A self-supplying hydrogen sulfide-promoting antibacterial hydrogel wound dressing, its preparation method and application

Abstract

This invention discloses a self-supplying hydrogen sulfide-based wound-healing antibacterial hydrogel wound dressing, its preparation method, and its application, relating to the field of medical dressing technology. The method involves uniformly dispersing CMCS and ε-PL-SATO in PBS buffer and placing them in tube A of a two-component syringe; uniformly dispersing OHA and NAC in PBS buffer and placing them in tube B of the two-component syringe; by squeezing the syringe plunger, the components in tubes A and B mix and react, and the resulting product is the self-supplying hydrogen sulfide-based wound-healing antibacterial hydrogel wound dressing. The hydrogel prepared by this invention uses oxidized hyaluronic acid and carboxymethyl chitosan as a matrix, loaded with an H2S donor (ε-PL-SATO) and its initiator NAC to achieve rapid gelation (gel time <30 seconds). This allows the hydrogel to be rapidly molded, making it suitable for clinical applications such as injection and wound treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical dressings, and particularly relates to a self-supplying hydrogen sulfide-promoting wound healing antibacterial hydrogel wound dressing and a preparation method and application thereof. BACKGROUND

[0002] Skin is the largest organ of the human body, protecting the body from external attacks, electrolyte loss, and water evaporation. The regeneration of normal wound skin mainly goes through four overlapping but different stages, including hemostasis, inflammation, cell proliferation, and matrix remodeling. However, the healing process of chronic wounds caused by bacterial infection often violates this schedule. Bacterial infection of wounds is now a major medical threat and challenge, especially Staphylococcus aureus-related skin wound infection, which often leads to serious consequences.

[0003] Although inflammation is a key stage in the wound healing process, the inflammatory response is considered a critical stage for the body to clear infected bacteria and create a tissue environment conducive to regeneration and repair, but long-term sustained inflammation can 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 can promote inflammation elimination and promote wound healing. Hydrogen sulfide (H2S) is a newly discovered gas signaling molecule, which shows great therapeutic potential in biomedical research due to its anti-inflammatory effect. As a small molecule signaling molecule, H2S can immediately exert a therapeutic effect by penetrating the cell membrane. One of the mechanisms of its therapeutic effect is to promote the transformation of macrophage phenotype to M2 macrophages. However, the commonly used H2S donors will uncontrollably release H2S, which will cause acute cytotoxicity and waste of drugs.

[0005] Therefore, if an injectable self-healing hydrogel dressing can resist bacteria at the wound site and self-supply controlled release of hydrogen sulfide gas, it is expected to promote the healing process of infected wounds. However, how to prepare a hydrogel dressing that can control the release of hydrogen sulfide gas is also a challenge. SUMMARY

[0006] In order to solve the problems in the background art, the present application provides a self-supplying hydrogen sulfide promoting wound healing antibacterial hydrogel wound dressing and a preparation method and application thereof. The present application improves the poor solubility of S-aryl thiohydroxamic acid (SATO) by coupling the H2S donor (ε-PL-SATO) of ε-polylysine and S-aryl thiohydroxamic acid (SATO). The donor can release H2S responsively under the action of N-acetyl cysteine (NAC).

[0007] In order to achieve the above-mentioned purposes, the first object of the present application is to provide a preparation method of a self-supplying hydrogen sulfide promoting wound healing antibacterial hydrogel wound dressing, characterized in that the method comprises the following steps:

[0008] Under ice bath conditions, a sodium hydroxylamine-O-sulfonate solution and a sodium thio-benzoate solution are prepared respectively;

[0009] Under ice bath conditions, the sodium hydroxylamine-O-sulfonate solution is added to the sodium thio-benzoate solution, and stirred for 20-40 minutes to obtain S-benzoyl thiohydroxylamine;

[0010] The S-benzoyl thiohydroxylamine is added to dichloromethane, 4-formyl benzoic acid and TFA are added, and the mixture is reacted at room temperature for 1-5 hours, and then purified to obtain SATO powder;

[0011] The SATO, EDC·HCl and NHS are dissolved in a mixed solvent of water and DMSO, uniformly mixed, and then ε-PL is added. After stirring and reacting at room temperature for 24-36 hours, the mixture is purified to obtain ε-PL-SATO;

[0012] The hyaluronic acid is dispersed in water, NaIO4 is added, and the mixture is stirred for 4-6 hours. Then, ethylene glycol is added to terminate the oxidation reaction, and the reactants are purified to obtain OHA;

[0013] The CMCS and ε-PL-SATO are uniformly dispersed in PBS buffer and placed in the A tube of a two-component syringe. The OHA and NAC are uniformly dispersed in PBS buffer and placed in the B tube of the two-component syringe. The components in the A tube and the B tube are mixed by pressing the push handle of the syringe, and the obtained product is a self-supplying hydrogen sulfide promoting wound healing antibacterial hydrogel wound dressing.

[0014] Preferably, the sodium hydroxylamine-O-sulfonate solution is prepared by adding sodium hydroxylamine-O-sulfonate to a sodium hydroxide solution; and the sodium thio-benzoate solution is prepared by adding thio-benzoic acid to a sodium hydroxide solution. The concentration of the sodium hydroxylamine-O-sulfonate solution is 1-2 mol / L, and the concentration of the sodium thio-benzoate solution is 0.5-1 mol / L.

[0015] Preferably, when preparing S-benzoyl thiohydroxylamine, the mass ratio of the hydroxylamine-O-sodium sulfonate solution to the sodium thio-benzoate solution is 1:1-2.

[0016] Preferably, when preparing SATO powder, the mass ratio of S-benzoyl thiohydroxylamine to 4-formylbenzoic acid is 1:1-2.

[0017] Preferably, when preparing SATO powder, after 1-5 hours of reaction, the reactants are filtered, the solvent is removed under reduced pressure, the crude product is recrystallized from ethyl acetate to obtain a crude product; and then the crude product is dissolved in dichloromethane and purified by column chromatography to obtain SATO powder.

[0018] Preferably, the mass ratio of the SATO, EDC·HCl, NHS and epsilon-PL is 3:4.6:2.3:1-2; and the volume ratio of water to DMSO in the mixed solvent is 4-5:1.

[0019] Preferably, the mass ratio of the hyaluronic acid to NaIO4 is 1-2:1.

[0020] Preferably, the mass ratio of the CMCS to epsilon-PL-SATO is 0.6-6:1; and the mass ratio of the OHA to NAC is 0.6-6:1.

[0021] A second object of the present application is to provide a self-supplying hydrogen sulfide-promoting wound healing antibacterial hydrogel wound dressing.

[0022] A third object of the present application is to provide a self-supplying hydrogen sulfide-promoting wound healing antibacterial hydrogel wound dressing for use in the preparation of an infectious wound treatment drug.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] The present application provides a self-supplying hydrogen sulfide-promoting wound healing antibacterial hydrogel wound dressing, a preparation method and application thereof. The present application improves the poor solubility of SATO by coupling epsilon-polylysine and S-aryl thiooxime (SATO) to form a H2S donor (epsilon-PL-SATO). The donor can release H2S responsively under the action of N-acetylcysteine (NAC).

[0025] The hydrogel prepared by the present application is based on oxidized hyaluronic acid and carboxymethyl chitosan as a matrix, and loaded with a H2S donor (epsilon-PL-SATO) and its initiator NAC to achieve rapid gelation (gelation time <30 seconds). This enables the hydrogel to be quickly formed, which is suitable for clinical applications such as injection and wound treatment.

[0026] The hydrogel prepared in this invention releases H2S by regulating the phosphatidylinositol 3-kinase (PI3K) / protein kinase B (Akt) signaling pathway and inhibiting the activation of nuclear factor κB (NF-κB), and promoting the polarization of macrophages from M1 type to anti-inflammatory and proliferative M2 type, thereby reducing the inflammatory response at the wound site and achieving anti-inflammatory and wound repair effects.

[0027] The hydrogel prepared by this invention inhibits bacterial growth at the wound site by utilizing the broad-spectrum antibacterial properties of chitosan and polylysine, reducing the risk of infection and providing a good sterile environment for wound healing.

[0028] The hydrogel prepared in this invention releases H2S, which not only reduces inflammation but also promotes angiogenesis and collagen deposition, accelerating wound healing. This innovation combines the multiple roles of H2S in wound regeneration.

[0029] The hydrogel of this invention is designed with good biocompatibility, rapid gelation characteristics and injectability, and is suitable for different types and shapes of wounds, including infected wounds and acute wounds.

[0030] This invention designs an H2S donor containing ε-polylysine-S-arylthiooxime (ε-PL-SATO). By coupling ε-polylysine with S-arylthiooxime (SATO), the H2S donor ε-PL-SATO is synthesized, which can respond to the action of an NAC initiator to control the slow release of H2S. This donor reacts with carboxymethyl chitosan, oxidized hyaluronic acid, and an N-acetylcysteine ​​(NAC) initiator to form a hydrogel, enabling long-term, sustained release of H2S at the wound site.

[0031] This invention provides a technical solution that allows for the slow release of H2S from the hydrogel, addressing its low bioavailability and short duration of action. Furthermore, wound treatment requires materials with rapid prototyping capabilities to quickly close wounds and improve patient comfort. The hydrogel system of this invention uses oxidized hyaluronic acid and carboxymethyl chitosan as a matrix, enabling rapid gelation within 30 seconds, making it suitable for rapid clinical application. Through this technology, the hydrogel can quickly cover the wound, forming a protective barrier and avoiding the risk of bacterial infection.

[0032] The present application 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 release H2S for a long time and continuously and slowly, and by virtue of the regulatory effect of H2S on the phenotype of macrophages, the PI3K / Akt signal pathway is adjusted and the activation of NF-κB is inhibited, so as to promote the polarization of macrophages to the anti-inflammatory and proliferation-promoting M2 type, and further reduce the inflammatory response at the wound site. Finally, the released H2S can promote angiogenesis and collagen deposition, help the damaged tissue to restore its structure and function, accelerate the healing of the wound, and achieve effective gas treatment of infected wounds. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 is a schematic diagram of the treatment of skin damage by OC@ε-PL-SATO hydrogel in the rat infected wound model in the present application.

[0034] Figure 2 is a synthesis schematic diagram of the hydrogen sulfide donor ε-Poly-L-lysine-SATO (ε-PL-SATO) in the present application.

[0035] Figure 3 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 application.

[0036] Figure 4 is a nuclear magnetic resonance, ultraviolet, and hydrogen sulfide release capacity diagram of the hydrogen sulfide donor in the present application: wherein Figure 4 A is the nuclear magnetic resonance hydrogen spectrum of ε-Poly-L-lysine (ε-PL) and ε-Poly-L-lysine-SATO (ε-PL-SATO), Figure 4 B is the UV-Vis spectrum of ε-PL, ε-PL-SATO, and SATO, Figure 4 C is the hydrogen sulfide release performance of ε-PL-SATO and SATO.

[0037] Figure 5 is a synthesis schematic diagram, nuclear magnetic resonance, and infrared spectrum of the chitosan-hyaluronic acid hydrogel in the present application: wherein Figure 5 A is the synthesis process of oxidized hyaluronic acid (OHA) and the schematic diagram of the generation of OHA-CMCS hydrogel by Schiff base reaction of OHA with carboxymethyl chitosan (CMCS), Figure 5 B is the nuclear magnetic resonance hydrogen spectrum of HA and OHA, Figure 5 C is the infrared spectrum of CMCS, HA, OHA, and OHA-CMCS.

[0038] Figure 6OC@ε-PL-SATO hydrogel preparation process diagram of the present application.

[0039] Figure 7 OC@ε-PL-SATO hydrogel prepared by the present application through needle injection.

[0040] Figure 8 Micro-morphology diagram of OC hydrogel with different composition ingredients in Example 1.

[0041] Figure 9 H2S release performance of OC@ε-PL-SATO hydrogel of the present application.

[0042] Figure 10 Rheological performance experimental results in Experimental Example 2, wherein, Figure 10 A is the amplitude sweep experimental results of OC series hydrogel, Figure 10 B is the frequency sweep experimental results of OC series hydrogel, Figure 10 C is the OC series hydrogel viscosity change with shear rate fold line chart, Figure 10 D is the alternating strain sweep experimental results of OC@ε-PL-SATO hydrogel, Figure 10 E is the self-repairing macroscopic photo of OC@ε-PL-SATO.

[0043] Figure 11 Biocompatibility experimental results of OC hydrogel with different composition ingredients in Experimental Example 2, wherein, Figure 11 A is the OC series hydrogel survival rate column chart of L929 cells, Figure 11 B is the hemolysis rate of OC@ε-PL-SATO hydrogel to red blood cells, Figure 11 C is the OC series hydrogel scratch repair ability picture of L929 cells.

[0044] Figure 12 Antibacterial effect on E. coli and S. aureus in Experimental Example 2, wherein, Figure 12 A is the OC series hydrogel inhibition ability fold line chart of E. coli, Figure 12 B is the OC series hydrogel inhibition ability fold line chart of S. aureus, Figure 12 C is the antibacterial picture of OC series hydrogel.

[0045] Figure 13 OC hydrogel with different composition ingredients in Experimental Example 2 Polarization performance experimental results of RAW264.7 cells, wherein, Figure 13 A&B&C are flow detection and quantitative statistics of polarized RAW264.7 cells, wherein, Figure 13D is the ELISA analysis result of polarized RAW264.7 cells, wherein, Figure 13 E is a schematic diagram of the regulation process of the OC@epsilon-PL-SATO of the application on the inflammatory microenvironment of the wound.

[0046] Figure 14 The hemostatic performance of the OC@epsilon-PL-SATO hydrogel prepared in the application.

[0047] Figure 15 A&B&C are the treatment effects of the OC@epsilon-PL-SATO hydrogel with high efficiency in promoting wound healing in experimental example 4 on the infected wounds of rats, wherein Figure 15 A is a photo of the skin wound condition of rats in each group at each time point, Figure 15 B is a schematic diagram of the wound healing process, Figure 15 C is the wound healing rate at different time points after treatment.

[0048] Figure 16 A schematic diagram of H&E and Masson staining of the representative section of the new tissue of the wound after treatment in each group in experimental example 4. DETAILED DESCRIPTION

[0049] In order to enable those skilled in the art to better understand the technical solutions of the application and to implement them, the application will be further described below in conjunction with specific embodiments and drawings, but the embodiments are not limiting of the application.

[0050] The purpose of the application is to safely and efficiently deliver hydrogen sulfide gas to the action site, 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 application designs a delivery strategy of self-supplying hydrogen sulfide hydrogel based on chitosan-hyaluronic acid, enhances the advantages of H2S in slow-release penetration of wound tissue, enhances macrophage polarization, and overcomes the local inflammatory microenvironment, optimizes the hydrogen sulfide release performance of the hydrogel using chemical cross-linking, biological branch connection, and click chemistry technology, constructs an intelligent hydrogel dressing with long-acting slow-release H2S, resists bacterial infection at the wound, improves the wound microenvironment, can effectively regulate the transformation of macrophages from M1 type to M2 type and fibroblast proliferation, inhibit excessive inflammatory response, accelerate tissue regeneration, and ultimately realize the overall optimization of infected wound healing through the "multiple" regulation mechanism.

[0051] In order to achieve the above-mentioned purpose, the first aspect of the application provides a preparation method of a self-supplying hydrogen sulfide promoting wound healing antibacterial hydrogel wound dressing, comprising the following steps:

[0052] Under ice bath conditions, hydroxylamine-O-sulfonic acid sodium solution and sodium thio-benzoate solution are prepared respectively;

[0053] Sodium hydroxylamine-O-sulfonate solution is prepared by adding hydroxylamine-O-sulfonate to sodium hydroxide solution; sodium thiobenzoate solution is prepared by adding thiobenzoic acid to sodium hydroxide solution; the concentration of sodium hydroxylamine-O-sulfonate solution is 1-2 mol / L; the concentration of sodium thiobenzoate solution is 0.5-1 mol / L.

[0054] S-phenylcarbonyl thiohydroxylamine is added into dichloromethane, 4-formylbenzoic acid and TFA are added, and after reaction at room temperature for 1-5 hours, Sato powder is obtained through purification.

[0055] Sato, EDC·HCl and NHS (N-hydroxysuccinimide) are dissolved in a mixed solvent of water and DMSO, ε-PL is added after uniform mixing, and after stirring and reaction at room temperature for 24-36 hours, ε-PL-Sato is obtained through purification.

[0056] Hyaluronic acid is dispersed in water, NaIO4 is added, and after stirring for 4-6 hours, the oxidation reaction is terminated by adding ethylene glycol, and the reactants are purified to obtain OHA.

[0057] Carboxymethyl chitosan (CMCS) and ε-PL-Sato are uniformly dispersed in PBS buffer and placed in the A tube of a two-component syringe, OHA and NAC are uniformly dispersed in PBS buffer and placed in the B tube of the two-component syringe, and the components in the A tube and the B tube are mixed by squeezing the push handle of the syringe, and the obtained product is a self-supplying hydrogen sulfide promoting wound healing antibacterial hydrogel wound dressing (OC@ε-PL-Sato).

[0058] The sodium hydroxylamine-O-sulfonate solution is prepared by adding hydroxylamine-O-sulfonate to sodium hydroxide solution; the sodium thiobenzoate solution is prepared by adding thiobenzoic acid to sodium hydroxide solution; the concentration of the sodium hydroxylamine-O-sulfonate solution is 1-2 mol / L; the concentration of the sodium thiobenzoate solution is 0.5-1 mol / L.

[0059] In the preparation of S-phenylcarbonyl thiohydroxylamine, the mass ratio of the sodium hydroxylamine-O-sulfonate solution to the sodium thiobenzoate solution is 1:1-2, preferably 1:1.

[0060] In the preparation of Sato powder, the mass ratio of S-phenylcarbonyl thiohydroxylamine to 4-formylbenzoic acid is 1:1-2, preferably 1:1, and TFA acts as a catalyst so only a small amount is needed.

[0061] In the preparation of Sato powder, after 1-5 hours of reaction, the reactants are filtered, the solvent is removed under reduced pressure, the crude product is recrystallized from ethyl acetate to obtain a crude product, the crude product is dissolved in dichloromethane, and column chromatography is used for purification to obtain Sato powder.

[0062] The mass ratio of the 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.

[0063] The mass ratio of the hyaluronic acid and NaIO4 is 1-2:1.

[0064] The mass ratio of the CMCS and ε-PL-SATO is 0.6-6:1, preferably 5:1; the mass ratio of the OHA and NAC is 0.6-6:1, preferably 5:1.

[0065] In an embodiment, a method for preparing a self-supplying hydrogen sulfide sustained-release hydrogel wound dressing, comprising the following steps:

[0066] (1) Synthesis of SATO:

[0067] 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 a NaOH solution to form a thiobenzoic acid sodium solution in a certain proportion;

[0068] Under ice bath conditions, the sodium hydroxylamine-O-sulfonate solution is added dropwise to the sodium thiobenzoate solution and stirred for 30 minutes to form a large amount of white precipitate, which is collected by filtration, washed with ultrapure water, and freeze-dried overnight to obtain S-benzoyl thiohydroxylamine (SBTHA);

[0069] Molecular sieves and a stirring rod are added to a reaction bottle, then SBTHA is added, followed by the addition of anhydrous CH2Cl2, 4-formylbenzoic acid is added to the reaction bottle, then TFA is added, the mixture is sealed, and the mixture is allowed to react at room temperature for 1-5 hours, the reaction mixture is filtered, the solvent is removed under reduced pressure, the crude product is recrystallized from ethyl acetate, and finally the crude product is dissolved in CH2Cl2 and purified by column chromatography to obtain white SATO powder.

[0070] (2) Synthesis of hydrogen sulfide donor ε-PL-SATO:

[0071] SATO, EDC-HCl and NHS are dissolved in a mixed solvent of water and DMSO (H2O / DMSO, v / v = 4 / 1), stirred for 0.5 hours to pre-activate SATO, then ε-PL is added to the solution and stirred at room temperature, after 36 hours of reaction, the solution is transferred to a dialysis bag (MWCO: 3500 Da) and dialyzed in ultrapure water for 3 days, then the obtained solution is freeze-dried and redissolved in methanol, then dialyzed in methanol (MWCO: 3500 Da), and finally the mixture is vacuum dried to obtain ε-PL-SATO, which is a light yellow transparent crystal.

[0072] (3) Synthesis of precursor of hydrogel matrix OHA:

[0073] Hyaluronic acid (HA) was dissolved in distilled water, then NaIO4 was added to the solution and stirred for 6 hours. Then glycol was added to terminate the oxidation reaction. The resulting solution was dialyzed with deionized water (MWCO: 3500 Da) for 3 days, freeze-dried to obtain OHA.

[0074] (4) Preparation of OC@ε-PL-SATO hydrogel self-supplying hydrogen sulfide:

[0075] The CMCS solution and the ε-PL-SATO solution were placed in the A tube of the double-component syringe, and the OHA solution and the NAC solution were placed in the B tube of the double-component syringe. By pressing the push handle of the syringe, the components in the AB tube were mixed, rapidly reacted, and the resulting product was the OC@ε-PL-SATO hydrogel.

[0076] The preparation of the CMCS solution and the ε-PL-SATO solution: 1 g of carboxymethyl chitosan (CMCS) was dissolved in 20 mL of PBS solution to prepare 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 ratio of the two solutions was CMCS solution: ε-PL-SATO solution = 4:1.

[0077] The preparation of the OHA solution and the NAC solution: 1 g of oxidized hyaluronic acid (OHA) was dissolved in 20 mL of PBS solution to prepare a 5% OHA solution; 1 g of acetylcysteine (NAC) was dissolved in 20 mL of PBS solution to prepare a 5% OHA solution.

[0078] The ratio of the two solutions was OHA solution: NAC solution = 4:1.

[0079] The second aspect of the present application provides a wound dressing of a hydrogel self-supplying hydrogen sulfide promoting wound healing and antibiosis. The present application provides a hydrogel wound dressing with self-supplying and slow-release hydrogen sulfide, the matrix of which is oxidized hyaluronic acid (OHA) and carboxymethyl chitosan (CMCS); a traditional hydrogen sulfide donor SATO is chemically grafted to polylysine ε-Poly-L-lysine to become a new hydrogen sulfide donor ε-Poly-L-lysine-SATO (ε-PL-SATO), and the ε-PL-SATO and its initiator acetylcysteine (NAC) are loaded into the hydrogel matrix.

[0080] The dressing can load a new hydrogen sulfide donor and its initiator, and can be injected at the wound site through a syringe, can block and treat any shaped wound, improves the delivery efficiency and bioavailability of hydrogen sulfide, helps the healing of infected wounds, improves the wound microenvironment, promotes macrophage polarization and fibroblast proliferation, inhibits excessive inflammatory response, and accelerates tissue regeneration.

[0081] The third aspect of the present application provides a self-supplied hydrogen sulfide promoting wound healing antibacterial hydrogel wound dressing for preparing an infected wound treatment drug.

[0082] The multifunctional hydrogel wound dressing OC@epsilon-PL-SATO prepared by the present application can be injected and filled into any shaped wound site, 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 pro-inflammatory M1 type to anti-inflammatory proliferative M2 type and promote angiogenesis, reduce the inflammatory level at the wound site, and promote wound healing. In addition, the multifunctional hydrogel wound dressing of the present application also has excellent antibacterial performance. Briefly, the carboxymethyl chitosan in the hydrogel matrix is a natural polymer with broad-spectrum antibacterial effect, and the antibacterial performance of the hydrogel is further improved by introducing the natural antibacterial polypeptide polylysine to branch the SATO, thereby enhancing the treatment effect of the hydrogel on infected wounds.

[0083] It should be noted that the experimental methods used in the present application are conventional methods unless otherwise specified; the reagents and materials used are commercially available unless otherwise specified.

[0084] Example 1

[0085] The preparation process of the OC@epsilon-PL-SATO hydrogel includes the following steps:

[0086] 1) Preparation of hydrogen sulfide donor epsilon-PL-SATO:

[0087] 1-1) Synthesis of SATO

[0088] 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. Then, thiobenzoic acid (1.11 g, 8 mmol) was added to 16 mL of 0.5M NaOH to form a 0.5M sodium thiobenzoate solution.

[0089] Under ice-bath conditions, 10 mL of 1 M hydroxylamine-O-sulfonic acid sodium salt was added dropwise to 0.5 M sodium thiobenzoate stirring for 30 min, forming a large amount of white precipitate. The precipitate was collected by filtration, washed with ultrapure water, and freeze-dried overnight without further purification. Molecular sieves and a stir bar were first added to the reaction vial, followed by S-benzoylthiohydroxylamine (SBTHA) (500 mg, 3.2 mmol) and then 8 mL of anhydrous CH2Cl2. In a separate vial, 510 mg of 4-formylbenzoic acid (3.4 mmol) was added, followed by 25 μL of TFA. The vial was sealed and the mixture was allowed to react at room temperature for 1-5 hours. The reaction mixture was filtered, and the solvent was removed under reduced pressure. The crude product was recrystallized from ethyl acetate to obtain a white powder. Finally, the crude product was dissolved in CH2Cl2and purified by column chromatography to obtain a white SATO powder, with the structure as follows:

[0090]

[0091] 1-2) Synthesis of hydrogen sulfide donor ε-PL-SATO by amide condensation reaction of SATO onto ε-polylysine, see Figure 2 as shown below, specifically including:

[0092] SATO, 460 mg EDC-HCl, and 230 mg NHS were dissolved in 20 mL of mixed solvent (H2O / DMSO, v / v = 4 / 1) and stirred for 0.5 hours to pre-activate the carboxyl group of SATO. Subsequently, 200 mg of ε-PL was added to the solution, which was stirred at room temperature for 36 hours. The solution was then transferred to a dialysis bag (MWCO: 3500 Da) and dialyzed in ultrapure water for 3 days. The resulting solution was freeze-dried, redissolved in methanol, and dialyzed in methanol (MWCO: 3500 Da), and finally vacuum-dried to obtain ε-PL-SATO as a light yellow transparent crystal. The structure is as follows:

[0093]

[0094] 2) Synthesis of oxidized hyaluronic acid OHA

[0095] First, 4 g of HA was dissolved in 200 mL of distilled water at a temperature of 25°C. Subsequently, 2 g of NaIO4was added to the solution and stirred for another 6 hours. 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 as a white sponge-like solid by freeze-drying, with the structure as follows:

[0096]

[0097] 3) Preparation of OC@ε-PL-SATO hydrogel

[0098] 3-1) Preparation of hydrogel precursor A solution

[0099] The main components of the hydrogel precursor A solution are carboxymethyl chitosan (CMCS), ε-PL-SATO and PBS buffer. The preparation method is as follows: first take 10 mL of PBS buffer, add 0.5 g of carboxymethyl chitosan to it, then add 0.1 g of ε-PL-SATO solid to it, and the obtained liquid is the hydrogel precursor A solution.

[0100] 3-2) Preparation of hydrogel precursor B solution

[0101] The main components of the hydrogel precursor B solution 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 to it, then add 0.1 g of acetylcysteine solid to it, and the obtained liquid is the hydrogel precursor B solution.

[0102] 3-3) Formation of OC@ε-PL-SATO hydrogel

[0103] First, the prepared hydrogel precursor A solution is placed in the A tube of the double-component syringe, and the prepared hydrogel precursor B solution is placed in the B tube of the double-component syringe. Then, press the plunger of the syringe to mix the A and B solutions in the mixing nozzle, and extrude at the action site to form the OC@ε-PL-SATO hydrogel.

[0104] Preparation of OC series hydrogel

[0105] The functionalized OHA-CMCS (abbreviated as OC) based hydrogel is prepared by the same method as described above, and the corresponding components are listed as shown in Table 1 below, and the obtained hydrogel is named as OC, OC@ε-PL and OC@ε-PL-SATO respectively.

[0106] Table 1 is the corresponding components of OC, OC@ε-PL and OC@ε-PL-SATO hydrogel

[0107]

[0108] Among them, the synthesis method of the OC hydrogel matrix includes:

[0109] First, 1.0 g of CMCS solid is dissolved in 20 mL of PBS buffer to form a 5% CMCS solution. 1.0 g of OHA solid is dissolved in 20 mL of PBS buffer to form a 5% OHA solution. Take 0.5 mL of the 5% OHA solution and add it to an equal volume of the 5% CMCS solution, vortex to mix, and let stand for 1 minute. A colorless transparent solid is formed, which is the OHA-CMCS hydrogel. The structure is as follows:

[0110]

[0111] The synthesis method of the OC@ε-PL-SATO hydrogel matrix comprises:

[0112] First, 1.0 g of CMCS solid is dissolved in 20 mL of PBS buffer to form 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. Mix the CMCS solution and the ε-PL-SATO solution in a volume ratio of 4:1 to form A liquid.

[0113] Dissolve 1.0 g of OHA solid 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 B liquid.

[0114] Take 0.5 mL of A liquid and add it to an equal volume of B liquid, vortex to mix, and let stand for 1 minute. A colorless transparent solid is formed, which is the OC@ε-PL-SATO hydrogel.

[0115] The synthesis method of the OC@ε-PL hydrogel matrix comprises:

[0116] First, 1.0 g of CMCS solid is dissolved in 20 mL of PBS buffer to form a 5% CMCS solution. 1 g of ε-PL is dissolved in 20 mL of PBS solution to prepare a 5% ε-PL solution. Mix the CMCS solution and the ε-PL solution in a volume ratio of 4:1 to form A liquid.

[0117] Dissolve 1.0 g of OHA solid 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 B liquid.

[0118] Take 0.5 mL of A liquid and add it to an equal volume of B liquid, vortex to mix, and let stand for 1 minute. A colorless transparent solid is formed, which is the OC@ε-PL hydrogel.

[0119] Experimental Example 2

[0120] The products obtained in each step of Example 1 were subjected to 1 The morphology, composition and chemical bonds of the product were systematically studied by means of physical and chemical test analysis techniques such as H NMR, UV-Vis, FTIR, SEM, etc., and the results are as follows:

[0121] The ε-PL-SATO obtained in the examples was characterized by nuclear magnetic resonance hydrogen spectrum and ultraviolet-visible spectrum.

[0122] As shown in Figure 4 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 combination with SATO, a plurality of new peaks at about 7.2-8.3 ppm appeared in the spectrum of ε-PL-SATO, which is related to the benzene protons on SATO, indicating that ε-PL-SATO is successfully synthesized. The ultraviolet-visible spectrum further confirms the conjugation of ε-PL-SATO, and the characteristic absorbance peak of the SATO group (about 320 nm) in the spectra of SATO and ε-PL-SATO reveals this point. Figure 4 B).

[0123] The performance of ε-PL-SATO in generating hydrogen sulfide with trigger NAC was detected by a hydrogen sulfide detection kit, as shown in Figure 4 C, the synthesized hydrogen sulfide donor ε-PL-SATO, compared with the original SATO, the hydrogen sulfide generation efficiency is increased by nearly 5 times. The above results confirm the successful synthesis of ε-PL-SATO and can efficiently generate hydrogen sulfide gas.

[0124] The hydrogel precursor and hydrogel matrix obtained in the examples were characterized by nuclear magnetic resonance hydrogen spectrum and FTIR. Figure 5 A shows the synthesis process of OHA-CMCS hydrogel; as shown in Figure 5 B, compared with HA, three new proton signals at 4.9, 5.0 and 5.1 ppm are observed in the OHA nuclear magnetic spectrum, which correspond to the hemiacetal proton and the ortho hydroxyl group (Figure 5C). FTIR further confirms the successful synthesis of OHA and the reaction between CMCS and OHA, and compared with free HA, the clear appearance of the 1730 cm -1 Place aldehyde C=O bond stretching vibration peak in the spectrum of OHA confirms that HA has been ring opening and OHA synthesis is successful. At the same time, the 3430 cm -1The most prominent feature peaks at 1606 cm-1 are attributed to O-H and N-H (amine) stretching vibrations. -1 The typical peak at 1500-1600 cm-1 represents the stretching vibration of C=O group in CMCS, which is correctly shown in its FTIR spectrum. -1 After the hydrogel is synthesized, the narrow peak at 1500-1600 cm-1 becomes wide, and the aldehyde group peak of OHA disappears, indicating that the-CHO of OHA is successfully combined with the-NH2 of CMCS and forms a hydrogel.

[0125] The material properties of the OC@ε-PL-SATO hydrogel in the examples are characterized by gelation experiments, injection experiments, scanning electron microscopy, and in vitro hydrogen sulfide release experiments.

[0126] As shown in Figure 6 , the precursor A liquid (CMCS, ε-PL-SATO) and the precursor B liquid (OHA, NAC) of the OC@ε-PL-SATO hydrogel can quickly form a hydrogel within 30 seconds after mixing at room temperature. The above results show that the OC@ε-PL-SATO hydrogel has the characteristics of ultra-fast gelation.

[0127] As shown in Figure 7 , the OC@ε-PL-SATO hydrogel can be injected through a needle after mixing with a two-component syringe and can be written into any shape. The above results confirm that the OC@ε-PL-SATO hydrogel has good injectability.

[0128] As shown in Figure 8 , the porous structure of the hydrogel is studied using a scanning electron microscope. The three hydrogels all present a loose and porous network structure, and the pore size of the OC@ε-PL-SATO hydrogel is relatively large, which is beneficial to wound healing.

[0129] As shown in Figure 9 , the OC@ε-PL-SATO hydrogel of the present application can effectively release H2S for about 24 hours after injection. As shown in Figure 3 , the schematic diagram of hydrogen sulfide donor ε-Poly-L-lysine-SATO (ε-PL-SATO) releasing H2S.

[0130] The rheological properties of the OC@ε-PL-SATO hydrogel are analyzed by rheological experiments. Dynamic strain sweep tests are performed using an Anton Paar rotational rheometer to estimate the gel-sol transition point and the linear viscoelastic region (LVR). Figure 10A). With a significant increase in shear strain, the loss modulus (G'') exceeds the storage modulus (G'), indicating that a gel-sol phase transition has occurred due to the structural destruction of the post-stabilized hydrogel under large deformation. In frequency scanning experiments ( Figure 10 In B), G' in all post-stabilized hydrogels consistently exceeded the corresponding G'' in the frequency range of 0.1–100 Hz, indicating stable hydrogel structure and behavior similar to viscoelastic gels. Simultaneously, the storage modulus of the OC hydrogel with added hydrogen sulfide donor ε-PL-SATO increased from approximately 100 Pa to 1000 Pa, indicating a stronger cross-linking network in OC@ε-PL and OC@ε-PL-SATO hydrogels compared to the standard OC hydrogel. This is mainly attributed to the formation of a double cross-linking of CMCS, ε-PL, and OHA, significantly improving its mechanical properties. Hydrogels with excellent self-healing properties and shape-adaptive characteristics show promising 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 (…). Figure 10 (C) This demonstrates the shear-thinning ability of the gel, consistent with previous macroscopic injectable experiments. To evaluate the self-healing ability of these hydrogels, OC@ε-PL-SATO hydrogel was selected as a model sample for rheological recovery and macroscopic self-healing tests. First, we performed continuous strain scanning on the OC@ε-PL-SATO hydrogel to determine its self-healing behavior based on the strain points that led to hydrogel collapse. The hydrogel network was disrupted at large strains (300%), while it began to repair itself once lower strains (1%) were applied. Figure 10 D). Even after three alternating cycles, the values ​​of G' and G'' almost recovered to their original values, indicating that all three hydrogels have good self-healing capabilities. On the other hand, the functional OC@ε-PL-SATO hydrogel cut into a semi-disc shape can self-heal to form a new disc-shaped hydrogel after incubation at 37°C for 30 minutes. Figure 10 E), and it can maintain its complete structure without obvious cracks, which once again proves its excellent self-healing properties.

[0131] Experimental Example 3

[0132] In vitro biological evaluation of OC@ε-PL-SATO hydrogel

[0133] 3.1 Cytotoxicity

[0134] The in vitro biocompatibility of hydrogels with L929 fibroblasts was determined according to the national standard (GB / T 16886.5-2003 / ISO 10993-5 1999) using the extraction 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, L929 cells inoculated in a 96-well plate were incubated with the above extract (200 μL) at 37°C, 5% CO2 for 24 h and 48 h, respectively. The control group was replaced with fresh DMEM. According to the manufacturer's procedure, the cell viability was detected by CCK-8 kit, and the absorbance of samples at 450 nm was measured by a microplate reader. The results are shown in Figure 11 As shown in Fig. 2A, the viability of L929 cells treated with different groups of OC hydrogels had no difference with PBS-treated cells (control) at 24 h and 48 h. Fresh red blood cells were collected from SD rats, resuspended with 2% volume concentration of normal saline, and incubated with OC@ε-PL-SATO hydrogel, normal saline (negative control), 1% Triton X-100 (positive control) and ddH2O (positive control) at 37°C for 3 h, centrifuged at 2000 rpm for 15 min, and the absorbance of supernatant at 545 nm was detected by a microplate reader. The results are shown in Figure 11 As shown in Fig. 2B, OC@ε-PL-SATO hydrogel showed good blood compatibility, with a hemolysis rate of less than 4%, and the supernatant of red blood cell suspension treated with hydrogel was colorless and transparent by visual inspection. Figure 11

[0135] 3.2 Cell scratch and migration performance

[0136] On the other hand, the effect of hydrogel on L929 cell migration was observed by cell scratch experiment. First, a scratch was made in the 6-well plate inoculated with L929 cells with a p200 pipette gun head. Then, 2 mL of hydrogel extract was added and incubated for 48 hours. The cell migration image was taken by inverted microscope. As shown in Fig. 3A, Figure 11 As shown in Fig. 3C, the cell migration speed of OC@ε-PL-SATO hydrogel was visually faster than other groups after 48 h of co-culture, revealing the excellent in vitro wound healing performance of functional OC@ε-PL-SATO hydrogel.

[0137] 3.3 In vitro antibacterial performance

[0138] The above three kinds of hydrogels were respectively injected into sterile EP tubes at 400 μg, and then 400 μL of 1 × 10 5 ​E. coli or S. aureus at 107 CFU / mL were added to the surface of the hydrogels described above, and after incubation for 0, 6, 12, and 24 h, the bacterial solution samples were transferred to a 96-well plate, and the absorbance value at 600 nm was recorded using a microplate reader to determine the growth of the bacteria. After the bacterial solution samples were co-incubated with the hydrogels for 24 h, they were diluted to an appropriate concentration and spread on agar plate medium, which was incubated at 37 °C for 16 h, after which the colonies were counted and photographed. The antibacterial curves showed that the growth of the two types of bacteria was significantly inhibited after 24 h of incubation in the OC series of hydrogels (Fig. 12A and B), which was attributed to the inherent antibacterial properties of CMCS. At the same time, the OD600 of S. aureus and E. 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, indicating that it had more excellent bactericidal performance, which was due to the fact that ε-PL itself is a broad-spectrum antibacterial peptide. Then, the colony formation inhibition test of E. coli and S. aureus further confirmed the excellent antibacterial performance of the ε-PL modified hydrogel, and there were almost no bacterial colonies on the agar plate compared with the control group and the OC hydrogel (Fig. 12C), again demonstrating its excellent in vitro antibacterial performance. Figure 12 C), again demonstrating its excellent in vitro antibacterial performance.

[0139] 3.4 In vitro promotion of macrophage polarization performance

[0140] RAW264.7 cells were seeded in a 6-well plate, pre-incubated with lipopolysaccharide (LPS, 1 μg / mL) to activate the pro-inflammatory M1 phenotype, and then treated with 3 kinds of hydrogel extracts (2 mL, OC, OC@ε-PL or OC@ε-PL-SATO) and IL-4 (20 ng / mL, positive control) for 48 h, respectively. Then, PE anti-mouse CD86 antibody and APC anti-mouse CD206 antibody were added and incubated with RAW264.7 cells for 30 min. Flow cytometry (BD FACS Celesta™, USA) and FlowJo software were used to analyze the changes in macrophage phenotype. As shown in Fig. 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 of 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 OC and OC@ε-PL groups lacking H2S production ability. This was consistent with the results of the average fluorescence intensity (MFI) analysis of CD206 in macrophages (Fig. 13C), indicating that M2 macrophages were effectively polarized.

[0141] The changes in cytokine secretion by macrophages were also investigated. Briefly, RAW264.7 cells seeded in 96-well plates were treated with the three hydrogel extracts plus LPS (1 pg / mL) and free LPS for 24 h, respectively. RAW264.7 cells seeded in 96-well plates without any additives were used as controls. The supernatants of the above treatments were measured for TNF-a, TGF-b, IL-10, and IL-1 b using ELISA kits. The results showed that in the case of macrophages first induced to M1 type by LPS, the secretion of the classical pro-inflammatory factors TNF-a and IL-1 b in RAW264.7 cells was significantly reduced after treatment with OC@e-PL-SATO hydrogel extract. In contrast, the levels of anti-inflammatory factors IL-10 and TGF-b detected by ELISA were increased compared to other treatments Figure 13 D). These results highlight the strong ability of OC@e-PL-SATO hydrogel to direct macrophages from pro-inflammatory M1 to anti-inflammatory M2 phenotypes (Fig. 13E).

[0142] Experimental Example 4

[0143] Evaluation of the pro-infectious wound healing properties of OC@e-PL-SATO hydrogel with self-supplying H2S ability, see Figure 1

[0144] 4.1 Hemostatic performance analysis in rats

[0145] The hemostatic performance of the hydrogels was determined by a rat tail transection bleeding model. The method was as follows: after the SD rats (~300 g, male) were anesthetized, 50% of the tail was cut off, and the wound was covered with gauze, 3M Tegaderm dressing and the above hydrogels, respectively, and the bleeding data during hemostasis were recorded. The wounds of the control group of rats were not treated in any way. Traditional hemostatic materials such as gauze and 3M Tegaderm were chosen as positive control groups, and rats without any treatment were used as a blank control. As shown in Figure 14 The hemostatic effect of the OC@e-PL-SATO hydrogel treatment group was significantly better than that of the other groups, and the hemostatic ability was better than that of the commercially available gauze and 3M Tegaderm™ materials.

[0146] 4.2 Construction of an infectious wound model in rats

[0147] 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 10 mm diameter wound was formed and infected with Staphylococcus aureus (10 pL, 10 6 CFU / mL) to form a rat infectious wound model.

[0148] 4.3 Hydrogel treatment intervention and histopathological analysis​

[0149] The above infection model rats were divided into five groups, 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, respectively. The results are shown in Figure 15 OC@ε-PL-SATO hydrogel group showed the best wound healing degree and the fastest healing speed at all treatment times Figure 16 A-C). Notably, by day 7, the wound contraction of the OC@ε-PL-SATO group increased by nearly 23% compared to the 3M dressing group. By day 14, the wound of the OC@ε-PL-SATO hydrogel group was almost completely closed (wound contraction rate of 99%), while the wound contraction rate of the OC@ε-PL group was 94%. In contrast, the other groups still had obvious residual wound areas. The H&E and Masson staining results further confirmed the excellent wound healing performance of the OC@ε-PL-SATO hydrogel ​ ). Compared with the control group, the healing effect of the OC@ε-PL-SATO hydrogel group was better, and the regenerated skin tissue structure was complete, with complete epithelial and dermal layers, increased new blood vessels and hair follicle formation. In addition, the thickest collagen bundles were observed in the wound tissue treated with OC@ε-PL-SATO hydrogel, which were dark blue, indicating that the damaged tissue was recovering and maturing.

[0150] Example 5

[0151] The same as Example 1, except that the synthesis method of OC@ε-PL-SATO hydrogel was changed, including

[0152] First, 0.6 g of CMCS solid was dissolved in 20 mL of PBS buffer to form a 3% 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 A solution. 0.6 g of OHA solid was dissolved in 20 mL of PBS buffer to form a 3% 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 B solution. 0.5 mL of A solution was added to an equal volume of B solution, vortexed and mixed, and left to stand for 1 minute, forming a colorless transparent solid, which was the OC@ε-PL-SATO hydrogel.

[0153] Example 6

[0154] The same as example 1, except that the synthesis method of OC@ε-PL-SATO hydrogel is changed, including

[0155] First, 0.8g of CMCS solid is weighed and dissolved in 20mL of PBS buffer to form a 4% CMCS solution. 1g of ε-PL-SATO is dissolved in 20mL of PBS solution to prepare a 5% ε-PL-SATO solution. The CMCS solution and the ε-PL-SATO solution are mixed in a volume ratio of 4:1 to serve as A liquid. 0.8g of OHA solid is weighed and dissolved in 20mL of PBS buffer to form a 4% OHA solution. 1g of NAC is dissolved in 20mL of PBS solution to prepare a 5% NAC solution. The OHA solution and the NAC solution are mixed in a volume ratio of 4:1 to serve as B liquid. 0.5mL of A liquid is added to an equal volume of B liquid, vortexed and mixed, and left to stand for 1 minute, forming a colorless transparent solid, which is OC@ε-PL-SATO hydrogel.

[0156] Example 7

[0157] The same as example 1, except that the synthesis method of OC@ε-PL-SATO hydrogel is changed, including

[0158] First, 1.2g of CMCS solid is weighed and dissolved in 20mL of PBS buffer to form a 6% CMCS solution. 1g of ε-PL-SATO is dissolved in 20mL of PBS solution to prepare a 5% ε-PL-SATO solution. The CMCS solution and the ε-PL-SATO solution are mixed in a volume ratio of 4:1 to serve as A liquid. 1.2g of OHA solid is weighed and dissolved in 20mL of PBS buffer to form a 6% OHA solution. 1g of NAC is dissolved in 20mL of PBS solution to prepare a 5% NAC solution. The OHA solution and the NAC solution are mixed in a volume ratio of 4:1 to serve as B liquid. 0.5mL of A liquid is added to an equal volume of B liquid, vortexed and mixed, and left to stand for 1 minute, forming a colorless transparent solid, which is OC@ε-PL-SATO hydrogel.

[0159] The effects achieved by the present application are as follows:

[0160] 1. Slow and sustained release of hydrogen sulfide gas

[0161] The hydrogen sulfide gas release ability of OC@e-PL-SATO hydrogel was detected in vitro, and the H2S real-time release behavior of the hydrogel was monitored using a H2S content detection kit and WSP-5 hydrogen sulfide fluorescent probe. The results show that the negative control hydrogel group (OC, OC@e-PL, OC@e-PL-SATO without NAC) cannot produce H2S without the H2S donor e-PL-SATO and the initiator NAC. The finally formed functional hydrogel combination OC@e-PL-SATO (tube A contains CMCS and H2S donor e-PL-SATO, and tube B contains OHA and initiator NAC) can significantly produce H2S, which is detected by H2S fluorescent probe WSP-5 and presents bright green fluorescence. Further quantitative detection of the release of H2S in OC@e-PL-SATO hydrogel using a H2S content detection kit shows that the release of H2S in OC@e-PL-SATO hydrogel reaches a peak within 6 h, about 7 μM, and the release behavior of H2S can last for 48 h, which is consistent with the results of short-term and long-term H2S fluorescence imaging detection, revealing that the functionalized OC@e-PL-SATO hydrogel has the characteristics of self-supply and sustained H2S release.

[0162] 2. Good biosafety.

[0163] L929 was used as a model cell to study the biosafety of the hydrogel, and the results of the cytotoxicity experiment and the live / dead cell staining experiment show that the extract of OC@e-PL-SATO hydrogel was co-incubated with L929 cells for 24 hours and 48 hours, respectively, and the cell viability was studied using a CCK8 kit. There was no significant difference in cell activity between the three hydrogel treatment groups and the control group regardless of the incubation time, indicating good biocompatibility. The live / dead staining experiment further proved the excellent cell compatibility of the OC series hydrogel, with a large number of live cells labeled as green fluorescence and a small amount of dead cells labeled as red fluorescence.

[0164] Further, rat red blood cells were used as model cells to study the blood safety of the hydrogel, and the results of the hemolysis experiment show that OC@e-PL-SATO hydrogel exhibits good blood compatibility with a hemolysis rate of less than 4%, and the supernatant of the red blood cell suspension treated with the hydrogel is colorless and transparent by visual inspection. In addition, the red blood cell morphology is still natural after being treated with OC@e-PL-SATO hydrogel for 3 hours, similar to the shape of healthy red blood cells, which again confirms that OC@e-PL-SATO hydrogel has good biocompatibility.

[0165] 3. Good antibacterial properties.

[0166] The antibacterial effect of the hydrogels was studied using Staphylococcus aureus and Escherichia coli, common wound infection bacteria. The inhibition curve and co-incubation plating results showed that the growth of the 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 more excellent bactericidal performance, which is due to the fact that ε-PL itself is a broad-spectrum antibacterial peptide. Compared with the control group and the OC series hydrogels, almost no bacterial colonies were formed on the agar plate.

[0167] 4. Regulate the polarization of macrophages to M2 anti-inflammatory and proliferation-promoting phenotype.

[0168] An in vitro RAW264.7 cell inflammation model was constructed, and the results of flow cytometry and ELISA determination showed that interleukin 4 (IL-4), as a positive control, effectively induced macrophages to polarize to the M2 phenotype, which was manifested as the red shift of 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 average fluorescence intensity (MFI) analysis of CD206 in macrophages, indicating that M2 macrophages are effectively polarized.

[0169] 5. Effective in vivo hemostatic performance

[0170] A tail transection bleeding model was established, and traditional hemostatic materials such as gauze and 3M Tegaderm were used as positive control groups, and rats without any treatment were blank controls. The hemostatic effect of the OC@ε-PL-SATO hydrogel treatment group was significantly better than that of the other groups, with the least amount of bleeding, the shortest hemostatic time, and better hemostatic ability than commercially available gauze and 3M Tegaderm materials.

[0171] 6. Effective in vivo wound healing effect

[0172] An infectious 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, and by the 7th day, the wound contraction of the OC@ε-PL-SATO group increased by nearly 23% compared with the 3M dressing group. By the 14th day, the wound of the OC@ε-PL-SATO hydrogel group was almost completely closed (the wound contraction rate was 99%), while the wound contraction rate of the OC@ε-PL group was 94%. In contrast, the other groups still had obvious residual wound areas. H&E and Masson staining results showed that the healing effect of the OC@ε-PL-SATO hydrogel group was better than that of the control group, the regenerated skin tissue structure was complete, the epithelial layer and dermal layer were complete, and the formation of new blood vessels and hair follicles increased. In addition, the thickest collagen bundles were observed in the wound tissue treated with the OC@ε-PL-SATO hydrogel, which were deep blue, indicating that the damaged tissue was recovering and maturing. The OC@ε-PL-SATO hydrogel with self-supplying H2S properties has a better therapeutic effect than the 3M dressing.

[0173] In summary, the present application uses a dual-component syringe to prepare a dual-component functional hydrogel. The precursor A liquid (CMCS, ε-PL-SATO) and the 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, can release H2S, and has antibacterial properties. In addition, in vitro experiments and transcriptome analysis show that the functional hydrogel can effectively promote the polarization of macrophages to the anti-inflammatory M2 phenotype caused by H2S, and reveal the related mechanisms mediated by the PI3K / Akt and NF-κB pathways. In vivo experiments confirm the effectiveness of the OC@ε-PL-SATO hydrogel in promoting wound healing in bacterial infection by regulating the inflammatory microenvironment, accompanied by good hemostasis, angiogenesis and collagen deposition. In summary, the OC@ε-PL-SATO hydrogel with rapid gelation characteristics and sustained release of H2S functions provides an effective therapeutic platform for chronic wound and bacterial infection wound management, and is a new type of wound healing promoting hydrogel dressing.

[0174] The preferred embodiments and their effects are described. However, once the basic inventive concept is known, those skilled in the art can make further changes and modifications to the embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0175] While embodiments of the application have been shown and described, it is to be understood that the embodiments described are merely exemplary of the principles and application of the present application. Numerous modifications and adaptions can be effected without departing from the spirit and scope of the present application, which is not limited to the exact construction and arrangement described. It is intended, therefore, to cover all modifications and adaptions that fall within the scope of the claims and their equivalents.

Claims

1. A method of preparing a pro-wound healing, antibacterial hydrogel wound dressing that self-supplies hydrogen sulfide, characterized in that, The method comprises the following steps: Sodium hydroxylamine-O-sulfonate solution and sodium thiobenzoate solution are respectively prepared under ice bath condition; Sodium hydroxylamine-O-sulfonate solution is added into sodium thiobenzoate solution under ice bath condition, and stirred for 20-40 minutes to obtain S-benzoylthiohydroxylamine; S-benzoylthiohydroxylamine is added into dichloromethane, 4-formylbenzoic acid and TFA are added, and the mixture is reacted at room temperature for 1-5 hours to obtain SATO powder after purification; SATO, EDC·HCl and NHS are dissolved in a mixed solvent of water and DMSO, uniformly mixed, and then ε-PL is added, and the mixture is stirred and reacted at room temperature for 24-36 hours to obtain ε-PL-SATO after purification; Hyaluronic acid is dispersed in water, NaIO4 is added, and the mixture is stirred for 4-6 hours, followed by adding ethylene glycol to terminate the oxidation reaction, and the reaction product is purified to obtain OHA; CMCS and ε-PL-SATO are uniformly dispersed in PBS buffer and placed in a tube of a two-component syringe, OHA and NAC are uniformly dispersed in PBS buffer and placed in another tube of the two-component syringe, and the components in the two tubes are mixed by pressing the plunger of the syringe, so that the obtained product is a self-supplying hydrogen sulfide promoting wound healing antibacterial hydrogel wound dressing.

2. A process for the preparation of a self-supplying hydrogen sulfide wound healing promoting antibacterial hydrogel wound dressing according to claim 1, characterized in that, The sodium hydroxylamine-O-sulfonate solution is prepared by adding sodium hydroxylamine-O-sulfonate into sodium hydroxide solution, and the sodium thiobenzoate solution is prepared by adding sodium thiobenzoate into sodium hydroxide solution; 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 of claim 1, wherein the method is characterized by, In the preparation of S-benzoylthiohydroxylamine, the mass ratio of the sodium hydroxylamine-O-sulfonate solution to the sodium thiobenzoate solution is 1:1-2.

4. The process for the preparation of a self-supplying hydrogen sulfide wound healing and antibacterial hydrogel wound dressing of claim 1, characterized in that, In the preparation of SATO powder, the mass ratio of S-benzoylthiohydroxylamine to 4-formylbenzoic acid is 1:1-2.

5. The process for the preparation of a self-supplying hydrogen sulfide wound healing and antibacterial hydrogel wound dressing of claim 1, characterized in that, In the preparation of SATO powder, after 1-5 hours of reaction, the reaction product is filtered, the solvent is removed under reduced pressure, the crude product is recrystallized from ethyl acetate to obtain a crude product, the crude product is dissolved in dichloromethane, and the solution is purified by column chromatography to obtain SATO powder.

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

1.

7. The process for the preparation of a self-supplying hydrogen sulfide wound healing and antibacterial hydrogel wound dressing of claim 1, characterized in that, The mass ratio of the hyaluronic acid to NaIO4 is 1-2:

1.

8. The process for the preparation of a self-supplying hydrogen sulfide wound healing and antibacterial hydrogel wound dressing according to claim 1, characterized in that, The mass ratio of the CMCS to ε-PL-SATO is 0.6-6:1, and the mass ratio of the OHA to NAC is 0.6-6:

1.

9. A self-supplying hydrogen sulfide promoting wound healing antibacterial hydrogel wound dressing prepared by the method of any one of claims 1-8.

10. Use of the self-supplying hydrogen sulfide promoting wound healing antibacterial hydrogel wound dressing of claim 9 in the preparation of an infected wound treatment drug.

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