Multifunctional composite hydrogel for integrally treating infected wounds as well as preparation method and application of multifunctional composite hydrogel

Abstract

The invention discloses multifunctional composite hydrogel for integrally treating infected wounds as well as a preparation method and application of the multifunctional composite hydrogel, and belongs to the technical field of medicines. The multifunctional composite hydrogel for integrally treating infected wounds is prepared from the following raw materials: polyhexamethylene biguanide grafted silk fibroin, acrylamide, N-isopropylacrylamide, salidroside, N, N-dimethylformamide, polyhexamethylene biguanide grafted polyhexamethylene biguanide grafted polyhexamethylene biguanide grafted polyhexamethylene biguanide grafted polyhexamethylene biguanide grafted polyhexamethylene biguanide grafted polyhexamethylene The composite hydrogel is prepared from N, N '-methylene bisacrylamide and water, has multiple functions including high air permeability, high tensile resistance, high adhesion, injectability, self-healing property and multiple responsiveness, and meets the mechanical property requirement of wound dressing and the biological requirement for promoting healing of infected wounds. The composite hydrogel has treatment effects in different stages of wound healing, and can integrally and efficiently solve the problems of slow healing of infected wounds, high scar formation risk and the like.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a multifunctional composite hydrogel for integrated treatment of infected wounds, and a preparation method and application thereof. Background Art

[0002] With the overuse of antibiotics and increasing bacterial resistance, the difficulty in treating infected wounds caused by bacteria (especially drug-resistant bacteria) has become a major challenge in global public health. The complex microenvironment of infected wounds, characterized by hypoxia, excessive wound exudate, accumulation of reactive oxygen species, overexpression of inflammatory cytokines, and impaired vascular regeneration, further hinders wound healing, making the treatment of infected wounds increasingly challenging. Therefore, the development of multifunctional wound dressings that combine antibacterial, anti-inflammatory, and antioxidant properties with the ability to promote wound repair is of great clinical significance. Hydrogels, due to their excellent hydrophilicity, biocompatibility, and three-dimensional network structure similar to that of the extracellular matrix, can effectively promote wound healing. Existing commercial hydrogel dressings have limited functionality, and most can only regulate a portion of the wound healing phase, failing to meet the needs of dynamic treatment of infected wounds throughout the entire healing process. Silk fibroin hydrogels, with their excellent biocompatibility, multiple bioactivities, high water absorption, and ability to regulate key wound healing signaling pathways, offer significant advantages in the wound dressing field. However, their weak antibacterial capacity and poor mechanical properties limit their application, making them unsuitable for the sole treatment of infected wounds. Therefore, the functionality and applicability of hydrogels can be significantly enhanced by combining silk fibroin with various polymers or incorporating bioactive molecules.

[0003] Wound healing requires close and orderly coordination of various stages. While skin wounds typically heal within a few days, larger wounds caused by infection may take weeks to heal, significantly increasing treatment costs. These chronic wounds often leave behind nonfunctional fibrotic scars that compromise tissue function. Therefore, the design and development of a novel wound dressing with strong antimicrobial activity and diverse biofunctionality is of great significance for the integrated clinical treatment of infected wounds. Summary of the Invention

[0004] The present invention proposes a multifunctional composite hydrogel for the integrated treatment of infected wounds, as well as its preparation method and application, which solves the problem of difficulty in wound healing due to infection. The multifunctional composite hydrogel of the present invention has multiple functions, including physical properties such as high air permeability, high tensile strength, high adhesion, injectability, self-healing and multiple responsiveness; as well as biological activities such as hemostasis, anti-oxidation, and promotion of angiogenesis. The multifunctional composite hydrogel of the present invention has a broad-spectrum and high-efficiency antibacterial activity, and can exert an antibacterial effect by changing the integrity of the bacterial cell plasma membrane. In addition, the multifunctional composite hydrogel of the present invention can exert anti-inflammatory activity through the NF-κB signaling pathway; it can also inhibit fibrosis by mediating the TGF-β / Smad3 / HMGB1 / RAGE signaling pathway, promote orderly wound healing, reduce the production of non-functional scars, and can treat infected wounds in an integrated manner at all stages.

[0005] To achieve the above objectives, the present invention provides the following technical solutions:

[0006] The present invention provides a multifunctional composite hydrogel for treating infected wounds in an integrated manner, which is composed of the following raw materials: modified silk fibroin, salidroside (Sal), acrylamide, N-isopropylacrylamide, N,N'-methylenebisacrylamide and water;

[0007] The modified silk fibroin is polyhexamethylene biguanide grafted silk fibroin.

[0008] The multifunctional composite hydrogel for the integrated treatment of infected wounds of the present invention has physical properties such as high air permeability, high tensile strength, high adhesion, injectability, self-healing and multiple responsiveness. It also has biological activities such as hemostasis, anti-oxidation, and angiogenesis promotion. At the same time, it has broad-spectrum and high-efficiency antibacterial activity and can exert antibacterial effects by changing the integrity of bacterial cell plasma membranes. The water vapor permeability of the multifunctional composite hydrogel for the integrated treatment of infected wounds of the present invention is 567.50-746.94 g / cm 2 ·24h, viscosity is 1.28-1.87×10 5 mPa·s, and the breaking strength is 65.51-248.16kPa.

[0009] Furthermore, the multifunctional composite hydrogel for treating infected wounds is composed of the following raw materials, calculated by weight: 1-2% modified silk fibroin, 1-5% salidroside, 8% acrylamide, 8% N-isopropylacrylamide, 0.8% N,N'-methylenebisacrylamide, and the balance water;

[0010] The modified silk fibroin is polyhexamethylene biguanide grafted silk fibroin.

[0011] Furthermore, the preparation method of the modified silk fibroin is as follows: glutaraldehyde is added to a silk fibroin (SF) aqueous solution, reacted for 0.5-2 hours, and a solution A is obtained; polyhexamethylene biguanide (PHMB) is added to the solution A, reacted for 8-12 hours, and a solution containing modified silk fibroin is obtained, that is, a solution containing polyhexamethylene biguanide grafted silk fibroin (SF-PHMB).

[0012] Furthermore, the concentration of the silk fibroin aqueous solution is 20 mg / mL;

[0013] And / or, the amount of glutaraldehyde added to the silk fibroin aqueous solution is 10 wt%;

[0014] And / or, the mass ratio of the silk fibroin to polyhexamethylene biguanide is (10-20):1.

[0015] Furthermore, the mass ratio of the silk fibroin to polyhexamethylene biguanide is 20:1.

[0016] The present invention also provides a method for preparing the above-mentioned integrated multifunctional composite hydrogel for treating infected wounds, comprising the following steps:

[0017] Acrylamide (AM), N-isopropylacrylamide (NIPAM), N,N'-methylenebisacrylamide (MBAA) and salidroside are added to a solution containing modified silk fibroin and reacted for 0.5 h to obtain solution C; ammonium persulfate (APS) is added to the solution C and reacted under heating conditions. After the reaction, a multifunctional composite hydrogel for treating infected wounds is obtained.

[0018] Furthermore, in the preparation method of a multifunctional composite hydrogel for the integrated treatment of infected wounds, the heating temperature is 60°C and the heating time is 0.5 h. Temperature is a key parameter in the hydrogel preparation process, directly affecting the polymerization rate, gel structure, and ultimate performance. APS has a moderate decomposition rate at 60°C, ensuring sufficient free radical generation to initiate polymerization while preventing runaway reaction. NIPAM and AM have suitable reactivity at this temperature, facilitating the formation of a uniform cross-linked network. MBAA, as a cross-linker, efficiently participates in polymerization at this temperature, forming a stable three-dimensional network structure. Furthermore, the addition of salidroside increases intermolecular hydrogen bonds, strengthens intermolecular interactions, and significantly enhances the water vapor permeability, viscosity, and breaking strength of the silk fibroin composite hydrogel. By precisely controlling the reaction temperature, the swelling ratio, mechanical strength, and thermoresponsiveness of the multifunctional composite hydrogel can be optimized to meet the needs of diverse application scenarios.

[0019] The present invention also provides the use of the above-mentioned integrated multifunctional composite hydrogel for treating infected wounds in the preparation of a medicine for treating wound infections.

[0020] The present invention also provides the use of the above-mentioned integrated multifunctional composite hydrogel for treating infected wounds in the preparation of a TGF-β / Smad3 signaling pathway inhibitor and / or an angiogenesis promoter.

[0021] The present invention also provides an integrated medicine for treating wound infection, the active ingredient of which is the above-mentioned multifunctional composite hydrogel for integrated treatment of infected wounds.

[0022] The present invention successfully constructed a composite hydrogel based on functionalized modified silk fibroin with excellent biocompatibility by modifying and controlling silk fibroin at the molecular level and loading it with the multi-active drug salidroside. The composite hydrogel has therapeutic effects at different stages of wound healing and can treat MRSA-infected wounds in an integrated manner throughout the entire wound healing process. The composite hydrogel has multiple functions, including physical properties such as high breathability, high tensile strength, high adhesion, injectability, self-healing, and multiple responsiveness; as well as biological activities such as hemostasis, antibacterial, antioxidant, anti-inflammatory, angiogenesis promotion, and fibrosis inhibition. It is highly consistent with the physical performance requirements of wound dressings and the biological needs of promoting the healing of infected wounds.

[0023] Compared with the prior art, the present invention has the following advantages and technical effects:

[0024] (1) The multifunctional composite hydrogel for the integrated treatment of infected wounds of the present invention uses polyhexamethylene biguanide grafted silk fibroin with acrylamide and N-isopropylacrylamide as the gel matrix, and can prepare a hydrogel that meets the requirements of wound dressings, such as high air permeability, high tensile strength, high adhesion, injectability, self-healing and multiple responsiveness, thereby solving the problem of poor mechanical properties of silk fibroin hydrogel.

[0025] (2) The multifunctional composite hydrogel for the integrated treatment of infected wounds of the present invention can exert an antibacterial effect by destroying the integrity of bacterial cell membranes and interfering with bacterial metabolism; the antibacterial rates against Escherichia coli, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus are all over 99.9%, which solves the problem of poor antibacterial performance of silk fibroin hydrogel.

[0026] (3) The multifunctional composite hydrogel for the integrated treatment of infected wounds of the present invention has good biocompatibility, hemostatic activity and antioxidant activity, and can be applied to wounds as a wound dressing; in addition, the composite hydrogel has a good swelling rate, can quickly adsorb red blood cells and effectively block bleeding wounds during the healing process of infected wounds, and absorb wound exudates to prevent the accumulation of exudates in the wound bed.

[0027] (4) The multifunctional composite hydrogel for the integrated treatment of infected wounds of the present invention can promote HUVECs tube formation and accelerate angiogenesis. In terms of anti-inflammatory, the composite hydrogel significantly inhibits the secretion of inflammatory cytokines such as IL-1β, IL-6, and TNF-α by RAW264.7 cells under LPS stimulation by regulating the NF-κB signaling pathway, thus solving the problem of weak anti-inflammatory activity of traditional wound dressings.

[0028] (5) The multifunctional composite hydrogel for the integrated treatment of infected wounds of the present invention can regulate fibroblast fibrosis, inhibit fibrosis by mediating the TGF-β / Smad3 / HMGB1 / RAGE signaling pathway, reshape the extracellular matrix, promote orderly wound healing, and reduce the formation of non-functional scars.

[0029] (6) The multifunctional composite hydrogel for the integrated treatment of infected wounds of the present invention contains salidroside. Compared with the hydrogel without salidroside, the multifunctional composite hydrogel for the integrated treatment of infected wounds of the present invention has a more obvious effect in promoting the healing of infected wounds. The wound can be completely healed in 14 days, and the regenerated granulation tissue is arranged in an orderly manner, which significantly reduces the formation of non-functional scars. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0031] Figure 1 Characterization diagrams of silk fibroin (SF), polyhexamethylene biguanide (PHMB), modified silk fibroin (SF-PHMB) in Example 3, the composite hydrogel (SP-P(AM-NIPAM)) in Comparative Example 1, and the multifunctional composite hydrogel for integrated treatment of infected wounds (SP-P(AM-NIPAM)@Sal5%) in Example 3; wherein, A is the H NMR spectrum of SF and SF-PHMB, B is the H NMR infrared spectrum of SF, PHMB, and SF-PHMB, and C is the SEM image of SP-P(AM-NIPAM) and SP-P(AM-NIPAM)@Sal5%;

[0032] Figure 2Rheological properties of the composite hydrogel (SP-P (AM-NIPAM)) in Comparative Example 1 and the multifunctional composite hydrogel (SP-P (AM-NIPAM) @ Sal5%) for integrated treatment of infected wounds in Example 3, wherein A is strain sweep, B is frequency sweep, C is temperature sweep, D is strain step sweep, G'SP-P (AM-NIPAM) represents the storage modulus of SP-P (AM-NIPAM), G"SP-P (AM-NIPAM) represents the loss modulus of SP-P (AM-NIPAM), G'SP-P (AM-NIPAM) @ Sal5% represents the storage modulus of SP-P (AM-NIPAM) @ Sal5%, and G"SP-P (AM-NIPAM) @ Sal5% represents the loss modulus of SP-P (AM-NIPAM) @ Sal5%;

[0033] Figure 3 The release rate of the multifunctional composite hydrogel for treating infected wounds in Example 3 under different conditions, MMP-2 represents matrix metalloproteinase-2;

[0034] Figure 4 The antioxidant properties of the blank group (control group), the model group (LPS group), the composite hydrogel (SP-P (AM-NIPAM)) and salidroside (Sal) in comparative example 1, and the multifunctional composite hydrogel for treating infected wounds (SP-P (AM-NIPAM) @ Sal5%) in Example 3, where A is the SOD level, B is the MDA level, C is the GSH content, and D is the GSH / GSSG ratio.

[0035] Figure 5 The hemostatic properties of the blank group (control group), the composite hydrogel (SP-P(AM-NIPAM)) in comparative example 1, and the multifunctional composite hydrogel for treating infected wounds (SP-P(AM-NIPAM)@Sal5%) in example 3 are shown, where A is the BCI index, B is the red blood cell adsorption rate, C is the blood loss, and D is the hemostasis graph;

[0036] Figure 6 The angiogenesis-promoting performance of the blank group (control group), the composite hydrogel (SP-P(AM-NIPAM)) and salidroside (Sal) in comparative example 1, and the multifunctional composite hydrogel (SP-P(AM-NIPAM)@Sal) for treating infected wounds in Example 3, wherein A is the tubule map, B is the number of blood vessels, and C is the length of blood vessels;

[0037] Figure 7The antibacterial properties of the blank group (control group), the composite hydrogel (SP-P(AM-NIPAM)) and salidroside (Sal) in comparative example 1, and the multifunctional composite hydrogel for treating infected wounds (SP-P(AM-NIPAM)@Sal5%) in example 3, wherein A is E. coli; B is S. aureus; and C is MRSA.

[0038] Figure 8 The anti-inflammatory properties of the blank group (control group), the model group (LPS group), the composite hydrogel (SP-P (AM-NIPAM)), salidroside (Sal) in comparative example 1, and the multifunctional composite hydrogel for treating infected wounds (SP-P (AM-NIPAM) @ Sal5%) in Example 3, wherein A is HMGB1, B is TNF-α, C is IL-1β, and D is IL-6;

[0039] Figure 9 The anti-fibrotic properties of the model group (LPS group) and the composite hydrogel (SP-P(AM-NIPAM)) and salidroside (Sal) in comparative example 1 and the multifunctional composite hydrogel for integrated treatment of infected wounds (SP-P(AM-NIPAM)@Sal5%) in Example 3 were compared, wherein A represents the TGF-β / Smad3 pathway and B represents the HMGB1 / RAGE pathway.

[0040] Figure 10 The figures show the healing effects of the blank group (control group), the vancomycin group (Van group), the composite hydrogel (SP-P(AM-NIPAM)) and salidroside (Sal) in Comparative Example 1, and the multifunctional composite hydrogel for treating infected wounds (SP-P(AM-NIPAM)@Sal5%) in Example 3, where A is the wound healing image at the same time, B is the wound area, and C is the wound healing rate.

[0041] Figure 11 The in vivo antibacterial effects of the blank group (control group), the vancomycin group (Van group), the composite hydrogel (SP-P(AM-NIPAM)) and salidroside (Sal) in Comparative Example 1, and the multifunctional composite hydrogel for integrated treatment of infected wounds (SP-P(AM-NIPAM)@Sal5%) in Example 3, wherein A is the observation result of wound colony plates at different times, and B is the number of wound colonies at different times;

[0042] Figure 12The tissue Masson staining analysis images (1000 μm) of the blank group (control group), vancomycin group (Van group), the composite hydrogel (SP-P(AM-NIPAM)) in comparative example 1, salidroside (Sal), and the multifunctional composite hydrogel for integrated treatment of infected wounds in Example 3 (SP-P(AM-NIPAM)@Sal5%). DETAILED DESCRIPTION

[0043] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0044] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0045] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0046] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0047] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0048] The embodiment of the present invention provides a multifunctional composite hydrogel for treating infected wounds in an integrated manner, which is composed of the following raw materials: modified silk fibroin, salidroside, acrylamide, N-isopropylacrylamide, N,N'-methylenebisacrylamide and water;

[0049] The modified silk fibroin is polyhexamethylene biguanide grafted silk fibroin.

[0050] The multifunctional composite hydrogel for the integrated treatment of infected wounds of the present invention has high air permeability, high tensile strength, high adhesion, injectability, self-healing and multiple responsiveness. At the same time, it has broad-spectrum and efficient antibacterial activity. By utilizing the cationic polymer polyhexamethylene biguanide (PHMB), it can combine with the negatively charged bacterial cell membrane, change the integrity of the bacterial cytoplasmic membrane and give the hydrogel excellent antibacterial effect. By loading salidroside, the hydrogel is given biological activities such as hemostasis, anti-oxidation, and promotion of angiogenesis; at the same time, the addition of salidroside increases the intermolecular hydrogen bonds, enhances the intermolecular interaction force, and significantly increases the water vapor permeability, viscosity and breaking strength of the silk fibroin composite hydrogel. The water vapor permeability of the multifunctional composite hydrogel for the integrated treatment of infected wounds of the present invention is 567.50-746.94g / cm 2 ·24h, viscosity is 1.28-1.87×10 5 mPa·s, and a breaking strength of 65.51-248.16 kPa, with excellent mechanical properties that meet the performance requirements of wound dressings.

[0051] In a preferred embodiment of the present invention, the multifunctional composite hydrogel for the integrated treatment of infected wounds is composed of the following raw materials, calculated by mass percentage: 1-2% modified silk fibroin, 1-5% salidroside, 4-12% acrylamide, 4-12% N-isopropylacrylamide, 0.2-0.8% N,N'-methylenebisacrylamide, and the balance is water; more preferably, it is composed of the following raw materials, calculated by mass percentage: 2% modified silk fibroin, 1-5% salidroside, 8% acrylamide, 8% N-isopropylacrylamide, 0.8% N,N'-methylenebisacrylamide, and the balance is water;

[0052] The modified silk fibroin is polyhexamethylene biguanide grafted silk fibroin.

[0053] In a preferred embodiment of the present invention, a modified silk fibroin preparation method comprises: adding glutaraldehyde to a 20 mg / mL silk fibroin aqueous solution, wherein the amount of glutaraldehyde added to the silk fibroin aqueous solution is 10 wt%, and reacting at room temperature for 0.5-2 hours to obtain a solution A; and adding polyhexamethylene biguanide to solution A, wherein the mass ratio of polyfibroin to polyhexamethylene biguanide is (1-20):1, and reacting at room temperature for 8-12 hours to obtain a solution containing the modified silk fibroin. More preferably, the mass ratio of silk fibroin to polyhexamethylene biguanide is 20:1.

[0054] An embodiment of the present invention further provides a method for preparing the above-mentioned integrated multifunctional composite hydrogel for treating infected wounds, comprising the following steps:

[0055] Acrylamide (AM), N-isopropylacrylamide (NIPAM), N,N'-methylenebisacrylamide (MBAA) and salidroside were added to a solution containing modified silk fibroin and reacted for 0.5 h to obtain solution C; ammonium persulfate (APS) was added to solution C and reacted at 60°C for 0.5 h. After the reaction, a multifunctional composite hydrogel for the integrated treatment of infected wounds was obtained.

[0056] In the preparation process of hydrogels, temperature is one of the key parameters, which directly affects the polymerization rate, gel structure and final performance. The decomposition rate of APS at 60°C is moderate, which can not only ensure the generation of sufficient free radicals to initiate polymerization, but also avoid reaction runaway; NIPAM and AM have suitable reactivity at this temperature, which is conducive to the formation of a uniform cross-linked network; MBAA, as a cross-linking agent, can efficiently participate in polymerization at this temperature to form a stable three-dimensional network structure. At the same time, the addition of salidroside increases intermolecular hydrogen bonds, enhances intermolecular interactions, and improves the mechanical properties of the hydrogel. By precisely controlling the reaction temperature, the swelling rate, mechanical strength and thermosensitive response performance of the multifunctional composite hydrogel can be optimized to meet the needs of different application scenarios.

[0057] The multifunctional composite hydrogel for treating infected wounds provided by the embodiments of the present invention can be used to prepare drugs for treating infected wounds. For example, the multifunctional composite hydrogel for treating infected wounds provided by the present invention can be used to prepare dressings for treating infected wounds.

[0058] The multifunctional composite hydrogel for the integrated treatment of infected wounds provided by the embodiments of the present invention can be used to prepare TGF-β / Smad3 signaling pathway inhibitors and / or angiogenesis promoters. The multifunctional composite hydrogel for the integrated treatment of infected wounds provided by the present invention can significantly downregulate the expression of TGF-β / smad3 and significantly promote angiogenesis; therefore, it can be used to prepare angiogenesis promoters and TGF-β / Smad3 signaling pathway inhibitors.

[0059] An embodiment of the present invention further provides a drug for treating wound infection, the active ingredient of which is the above-mentioned integrated multifunctional composite hydrogel for treating infected wounds.

[0060] The present invention prepares a multifunctional composite hydrogel for the integrated treatment of infected wounds that exhibits excellent swelling rate, water vapor permeability, porosity, and high tensile strength, self-healing, and adhesion properties. This composite hydrogel exhibits excellent biocompatibility, hemostatic activity, and antioxidant activity, and can promote tissue and blood vessel regeneration. Furthermore, the multifunctional composite hydrogel for the integrated treatment of infected wounds exhibits broad-spectrum and highly effective antibacterial activity, exerting its antibacterial effect by altering the integrity of bacterial cytoplasmic membranes. Furthermore, the multifunctional composite hydrogel for the integrated treatment of infected wounds can exert anti-inflammatory activity through the NF-κB signaling pathway and can inhibit fibrosis by mediating the TGF-β / Smad3 / HMGB1 / RAGE signaling pathway, promoting orderly wound healing and reducing the formation of non-functional scars. This hydrogel exhibits broad application prospects in rapid adhesion and sealing of infected wounds, hemostasis and antibacterial properties, anti-inflammatory and antioxidant properties, and promoting healing of infected wounds.

[0061] Unless otherwise specified, the room temperature in the present invention is 25±2°C.

[0062] All raw materials used in the examples of the present invention were purchased commercially. For example, acrylamide, N-isopropylacrylamide, salidroside, glycyrrhizin, and arctigenin were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; polyhexamethylene biguanide hydrochloride was purchased from Bid Pharmaceutical Technology Co., Ltd.; monoclonal antibodies TGF-β, smad3, p-smad3, α-SMA, COL1, P65, p-P65, IL-1β, HMGB1, and GAPDH, and goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Wuhan Abotek Co., Ltd.; matrix metalloproteinase-2 was purchased from Wuhan Sanying Biotechnology Co., Ltd.; DEMM culture medium was purchased from Thermo Fisher Scientific; fetal bovine serum was purchased from Serena Life Science Technology Development (Shanghai) Co., Ltd.; all chemical reagents were of analytical grade.

[0063] In the embodiment of the present invention, the complete culture medium is DEMM culture medium supplemented with 10 wt % fetal bovine serum.

[0064] It should be pointed out that the matters not described in detail in the present invention are conventional operating means in this field and are not the focus of the present invention.

[0065] The technical solution of the present invention is further illustrated by the following examples.

[0066] Examples 1-3

[0067] A method for preparing a multifunctional composite hydrogel for treating infected wounds in an integrated manner, comprising the following steps:

[0068] (1) A 25 wt% glutaraldehyde solution was added dropwise to 1 mL of a 20 mg / mL silk fibroin aqueous solution (the amount of glutaraldehyde added to the silk fibroin aqueous solution was 10 wt%) under stirring at a stirring speed of 1000 r / min and a titration speed of 1 mL / min. The mixture was reacted at room temperature for 1 h to obtain solution A.

[0069] (2) polyhexamethylene biguanide was added dropwise to solution A (the mass ratio of silk fibroin to polyhexamethylene biguanide was 20:1) under stirring at a stirring speed of 1000 r / min and a titration speed of 1 mL / min. The mixture was reacted at room temperature for 10 h to obtain solution B, which was a solution containing modified silk fibroin (polyhexamethylene biguanide grafted silk fibroin);

[0070] (3) Acrylamide, N-isopropylacrylamide, N,N'-methylenebisacrylamide and salidroside were added to solution B under stirring conditions at a speed of 1000 r / min and a titration speed of 1 mL / min, and reacted at room temperature for 0.5 h to obtain solution C; 10 μL of ammonium persulfate was dropped into solution C under stirring conditions at a speed of 1000 r / min and a titration speed of 1 mL / min, and reacted at 60°C for 0.5 h to obtain a multifunctional composite hydrogel for the integrated treatment of infected wounds.

[0071] The mass percentages of the components in the multifunctional composite hydrogels of Examples 1-3 are shown in Tables 1-3.

[0072] Table 1 Example 1

[0073]

[0074] Table 2 Example 2

[0075]

[0076]

[0077] Table 3 Example 3

[0078]

[0079] Example 4

[0080] The preparation method of the multifunctional composite hydrogel for integrated treatment of infected wounds is the same as that of Example 3, except that the concentration of the silk fibroin aqueous solution is 10 mg / mL (ie, the mass ratio of silk fibroin to polyhexamethylene biguanide in this example is 10:1).

[0081] Comparative Example 1

[0082] The preparation method of the hydrogel was the same as that of Example 3, except that salidroside was not added and the mixture was made up to 100% with water. The obtained hydrogel was designated as SP-P(AM-NIPAM).

[0083] Comparative Example 2

[0084] This comparative example provides a method for preparing arginine-grafted silk fibroin, the steps of which are as follows:

[0085] A 25 wt% glutaraldehyde solution was added to 1 mL of a 20 mg / mL silk fibroin aqueous solution (the amount of glutaraldehyde added to the silk fibroin aqueous solution was 10 wt%) under stirring at a stirring speed of 1000 r / min and a titration speed of 1 mL / min. The mixture was reacted at room temperature for 1 h to obtain solution A.

[0086] Arginine was added to solution A under stirring conditions (the mass ratio of silk fibroin to arginine was 20:1), the stirring speed was 1000 r / min, the titration speed was 1 mL / min, and the reaction was carried out at room temperature for 10 h to obtain solution B, which was the arginine-grafted silk fibroin solution.

[0087] Comparative Example 3

[0088] This comparative example provides a method for preparing metformin grafted silk fibroin, the steps of which are as follows:

[0089] A 25 wt% glutaraldehyde solution was added to 1 mL of a 20 mg / mL silk fibroin aqueous solution (the amount of glutaraldehyde added to the silk fibroin aqueous solution was 10 wt%) under stirring at a stirring speed of 1000 r / min and a titration speed of 1 mL / min. The mixture was reacted at room temperature for 1 h to obtain solution A.

[0090] Metformin was added to solution A under stirring conditions (the mass ratio of silk fibroin to metformin was 20:1), the stirring speed was 1000 r / min, the titration speed was 1 mL / min, and the reaction was carried out at room temperature for 10 h to obtain solution B, which was a solution of metformin-grafted silk fibroin.

[0091] Comparative Example 4

[0092] The preparation method of the composite hydrogel provided in this comparative example is the same as that in Example 3, except that the concentration of the silk fibroin aqueous solution is 100 mg / mL (ie, the mass ratio of silk fibroin to polyhexamethylene biguanide in this comparative example is 100:1).

[0093] Comparative Example 5

[0094] The preparation method of the composite hydrogel provided in this comparative example is the same as that in Example 3, except that salidroside is replaced by glycyrrhizin.

[0095] Comparative Example 6

[0096] The preparation method of the composite hydrogel provided in this comparative example is the same as that in Example 3, except that salidroside is replaced by arctigenin.

[0097] Taking the modified silk fibroin in Example 3 and Comparative Examples 2-3 as examples, the effects of different guanidine compounds grafted onto silk fibroin on the antibacterial effect of MRSA were verified. The experimental process was as follows: first, the density of the cultured bacterial suspension was tested by Nano-drop, and the density of the bacterial suspension was diluted to 10 using LB medium as the solvent. 8 CFU / mL; Take 100 μL of the cultured bacterial suspension in the solid culture medium, spread it evenly, add 50 μL of 0.2 mg / mL SF or SF-PHMB to the agar plate well, and incubate in a 37°C incubator for 20-24 hours. After the incubation, take out the culture plate and place it under the microscope to observe the inhibition zone and measure the diameter of the inhibition zone (as a blank control). In addition, the density of the cultured bacterial suspension was tested by Nano-drop, and the bacterial suspension density was diluted to 10 using LB medium as the solvent. 6 CFU / mL; Take a 24-well plate, add 500 μL of 0.4 mg / mL modified silk fibroin solution in Example 3, Comparative Example 2-Comparative Example 3 to each plate, add 500 μL PBS to the control group, then add 500 μL of diluted bacterial suspension to each well, place the plate in a shaker, and incubate at 37°C for 12 hours. After 12 hours, take out the plate, take out 100 μL of the bacterial suspension from each well of the 24-well plate, transfer it to a pre-sterilized 1.5 mL centrifuge tube, add 900 μL sterile PBS to dilute it 10 times, and then continue to dilute it 10 times. 8 Take 100 μL of the final dilution of 10 8 The bacterial suspension was evenly spread on solid culture medium and incubated for 18-24 hours. The suspension was removed, plated, photographed, and counted, and the antibacterial rate of the sample was calculated. Antibacterial rate (%) = (number of colonies on control culture medium - number of colonies on sample culture medium) / number of colonies on control culture medium × 100%. Specific results are shown in Table 4.

[0098] Table 4 Antibacterial effects of different guanidine compounds grafted onto silk fibroin against MRSA

[0099]

[0100] As shown in Table 4, polyhexamethylene biguanide-grafted silk fibroin exhibited the best antibacterial effect. Polyhexamethylene biguanide (PHMB) is a strongly cationic polymer that binds to negatively charged bacterial cell membranes, causing cytoplasmic leakage and ultimately killing the bacteria. PHMB also exhibits strong antibacterial activity, low cytotoxicity, and good biocompatibility. Therefore, polyhexamethylene biguanide-modified silk fibroin (SF-PHMB) was used in subsequent experiments.

[0101] Using the modified silk fibroin prepared in Examples 3-4 and Comparative Example 4 as examples, the effect of the amount of polyhexamethylene biguanide added on the grafting efficiency was verified (the mass ratio of silk fibroin to polyhexamethylene biguanide was 20:1, 10:1, and 100:1). The PHMB content was determined according to the "Hygiene Requirements for Guanidine Disinfectants" (GB / T26367-2020). PHMB was dissolved in deionized water to prepare working solutions of 1, 2, 4, 8, and 10 mg / mL. PHMB solution, sodium acetate trihydrate solution (10 wt%), and Eosin Y solution (0.024 wt%) were mixed in a volume ratio of 2:1:2.5:19.5. The absorbance was measured at a wavelength of 545 nm using a microplate reader. A standard curve was plotted and the PHMB content was calculated. PHMB reaction rate (%) = (total PHMB content - PHMB content in the supernatant) / total PHMB content × 100%.

[0102] The free amino group concentration was determined using the o-phthalaldehyde method. Dissolve 40 mg of o-phthalaldehyde in 1 mL of methanol, add 25 mL of 100 mmol / L sodium tetraborate, 2.5 mL of 10 wt% SDS, and 100 μL of β-mercaptoethanol, and dilute to 50 mL with water to prepare the o-phthalaldehyde (OPA) derivatization reagent. Take 150 μL of each L-lysine standard solution (1, 2, 4, 8, 10 mmol / L), add 3 mL of the OPA derivatization reagent, react at room temperature for 2 minutes, and measure the absorbance at 340 nm. A standard curve was drawn based on the absorbance corresponding to different L-lysine concentrations. Free amino group reaction rate (%) = (total free amino group content - free amino group content in the supernatant) / total free amino group content × 100%. See Table 5 for specific results.

[0103] Table 5 Effect of different amounts of polyhexamethylene biguanide added on grafting rate

[0104]

[0105] It can be seen from Table 5 that when the mass ratio of silk fibroin to polyhexamethylene biguanide is 20:1, the grafting effect of polyhexamethylene biguanide is the best.

[0106] Taking the composite hydrogels prepared in Example 3 and Comparative Examples 5-6 as examples, the cell migration promoting effect of the composite hydrogels loaded with different active ingredients was verified. The experimental process was as follows: DMEM complete medium was replaced with DMEM medium containing 2 wt% FBS. The purpose was to use a medium containing only low concentrations of serum to maintain the basic survival of the cells and eliminate the effect of serum on cell migration. L929 cells were cultured at a rate of 1×10 5 Cells were cultured in a 6-well plate at a density of 100 μL / well at 37°C and 5% CO2 until confluence. A 200 μL pipette tip was used to gently scratch the bottom of the plate perpendicularly to form a scratch. The old culture medium was then aspirated and the detached cells were washed with PBS. The washing step was repeated three times. 2 mL of the hydrogel extract containing 2 wt% FBS with different active ingredients at a loading concentration of 200 μM was added. The control group was treated with 2 wt% FBSDMEM culture medium. The cells were cultured for 24 hours. During this period, the cells were observed and photographed under an inverted phase contrast microscope at 24 hours. The area of the scratch between cells was measured using ImageJ software, and the cell migration rate was calculated. The specific results are shown in Table 6.

[0107] Table 6 Effect of composite hydrogels loaded with different active ingredients on promoting cell migration

[0108]

[0109] It can be seen from Table 6 that salidroside has the best effect in promoting cell migration.

[0110] The physical properties (viscosity, water vapor permeability, and breaking strength) of the multifunctional composite hydrogels for the integrated treatment of infected wounds prepared in Examples 1 to 3 were tested in accordance with the standard YY / T 1293.4-2016 “Contact Wound Dressings Part 4: Hydrocolloid Dressings”. The results are shown in Table 7.

[0111] Table 7 Physical properties of multifunctional composite hydrogel

[0112]

[0113] It can be seen from Table 7 that with the increase of salidroside content, the performance of the multifunctional composite hydrogel for the integrated treatment of infected wounds is enhanced.

[0114] Experimental Example 1 Rheological properties of multifunctional composite hydrogel for integrated treatment of infected wounds

[0115] Experimental method: The measurements were performed using a parallel plate rheometer at 25°C. The gap was maintained at 1 mm in all experiments. All hydrogels were formed on 6-well plates and strain amplitude scans were performed after incubation for 0.5 h to determine the linear viscoelastic range of all prepared hydrogels. During the scan, the angular frequency was set to 10 rad / s and the strain amplitude was set to 0.001-10%. Subsequently, all hydrogel samples were subjected to frequency sweep measurements with the strain amplitude (strain) set to 1% and the angular frequency range from 0.01 to 100 rad / s. Based on this, the storage modulus (G) and loss modulus (G") were plotted as a function of angular frequency.

[0116] The measurements were performed using a parallel-plate rheometer. The gap was maintained at 1 mm in all experiments. All hydrogels were formed in 6-well plates and subjected to strain amplitude sweeps after 0.5 h of incubation, set to 1% strain, over a temperature range of 25 to 60°C. The storage modulus (G′) and loss modulus (G″) were plotted as a function of temperature.

[0117] To further investigate the self-healing properties of the hydrogel, a continuous step-strain experiment was performed using a rheometer. At a constant angular frequency (10 rad / s), the strain was gradually increased from 1% to 300%, causing the gel to break. The strain was then reduced to 1% and a sweep test was performed to analyze the recovery of the hydrogel's elastic modulus. This process was repeated to monitor changes in the storage and loss moduli, with each stage lasting 120 seconds.

[0118] The results are as follows Figure 2 As shown in the figure, under the strain of 0.001%-10% and the frequency of 1-1000Hz, the G′ of the silk fibroin composite hydrogel is always greater than G″, and the viscoelasticity of the hydrogel is still maintained. The internal structure has not collapsed, and the storage modulus of SP-P(AM-NIPAM) is always higher than the storage modulus of SP-P(AM-NIPAM)@Sal 5%, which confirms that the elasticity and compressive strength of SP-P(AM-NIPAM) hydrogel are higher than those of SP-P(AM-NIPAM)@Sal 5% hydrogel. Under the temperature change of 25-50℃, the G′ of the silk fibroin composite hydrogel is always greater than G″, indicating that the hydrogel will not be destroyed under this temperature change, which is beneficial to the practical application of the hydrogel. As the temperature increases, the storage modulus of the silk fibroin composite hydrogel increases. This is because the higher the temperature, the tighter the molecular chains inside the hydrogel are entangled, which increases the elasticity of the hydrogel. As shown in the figure, the G′ of the silk fibroin composite hydrogel is always greater than G″, indicating that the hydrogel will not be destroyed under this temperature change, which is beneficial to the practical application of the hydrogel. As the temperature increases, the storage modulus of the silk fibroin composite hydrogel increases. This is because the higher the temperature, the tighter the molecular chains inside the hydrogel are entangled, which increases the elasticity of the hydrogel. Figure 2As shown in Figure (D), the self-healing properties of the hydrogel were evaluated through continuous step strain testing. When the hydrogel was subjected to a 1500% strain for 5 minutes, its internal structure changed, and the storage modulus became higher than the loss modulus. When the applied strain was stepped to 1%, the hydrogel returned to its initial state, demonstrating that the synthesized SP-P(AM-NIPAM)@Sal 5% hydrogel exhibited excellent self-healing properties.

[0119] Experimental Example 2 Release rate of multifunctional composite hydrogel for integrated treatment of infected wounds under different conditions

[0120] Experimental Method: Weigh 1g of each SP-P(AM-NIPAM)@Sal 5% hydrogel and place the hydrogel into a 3500D dialysis bag. Tie the ends of the dialysis bag with string. Place the bag in 50mL of PBS solution at pH 7.2, 5.4, and a MMP-2 concentration of 100ng / mL, respectively. Place the bag in a thermostatic oscillator at 100rpm and 37°C. 3) At predetermined time points, aspirate 1mL of sample solution for content determination and add 1mL of fresh PBS, maintaining the PBS volume in the centrifuge tube constant. In vitro release curves are plotted.

[0121] The results are as follows Figure 3As shown in the release experiment of SP-P(AM-NIPAM)@Sal 5% hydrogel, the release rate of salidroside was slow at pH = 7.4, with a cumulative release rate of 34.455±0.43% of the drug within 24 hours and a cumulative release rate of 83.76±0.75% at 168 hours, basically reaching a state of equilibrium. The sustained release process of SP-P(AM-NIPAM)@Sal 5% hydrogel shows that salidroside is encapsulated in the hydrophobic region within the hydrogel and gradually released into the hydrogel and surrounding medium. The inherent water absorption and swelling of the hydrogel promotes the release of the drug from the hydrophobic region, which is beneficial for the hydrogel to provide long-lasting anti-inflammatory and antioxidant effects when applied to infected wounds. Under acidic conditions of pH 5.4, the release process of the SP-P(AM-NIPAM)@Sal 5% hydrogel accelerated, releasing 53.45±1.17% of the drug within 24 hours, a higher cumulative release rate than at pH 7.4, and reaching nearly equilibrium at 144 hours. This is because the lower pH accelerates the hydrogel's water absorption and swelling, leading to more rapid drug exudation. Under acidic conditions of pH 5.4 and with the introduction of MMP-2 into the release medium, the release process of the SP-P(AM-NIPAM)@Sal 5% hydrogel was further accelerated, reaching a cumulative release rate of 63.60±0.44% within 24 hours and approaching equilibrium at 96 hours. The SP-P(AM-NIPAM)@Sal 5% hydrogel exhibits pH- and MMP-2-responsive release, facilitating rapid release of the active ingredient, salidroside, at the site of infection.

[0122] Experimental Example 3 Antioxidant properties of multifunctional composite hydrogel for integrated treatment of infected wounds

[0123] Taking the multifunctional composite hydrogel for treating infected wounds prepared in Example 3 (denoted as SP-P(AM-NIPAM)@Sal5%) as an example, the antioxidant properties of the multifunctional composite hydrogel for treating infected wounds of the present invention were tested. The test method is as follows: L929 cells in the logarithmic growth phase were cultured at a rate of 1×10 5Cells were seeded at a density of 1 mL / well in a 12-well plate and incubated in a 37°C, 5% CO2 incubator for 24 hours. The old culture medium was then discarded, and the experimental group was treated with 1 mL of the multifunctional composite hydrogel extract for the integrated treatment of infected wounds, while the blank group (control group) and the model group (LPS group) were treated with 1 mL of complete culture medium. After 24 hours, the old culture medium was discarded, and the model and experimental groups were treated with 1 mL of complete culture medium containing 1 μg / mL LPS (lipopolysaccharide), while the normal group was treated with 1 mL of complete culture medium and incubated at 37°C, 5% CO2 for 24 hours. After the incubation period, all liquid was discarded, and the cells were washed twice with PBS. The cells were lysed with lysis buffer in an ice-water bath, centrifuged (12,000 rpm, 5 minutes), and the supernatant was collected. Subsequent procedures were performed according to the instructions of the GSH, MDA, and SOD kits to measure GSH and MDA levels and SOD activity in L929 cells.

[0124] The present invention evaluated the antioxidant properties of the hydrogel by detecting the levels of MDA, SOD and GSH in L929 cells induced by LPS before and after hydrogel treatment. Figure 4 As shown. With the introduction of salidroside, the antioxidant activity of SP-P(AM-NIPAM)@Sal5% hydrogel was enhanced. Under LPS stimulation, the SOD level in L929 cells decreased significantly and the MDA level increased significantly. However, after treatment with SP-P(AM-NIPAM)@Sal 5% hydrogel, the SOD level of L929 cells returned to normal levels and the MDA expression of L929 cells was reduced (P < 0.001). To further explore the protective mechanism of the hydrogel, the expression of GSH was detected before and after cell treatment. LPS stimulation significantly reduced the expression of the antioxidant molecule GSH in L929 cells, and the average GSH level decreased to 6.99 μM. In contrast, after treatment with SP-P(AM-NIPAM) and SP-P(AM-NIPAM)@Sal 5% hydrogels, the average GSH level increased by 7.94 and 9.97 μM, respectively, increasing the expression of the antioxidant molecule GSH in L929 cells (P < 0.001).

[0125] Experimental Example 4: Hemostatic and angiogenic properties of multifunctional composite hydrogel for integrated treatment of infected wounds

[0126] The test method is as follows:

[0127] Gently drip 50 μL of sodium citrate anticoagulated sheep blood onto the surface of a 5 mm wide and 5 mm long rectangular hydrogel material. The sample was then placed in a 37°C constant temperature shaker and incubated for 10 minutes. An equal amount of deionized water was then slowly added along the wall of the culture dish and incubated for another 2 minutes. The color and state of the solution were recorded. Separately, 50 μL of sodium citrate anticoagulated sheep blood was added to an equal amount of deionized water and incubated for the same 2 minutes as a blank control group. Finally, the supernatant of each sample was measured for absorbance at a wavelength of 540 nm using a microplate reader, and the in vitro coagulation index was calculated.

[0128] 100 μL of hydrogel was added to a 96-well plate, and then 50 μL of citrated whole blood was added to the hydrogel surface. The cells were incubated on a shaker at 37°C for 10 minutes, and non-adherent erythrocytes were washed with PBS. The hydrogel was then transferred to a centrifuge tube containing 3 mL of deionized water and incubated at 37°C for 30 minutes to lyse the adherent erythrocytes. OD was measured at 540 nm, and a control of 50 μL of blood added to 3 mL of deionized water was used.

[0129] SD rats were anesthetized with 2wt% sodium pentobarbital solution as the anesthetic, and the dosage volume was 0.2mL / 100g body weight. The fur at the right lower abdomen of the injection site was wiped and disinfected with alcohol. Insert the needle with the needle tip bevel upward into the abdominal cavity, fix the needle, withdraw to confirm that the needle position is correct, and slowly inject the liquid. After the rat is fully anesthetized, remove the hair in the surgical area with a razor and disinfect it with iodine tincture, and then deiodinate with alcohol. Fully expose the rat's abdomen, cut the abdominal epidermis at the xiphoid process with tissue scissors, clamp the epithelial layer with tissue forceps, cut the muscle fascia layer by layer, open the abdominal cavity, wipe off excess blood with gauze, and remove the liver from the abdominal cavity to fully expose it. Construction of the liver laceration model: Clean gauze and quantitative filter paper were placed under the exposed liver. Use a scalpel to cut the liver to create a laceration. The incision is 3mm deep and 1cm long. After the incision is completed, the hydrogel material is immediately injected into the surface of the liver laceration. Observe the bleeding of the laceration, measure the bleeding time and weigh the amount of bleeding.After the experiment is completed, all animals are euthanized and safely handled.

[0130] A live / dead cell assay was used to assess cell viability. HUVECs were plated at 1 × 10 4 Cells were seeded at a density of 10 cells / mL in 24-well plates. After 24 hours of culture, live and dead cell assays were performed according to the manufacturer's protocol. Stained samples were observed under a fluorescence microscope.

[0131] Tube formation assay was performed to evaluate the angiogenesis-promoting ability of the hydrogel. Matrigel (10 mg / mL) was added to a 96-well plate and solidified at 37°C for 30 min. HUVECs were seeded in a 96-well plate (2 × 10 3The cells were incubated with the hydrogel extracts on Matrigel plates (per well of a custom-made 96-well microplate) for 8 hours. After 8 hours, the angiogenesis was observed under a microscope and the results were quantified and statistically analyzed using ImageJ software.

[0132] The present invention evaluates the hemostatic effect of the hydrogel and first determines the coagulation index of the hydrogel. The results are as follows: Figure 5 As shown, both hydrogels (hydrogels without active ingredient loading and hydrogels loaded with active ingredient (salidroside)) have excellent hemostatic activity. Moreover, with the introduction of salidroside, the coagulation index of SP-P(AM-NIPAM)@Sal 5% hydrogel is less than that of SP-P(AM-NIPAM) hydrogel, and the coagulation activity is stronger. The red blood cell adsorption rate of the two hydrogels was further measured. The results showed that both hydrogels can capture and adsorb red blood cells. In addition, the introduction of salidroside increased the red blood cell adsorption rate of the hydrogel. Finally, the in vivo hemostatic activity of the two hydrogels was measured. In the rat liver incision model, the hydrogel was injected at the wound. The wound bleeding volume of the blank treatment group was 638.3±149.6 mg, and the wound bleeding volume of the SP-P(AM-NIPAM)@Sal 5% hydrogel treatment group was reduced to 214.3±33.5 mg. The hydrogel quickly coagulated and reduced the amount of bleeding in the liver (P<0.001). In addition, due to the excellent adhesion effect of the multifunctional composite hydrogel for integrated treatment of infected wounds, it can adhere to the wound to prevent delayed bleeding.

[0133] The present invention also explores the angiogenesis-promoting effect of the multifunctional composite hydrogel for integrated treatment of infected wounds, such as Figure 6 As shown, the multifunctional composite hydrogel for the integrated treatment of infected wounds of the present invention can promote the formation of tubes by HUVECs cells, significantly increasing the number and length of newly formed blood vessels. The introduction of salidroside enhances the tube-forming ability of the multifunctional composite hydrogel for the integrated treatment of infected wounds, demonstrating its excellent ability to promote angiogenesis.

[0134] Experimental Example 5 Antibacterial properties of multifunctional composite hydrogel for integrated treatment of infected wounds

[0135] Experimental method: The plate colony count method was used to determine the lethality curve of the hydrogel against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and MRSA. The specific process was as follows: the bacteria were inoculated in 100 mL of LB medium and cultured on a constant temperature shaker at 37°C for 18 hours, centrifuged at 4°C and 4000g, and diluted to 10 with 0.85wt% sterile saline solution. 6CFU / mL: Add the hydrogel to a diluted bacterial suspension to a concentration of 0.1 mg / mL and incubate on a shaker (37°C, 150 rpm). Remove the bacterial suspension at different time points and perform a gradient dilution. Then, spread 100 μL of the diluted bacterial suspension on plate count agar. After 24 hours, count the colonies on the plate at each time point.

[0136] The present invention selected E. coli, S. aureus and MRSA as representative bacteria to investigate the antibacterial properties of the hydrogel. Figure 7 As shown. Compared with the PBS solution (Control) group, the multifunctional composite hydrogel group for treating infected wounds of Example 3 and the hydrogel group of Comparative Example 1 both exhibited excellent antibacterial properties. When the hydrogel addition ratio was 10wt%, the composite hydrogel of Comparative Example 1 was able to completely kill the three bacteria in 4 hours, while the multifunctional composite hydrogel for treating infected wounds of Example 3 was able to completely kill the three bacteria in 2 hours, demonstrating that the addition of salidroside further enhanced the antibacterial activity.

[0137] Test Example 6 Determination of anti-inflammatory activity of multifunctional composite hydrogel for integrated treatment of infected wounds

[0138] Experimental method: RAW264.7 macrophages were seeded in 12-well plates (1×10 5 / well), cultured at 37 ° C, 5% CO2 for 24 h to allow the cells to adhere, and continued to culture at 37 ° C, 5% CO2 for 24 h. Then, the old culture medium was aspirated and the following treatments were performed: the control group did not receive any treatment, the SP-P (AM-NIPAM) group was added with 1 mL of the hydrogel extract with an LPS concentration of 1 μg / mL, the Sal group was added with 1 mL of DMEM medium with a salidroside concentration of 200 μM and an LPS concentration of 1 μg / mL, and the SP-P (AM-NIPAM) @ Sal 5% group was added with 1 mL of the hydrogel extract with a salidroside concentration of 200 μM and an LPS concentration of 1 μg / mL. Then, culture was continued for 24 h, and the cell culture supernatant was collected and the release levels of IL-1β, II-6, TNF-α, and HMGB1 were determined according to the kit instructions.

[0139] Chronic wounds caused by infection are characterized by persistent inflammation. The migration and activation of macrophages in chronic wounds are greatly hindered, leading to overexpression of inflammatory cytokines such as TNF-α, IL-1β and IL-6. The present invention evaluates the anti-inflammatory activity of the hydrogel by measuring the expression of inflammatory cytokines HMGB1, TNF-α, IL-1β and IL-6 secreted by RAW264.7 cells. Figure 8As shown, compared with the LPS model group, the hydrogel can reduce the secretion of IL-1β and IL-6 mediated by LPS and reduce the direct stimulation of LPS on cells. The introduction of salidroside can further enhance the anti-inflammatory effect of the hydrogel. The hydrogel has a more significant effect on reducing the secretion of various inflammatory cytokines, indicating that salidroside plays an important role in the anti-inflammatory effect of silk fibroin composite hydrogel.

[0140] Experimental Example 7 Determination of anti-fibrotic activity of multifunctional composite hydrogel for integrated treatment of infected wounds

[0141] Experimental method: L929 cells in the logarithmic growth phase were cultured at a rate of 1×10 5 The cells were seeded into 12-well plates at a density of 1 mL / well and cultured in a 37°C, 5% CO2 incubator for 24 hours. The old culture medium was then discarded, and the SP-P(AM-NIPAM) group was added with 1 mL of the hydrogel extract at an LPS concentration of 1 μg / mL. The Sal group was added with 1 mL of DMEM medium containing 200 μM salidroside and 1 μg / mL LPS. The SP-P(AM-NIPAM)@Sal 5% group was added with 200 μM salidroside and 1 mL of the hydrogel extract at an LPS concentration of 1 μg / mL, and then cultured for another 24 hours. The control group was added with 1 mL of complete culture medium and incubated at 37°C, 5% CO2 for 24 hours. After incubation, all liquid was aspirated and washed twice with PBS. The cells were lysed with lysis buffer in an ice-water bath, centrifuged (12,000 r / min, 5 min), and the supernatant was collected for Western Blot analysis.

[0142] The TGF-β / Smad3 signal transduction pathway is closely related to skin fibrosis. The TGF-β / Smad3 signal transduction pathway acts on fibroblasts, causing them to differentiate into myofibroblasts, leading to an increase in α-SMA and type I collagen. The present invention explores the mechanism by which hydrogels inhibit fibrosis and measures the inhibitory effect of hydrogels on fibrosis before and after LPS treatment. Figure 9 As shown, the multifunctional composite hydrogel for treating infected wounds in the present invention can inhibit the fibrosis of fibroblasts by inhibiting the HMGB1 / RAGE / TGF-β / Smad3 signaling pathway, and can be used to promote scarless healing of infected wounds.

[0143] Experimental Example 8 In vivo pharmacodynamics of multifunctional composite hydrogel for integrated treatment of infected wounds

[0144] Experimental method: SD rats were anesthetized by intraperitoneal injection and then their back hair was shaved with an electric shaver. Then, a circular wound with a diameter of approximately 6 mm was made on the back of each rat. To simulate the infection environment, 100 μL of a suspension containing methicillin-resistant Staphylococcus aureus (1×106 CFU / mL) was dripped onto the wound. After the experimental group was rinsed with normal saline, they were treated with the composite hydrogel of Comparative Example 1, salidroside and the multifunctional composite hydrogel for treating infected wounds of Example 3. In the control group, the experimental subjects were only rinsed with normal saline and no other treatment measures were applied. All operating procedures performed in the present invention strictly followed the Guidelines for the Care and Use of Laboratory Animals formulated by the National Research Council. The wounds of different groups were photographed and recorded with a camera at 0d, 3d, 5d, 7d and 14d after surgery, and the wound area was quantified by Image J software for comparison.

[0145] On days 1, 5, 7, and 14, rats were euthanized by inhalation of an overdose of isoflurane, and wound tissues were removed. The tissues were ground and evenly spread on TSB solid culture medium. The number of colonies was counted after incubation at 37°C for 24 hours.

[0146] On the fourteenth day of the experiment, the tissues were fixed with 4 wt % paraformaldehyde solution, and then sectioned, stained with H&E and Masson staining, and other pathological indicators were detected, and immunofluorescence staining analysis was performed.

[0147] The wound morphology on the 3rd, 5th, 7th and 14th days after surgery was as follows Figure 10 As shown, the wound surface gradually decreased over time in all treatment groups. The hydrogel possesses excellent moisturizing properties and an ideal water vapor permeability, maintaining the moist environment necessary for wound healing, promoting granulation tissue proliferation, and accelerating skin wound healing. SP-P(AM-NIPAM)@Sal 5% demonstrated the best therapeutic effect across all time periods, demonstrating a significant advantage in promoting wound healing in MRSA-infected patients. Its moist environment and excellent antibacterial, anti-inflammatory, and antioxidant properties further promoted wound healing.

[0148] Bacteria were isolated from the wound surface on days 1, 5, 7, and 14 after treatment, and the number of colonies was calculated by plate counting method, e.g. Figure 11 As shown, the number of bacterial colonies in each treatment group decreased over time. The wound treated with 5% SP-P(AM-NIPAM)@Sal hydrogel exhibited the lowest bacterial count. The in vivo antibacterial rate exceeded 99% at all time points, surpassing that of the positive control vancomycin-treated group, demonstrating a long-term in vivo antibacterial effect.

[0149] The wound tissue was examined histologically to observe the regeneration process of the wound in each group and to evaluate the healing effect of the hydrogel on the infected wound in vivo. Figure 12) showed that while the control group had not yet completed epithelialization, the hydrogel group showed significant new granulation tissue and regenerated hair follicles, accelerating the formation of skin appendages such as sebaceous glands. The newly formed collagen fibers were densely and orderly arranged, and the wound area returned to normal tissue, demonstrating that the hydrogel can promote the healing of infected wounds and inhibit fibrosis, achieving the best therapeutic effect in promoting the repair of infected wounds.

[0150] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A multifunctional composite hydrogel for integrated treatment of infected wounds, characterized in that: The invention is composed of the following raw materials: modified silk fibroin, salidroside, acrylamide, N-isopropylacrylamide, N,N'-methylenebisacrylamide and water; The modified silk fibroin is polyhexamethylene biguanide grafted silk fibroin.

2. The multifunctional composite hydrogel for treating infected wounds according to claim 1, characterized in that: The invention is composed of the following raw materials according to mass percentage: 1-2% modified silk fibroin, 1-5% salidroside, 4%-12% acrylamide, 4-12% N-isopropylacrylamide, 0.2-0.8% N,N'-methylenebisacrylamide, and the balance is water; The modified silk fibroin is polyhexamethylene biguanide grafted silk fibroin.

3. The multifunctional composite hydrogel for treating infected wounds according to claim 1, characterized in that: The modified silk fibroin preparation method comprises: adding glutaraldehyde to a silk fibroin aqueous solution, reacting for 0.5-2 hours to obtain solution A; adding polyhexamethylene biguanide to the solution A, reacting for 8-12 hours to obtain a solution containing the modified silk fibroin.

4. The multifunctional composite hydrogel for treating infected wounds according to claim 3, characterized in that: The concentration of the silk fibroin aqueous solution is 20 mg / mL; And / or, the amount of glutaraldehyde added to the silk fibroin aqueous solution is 10 wt%; And / or, the mass ratio of the silk fibroin to polyhexamethylene biguanide is (10-20):

1.

5. The multifunctional composite hydrogel for treating infected wounds according to claim 4, characterized in that: The mass ratio of the silk fibroin to polyhexamethylene biguanide is 20:

1.

6. A method for preparing the multifunctional composite hydrogel for treating infected wounds according to any one of claims 1 to 5, characterized in that: The following steps are involved: Acrylamide, N-isopropylacrylamide, N,N'-methylenebisacrylamide and salidroside are added to a solution containing modified silk fibroin and reacted for 0.5 h to obtain solution C; ammonium persulfate is added to the solution C and reacted under heating conditions. After the reaction, a multifunctional composite hydrogel for treating infected wounds is obtained.

7. The method for preparing the multifunctional composite hydrogel for treating infected wounds according to claim 6, characterized in that: The heating temperature is 60° C. and the heating time is 0.5 h.

8. Use of the integrated multifunctional composite hydrogel for treating infected wounds according to any one of claims 1 to 5 in the preparation of a drug for treating wound infections.

9. Use of the multifunctional composite hydrogel for treating infected wounds according to any one of claims 1 to 5 in the preparation of a TGF-β / Smad3 signaling pathway inhibitor and / or an angiogenesis promoter.

10. An integrated drug for treating wound infection, characterized in that: The active ingredient is the multifunctional composite hydrogel for treating infected wounds as described in any one of claims 1 to 5.

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