Antibacterial hydrogel wound dressing capable of self-supplying hydrogen sulfide and promoting wound healing as well as preparation method and application of antibacterial hydrogel wound dressing

Abstract

The invention discloses a hydrogen sulfide self-supply antibacterial hydrogel wound dressing for promoting wound healing as well as a preparation method and application thereof, and relates to the technical field of medical dressings. The method comprises the following steps: uniformly dispersing CMCS and epsilon-PL-SATO in a PBS buffer solution, placing the PBS buffer solution in a tube A of a two-component injector, uniformly dispersing OHA and NAC in the PBS buffer solution, and placing the PBS buffer solution in a tube B of the two-component injector; the components in the tube A and the tube B are mixed and reacted by extruding a push handle of an injector, and an obtained product is the antibacterial hydrogel wound dressing capable of promoting wound healing and self-supplying hydrogen sulfide. According to the hydrogel prepared by the invention, oxidized hyaluronic acid and carboxymethyl chitosan are taken as matrixes, and an H2S donor (epsilon-PL-SATO) and an initiator NAC thereof are loaded to realize rapid gelation (the gelation time is lt: 30 seconds). The hydrogel can be rapidly formed and is suitable for clinical application such as injection and wound treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of medical dressings, and in particular relates to an antibacterial hydrogel wound dressing that promotes wound healing and can self-supply hydrogen sulfide, and a preparation method and application thereof. Background Art

[0002] Skin is the largest organ in the human body, protecting it from external aggression, electrolyte loss, and evaporation of body water. Skin regeneration in normal wounds mainly undergoes four overlapping but distinct phases, including hemostasis, inflammation, cell proliferation, and matrix remodeling. However, the healing process of chronic wounds caused by bacterial infection often violates this schedule. Wound-prone bacterial infection has now become a major medical threat and challenge, especially Staphylococcus aureus-related skin wound infection, which often leads to serious consequences.

[0003] Although inflammation is a critical stage in the wound healing process and the inflammatory response is considered to be a key stage for the body to eliminate infectious bacteria and create a tissue environment conducive to regeneration and repair, long-term inflammation may still cause tissue damage and delay wound healing.

[0004] Macrophages are one of the most important inflammatory cells in the wound healing process. They can be activated into two different phenotypes: M1 phenotype (inflammatory phenotype) and M2 phenotype (anti-inflammatory phenotype). In infected wounds, macrophage dysfunction leads to a persistent inflammatory environment and hinders tissue repair. Therefore, drugs that can adjust macrophages from M1 to M2 may promote inflammation resolution and promote wound healing. Hydrogen sulfide (H2S) is a newly discovered gaseous signaling molecule that shows great therapeutic potential in biomedical research due to its anti-inflammatory effect. As a small molecule signaling molecule, H2S can penetrate the cell membrane and exert an immediate therapeutic effect. One of the mechanisms of its therapeutic effect is to promote the transformation of macrophage phenotype to M2 macrophages. However, commonly used H2S donors release H2S uncontrolled, which can lead to acute cytotoxicity and drug waste.

[0005] Therefore, if the injectable self-healing hydrogel dressing can fight against bacteria that infect the wound and can self-supply slow-release hydrogen sulfide gas, it is expected to promote the healing process of infected wounds. However, how to prepare a hydrogel dressing that can slowly and controlled release hydrogen sulfide gas is also a challenge. Summary of the invention

[0006] In order to solve the deficiencies in the above-mentioned background technology, the present invention mainly focuses on the low bioavailability of traditional hydrogen sulfide donors, such as uncontrolled release rate in physiological environment. The present invention provides a wound healing antibacterial hydrogel wound dressing that self-supplies hydrogen sulfide, and a preparation method and application thereof. The present invention improves the extremely poor solubility of SATO by coupling ε-polylysine with S-arylthioxime (SATO) to form an H2S donor (ε-PL-SATO), and the donor can responsively release H2S under the action of N-acetylcysteine ​​(NAC).

[0007] In order to achieve the above objectives, the first objective of the present invention is to provide a method for preparing a wound healing-promoting antibacterial hydrogel wound dressing that self-supplies hydrogen sulfide, characterized in that it comprises the following steps: Under ice bath conditions, prepare sodium hydroxylamine-O-sulfonate solution and sodium thiobenzoate solution respectively; Under ice bath conditions, add the sodium hydroxylamine-O-sulfonate solution to the sodium thiobenzoate solution and stir for 20 to 40 minutes to obtain S-benzoylthiohydroxylamine; S-benzoylthiohydroxylamine is added to dichloromethane, and then 4-formylbenzoic acid and TFA are added, reacting at room temperature for 1 to 5 hours, and then purifying to obtain SATO powder; SATO, EDC·HCl and NHS were dissolved in a mixed solvent of water and DMSO, and after uniform mixing, ε-PL was added, and the mixture was stirred at room temperature for 24 to 36 hours, and then ε-PL-SATO was obtained by purification; Disperse hyaluronic acid in water, add NaIO4, stir for 4-6 hours, then add ethylene glycol to terminate the oxidation reaction, and purify the reactant to obtain OHA; CMCS and ε-PL-SATO are uniformly dispersed in PBS buffer and placed in tube A of a two-component syringe, and OHA and NAC are uniformly dispersed in PBS buffer and placed in tube B of a two-component syringe; by squeezing the push handle of the syringe, the components of tubes A and B are mixed and reacted, and the resulting product is an antibacterial hydrogel wound dressing that promotes wound healing and self-supplies hydrogen sulfide.

[0008] Preferably, the sodium hydroxylamine-O-sulfonate solution is prepared by adding hydroxylamine-O-sulfonate to a sodium hydroxide solution; the sodium thiobenzoate solution is prepared by adding thiobenzoic acid to a sodium hydroxide solution; wherein the concentration of the sodium hydroxylamine-O-sulfonate solution is 1-2 mol / L; and the concentration of the sodium thiobenzoate solution is 0.5-1 mol / L.

[0009] Preferably, when preparing S-benzoylthiohydroxylamine, the mass ratio of the sodium hydroxylamine-O-sulfonate solution to the sodium thiobenzoate solution is 1:1-2.

[0010] Preferably, when preparing SATO powder, the mass ratio of S-benzoylthiohydroxylamine to 4-formylbenzoic acid is 1:1-2.

[0011] Preferably, when preparing SATO powder, after reacting for 1 to 5 hours, the reactant is filtered, the solvent is removed under reduced pressure, and the crude product is recrystallized from ethyl acetate to obtain a crude product; the crude product is then dissolved in dichloromethane and purified by column chromatography to obtain SATO powder.

[0012] Preferably, the mass ratio of SATO, EDC·HCl, NHS and ε-PL is 3:4.6:2.3:1-2; and the volume ratio of water and DMSO in the mixed solvent is 4-5:1.

[0013] Preferably, the mass ratio of hyaluronic acid to NaIO4 is 1-2:1.

[0014] Preferably, the mass ratio of the CMCS to the ε-PL-SATO is 0.6-6:1; the mass ratio of the OHA to the NAC is 0.6-6:1.

[0015] The second object of the present invention is to provide an antibacterial hydrogel wound dressing that promotes wound healing and self-supplies hydrogen sulfide.

[0016] The third object of the present invention is to provide an antibacterial hydrogel wound dressing that promotes wound healing and self-supplies hydrogen sulfide for use in the preparation of infectious wound treatment drugs.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides an antibacterial hydrogel wound dressing that promotes wound healing and can self-supply hydrogen sulfide, as well as a preparation method and application thereof. The present invention improves the extremely poor solubility of SATO by coupling ε-polylysine with S-arylthioxime (SATO) through a H2S donor (ε-PL-SATO), and the donor can responsively release H2S under the action of N-acetylcysteine ​​(NAC).

[0018] The hydrogel prepared by the present invention is composed of oxidized hyaluronic acid and carboxymethyl chitosan as the matrix, and is loaded with H2S donor (ε-PL-SATO) and its initiator NAC to achieve rapid gelation (gel time <30 seconds). This enables the hydrogel to be quickly formed and is suitable for clinical applications such as injection and wound treatment.

[0019] The H2S released by the hydrogel prepared by the present invention regulates the phosphatidylinositol 3-kinase (PI3K) / protein kinase B (Akt) signaling pathway and inhibits the activation of nuclear factor κB (NF-κB), and promotes the polarization of macrophages from the M1 type to the anti-inflammatory and pro-proliferative M2 type, thereby reducing the inflammatory response at the wound and achieving the effects of anti-inflammatory and promoting wound repair.

[0020] The hydrogel prepared by the invention inhibits bacterial growth at the wound by utilizing the broad-spectrum antibacterial properties of chitosan and polylysine, reduces the risk of infection, and provides a good sterile environment for wound healing.

[0021] The H2S released by the hydrogel prepared by the present invention not only reduces inflammation, but also promotes angiogenesis and collagen deposition, accelerating wound healing. This innovation combines the multiple effects of H2S in wound regeneration.

[0022] The hydrogel of the present invention is designed to have good biocompatibility, rapid gelation properties and injectability, and can be suitable for wounds of different types and shapes, including infected wounds and acute wounds.

[0023] The present invention designs a H2S donor containing ε-polylysine-S-arylthioxime (ε-PL-SATO), and synthesizes the H2S donor ε-PL-SATO by coupling ε-polylysine with S-arylthioxime (SATO), which can respond to the action of NAC initiator and control the slow release of H2S. The donor reacts with carboxymethyl chitosan, oxidized hyaluronic acid and N-acetylcysteine ​​(NAC) initiator to form a hydrogel, which can achieve long-term and sustained release of H2S at the wound site.

[0024] The present invention provides a technical solution, and H2S is slowly released from the hydrogel, solving the problems of low bioavailability and short action time. Then, wound treatment requires the material to have the ability to form quickly, which can quickly seal the wound and improve the comfort of the patient. The hydrogel system of the present invention uses oxidized hyaluronic acid and carboxymethyl chitosan as the matrix, which can quickly gel within 30 seconds and is suitable for rapid clinical application. Through this technology, the hydrogel can quickly cover the wound, form a protective barrier, and avoid the risk of bacterial infection.

[0025] The present invention inhibits bacterial growth at the wound site through the broad-spectrum antibacterial properties of chitosan and polylysine in the hydrogel, reduces the risk of infection, and provides a good sterile environment for wound healing. Subsequently, the system can slowly release H2S for a long time and continuously. By regulating the phenotype of macrophages, by regulating the PI3K / Akt signaling pathway and inhibiting the activation of NF-κB, macrophages are promoted to polarize toward the anti-inflammatory and pro-proliferative M2 type, further reducing the inflammatory response at the wound site. Finally, the released H2S can promote angiogenesis and collagen deposition, help damaged tissues restore their structure and function, accelerate wound healing, and achieve effective gas therapy for infected wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1Schematic diagram of OC@ε-PL-SATO hydrogel treating skin damage in the rat infected wound model of the present invention.

[0027] Figure 2 Schematic diagram of the synthesis of the hydrogen sulfide donor ε-Poly-L-lysine-SATO (ε-PL-SATO) in the present invention.

[0028] Figure 3 It is a schematic diagram of the release of H2S by the hydrogen sulfide donor ε-Poly-L-lysine-SATO (ε-PL-SATO) in the present invention.

[0029] Figure 4 The nuclear magnetic resonance, ultraviolet and hydrogen sulfide release capacity diagrams of the hydrogen sulfide donor in the present invention are: Figure 4 A is the H NMR spectrum of ε-Poly-L-lysine (ε-PL) and ε-Poly-L-lysine-SATO (ε-PL-SATO). Figure 4 B is the UV-Vis spectra of ε-PL, ε-PL-SATO and SATO, Figure 4 C is the hydrogen sulfide release performance of ε-PL-SATO and SATO.

[0030] Figure 5 The synthesis schematic diagram, NMR and IR spectra of the chitosan-hyaluronic acid hydrogel of the present invention are shown in FIG. Figure 5 A is a schematic diagram of the synthesis process of oxidized hyaluronic acid (OHA) and its reaction with carboxymethyl chitosan (CMCS) through Schiff base to generate OHA-CMCS hydrogel. Figure 5 B is the H NMR spectrum of HA and OHA, Figure 5 C is the infrared spectra of CMCS, HA, OHA and OHA-CMCS.

[0031] Figure 6 It is a schematic diagram of the gelation process of the OC@ε-PL-SATO hydrogel of the present invention.

[0032] Figure 7 This is a picture of the OC@ε-PL-SATO hydrogel prepared in the present invention being injected through a needle.

[0033] Figure 8 This is a microscopic morphology of OC hydrogels with different components in Example 1.

[0034] Fig. 9 This is the H2S release performance of the OC@ε-PL-SATO hydrogel of the present invention.

[0035] Fig.10 is the rheological properties experimental result in Experimental Example 2, wherein: Fig.10 A is the amplitude scanning experimental result of OC series hydrogels. Fig.10 B is the frequency scanning experimental result of OC series hydrogels. Fig.10 C is the line graph of the viscosity of OC series hydrogels changing with shear rate. Fig.10 D is the alternating strain scanning experimental result of OC@ε-PL-SATO hydrogel. Fig.10 E is the self-healing macroscopic photograph of OC@ε-PL-SATO.

[0036] Fig.11 The biocompatibility test results of OC hydrogels with different components in Experimental Example 2, where: Fig.11 A is the bar graph of the survival rate of L929 cells by OC series hydrogels. Fig.11 B is the hemolysis rate of OC@ε-PL-SATO hydrogel on red blood cells, Fig.11 C is a picture of the scratch repair ability of OC series hydrogels on L929 cells.

[0037] Fig.12 The antibacterial effect of the compound on Escherichia coli (E.coli) and Staphylococcus aureus (S. aureus) in Experimental Example 2 is shown in FIG. Fig.12 A is the line graph of the inhibitory ability of OC series hydrogels on Escherichia coli. Fig.12 B is the line graph of the inhibitory ability of OC series hydrogels on Staphylococcus aureus. Fig.12 C is the antibacterial picture of OC series hydrogel.

[0038] Fig.13 The results of the experimental study on the polarization performance of OC hydrogels with different compositions on RAW264.7 cells in Experimental Example 2 are as follows: Fig.13 A&B&C are flow cytometry detection and quantitative statistics of polarized RAW264.7 cells, where: Fig.13 D is the ELISA analysis result of polarized RAW264.7 cells, where Fig.13 E is a schematic diagram of the regulation process of OC@ε-PL-SATO of the present invention on the inflammatory microenvironment of the wound.

[0039] Fig.14 The hemostatic property of OC@ε-PL-SATO hydrogel prepared in the present invention.

[0040] Fig.15 A&B&C are the therapeutic effects of OC@ε-PL-SATO hydrogel, which is highly effective in promoting wound healing, on infected wounds in rats in Experimental Example 4. Fig.15 A is the photos of skin wounds of rats in each group at each time point. Fig.15 B is a schematic diagram of the wound healing process. Fig.15C is the wound healing rate at different time points after treatment.

[0041] Fig.16 Schematic diagram of H&E and Masson staining of representative sections of newly formed tissue on the wound surface after wound treatment in each group of Experimental Example 4. DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings, but the embodiments are not intended to limit the present invention.

[0043] The purpose of the present invention is to safely and efficiently deliver hydrogen sulfide gas to the site of action, improve the utilization efficiency of hydrogen sulfide in wound repair, and how to coordinate with other treatment strategies (such as antibacterial and anti-inflammatory). The present invention enhances the advantages of H2S in wound tissue sustained-controlled release and penetration, enhancing macrophage polarization and overcoming the local inflammatory microenvironment by designing a delivery strategy of self-supplied hydrogen sulfide hydrogel based on chitosan-hyaluronic acid, and optimizes the release performance of hydrogel hydrogen sulfide, constructs a smart hydrogel dressing with long-term sustained release of H2S, fights bacterial infection at the wound, improves the wound microenvironment, can effectively regulate the transformation of macrophages from M1 to M2 and fibroblast proliferation, inhibits excessive inflammatory response, accelerates tissue regeneration, and finally achieves comprehensive optimization of infectious wound healing through "multiple" regulatory mechanisms.

[0044] In order to achieve the above object, the first aspect of the present invention provides a method for preparing an antibacterial hydrogel wound dressing that promotes wound healing and self-supplies hydrogen sulfide, comprising the following steps: Under ice bath conditions, prepare sodium hydroxylamine-O-sulfonate solution and sodium thiobenzoate solution respectively; Under ice bath conditions, add the sodium hydroxylamine-O-sulfonate solution to the sodium thiobenzoate solution and stir for 20 to 40 minutes to obtain S-benzoylthiohydroxylamine; S-benzoylthiohydroxylamine is added to dichloromethane, and then 4-formylbenzoic acid and TFA are added, reacting at room temperature for 1 to 5 hours, and then purifying to obtain SATO powder; SATO, EDC·HCl and NHS (N-hydroxysuccinimide) were dissolved in a mixed solvent of water and DMSO, and then ε-PL was added after uniform mixing. After stirring at room temperature for 24 to 36 hours, ε-PL-SATO was obtained after purification. Disperse hyaluronic acid in water, add NaIO4, stir for 4-6 hours, then add ethylene glycol to terminate the oxidation reaction, and purify the reactant to obtain OHA; Carboxymethyl chitosan (CMCS) and ε-PL-SATO were evenly dispersed in PBS buffer and placed in tube A of a two-component syringe. OHA and NAC were evenly dispersed in PBS buffer and placed in tube B of a two-component syringe. By squeezing the push handle of the syringe, the components of tubes A and B were mixed and reacted, and the resulting product was an antibacterial hydrogel wound dressing (OC@ε-PL-SATO) that promotes wound healing and self-supply of hydrogen sulfide.

[0045] The sodium hydroxylamine-O-sulfonate solution is prepared by adding hydroxylamine-O-sulfonate to a sodium hydroxide solution; the sodium thiobenzoate solution is prepared by adding thiobenzoic acid to a sodium hydroxide solution; wherein the concentration of the sodium hydroxylamine-O-sulfonate solution is 1-2 mol / L; and the concentration of the sodium thiobenzoate solution is 0.5-1 mol / L.

[0046] When preparing S-benzoylthiohydroxylamine, the mass ratio of the sodium hydroxylamine-O-sulfonate solution to the sodium thiobenzoate solution is 1:1-2, preferably 1:1.

[0047] When preparing SATO powder, the mass ratio of S-benzoylthiohydroxylamine to 4-formylbenzoic acid is 1:1-2, preferably 1:1, wherein TFA plays a catalytic role so only a trace amount is required.

[0048] When preparing SATO powder, after reacting for 1 to 5 hours, the reactant is filtered, the solvent is removed under reduced pressure, and the crude product is recrystallized from ethyl acetate to obtain a crude product; the crude product is then dissolved in dichloromethane and purified by column chromatography to obtain SATO powder.

[0049] The mass ratio of SATO, EDC·HCl, NHS and ε-PL is 3:4.6:2.3:1-2; the volume ratio of water and DMSO in the mixed solvent is 4-5:1.

[0050] The mass ratio of the hyaluronic acid to NaIO4 is 1-2:1.

[0051] The mass ratio of the CMCS to the ε-PL-SATO is 0.6-6:1, preferably 5:1; the mass ratio of the OHA to the NAC is 0.6-6:1, preferably 5:1.

[0052] In one embodiment, a method for preparing a hydrogel wound dressing that self-supplies slow-release hydrogen sulfide comprises the following steps: (1) Synthetic SATO: Under ice bath conditions, hydroxylamine-O-sulfonate is added to a sodium hydroxide solution to form a sodium hydroxylamine-O-sulfonate solution; at the same time, thiobenzoic acid is added to the NaOH solution in a certain proportion to form a sodium thiobenzoate solution; Under ice bath conditions, the sodium hydroxylamine-O-sulfonate solution was added dropwise to the sodium thiobenzoate solution and stirred for 30 minutes to form a large amount of white precipitate, which was collected by filtration, washed with ultrapure water, and freeze-dried overnight to obtain S-benzoylthiohydroxylamine (SBTHA); Molecular sieves and a stirrer were added to the reaction bottle, and then SBTHA was added, followed by anhydrous CH2Cl2. 4-Formylbenzoic acid was added to the reaction bottle, and then TFA was added. The vial was sealed and the mixture was allowed to react at room temperature for 1-5 hours. The reaction mixture was filtered, the solvent was removed under reduced pressure, and the crude product was recrystallized from ethyl acetate. Finally, the crude product was dissolved in CH2Cl2 and purified by column chromatography to obtain white SATO powder.

[0053] (2) Synthesis of hydrogen sulfide donor ε-PL-SATO: SATO, EDC·HCl and NHS were dissolved in a mixed solvent of water and DMSO (H2O / DMSO, v / v = 4 / 1) and stirred for 0.5 h to pre-activate SATO. Subsequently, ε-PL was added to the solution and stirred at room temperature. After reacting for 36 h, the solution was transferred to a dialysis bag (MWCO: 3500 Da) and dialyzed in ultrapure water for 3 days. The resulting solution was then freeze-dried and redissolved in methanol and then dialyzed in methanol (MWCO: 3500 Da). Finally, the mixture was vacuum dried to obtain ε-PL-SATO as pale yellow transparent crystals.

[0054] (3) OHA, the precursor material for synthesizing the hydrogel matrix: Hyaluronic acid (HA) was dissolved in distilled water, and then NaIO4 was added to the solution and stirred for another 6 hours. Ethylene glycol was then added to terminate the oxidation reaction. The resulting solution was dialyzed against deionized water (MWCO: 3500 Da) for 3 days and freeze-dried to obtain OHA.

[0055] (4) Preparation of OC@ε-PL-SATO hydrogel with self-supplied hydrogen sulfide: The CMCS solution and ε-PL-SATO solution were placed in the A tube of the two-component syringe, and the OHA solution and NAC solution were placed in the B tube of the two-component syringe. By squeezing the push handle of the syringe, the components in the AB tubes were mixed and reacted quickly, and the resulting product was OC@ε-PL-SATO hydrogel.

[0056] The preparation of CMCS solution and ε-PL-SATO solution is as follows: 1 g of carboxymethyl chitosan (CMCS) is dissolved in 20 mL of PBS solution to prepare a 5% CMCS solution; 1 g of ε-PL-SATO is dissolved in 20 mL of PBS solution to prepare a 5% ε-PL-SATO solution. The ratio of the two solutions is CMCS solution: ε-PL-SATO solution = 4:1.

[0057] Preparation of OHA solution and NAC solution: dissolve 1 g of oxidized hyaluronic acid (OHA) in 20 mL of PBS solution to prepare a 5% OHA solution; dissolve 1 g of acetylcysteine ​​(NAC) in 20 mL of PBS solution to prepare a 5% OHA solution.

[0058] The ratio of the two solutions is OHA solution: NAC solution = 4:1.

[0059] The second aspect of the present invention provides an antibacterial hydrogel wound dressing that promotes wound healing and self-supplies hydrogen sulfide. The present invention provides a hydrogel wound dressing that self-supplies and slowly releases hydrogen sulfide, the matrix of which is oxidized hyaluronic acid (OHA) and carboxymethyl chitosan (CMCS); the traditional hydrogen sulfide donor SATO is chemically grafted onto polylysine ε-Poly-L-lysine to become a new hydrogen sulfide donor ε-Poly-L-lysine-SATO (ε-PL-SATO), and ε-PL-SATO and its initiator acetylcysteine ​​(NAC) are loaded into the hydrogel matrix.

[0060] The dressing can load a new type of hydrogen sulfide donor and its initiator, and can be injected into the wound through a syringe. It can seal and treat wounds of any shape, improve the delivery efficiency and bioavailability of hydrogen sulfide, help the healing of infectious wounds, improve the wound microenvironment, promote macrophage polarization and fibroblast proliferation, inhibit excessive inflammatory response, and accelerate tissue regeneration. A third aspect of the present invention provides an application of a wound healing-promoting antibacterial hydrogel wound dressing that self-supplies hydrogen sulfide in the preparation of a drug for treating infectious wounds.

[0061] The multifunctional hydrogel wound dressing OC@ε-PL-SATO prepared by the present invention can be injected and filled into wounds of any shape, slowly releases hydrogen sulfide gas at the wound site, can effectively regulate the inflammatory microenvironment at the wound site, promote the transformation of macrophages from the pro-inflammatory M1 type to the anti-inflammatory and proliferative M2 type and promote angiogenesis, reduce the level of inflammation at the wound site, and promote wound healing. In addition, the multifunctional hydrogel wound dressing of the present invention also has excellent antibacterial properties. In short, the carboxymethyl chitosan in the hydrogel matrix is ​​a natural polymer with a broad-spectrum antibacterial effect. By introducing the natural antibacterial polypeptide polylysine grafted SATO, the antibacterial properties of the hydrogel are further improved, and the therapeutic effect of the hydrogel on the treatment of infectious wounds is enhanced.

[0062] It should be noted that the experimental methods used in the present invention are all conventional methods unless otherwise specified; the reagents and materials used are all commercially available unless otherwise specified.

[0063] Example 1 The preparation process of OC@ε-PL-SATO hydrogel includes the following steps: 1) Preparation of hydrogen sulfide donor ε-PL-SATO: 1-1) Synthesis of SATO Under ice bath conditions, hydroxylamine-O-sulfonate (1.13 g, 10 mmol) was added to 10 ml of 1M sodium hydroxide to form a 1M sodium hydroxylamine-O-sulfonate solution. Thiobenzoic acid (1.11 g, 8 mmol) was then added to 16 mL of 0.5M NaOH to form a 0.5M sodium thiobenzoate solution in a certain ratio.

[0064] Under ice bath conditions, 10 ml of 1M sodium hydroxylamine-O-sulfonate was added dropwise to 0.5M sodium thiobenzoate and stirred for 30 minutes to form a large amount of white precipitate. The precipitate was collected by filtration, washed with ultrapure water, and freeze-dried overnight without further purification. First, molecular sieves and a stirrer were added to the reaction bottle, and then S-benzoylthiohydroxylamine (SBTHA) (500 mg, 3.2 mmol) was added. Then 8 mL of anhydrous CH2Cl2 was added, and 510 mg of 4-formylbenzoic acid (3.4 mmol) was added to the vial, and then 25 μL of TFA was added. The vial was sealed and the mixture was allowed to react at room temperature for 1-5 hours. The reaction mixture was filtered, the solvent was removed under reduced pressure, and the crude product was recrystallized from ethyl acetate to obtain a white powder. Finally, the crude product was dissolved in CH2Cl2 and purified by column chromatography to obtain a white SATO powder with the following structure:

[0065] 1-2) Synthesis of hydrogen sulfide donor ε-PL-SATO, SATO was grafted onto ε-polylysine via amide condensation reaction, see Figure 2 As shown, specifically including: 300 mg SATO, 460 mg EDC·HCl and 230 mg NHS were dissolved in 20 mL mixed solvent (H2O / DMSO, v / v = 4 / 1) and stirred for 0.5 h to pre-activate the carboxyl groups of SATO. Subsequently, 200 mg of ε-PL was added to the solution and stirred at room temperature. After reacting for 36 h, the solution was transferred to a dialysis bag (MWCO: 3500 Da) and dialyzed in ultrapure water for 3 days. The resulting solution was freeze-dried and redissolved in methanol, then dialyzed against methanol (MWCO: 3500Da), and finally vacuum dried to obtain ε-PL-SATO as light yellow transparent crystals. The structure is as follows:

[0066] 2) Synthesis of oxidized hyaluronic acid (OHA) First, 4 g of HA was dissolved in 200 mL of distilled water at 25°C. Subsequently, 2 g of NaIO4 was added to the solution and stirred for another 6 h. Ethylene glycol (20 mL) was then added to terminate the oxidation reaction. The resulting solution was dialyzed against deionized water (MWCO: 3500 Da) for 5 days and OHA was obtained by freeze drying as a white spongy solid with the following structure:

[0067] 3) Preparation of OC@ε-PL-SATO hydrogel 3-1) Preparation of hydrogel precursor A solution The main components of hydrogel precursor A liquid are carboxymethyl chitosan (CMCS), ε-PL-SATO and PBS buffer. The preparation method is as follows: first, take 10 mL PBS buffer, add 0.5 g of carboxymethyl chitosan, and then add 0.1 g of ε-PL-SATO solid to it, and the resulting liquid is hydrogel precursor A liquid.

[0068] 3-2) Preparation of hydrogel precursor B solution The main components of the hydrogel precursor B liquid are oxidized hyaluronic acid (OHA), acetylcysteine ​​(NAC), and PBS buffer. The preparation method is as follows: first, take 10 mL of PBS buffer, add 0.5 g of oxidized hyaluronic acid, and then add 0.1 g of acetylcysteine ​​solid to it. The resulting liquid is the hydrogel precursor B liquid.

[0069] 3-3) Formation of OC@ε-PL-SATO hydrogel First, the prepared hydrogel precursor liquid A was placed in the A tube of the two-component syringe, and the prepared hydrogel precursor liquid B was placed in the B tube of the two-component syringe. Then, the plunger of the syringe was pressed to mix the A and B liquids in the mixing nozzle and squeezed out at the action site to form the OC@ε-PL-SATO hydrogel.

[0070] Preparation of OC series hydrogels Functionalized OHA-CMCS (abbreviated as OC)-based hydrogels were prepared by the same method as described above, and the corresponding components were listed as shown in Table 1. The obtained hydrogels were named OC, OC@ε-PL, and OC@ε-PL-SATO, respectively.

[0071] Table 1 The corresponding components of OC, OC@ε-PL and OC@ε-PL-SATO hydrogels

[0072] The OC hydrogel matrix synthesis method comprises: First, weigh 1.0g of CMCS solid and dissolve it in 20mL PBS buffer to form a 5% CMCS solution. Weigh 1.0g of OHA solid and dissolve it in 20mL PBS buffer to form a 5% OHA solution. Take 0.5mL of 5% OHA solution and add it to an equal volume of 5% CMCS solution, vortex to mix, and let it stand for 1 minute to form a colorless transparent solid, which is OHA-CMCS hydrogel. The structure is as follows:

[0073] The OC@ε-PL-SATO hydrogel matrix synthesis method comprises: First, 1.0 g of CMCS solid was weighed and dissolved in 20 mL of PBS buffer to form a 5% CMCS solution. 1 g of ε-PL-SATO was dissolved in 20 mL of PBS solution to prepare a 5% ε-PL-SATO solution. The CMCS solution and the ε-PL-SATO solution were mixed at a volume ratio of 4:1 to form solution A.

[0074] Weigh 1.0 g of OHA solid and dissolve it in 20 mL of PBS buffer to form a 5% OHA solution. Dissolve 1 g of NAC in 20 mL of PBS solution to prepare a 5% NAC solution. Mix the OHA solution and the NAC solution in a volume ratio of 4:1 to form Solution B.

[0075] Take 0.5 mL of solution A, add it to an equal volume of solution B, vortex to mix, and let it stand for 1 minute to form a colorless transparent solid, which is OC@ε-PL-SATO hydrogel.

[0076] The OC@ε-PL hydrogel matrix synthesis method comprises: First, 1.0 g of CMCS solid was weighed and dissolved in 20 mL of PBS buffer to form a 5% CMCS solution. 1 g of ε-PL was dissolved in 20 mL of PBS solution to prepare a 5% ε-PL solution. The CMCS solution and the ε-PL solution were mixed at a volume ratio of 4:1 to form solution A.

[0077] Weigh 1.0 g of OHA solid and dissolve it in 20 mL of PBS buffer to form a 5% OHA solution. Dissolve 1 g of NAC in 20 mL of PBS solution to prepare a 5% NAC solution. Mix the OHA solution and the NAC solution in a volume ratio of 4:1 to form Solution B.

[0078] Take 0.5 mL of solution A, add it to an equal volume of solution B, vortex to mix, and let it stand for 1 minute to form a colorless transparent solid, which is the OC@ε-PL hydrogel.

[0079] Experimental Example 2 The products obtained in each step of Example 1 were 1 The morphology, composition and chemical bonds of the product were systematically studied using modern physical and chemical testing and analysis techniques such as H NMR, UV-Vis, FTIR and SEM. The results are as follows: The present invention characterizes the ε-PL-SATO obtained in the embodiment by hydrogen nuclear magnetic resonance spectrum and ultraviolet visible spectrum.

[0080] like Figure 4 As shown in Figure A, the H-NMR spectrum of ε-PL shows typical characteristic peaks at 3.98 ppm, 3.76 ppm, 3.27 ppm, 3.03 ppm, 1.90 ppm, 1.60 ppm and 1.41 ppm. After combining with SATO, multiple new peaks around 7.2-8.3 ppm appeared in the ε-PL-SATO spectrum. These peaks are related to the benzene protons on SATO, indicating that the ε-PL-SATO was successfully synthesized. The UV-visible spectrum further confirmed the conjugation of ε-PL-SATO, which was revealed by the characteristic absorbance peak (about 320 nm) of the SATO group in the SATO and ε-PL-SATO spectra ( Figure 4 B).

[0081] The performance of ε-PL-SATO and the trigger agent NAC in producing hydrogen sulfide was tested by a hydrogen sulfide detection kit. Figure 4 As shown in Figure C, the hydrogen sulfide generation efficiency of the synthesized hydrogen sulfide donor ε-PL-SATO is nearly 5 times higher than that of the original SATO. The above results confirm that ε-PL-SATO was successfully synthesized and can efficiently generate hydrogen sulfide gas.

[0082] The present invention characterizes the hydrogel precursor and the hydrogel matrix obtained in the examples by hydrogen nuclear magnetic resonance spectrum and FTIR. Figure 5 As shown in A, the synthesis process of OHA-CMCS hydrogel; Figure 5 As shown in Figure 5B, compared with HA, three new proton signals at 4.9, 5.0, and 5.1 ppm were observed in the NMR spectrum of OHA, corresponding to the hemiacetal protons and the ortho-hydroxyl groups (Figure 5C). FTIR further confirmed the successful synthesis of OHA and the reaction between CMCS and OHA. Compared with free HA, a 1730 cm -1 The stretching vibration peak of the aldehyde-C=O bond at 3430 cm -1 The most prominent characteristic peak at 1606cm is attributed to OH and NH (amine) stretching vibrations. -1 The typical peak at 1500–1600 cm represents the stretching vibration of the C=O group in CMCS, which is correctly displayed in its FTIR spectrum. -1 The narrow peak at 240 nm widened and the aldehyde peak of OHA disappeared, indicating that the -CHO of OHA successfully combined with the -NH2 of CMCS to form a hydrogel. The above results confirmed the successful synthesis of OHA and the successful synthesis of the hydrogel matrix OHA-CMCS.

[0083] The present invention characterizes the material properties of the OC@ε-PL-SATO hydrogel in the embodiment through gelation experiments, injection experiments, scanning electron microscopy and hydrogen sulfide in vitro release experiments.

[0084] like Figure 6 As shown in the figure, the precursor A liquid (CMCS, ε-PL-SATO) and the precursor B liquid (OHA, NAC) of OC@ε-PL-SATO hydrogel can quickly form a hydrogel within 30 seconds after mixing at room temperature. The above results show that OC@ε-PL-SATO hydrogel has the characteristics of ultra-fast gelation.

[0085] like Figure 7 As shown, OC@ε-PL-SATO hydrogel can be mixed with a two-component syringe, injected through a needle, and written into any shape. The above results confirm that OC@ε-PL-SATO hydrogel has good injectability.

[0086] like Figure 8 As shown in the figure, the pore structure of the hydrogel was studied using scanning electron microscopy. The above three hydrogels all showed a loose and porous network structure. The pore size of OC@ε-PL-SATO hydrogel was relatively large, which was conducive to wound healing.

[0087] like Fig. 9 As shown in FIG. 1 , the OC@ε-PL-SATO hydrogel of the present invention can effectively release H2S for about 24 hours after injection. Figure 3 Schematic diagram of H2S release from hydrogen sulfide donor ε-Poly-L-lysine-SATO (ε-PL-SATO).

[0088] The present invention analyzes the rheological properties of OC@ε-PL-SATO hydrogel through rheological experiments. Dynamic strain scanning experiments were performed using Anton Paar rotational rheometer to estimate the gel-sol transition point and the linear viscoelastic region ( Fig.10 A). As the shear strain increases significantly, the loss modulus (G'') exceeds the storage modulus (G'), indicating that the gel-sol phase transition occurs due to the destruction of the structure of the post-stabilized hydrogel under large deformation. Fig.10 B), G' in all post-stabilized hydrogels is always greater than the corresponding G'' in the frequency range of 0.1-100 Hz, indicating that the hydrogel structure is stable and behaves similarly to a viscoelastic gel. At the same time, the storage modulus of the OC hydrogel with the addition of the hydrogen sulfide donor ε-PL-SATO increased from about 100Pa to 1000Pa, indicating that there is a stronger cross-linked network in the OC@ε-PL and OC@ε-PL-SATO hydrogels than in the OC hydrogel. This is mainly attributed to the formation of double cross-links by CMCS, ε-PL and OHA, which significantly improves its mechanical properties. The hydrogels with excellent autonomous healing properties and shape adaptive properties show good application prospects in the injection treatment of irregular wounds due to their shear thinning ability. The viscosity of the OC@ε-PL-SATO hydrogel decreases with increasing shear rate ( Fig.10 C), demonstrating the shear thinning ability of the gel, which is consistent with previous macroscopic injectable experimental results. In order to evaluate the self-healing ability of these hydrogels, OC@ε-PL-SATO hydrogels were selected as model samples for rheological recovery tests and macroscopic self-healing tests. First, we performed a continuous strain scan on the OC@ε-PL-SATO hydrogel to determine the self-healing behavior of the hydrogel based on the strain point that caused the hydrogel to collapse. The hydrogel network was destroyed under a larger strain (300%), while once a lower strain (1%) was applied, the hydrogel began to repair ( Fig.10 D). Even after three alternating repeated cycles, the values ​​of G' and G'' can almost recover to their original values, indicating that all three hydrogels have good self-healing ability. On the other hand, the functional OC@ε-PL-SATO hydrogel cut into semi-disc shape can self-heal to form a new disc-shaped hydrogel after incubation at 37°C for 30 min ( Fig.10E), and it can maintain its intact structure without obvious cracks, once again demonstrating its excellent self-healing properties.

[0089] Experimental Example 3 In vitro biological evaluation of OC@ε-PL-SATO hydrogel 3.1 Cytotoxicity The in vitro biocompatibility of the hydrogel with L929 fibroblasts was determined according to the national standard (GB / T16886.5-2003 / ISO 10993-53199) using the extract method. First, the hydrogel samples (OC, OC@ε-PL or OC@ε-PL-SATO) were incubated in DMEM complete medium for 24 h to prepare the extract (5 mg / mL). Next, the L929 cells seeded in a 96-well plate were incubated with the above extract (200 μL) at 37°C and 5% CO2 for 24 h and 48 h, respectively. The control group was replaced with fresh DMEM. According to the manufacturer's procedures, the cell viability was detected using the CCK-8 kit, and the absorbance of the sample at 450 nm was measured using an enzyme reader. The results are shown in Figure 2. Fig.11 As shown in A, the viability of L929 cells treated with OC hydrogels in different groups was not different from that of cells treated with PBS (control) at 24h and 48h. Red blood cells were collected from fresh blood of SD rats, resuspended in saline with a volume concentration of 2%, and incubated with OC@ε-PL-SATO hydrogel, saline (negative control), 1% Triton X-100 (positive control) and ddH2O (positive control) at 37°C for 3h, centrifuged at 2000rpm for 15min, and the absorbance of the supernatant at 545nm was detected by an enzyme-labeled instrument. The results are shown in Fig.11 As shown in B, Fig.11 As shown in B, OC@ε-PL-SATO hydrogel exhibited good blood compatibility with a hemolysis rate of less than 4%, and visual inspection confirmed that the supernatant of the hydrogel-treated red blood cell suspension was colorless and transparent.

[0090] 3.2 Cell scratch and migration performance On the other hand, the effect of hydrogel on L929 cell migration was observed by cell scratch test. First, a p200 pipette tip was used to create scratches in a 6-well plate seeded with L929 cells. Then, 2 mL of hydrogel extract was added and incubated for 48 hours. Cell migration images were taken with an inverted microscope. Fig.11 As shown in Figure 3C, after 48 h of co-culture, the cell migration speed of OC@ε-PL-SATO hydrogel was intuitively faster than that of other groups, revealing the excellent in vitro wound healing performance of functional OC@ε-PL-SATO hydrogel.

[0091] 3.3 In vitro antibacterial properties 400 μg of each of the three hydrogels were injected into a sterile EP tube, and then 400 μL of 1×10 5 CFU / mL of Escherichia coli or Staphylococcus aureus were added to the surface of the hydrogel. After incubation for 0, 6, 12, and 24 h, the bacterial samples were transferred to a 96-well plate and the absorbance at 600 nm was recorded by a microplate reader to determine the growth of bacteria. After the bacterial samples were co-incubated with the hydrogel for 24 h, they were diluted to an appropriate concentration and spread on an agar plate medium. After incubation at 37 °C for 16 h, the colonies were counted and photographed. The antibacterial curves showed that the growth of the two bacteria was significantly inhibited after 24 h of culture in the OC series hydrogels (Figure 12A and B), which was attributed to the inherent antibacterial properties introduced by CMCS. At the same time, the OD600 of Staphylococcus aureus and Escherichia coli in the ε-PL functionalized hydrogel group (OC@ε-PL and OC@ε-PL-SATO) was significantly lower than that in the OC group, which means that it has better bactericidal performance, because ε-PL itself is a broad-spectrum antimicrobial peptide. Then, the excellent antibacterial properties of the ε-PL modified hydrogels were further confirmed by the Escherichia coli and Staphylococcus aureus colony formation inhibition tests, and almost no bacterial colonies were formed on the agar plates compared with the control group and OC hydrogels ( Fig.12 C), again demonstrating its excellent in vitro antibacterial properties.

[0092] 3.4 In vitro macrophage polarization performance RAW264.7 cells were seeded in 6-well plates and pre-incubated with lipopolysaccharide (LPS, 1 μg / mL) to activate the pro-inflammatory M1 phenotype. They were then treated with three hydrogel extracts (2 mL, OC, OC@ε-PL, or OC@ε-PL-SATO) and IL-4 (20 ng / mL, positive control) for 48 h. PE anti-mouse CD86 antibody and APC anti-mouse CD206 antibody were then added and incubated with RAW264.7 cells for 30 min. Flow cytometry (BD FACS Celesta TM, USA) and FlowJo software were used to analyze the changes in macrophage phenotype. As shown in Figures 13A and B, interleukin 4 (IL-4), as a positive control, reasonably induced macrophage polarization to the M2 phenotype at a high level, which was manifested as a red shift in the histogram of macrophages expressing CD206. In addition, the number of macrophages expressing the M2 macrophage marker CD206 in the OC@ε-PL-SATO hydrogel treatment group was significantly higher than that in the OC and OC@ε-PL groups that lack the ability to produce H2S. This is consistent with the mean fluorescence intensity (MFI) analysis results of CD206 in macrophages (Figure 13C), indicating that M2 macrophages were effectively polarized.

[0093] At the same time, the changes in cytokines secreted by macrophages were studied. In brief, RAW264.7 cells seeded in 96-well plates were treated with three hydrogel extracts plus LPS (1 μg / mL) and free LPS for 24 hours. RAW264.7 cells seeded in 96-well plates without any additives were set as the control group, and the supernatants of the above treatment groups were measured using TNF-α, TGF-β, IL-10 and IL-1β ELISA kits. The results showed that when macrophages were first induced to the M1 type by LPS, the secretion of classic pro-inflammatory factors TNF-α and IL-1β in RAW264.7 cells was significantly reduced after treatment with OC@ε-PL-SATO hydrogel extract. In contrast, the levels of anti-inflammatory factors IL-10 and TGF-β detected by ELISA were increased compared with other treatments ( Fig.13 D). These results highlight the powerful ability of OC@ε-PL-SATO hydrogels to guide macrophage polarization from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype (Figure 13E).

[0094] Experimental Example 4 Evaluation of the pro-infective wound healing performance of OC@ε-PL-SATO hydrogel with self-supply of H2S, see Figure 1 shown.

[0095] 4.1 Analysis of hemostatic properties in rats The hemostatic properties of the hydrogel were determined using a rat tail-cut bleeding model. The method is as follows: After anesthetizing SD rats (~300g, male), 50% of the tail was cut off, and the wound was covered with gauze, 3M Tegaderm dressings, and the above-mentioned hydrogel, respectively, and the bleeding data during the hemostasis process was recorded. The wounds of the control group rats were not treated in any way. Traditional hemostatic materials such as gauze and 3M Tegaderm were selected as the positive control group, and rats that were not treated in any way were used as blank controls. Fig.14 As shown, the hemostatic effect of the OC@ε-PL-SATO hydrogel treatment group was significantly better than that of the other groups, and its hemostatic ability was better than that of commercially available gauze and 3M TegadermTM material.

[0096] 4.2 Construction of rat infected wound model First, a rat full-thickness skin incision model was established. Typically, the rats were anesthetized and the hair on the back of the rats was removed. Next, a wound with a diameter of 10 mm was formed and Staphylococcus aureus (10 μL, 10 6 CFU / mL) infection to form a rat infected wound model.

[0097] 4.3 Hydrogel therapeutic intervention and histopathological analysis The rats in the infection model were divided into five groups, with 4 rats in each group, and treated with normal saline (control), 3M Tegaderm dressing (positive control), OC hydrogel, OC@ε-PL hydrogel, and OC@ε-PL-SATO hydrogel, respectively. The wounds were monitored and photographed on days 0, 3, 7, 10, and 14. Fig.15 As shown, the OC@ε-PL-SATO hydrogel group showed the best wound healing degree and the fastest healing speed at all treatment times ( Fig.15 AC). Notably, by day 7, wound contraction in the OC@ε-PL-SATO group increased by nearly 23% compared with the 3M dressing group. By day 14, the wounds in the OC@ε-PL-SATO hydrogel group were almost completely closed (wound contraction rate of 99%), while the wound contraction rate in the OC@ε-PL group was 94%. In contrast, there were still significant residual wound areas in the other groups. H&E and Masson staining results further confirmed the excellent wound healing performance of OC@ε-PL-SATO hydrogel ( Fig.16 ). Compared with the control group, the OC@ε-PL-SATO hydrogel group showed better healing effects, with intact regenerated skin tissue structure, intact epithelial and dermal layers, and increased formation of new blood vessels and hair follicles. In addition, the thickest collagen bundles were observed in the wound tissue treated with OC@ε-PL-SATO hydrogel, which was dark blue, indicating that the damaged tissue was recovering and maturing.

[0098] Example 5 The same as Example 1, except that the synthesis method of OC@ε-PL-SATO hydrogel was changed, including First, weigh 0.6g of CMCS solid and dissolve it in 20mL of PBS buffer to form a 3% CMCS solution. Take 1g of ε-PL-SATO and dissolve it in 20mL of PBS solution to prepare a 5% ε-PL-SATO solution. Mix the CMCS solution and the ε-PL-SATO solution in a volume ratio of 4:1 as liquid A. Weigh 0.6g of OHA solid and dissolve it in 20mL of PBS buffer to form a 3% OHA solution. Take 1g of NAC and dissolve it in 20mL of PBS solution to prepare a 5% NAC solution. Mix the OHA solution and the NAC solution in a volume ratio of 4:1 as liquid B. Take 0.5 mL of liquid A, add it to an equal volume of liquid B, vortex to mix, and let it stand for 1 minute to form a colorless and transparent solid, which is OC@ε-PL-SATO hydrogel.

[0099] Example 6 The same as Example 1, except that the synthesis method of OC@ε-PL-SATO hydrogel was changed, including First, weigh 0.8g of CMCS solid and dissolve it in 20mL of PBS buffer to form a 4% CMCS solution. Take 1g of ε-PL-SATO and dissolve it in 20mL of PBS solution to prepare a 5% ε-PL-SATO solution. Mix the CMCS solution and the ε-PL-SATO solution in a volume ratio of 4:1 as liquid A. Weigh 0.8g of OHA solid and dissolve it in 20mL of PBS buffer to form a 4% OHA solution. Take 1g of NAC and dissolve it in 20mL of PBS solution to prepare a 5% NAC solution. Mix the OHA solution and the NAC solution in a volume ratio of 4:1 as liquid B. Take 0.5 mL of liquid A, add it to an equal volume of liquid B, vortex to mix, and let it stand for 1 minute to form a colorless and transparent solid, which is the OC@ε-PL-SATO hydrogel.

[0100] Example 7 The same as Example 1, except that the synthesis method of OC@ε-PL-SATO hydrogel was changed, including First, 1.2 g of CMCS solid was weighed and dissolved in 20 mL of PBS buffer to form a 6% CMCS solution. 1 g of ε-PL-SATO was dissolved in 20 mL of PBS solution to prepare a 5% ε-PL-SATO solution. The CMCS solution and the ε-PL-SATO solution were mixed at a volume ratio of 4:1 as liquid A. 1.2 g of OHA solid was weighed and dissolved in 20 mL of PBS buffer to form a 6% OHA solution. 1 g of NAC was dissolved in 20 mL of PBS solution to prepare a 5% NAC solution. The OHA solution and the NAC solution were mixed at a volume ratio of 4:1 as liquid B. 0.5 mL of liquid A was added to an equal volume of liquid B, vortexed to mix, and left for 1 minute to form a colorless and transparent solid, which was the OC@ε-PL-SATO hydrogel.

[0101] The effects that the present invention can achieve are as follows: 1. Slow and continuous release of hydrogen sulfide gas The hydrogen sulfide gas release ability of OC@ε-PL-SATO hydrogel was detected in vitro, and the real-time release behavior of H2S from the hydrogel was monitored using the H2S content detection kit and WSP-5 hydrogen sulfide fluorescent probe. The results showed that in the absence of H2S donor ε-PL-SATO and initiator NAC, the negative control hydrogel groups (OC, OC@ε-PL, OC@ε-PL-SATO without NAC) could not produce H2S. The functional hydrogel combination OC@ε-PL-SATO (tube A contains CMCS and H2S donor ε-PL-SATO, tube B contains OHA and initiator NAC) can significantly produce H2S, which is detected by the H2S fluorescent probe WSP-5, showing bright green fluorescence. The release of H2S in OC@ε-PL-SATO hydrogel was further quantitatively detected using a H2S content detection kit. The results showed that the release of H2S in OC@ε-PL-SATO hydrogel reached a peak of approximately 7 μM within 6 h, and the release of H2S could last for 48 h, which was consistent with the short-term and long-term H2S fluorescence imaging detection results, revealing that the functionalized OC@ε-PL-SATO hydrogel has self-supplied and sustained H2S release characteristics.

[0102] 2. Good biosafety.

[0103] L929 was used as a model cell to study the biosafety of the hydrogel. The results of cytotoxicity experiments and live-dead cell staining tests showed that the extracts of OC@ε-PL-SATO hydrogels were co-incubated with L929 cells for 24 hours and 48 hours, respectively, and the cell viability was studied using the CCK8 kit. Regardless of the incubation time, there was no significant difference in cell activity between the three hydrogel-treated groups and the control group, indicating good biocompatibility. The live / dead staining test further demonstrated the excellent cell compatibility of the OC series of hydrogels, as shown by a large number of live cells marked with green fluorescence and a very small number of dead cells marked with red fluorescence.

[0104] Furthermore, the blood safety of the hydrogel was studied using rat red blood cells as model cells. The results of the hemolysis experiment showed that the OC@ε-PL-SATO hydrogel exhibited good blood compatibility, with a hemolysis rate of less than 4%. Visual inspection confirmed that the supernatant of the red blood cell suspension treated with the hydrogel was colorless and transparent. In addition, after being treated with OC@ε-PL-SATO hydrogel for 3 hours, the red blood cell morphology still retained a natural biconcave disc shape, which was similar to the shape of healthy red blood cells, once again confirming that the OC@ε-PL-SATO hydrogel has good biocompatibility.

[0105] 3.Good antibacterial properties.

[0106] The antibacterial effect of the hydrogel was studied using Staphylococcus aureus and Escherichia coli, common wound infection bacteria. The results of the inhibition curve and co-incubation coating showed that the growth of the above two bacteria was significantly inhibited after 24 hours of culture in the OC series hydrogels. At the same time, the OD600 of Staphylococcus aureus and Escherichia coli in the ε-PL functionalized hydrogel group (OC@ε-PL and OC@ε-PL-SATO) was significantly lower than that in the OC group, which means that it has better bactericidal properties, which is because ε-PL itself is a broad-spectrum antimicrobial peptide. Compared with the control group and the OC series hydrogels, there was almost no bacterial colony formation on the agar plate.

[0107] 4. Regulate the polarization of macrophages toward M2 anti-inflammatory and pro-proliferative phenotype.

[0108] The RAW264.7 cell inflammation model was constructed in vitro, and the results of flow cytometry and ELISA assays showed that interleukin 4 (IL-4) as a positive control effectively induced macrophage polarization to the M2 phenotype, which was manifested as a red shift in macrophages expressing CD206 in the histogram. In addition, the number of macrophages expressing the M2 macrophage marker CD206 in the OC@ε-PL-SATO hydrogel treatment group was significantly higher than that in the negative control. This is consistent with the results of the mean fluorescence intensity (MFI) analysis of CD206 in macrophages, indicating that M2 macrophages are effectively polarized.

[0109] 5. Effective in vivo hemostatic properties A tail-broken bleeding model was established, with traditional hemostatic materials such as gauze and 3M Tegaderm as the positive control group, and rats without any treatment as the blank control. The hemostatic effect of the OC@ε-PL-SATO hydrogel-treated group was significantly better than that of the other groups, with the least bleeding, the shortest hemostatic time, and better hemostatic ability than commercially available gauze and 3M Tegaderm materials.

[0110] 6. Effective wound healing effect in vivo An infected full-thickness skin defect model was established, and animal experiments and quantitative statistical results showed that after covering the wound with different hydrogels or 3M Tegaderm™ (a commercial film dressing), the wound contraction area was monitored at different time points (0, 3, 7, 10, and 14 days). The OC@ε-PL-SATO hydrogel group showed the best degree of wound healing and the fastest healing speed at all treatment times. By day 7, the wound contraction in the OC@ε-PL-SATO group increased by nearly 23% compared with the 3M dressing group. By day 14, the wound in the OC@ε-PL-SATO hydrogel group was almost completely closed (wound contraction rate of 99%), while the wound contraction rate in the OC@ε-PL group was 94%. In contrast, there was still a significant residual wound area in the other groups. H&E and Masson staining results showed that the OC@ε-PL-SATO hydrogel group had a better healing effect than the control group, with intact regenerated skin tissue structure, intact epithelial and dermal layers, and increased formation of new blood vessels and hair follicles. In addition, the thickest collagen bundles were observed in the wound tissue treated with OC@ε-PL-SATO hydrogel, which was dark blue, indicating that the damaged tissue was recovering and maturing. OC@ε-PL-SATO hydrogel with self-supply of H2S had better therapeutic effect than 3M dressing.

[0111] In summary, the present invention uses a two-component syringe to prepare a two-component functional hydrogel. Precursor A liquid (CMCS, ε-PL-SATO) and precursor B liquid (CMCS, ε-PL-SATO) can quickly form a hydrogel within 30 seconds after mixing at room temperature. The OC@ε-PL-SATO hydrogel has a short gelation time. The hydrogel can both slowly release H2S and be antibacterial. In addition, in vitro experiments and transcriptome analysis showed that the functional hydrogel can effectively promote the polarization of macrophages to the anti-inflammatory M2 phenotype caused by H2S, and revealed the related mechanisms mediated by PI3K / Akt and NF-κB pathways. In vivo experiments confirmed the effectiveness of OC@ε-PL-SATO hydrogel in promoting the healing of bacterial-infected wounds by regulating the inflammatory microenvironment, accompanied by good hemostasis, angiogenesis and collagen deposition. In general, OC@ε-PL-SATO hydrogel with rapid gelation characteristics and sustained release of H2S function provides an effective therapeutic platform for the management of chronic wounds and bacterial-infected wounds, and is a new type of hydrogel dressing that promotes wound healing.

[0112] The present invention describes preferred embodiments and their effects. However, those skilled in the art may make additional changes and modifications to these embodiments once they are aware of the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0113] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a wound healing-promoting antibacterial hydrogel wound dressing that self-supplies hydrogen sulfide, characterized in that: The following steps are involved: Under ice bath conditions, prepare sodium hydroxylamine-O-sulfonate solution and sodium thiobenzoate solution respectively; Under ice bath conditions, add the sodium hydroxylamine-O-sulfonate solution to the sodium thiobenzoate solution and stir for 20 to 40 minutes to obtain S-benzoylthiohydroxylamine; S-benzoylthiohydroxylamine is added to dichloromethane, and then 4-formylbenzoic acid and TFA are added, reacting at room temperature for 1 to 5 hours, and then purifying to obtain SATO powder; SATO, EDC·HCl and NHS were dissolved in a mixed solvent of water and DMSO, and after uniform mixing, ε-PL was added, and the mixture was stirred at room temperature for 24 to 36 hours, and then ε-PL-SATO was obtained by purification; Disperse hyaluronic acid in water, add NaIO4, stir for 4-6 hours, then add ethylene glycol to terminate the oxidation reaction, and purify the reactant to obtain OHA; CMCS and ε-PL-SATO are uniformly dispersed in PBS buffer and placed in tube A of a two-component syringe, and OHA and NAC are uniformly dispersed in PBS buffer and placed in tube B of a two-component syringe; by squeezing the push handle of the syringe, the components of tubes A and B are mixed and reacted, and the resulting product is an antibacterial hydrogel wound dressing that promotes wound healing and self-supplies hydrogen sulfide.

2. The method for preparing the wound healing-promoting antibacterial hydrogel wound dressing with self-supply of hydrogen sulfide according to claim 1, characterized in that: The sodium hydroxylamine-O-sulfonate solution is prepared by adding hydroxylamine-O-sulfonate to a sodium hydroxide solution; the sodium thiobenzoate solution is prepared by adding thiobenzoic acid to a sodium hydroxide solution; wherein the concentration of the sodium hydroxylamine-O-sulfonate solution is 1-2 mol / L; and the concentration of the sodium thiobenzoate solution is 0.5-1 mol / L.

3. The method for preparing the wound healing-promoting antibacterial hydrogel wound dressing with self-supply of hydrogen sulfide according to claim 1, characterized in that: When preparing S-benzoylthiohydroxylamine, the mass ratio of the sodium hydroxylamine-O-sulfonate solution to the sodium thiobenzoate solution is 1:1-2.

4. The method for preparing the wound healing-promoting antibacterial hydrogel wound dressing with self-supply of hydrogen sulfide according to claim 1, characterized in that: When preparing SATO powder, the mass ratio of S-benzoylthiohydroxylamine to 4-formylbenzoic acid is 1:1~2.

5. The method for preparing the wound healing-promoting antibacterial hydrogel wound dressing with self-supply of hydrogen sulfide according to claim 1, characterized in that: When preparing SATO powder, after reacting for 1 to 5 hours, the reactant is filtered, the solvent is removed under reduced pressure, and the crude product is recrystallized from ethyl acetate to obtain a crude product; the crude product is then dissolved in dichloromethane and purified by column chromatography to obtain SATO powder.

6. The method for preparing the wound healing-promoting antibacterial hydrogel wound dressing with self-supply of hydrogen sulfide according to claim 1, characterized in that: The mass ratio of SATO, EDC·HCl, NHS and ε-PL is 3:4.6:2.3:1-2; the volume ratio of water and DMSO in the mixed solvent is 4-5:

1.

7. The method for preparing the wound healing-promoting antibacterial hydrogel wound dressing with self-supply of hydrogen sulfide according to claim 1, characterized in that: The mass ratio of the hyaluronic acid to NaIO4 is 1-2:

1.

8. The method for preparing the wound healing-promoting antibacterial hydrogel wound dressing with self-supply of hydrogen sulfide according to claim 1, characterized in that: The mass ratio of the CMCS to the ε-PL-SATO is 0.6-6:1; the mass ratio of the OHA to the NAC is 0.6-6:

1.

9. An antibacterial hydrogel wound dressing for promoting wound healing and self-supplied with hydrogen sulfide, prepared by the method according to any one of claims 1 to 8.

10. Use of the wound healing-promoting antibacterial hydrogel wound dressing with self-supply of hydrogen sulfide according to claim 9 in the preparation of a drug for treating infectious wounds.

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