mimic peptide to mixture i to obtain mixture ii; S4, the composite core is immersed in the mixed solution ii for thermally induced crosslinking to obtain the composite; S5. Hyaluronic acid was dissolved in PBS to prepare a hyaluronic acid solution. 3-(acrylamido)phenylboronic acid, EDC and NHS were added to the hyaluronic acid solution. The reaction was carried out in the dark, purified by dialysis, and freeze-dried to obtain oxidized hyaluronic acid. S6, oxidized hyaluronic acid and its inclusion complex are dissolved in PBS to form a pregel solution. The complex prepared in step S4 is completely immersed in the pregel solution, and after being removed, it is cross-linked by ultraviolet light irradiation to obtain a composite hydrogel that promotes wound healing.

2. The method for preparing a composite hydrogel for promoting wound healing according to claim 1, characterized in that, In step (1), the ratio of the silk fibroin fiber to the lithium bromide solution is 1g:10mL; In step (3), the parameters for electrospinning are set as follows: injection speed 0.5-1 mL / h, voltage 15-20 kV, receiving distance 12-20 cm, and ambient humidity controlled at 40-50%.

3. The method for preparing a composite hydrogel for promoting wound healing according to claim 1, characterized in that, In step (a), the ratio of hydroxypropyl-β-cyclodextrin to deionized water is 1g:100-150mL; In step (b), the ratio of curcumin to anhydrous ethanol is 1g:10-20mL.

4. The method for preparing a composite hydrogel for promoting wound healing according to claim 1, characterized in that, In step S5, the ratio of the amount of 3-(acrylamido)phenylboronic acid, EDC, NHS and hyaluronic acid solution used is 0.1-0.15g:0.5g:0.3g:100mL.

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