Dual-mode antibacterial repair-promoting injectable hydrogel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to dual-mode antibacterial repair-promoting injectable hydrogel as well as a preparation method and application thereof. The dual-mode antibacterial and repair-promoting injectable hydrogel is prepared by encapsulating PES (Polyethylene Sulfonate)-RSNO (Reverse Synthesized Nitric Oxide) particles into P407 / P188 / Sal hydrogel, wherein the P407 / P188 / Sal hydrogel is the P407 / P188 / Sal hydrogel doped with PDA-MnO2 NPs and loaded with metformin chitosan-vanillin composite microspheres. The invention further discloses a preparation method of the dual-mode antibacterial and repair-promoting injectable hydrogel. The hydrogel disclosed by the invention can be used for promoting generation and reepithelialization of skin collagenous fiber cells at a diabetic wound and generation of skin appendages such as hair follicles, has an angiogenesis promoting effect and a certain anti-inflammatory effect, can be used for promoting the healing process of wound tissues, and can be used as an excellent wound dressing for wound repair.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a dual-mode antibacterial and repair-promoting injectable hydrogel as well as a preparation method and application thereof. BACKGROUND

[0002] Diabetic wounds, as a serious complication of diabetes, have caused a significant economic burden on the healthcare system and patients, mainly due to the high cost of wound care, including frequent hospitalization and dressing changes. The special local microenvironment of diabetic wound, such as long-term inflammation, poor angiogenesis and epithelial dysfunction, can lead to slow or even non-healing of the wound, increase the risk of bacterial infection, and even threaten life safety. At the same time, the pathological features of diabetic wounds (such as persistent hyperglycemia, neuropathy, and vasculopathy) often lead to deep ulceration, irregular wound surface, and formation of deep cavities or sinuses. This highly irregular wound morphology poses a great challenge to traditional wound dressings: solid or pre-formed dressings are difficult to achieve full adhesion to the wound surface, and are prone to form "dead space" in the concave part of the wound, leading to accumulation of exudate, bacterial growth and ineffective drug delivery to the deep lesion. Frequent dressing changes or forced filling can easily cause mechanical peeling of the fragile granulation tissue and new epithelium (secondary damage), further delaying the healing process.

[0003] Hydrogels, as wound dressings, are similar in structure to biological soft tissues, can cover wounds as a temporary barrier to prevent external infection, and induce skin tissue regeneration, and have become the most attractive wound dressing. However, most existing hydrogel materials only provide a passive repair environment and lack the ability to respond to complex wound environments (such as bacterial infection and tissue repair), which cannot meet the needs of rapid healing of bacterial diabetic wounds. At the same time, existing hydrogel antibacterial treatments mostly rely on chemical drugs (such as antibiotics), leading to the emergence of drug-resistant bacteria and increasing the difficulty of treating bacterial diabetic wounds.

[0004] Therefore, it is of great significance to develop a multifunctional hydrogel that can achieve efficient antibacterial and wound repair for the treatment of bacterial diabetic wounds. SUMMARY

[0005] In view of the above, the present application provides a dual-mode antibacterial and repair-promoting injectable hydrogel and a preparation method and application thereof. The present application is based on a response surface method to optimize a poloxamer 407 / poloxamer 188 / saline temperature-sensitive hydrogel system, and successfully constructs a hydrogel loaded with PDA-MnO2 nanoparticles, CVM microspheres and PES-RSNO particles, which has a dual-mode antibacterial and repair-promoting function. The PES-RSNO particles (release endogenous gas molecule NO to inhibit bacteria) and the PDA-MnO2 NPs (photothermal response to inhibit bacteria) achieve a dual-mode synergistic antibacterial effect of light and NO, and the antibacterial rate of Staphylococcus aureus and Escherichia coli reaches 99.9% in a short time, thereby reducing the risk of drug resistance. At the same time, the hydrogel material activates the pro-angiogenic signal, effectively improves the blood supply of the wound, and the released anti-inflammatory substances can reduce the local inflammatory response, thereby creating a favorable environment for tissue repair, and has innovative application potential in the treatment of infected wounds and the promotion of tissue repair.

[0006] In order to achieve the above-mentioned purpose, the present application realizes the following technical scheme: In a first aspect, the present application provides a dual-mode antibacterial and repair-promoting injectable hydrogel, wherein the dual-mode antibacterial and repair-promoting injectable hydrogel encapsulates PES-RSNO particles in a hydrogel matrix doped with PDA-MnO2 NPs and CVM microspheres, and the hydrogel matrix is composed of P407, P188 and Sal.

[0007] In a second aspect, the present application provides a preparation method of the dual-mode antibacterial and repair-promoting injectable hydrogel of the first aspect, comprising the following steps: S1, respectively weigh P407, P188 and Sal, dissolve them in pre-cooled deionized water, and stir until completely dissolved to obtain a transparent micellar solution: S2, add PES-RSNO particles, PDA-MnO2 NPs and CVM microspheres to the transparent micellar solution and vortex to disperse, and obtain the product.

[0008] In some embodiments of the present application, the step S1 specifically comprises: weighing Sal and placing it in a centrifugal tube, adding pre-cooled distilled water and stirring until completely dissolved, then adding pre-weighed P407 and P188 powders into the Sal solution, and placing it at 4±1℃ for 24-36h until a transparent micellar solution is obtained; in the transparent micellar solution, the amount of Sal is 0.5%-1%, w / v; the amount of P407 is 22%-24%, w / v; and the amount of P188 is 2%-3%, w / v. In some embodiments of the present application, the amount of Sal is 0.7±0.1%, w / v; the amount of P407 is 23±0.5%, w / v; and the amount of P188 is 2.5±0.1%, w / v.

[0009] In some embodiments of the present application, the amount of Sal is 0.73%, w / v; the amount of P407 is 23%, w / v; and the amount of P188 is 2.5%, w / v.

[0010] In some embodiments of the present application, the method for preparing the PES-RSNO microparticles in step S2 comprises: collecting the L. rhamnosus culture, adding deionized water to the culture, treating at 80±5℃ for 24±5 h, and centrifuging to separate an orange transparent liquid to obtain a PES mother liquor; dispersing RSNO powder in the PES mother liquor, stirring to mix uniformly to obtain a PES-RSNO solution, and freeze-drying the PES-RSNO solution to obtain PES-RSNO microparticles.

[0011] In some embodiments of the present application, the concentration of the PES mother liquor is 2 mg / mL, and the concentration of RSNO is 0.3 mg / mL.

[0012] In some embodiments of the present application, the preparation of RSNO comprises: dissolving thiomalic acid in deionized water, adding HCl and NaNO2 to the thiomalic acid solution, stirring in the dark under ice bath conditions for 20 minutes until a red solution is formed, pre-freezing the red solution at -40℃, and freeze-drying for 48 h to obtain pink RSNO powder.

[0013] In some embodiments of the present application, the concentration of thiomalic acid is 0.1875 g / mL.

[0014] In some embodiments of the present application, the concentration of HCl is 1 mol / L.

[0015] In some embodiments of the present application, the mass-volume ratio of HCl to NaNO2 is 2:0.69, mL: g.

[0016] In some embodiments of the present application, in step S2, the method for preparing PDA-MnO2 NPs comprises: adding MnO2 nanoparticles to Tris / HCl buffer solution, ultrasonic dispersion treatment, then adding dopamine hydrochloride, ultrasonic stirring at room temperature in the dark, centrifuging to collect the nanoparticle precipitate, and washing the nanoparticle precipitate with anhydrous ethanol and pure water respectively, and drying to obtain the PDA-MnO2 NPs; wherein the pH of the Tris / HCl buffer solution is 8.5±0.5.

[0017] In some embodiments of the present application, the concentration of the Tris / HCl buffer solution is 0.01 mol / L, and the pH is 8.5.

[0018] In some embodiments of the present application, the mass ratio of MnO2 nanoparticles to dopamine hydrochloride is 1:1.

[0019] In some embodiments of the present application, in step S2, the preparation method of the CVM microspheres comprises: dissolving chitosan in 1% acetic acid solution to obtain a chitosan solution, then dissolving metformin hydrochloride in the chitosan solution to obtain a drug-loaded aqueous phase solution; adding an emulsifier Span-80 into soybean oil to obtain an oil phase solution; adding the drug-loaded aqueous phase solution into the stirred oil phase solution to perform emulsification; then adding a vanillin crosslinking agent solution to perform reaction, to obtain a microsphere precipitate, which is washed and dried to obtain the CVM microspheres.

[0020] In some embodiments of the present application, the mass-volume ratio of chitosan to 1% acetic acid solution is 1:50, g:mL.

[0021] In some embodiments of the present application, the mass ratio of chitosan to metformin hydrochloride is 5:2.

[0022] In some embodiments of the present application, the content of the emulsifier Span-80 in the oil phase solution is 2 wt%.

[0023] In some embodiments of the present application, the volume ratio of the drug-loaded aqueous phase solution to the oil phase solution is 1:10.

[0024] In some embodiments of the present application, the concentration of the vanillin crosslinking agent is 2.5%.

[0025] In a third aspect of the present application, a pharmaceutical composition is provided, wherein the active ingredient of the pharmaceutical composition comprises the dual-mode antibacterial and repair-promoting injectable hydrogel described above.

[0026] In some embodiments of the present application, the pharmaceutical composition further comprises at least one pharmaceutical inactive ingredient.

[0027] In some embodiments of the present application, the pharmaceutical inactive ingredient can be a carrier, an excipient, a diluent, and the like commonly used in pharmacy, which is not specifically limited herein.

[0028] In a fourth aspect of the present application, the multifunctional temperature-sensitive hydrogel of the first aspect or the pharmaceutical composition of the third aspect is used for preparing a diabetic wound repair product.

[0029] In some embodiments of the present application, the diabetic wound repair product is for a bacterial diabetic wound.

[0030] In some embodiments of the present application, the bacteria include Staphylococcus aureus and Escherichia coli.

[0031] The beneficial technical effects of the one or more technical solutions described above are as follows: (1) Dual antibacterial mode: the Lactobacillus rhamnosus screened in the present application has excellent antibacterial and anti-inflammatory performance. L. rhamnosusProbiotics, the combination of probiotics and heat-responsive NO donors, the synergistic antibacterial effect of probiotics and NO is achieved by the photothermal antibacterial effect of PDA, and the drug resistance is reduced. Among them, the excellent antibacterial effect of the photothermal antibacterial synergistic effect of PES-RSNO microparticles and PDA-MnO2 nanoparticles is as follows: a large amount of heat is generated by the water gel through near-infrared light for a short time, and a large amount of NO is generated to achieve high-efficiency bacteriostatic effect; under the condition of no light, the low level of NO gas released by PES-RSNO interacts with PES to penetrate and destroy the integrity of the cell membrane, realizing long-term antibacterial. At the same time, the released NO gas plays a key role in the process of angiogenesis. It can promote the proliferation and migration of vascular endothelial cells, and then promote the formation of new blood vessels.

[0032] (2) Excellent antioxidant capacity and tissue repair capacity: The loading of PDA-MnO2 nanoparticles endows the hydrogel with antioxidant stress resistance and oxygen production function. The hydrogel loaded with PDA-MnO2 NPs has a rapid clearing effect on H2O2, which can effectively reduce the accumulation of active oxygen. At the same time, the hydrogel can effectively scavenge ROS in the in vitro environment, and protect the cells from oxidative damage, which is crucial for improving the hypoxic condition in the diabetic wound environment and helps to promote the healing process of diabetic wounds. Animal experiments show that the hydrogel described in the application significantly improves the healing rate of diabetic wounds (93.62% on the 14th day) and promotes collagen deposition (density of 80.82%), which can be used as an excellent wound dressing for wound repair. At the same time, under the action of the wound microenvironment, the hydrogel dressing can slowly release metformin, regulate the local and even systemic blood glucose level of the wound, and adjust the long inflammation period of the diabetic wound, which is crucial for the treatment of bacterial diabetic wounds.

[0033] (3) Excellent biocompatibility and low cytotoxicity: The temperature-sensitive hydrogel with adjustable gelation temperature is prepared by taking P407, P188 and Sal as the matrix, which has good biocompatibility and moisturizing property, no cytotoxicity, and can promote wound healing, and is suitable for long-term use.

[0034] (4) Adapt to the characteristics of the wound surface: The hydrogel described in the application has good injectability and good adaptability to various shapes of wounds, especially irregular wounds, which can achieve complete filling, and is easy to remove, which can avoid secondary damage to the wound and effectively avoid infection problems caused by the use of materials. BRIEF DESCRIPTION OF DRAWINGS

[0035] The drawings accompanying the specification of the present application form a part thereof, serve to provide further understanding of the present application, and together with the description of the exemplary embodiments of the present application and the explanation thereof, explain the present application, and do not constitute an improper limitation of the present application.

[0036] Figure 1 Coli and PES S. aureus Coli and PES S. aureus Coli and PES S. aureus Coli and PES S. aureus Coli and PES Figure 2 Coli and PES E. coli Coli and PES E. coli Coli and PES E. coli Coli and PES E. coli Coli and PES Figure 3 Coli and RSNO S. aureus Coli and RSNO S. aureus Coli and RSNO S. aureus Coli and RSNO S. aureus Coli and RSNO Figure 4 Coli and RSNO E. coli Coli and RSNO E. coli Coli and RSNO Figure 5 Coli and PES-RSNO S. aureus Coli and PES-RSNO E. coli Coli and PES-RSNO S. aureus Coli and PES-RSNO S. aureus Coli and PES-RSNO E. coli Coli and PES-RSNO E. coli Coli and PES-RSNO Figure 6 Water content of P407 / P188 / Sal thermosensitive hydrogel in the embodiments of the present application Figure 7The moisture retention rate of the P407 / P188 / Sal temperature-sensitive hydrogel in the embodiments of the present application; Figure 8 The swelling rate of the P407 / P188 / Sal temperature-sensitive hydrogel in the embodiments of the present application; Figure 9 The dissolution rate of the P407 / P188 / Sal temperature-sensitive hydrogel in the embodiments of the present application; Figure 10 The photothermal images of the temperature-sensitive hydrogels with different compositions in the embodiments of the present application under near-infrared light; Figure 11 The photothermal behaviors of different temperature-sensitive hydrogels in the embodiments of the present application under near-infrared light (a) Temperature change of different temperature-sensitive hydrogels with time under 808 nm near-infrared light (b) Photothermal stability of the temperature-sensitive hydrogels under 808 nm laser (1.0 W / cm 2 ) irradiation for 5 cycles; Figure 12 The NO release behaviors of the temperature-sensitive hydrogels in the embodiments of the present application under near-infrared light or without near-infrared light; Figure 13 The antibacterial effects of the hydrogels in the embodiments of the present application on S. aureus under near-infrared light, wherein (a) is a plate coating photograph of S. aureus after treatment under different conditions, and (b) is the colony number of S. aureus after treatment under different conditions; Figure 14 The antibacterial effects of the hydrogels in the embodiments of the present application on E. coli under near-infrared light, wherein (a) is a plate coating photograph of E. coli after treatment under different conditions, and (b) is the colony number of E. coli after treatment under different conditions; Figure 15 The antioxidant behaviors of the hydrogels in the embodiments of the present application, wherein (a) is the antioxidant property of the hydrogels determined by the titanium sulfate method, (b) is the antioxidant property of the hydrogels determined by the salicylic acid method, (c) is the antioxidant property of the hydrogels determined by the DPPH free radical scavenging experiment, and (d) is the antioxidant property of the hydrogels determined by the ABTS free radical scavenging experiment; Figure 16 The cell survival rates of the cells treated by the hydrogels in the embodiments of the present application in the presence of H2O2; Figure 17 The oxygen production behaviors of the PDA-MnO2NPs-loaded hydrogels in the embodiments of the present application; Figure 18 The cell survival rates of the cells treated by the hydrogels in the embodiments of the present application for 24 h and 48 h, respectively; Figure 19 The live / dead cell staining results of the cells treated by the hydrogels in the embodiments of the present application; Figure 20 (a) Hemolysis effect of different hydrogels, (b) Hemolysis rate of different hydrogels, for blood compatibility of different hydrogels in embodiments of the present application; Figure 21 (a) Tube formation images of HUVEC cells treated with hydrogels at different times, for tube formation images of HUVEC cells treated with hydrogels at different times in embodiments of the present application; Figure 22 (a) Wound healing changes, (b) Wound healing results simulating different healing times, (c) Wound healing rate, for wound healing experimental results in embodiments of the present application; Figure 23 (a) H&E staining images, (b) Epithelial regeneration thickness, for H&E staining results in embodiments of the present application; Figure 24 (a) Masson staining images, (b) Collagen deposition ratio, for Masson staining results in embodiments of the present application; Figure 25 (a) Immunohistochemical IL-6, TNF-α staining pictures of wounds on the 7th day, (b) Relative expression of IL-6, (c) Relative expression of TNF-α, for immunohistochemical IL-6, TNF-α staining results of wounds on the 7th day in embodiments of the present application; Figure 26 (a) Immunohistochemical CD31, α-SMA staining pictures of wounds on the 7th day, (b) Relative expression of CD31, (c) Relative expression of α-SMA, for immunohistochemical CD31, α-SMA staining results of wounds on the 7th day in embodiments of the present application; Figure 27 H&E staining pictures of important organs, for H&E staining pictures of important organs in embodiments of the present application. DETAILED DESCRIPTION

[0037] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0038] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, devices, components and / or combinations thereof, but do not preclude the presence or addition of one or more other features, steps, operations, devices, components and / or combinations thereof. It is to be understood that the scope of the present application is not limited to the specific specific embodiments described below; it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application.

[0039] In the following specific examples, the amount of raw material components are described in terms of measurement parameters, which can vary within a range of measurement precision unless otherwise specified. In terms of temperature and time parameters, acceptable variations are allowed due to instrument testing precision or operational precision.

[0040] Abbreviations and their full names used in this document are listed in Table 1.

[0041] Table 1 Abbreviations

[0042] Materials and reagents used in the examples Materials Lactobacillus rhamnosus CICC 6141 (ATCC 53103) was purchased from China General Microbiological Culture Collection Center. Escherichia coli (ATCC 25922) was stably cultured, passaged and cryopreserved in the laboratory of the Marine College of Shandong University. Staphylococcus aureus (ATCC 25923) was stably cultured, passaged and cryopreserved in the laboratory of the Marine College of Shandong University. Lactobacillus rhamnosus Escherichia coli Staphylococcus aureus

[0043] Mouse connective tissue L cell line 929 was obtained from the Cell Bank of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and was stably cultured, passaged and cryopreserved in the laboratory of the Marine College of Shandong University.

[0044] Materials and reagents used in the experiments are listed in Table 2.

[0045] Table 2 Materials and reagents used in the experiments

[0046] ​​​The data obtained from all experiments in the following examples were analyzed in depth, statistically processed and the pictures were optimized by GraphPad Prism 8.0 software. For the quantitative data, the mean ± standard deviation (Mean ± SD) was used to present. When comparing the differences between different groups of data, ANOVA analysis method was used to test the significant differences of each group of data. Specifically, when the p value is less than 0.05 (*p<0.05), 0.01 (**p<0.01), 0.001 (***p<0.001) and 0.0001 (****p<0.0001), it is considered that there is a significant difference between groups; and "ns" means no significant difference between groups.

[0047] Example 1 Preparation of gel matrix In this study, P407, P188 and Sal were selected as the basic materials for the preparation of gels, and the precursor solution of the gel was prepared by cold dissolution method.

[0048] The preparation method is as follows: 2.3 g of P407 powder, 0.25 g of P188, and 0.073 g of Sal powder are respectively dissolved in 10 mL of deionized water pre-cooled at 4°C, stirred and left overnight at 4°C until completely dissolved to obtain a transparent micellar solution. The CVM microspheres are added to the above solution at a final concentration of 5 mg / mL, and a vortex mixer is used for three-stage oscillation (30 s x 3 times, interval 2 min) to ensure uniform dispersion of the microspheres.

[0049] Example 2 Preparation of multifunctional temperature-sensitive hydrogel loaded with microparticles (1) Preparation of PDA-MnO2 nanoparticles First, 0.4844 g of Tris was dissolved in 400 mL of distilled water, and then 0.5 mol / L hydrochloric acid was added dropwise to adjust the pH of the solution to 8.5 (obtaining 0.01 mol / L Tris / HCl buffer). 300 mg of MnO2 nanoparticles were dispersed in 300 mL of buffer and ultrasonicated for 15 min, then 300 mg of hydrochloric acid dopamine was added and ultrasonicated for 10 min, and stirred at room temperature for 16 h in the dark. The precipitate was collected by centrifugation, and then the obtained nanoparticles were sequentially washed by centrifugation (8000 r, 10 min) with 20 mL of anhydrous ethanol and pure water, respectively, and the supernatant was discarded. Finally, the collected nanoparticle precipitate was placed in an oven and dried at 60°C for 12 h.

[0050] (2) Investigation of antibacterial performance of PES-RSNO microparticles and determination of the optimal synergistic ratio Preparation of PES: Lactobacillus rhamnosus (200 μL, concentration 10 8CFU / mL) were inoculated on MRS medium agar plates and incubated at 37 °C for 48 h. After incubation, the biofilm formed by probiotic bacteria was scraped from the agar plates and collected in 50 mL of reactor vessel. Then, 25 mL of deionized water was added to the vessel and treated at 80 °C for 24 h to extract the probiotic derivatives. The treated mixture was centrifuged at 13,000 g for 20 min at 4 °C to separate the orange transparent liquid (PES), which was pre-frozen at -80 °C for 24 h and then freeze-dried for 48 h to obtain the probiotic extract.

[0051] Preparation of RSNO: First, thiomalic acid (1.5 g) was dissolved in 8 mL of deionized water. Then, HC1 (2 mL, 1 mol / L) and NaN02(0.69 g) were added to the thiomalic acid solution and stirred in the dark for 20 min in an ice bath until a red solution was formed. Finally, the red solution was pre-frozen at -40 °C and freeze-dried for 48 h to obtain pink RSNO powder.

[0052] To screen the optimal antibacterial concentration of probiotic extract (PES) and RSNO and ensure the biosafety for the subsequent experiments, the antibacterial effect test and biocompatibility evaluation of PES and RSNO were performed. First, the PES solution with an initial concentration of about 2 mg / mL was prepared by hydrothermal synthesis at 80 °C for 24 h, and the mother liquor concentration was assumed to be 4X. Then, the PES solution was diluted step by step with deionized water, and the concentration after dilution was 2X, IX, and finally a series of solutions with different concentrations (including 0 mg / mL, IX, 2X, 4X) were obtained. The obtained RSNO powder was dissolved in pure water to prepare a series of RSNO solutions with different concentrations (including 0 mg / mL, 0.1 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 0.7 mg / mL) to evaluate the antibacterial efficacy of RSNO through antibacterial experiments.

[0053] These PES solutions and RSNO solutions with different concentrations were co-cultured with S. aureus (ATCC 29213) S. aureus ) and E. coli (ATCC 25922) E. coli ) for 12 h, and the antibacterial effects of the two were observed. The results are shown in Figures 1-4 , and the PES solutions with concentrations of 0 mg / mL to 2 mg / mL failed to effectively inhibit the growth of S. aureus, E. coli . The plate coating experiment and its quantitative analysis results showed that the PES solution had no significant inhibitory effect on the growth of S. aureus, E. coli . When the concentration of the RSNO solution reached 0.3 mg / mL, it initially showed the ability to inhibit bacteria, but the effect was not significant and only a small amount of S. aureus, E. coliFurther increasing the concentration of RSNO to 0.5 mg / mL, it can effectively inhibit the growth of S. aureus When the concentration of RSNO is increased to 0.7 mg / mL, it can inhibit the growth of E. coli .

[0054] S. aureus E. coli respectively, and co-cultured with deionized water, PES (2 mg / mL), RSNO (0.3 mg / mL) and PES-RSNO (PES 2 mg / mL, RSNO 0.3 mg / mL) for 12 h, the plate colony count photos and the quantitative statistics of CFU of the culture solution are shown in Figure 5 , it can be found that the antibacterial ability of PES treatment group and RSNO treatment group is weak, while the PES-RSNO treatment group has obvious inhibitory effect on the growth and reproduction of bacteria after co-cultured with S. aureus E. coli , and can effectively inhibit the proliferation of bacteria. Therefore, the concentrations of PES and RSNO are determined as 0.3 mg / mL and 2 mg / mL respectively.

[0055] The preparation of PES-RSNO is as follows: freeze-dried RSNO powder (0.3 mg) is dispersed in 1 mL PES mother liquor (2 mg / mL). The liquid is stirred in ice bath under light-proof conditions for 20 minutes to obtain PES-RSNO solution, and the obtained solution is pre-frozen at -40℃ and then freeze-dried to obtain PES-RSNO solid.

[0056] (3) Preparation of CVM microspheres Dissolve 0.04 g of chitosan CS powder in 2 mL of 1% acetic acid aqueous solution to obtain a chitosan solution, and then dissolve 0.016 g of metformin hydrochloride in the chitosan solution to prepare a drug-loaded aqueous solution. Add emulsifier Span-80 (2 wt%) to 20 mL of soybean oil and stir in a constant temperature water bath at 50℃ for 40 minutes to fully mix. Then, add the aqueous phase to the stirred oil phase solution, and stir with a magnetic stirrer for 30 min for emulsification. Thereafter, add 1 mL of prepared vanillin crosslinking agent (2.5%) solution dropwise with a syringe, and then react for 4 h. The obtained microspheres are precipitated and washed with 20 mL of petroleum ether, acetone, anhydrous ethanol and distilled water (3000 r, 10 min) in turn, and dried in an oven at 60℃ for 8 h after washing.

[0057] Example 3 Study of the properties of the multifunctional thermoresponsive hydrogel ​​​Four groups of hydrogels loaded with different active substances were prepared according to the optimized ratio of Example 1-2, including (1) P407 (23%, w / v) / P188 (2.5%, w / v) / Sal (0.73%, w / v) temperature-sensitive gel (PP); (2) P407 / P188 / Sal gel loaded with 1 mg / mL CVM microspheres (PM); (3) P407 / P188 / Sal gel loaded with 1 mg / mL CVM microspheres, 1 mg / mL PDA-MnO2 nanoparticles (PMM); (4) P407 / P188 / Sal gel loaded with 1 mg / mL CVM microspheres, 1 mg / mL PDA-MnO2 nanoparticles, and 2.3 mg / mL PES-RSNO microparticles (PMMR).

[0058] Appearance and microstructure of the gels. The P407 / P188 / Sal temperature-sensitive gel system and after drug loading, both in sol and gel states, exhibited uniform appearance without visible flocculation or precipitation, and uniform texture, which indicated that the optimized temperature-sensitive gel could simultaneously load PDA-MnO2 nanoparticles, CVM microspheres, and PES-RSNO while maintaining the uniformity of the gel solution. 1 mL of hydrogel solution of different groups was transferred to a 24-well plate, frozen at -80°C, and then treated by freeze-drying machine for 48 h to obtain freeze-dried hydrogels. Then the liquid nitrogen brittle fracture technique was used to obtain the internal cross section. These cross-sectioned gels were adhered to conductive tape and gold-plated using traditional sputtering technology to enhance conductivity. Image J software was used to measure the pore size of the hydrogels and to statistically analyze the pore size distribution to evaluate the microstructure. After statistics, the average pore sizes of the PP, PM, PMM, and PMMR groups were 1059.412 ± 80.13 nm, 703.598 ± 40.284 nm, 364.42 ± 25.29 nm, and 467.77 ± 31.842 nm, respectively. The addition of microspheres significantly reduced the pore size of the temperature-sensitive gel, and the three-dimensional network structure significantly improved the compactness. The addition of PDA-MnO2 nanoparticles reduced the pore size of the gel, which indicated that the higher the crosslinking density, the more compact the microstructure of the hydrogel. The addition of PES-RSNO microparticles made the pore size of the gel more loose and irregular, which may be due to the hydrophilicity of PES-RSNO microparticles.

[0059] Gelation temperature. The test tube inversion method was used to explore the setting point of P407 / P188 / Sal mixed system under different conditions. The test results showed that the gelation temperature of P407 / P188 / Sal temperature-sensitive gel system changed with the change of drug loading, the gelation temperature of PP gel was 34.1±0.3℃, the gelation temperature of PM gel was 33.5±0.1℃, the gelation temperature of PMM gel was 33.2±0.2℃, and the gelation temperature of PMMR gel was 33.3±0.1℃, which was lower than the body surface temperature and could meet the requirements of skin application.

[0060] Gelation time. The gelation time of the gel was determined at 37℃ by the test tube inversion method. Among them, the gelation time of PP gel was 100.0±8.5 s, the gelation time of PM gel was 69.5±3.1 s, the gelation time of PMM gel was 53.1±3.9 s, and the gelation time of PMMR gel was 56.3±6.0 s, which could quickly gel and meet the application requirements. With the increase of drug content and type in the gel system, the gelation time showed a trend of gradually shortening.

[0061] Viscosity. An ideal hydrogel should have good flowability and low viscosity at room temperature, and high viscosity and poor flowability at body temperature, so as to be fixed at the target position after injection. The gel solution was placed in a 50 mL beaker, and the viscosity of the four different gel precursor solutions was measured at room temperature (25℃) using a rotary viscometer, and the experimental results were recorded in turn. The results showed that the viscosity of PP gel at room temperature was 813.71±3.89 Pa·s, the viscosity of PM gel was 867.11±4.21 Pa·s, the viscosity of PMM gel was 884.21±5.42 Pa·s, and the viscosity of PMMR gel was 893.71±5.13 Pa·s. At room temperature, the temperature-sensitive gel has a suitable viscosity, which can ensure good needle passability and meet the application requirements of temperature-sensitive gel at room temperature.

[0062] pH performance. The measurement of pH value was carried out by placing the electrode in the gel solution, and the final pH value was obtained by averaging the readings of three samples and calculating the standard deviation (SD). The experimental results were recorded after determination, the pH of PP temperature-sensitive gel preparation was 6.51±0.15, the pH of PM temperature-sensitive gel preparation was 6.59±0.25, the pH of PMM temperature-sensitive gel preparation was 6.64±0.72, and the pH of PMMR temperature-sensitive gel preparation was 6.55±0.19, which was in the physiological pH (6.0-7.5) range, so it had no irritation to the skin mucosa and was suitable for use as a skin drug preparation.

[0063] Moisture content and moisture retention of the gels. The prepared hydrogel precursor solution was placed in an oven at 37 °C, and three groups of parallel samples were prepared, and their mass changes were monitored at predetermined time intervals until the weight remained stable, and the moisture content and moisture retention of the gels were calculated. From Figure 6 It can be seen from Table 1 that the moisture content of the gels is all above 75%, which is due to the high hydrophilicity of Sal main chain rich in hydroxyl groups, and the good moisture retention of P407 and P188, the combination of the three can make the composite gel combine more water molecules, and the moisture content is higher. The addition of microspheres, PDA-MnO2 nanoparticles and PES-RSNO microparticles has no significant effect on the moisture content of the temperature-sensitive gel, and the moisture content is basically consistent. This is because there are hydrogen bonds and other intermolecular forces between P407, P188 and Sal, thereby forming a dense network structure, and the network structure formed can intercept water molecules. The high moisture content of the hydrogel provides a moist environment for the wound, reducing friction on the wound. From Figure 7 It can be seen from Table 2 that the moisture retention rate gradually decreases with time, and the moisture retention ability is the lowest after 12 hours. The slope represents the water loss rate of the hydrogel, and the water loss rate of the hydrogel is basically consistent, with no large difference, proving that the moisture retention ability of the four groups of hydrogels with different compositions remains consistent.

[0064] Swelling properties. The swelling behavior of P407 / P188 / Sal composite hydrogel at 37 °C as a function of time. The dried hydrogel (100 mg) was weighed and recorded as W0, then immersed in PBS (pH = 7.4, 20 mL) at 37 °C, taken out every certain time, wiped off the surface water, weighed and recorded to calculate the swelling rate, and the results are shown in Figure 8 As shown in Table 3, within 0-15 min, the swelling ratio of P407 / P188 / Sal composite hydrogel rapidly increases, Sal contains polar groups carboxyl and hydroxyl, which affects the water absorption capacity of the hydrogel. Subsequently, the growth rate of the swelling ratio slows down and eventually tends to be flat, and the swelling ratio of the composite hydrogel gradually decreases after 1 h, which may be due to the gradual loosening of the network structure of the hydrogel caused by the swelling of the hydrogel, and with the extension of time, the network structure of the hydrogel tends to collapse, thereby causing the swelling ratio to decrease. In addition, there is no obvious difference in the swelling rate of all freeze-dried hydrogels.

[0065] Water solubility. In order to explore the degradable properties of P407 / P188 / Sal composite hydrogel, the dissolution characteristics of all freeze-dried hydrogels within 3 days were studied, and freeze-dried hydrogel blocks (n = 3) of the same weight (100 mg) were immersed in 20 mL of PBS solution (pH = 7.4) at 37 °C, and stirred at 100 rpm. The freeze-dried hydrogel was removed every 24 h, freeze-dried and weighed to calculate the dissolution rate, and the results are shown in Figure 9As shown, all hydrogels can gradually decompose in PBS solution, and the solubility of PP, PM, PMM, PMMR hydrogels reached 71.39%, 73.72%, 74.15% and 78.40% respectively on the first day. And the solubility of all freeze-dried composite hydrogels reached more than 90% within 3 days, which confirmed that the composite hydrogels have good solubility.

[0066] Photo-thermal conversion performance. The photo-thermal conversion ability of PDA-MnO2 NPs in the hydrogels under near-infrared laser irradiation was evaluated by photo-thermal experiments. 500 μL of different hydrogels (PP, PM, PMM, PMMR) were placed in 1.5 mL centrifuge tubes, and irradiated under near-infrared 808 nm light (808 nm, power density 1.0 W / cm 2 ) until the temperature reached the peak. Subsequently, the near-infrared laser was turned off to make the sample temperature fall to ambient temperature. The infrared thermal imager was used to monitor and record the temperature change, and the photo-thermal image was captured every 2 minutes. In addition, the photo-thermal performance test of the hydrogel sample was carried out for five cycles to study its photo-thermal stability. Each irradiation lasted for 10 minutes, followed by cooling for 10 minutes, and the temperature change of the hydrogel was monitored in real time to observe whether the peak temperature of the sample was consistent during irradiation. The results are shown in Figure 10 As can be seen, with the addition of PDA-MnO2 NPs, the hydrogel can begin to perform photo-thermal conversion, and the temperature rises significantly. As shown in Figure 11 (a), when the concentration of PDA-MnO2 NPs is set to 1 mg / mL, the temperature of PMMR hydrogel can rise to more than 50℃ after 10 minutes of 808 nm near-infrared light irradiation, which indicates that the hydrogel exhibits excellent photo-thermal conversion effect at this concentration. As shown in Figure 11 (b), in the cycle stability test, after five consecutive on-off laser irradiations, the temperature response of PDA-MnO2 NPs remains consistent, and the temperature peak does not decrease significantly, which indicates that PDA-MnO2 NPs embedded in the hydrogel maintains good photo-thermal stability. This stability is crucial for the multiple use and long-term operation of the hydrogel in practical applications, meeting the application requirements.

[0067] NO release test. The NO detection kit (Cat#A012-1-2, Nanjing Jiancheng Biological Engineering Institute) was used to determine the amount of NO released. 200 μL of PP, PM, PMM, PMMR hydrogels were placed in a 96-well plate, and 200 μL of deionized water was added as a control group. The experiment was divided into two groups, one group received 808 nm near-infrared light irradiation for 20 minutes, and the other group was placed in the dark for 20 minutes. Subsequently, the NO detection kit was used to detect the NO gas release of the gel. The results are shown in Figure 12As shown, compared with the control group (Control) and PM and PMM hydrogel groups, the PMMR hydrogel group can rapidly release NO gas (0.647 mM) under 808 nm near-infrared light irradiation. In addition, even under non-irradiation conditions, the PMMR hydrogel can continuously release a lower level of NO. This indicates that the encapsulation structure of the PMMR hydrogel does not hinder the release of NO by PES-RSNO microparticles. Therefore, the PMMR hydrogel not only has the ability to rapidly release NO, but also provides the possibility of achieving long-term antibacterial under non-light conditions and rapid photothermal / NO synergistic antibacterial under light conditions, which lays the foundation for the development of effective antibacterial treatment strategies.

[0068] Antibacterial performance test. Add 1 mL of PP, PM, PMM, and PMMR, four different hydrogel precursor solutions, respectively, into a 24-well plate. The experiment is divided into two groups, one group is irradiated under near-infrared light, and the other group is not irradiated under near-infrared light. Incubate the four groups of hydrogel precursor solutions at 37°C until the hydrogels are formed. The initial concentration of S. aureus and E. coli used is adjusted to OD600=0.2. Add 100 μL of bacterial suspension to the surface of the hydrogel, and incubate at 37°C for 30 minutes. Then, irradiate the hydrogel with an 808 nm laser (power density 1.0 W / cm 2 ) for 10 minutes. After treatment, dilute the bacterial suspension in the well plate with 0.85% sterile normal saline by 10 times, and take 20 μL of the diluted bacterial solution to spread on agar plates. After 24 h of incubation, count and record the number of colonies formed to evaluate the antibacterial activity of the hydrogel.

[0069] As Figure 12 shown, compared with other treatment groups, the PMMR hydrogel can effectively eliminate S. aureus after being treated with 808 nm near-infrared light. In addition, by quantitatively analyzing S. aureus the number of colonies, it is shown that the short-term photothermal sterilization effect of the PMMR hydrogel reaches an antibacterial rate of 99.12%. As Figure 13 shown, compared with other treatment groups, the PMMR hydrogel can effectively kill E. coli bacteria after being treated with near-infrared light. In addition, by quantitative analysis, it is found that the short-term photothermal sterilization effect of the PMMR hydrogel reaches an antibacterial rate of 99.58%. The above data show that the PMMR hydrogel exhibits excellent in vitro photothermal / NO synergistic rapid antibacterial effect, providing a scientific basis for developing photothermal / NO treatment strategies based on PMMR hydrogel to treat bacterial infectious wounds.

[0070] By comparing the morphology of bacteria treated with PMMR combined with near-infrared light (NIR) and PBS, significant differences can be observed, as Figure 14The control group, PP, PM and PMM were cultured without near-infrared light treatment. The control group, PP, PM and PMM were cultured with near-infrared light treatment. E. coli The bacterial surface was smooth and the cell wall structure was intact. In contrast, the bacteria treated with PMMR gel showed increased cell surface wrinkles and depressions under near-infrared light irradiation, and the activity was lost.

[0071] Antioxidant capacity. The ability of PDA-MnO2NPs-embedded hydrogel to scavenge H2O2 was evaluated using the titanium sulfate method. 5 mL of H2O2 solution was added to 1 mL of prepared hydrogel, respectively. Then, these samples were placed in a constant temperature shaker at 37°C for 30 min, while the control group without hydrogel was set. 1 mL of sample solution was mixed with 2 mL of titanium sulfate (Ti(SO4)2) solution, respectively, and then shaken for 30 min. The ultraviolet absorbance of the solution was measured at 405 nm wavelength using a UV spectrometer to evaluate the effect of hydrogel on scavenging H2O2 (n = 3). Through the experiment, we found that the hydrogel showed a significant effect on scavenging reactive oxygen species (ROS). Specifically, as shown in (a) of FIG. 6, after the addition of hydrogen peroxide (H2O2) in the hydrogel reaction system, we observed a significant decrease in the absorbance value of PDA-MnO2NPs-loaded hydrogel at 405 nm after 30 minutes of incubation, which was lower than the ultraviolet absorption values of the control group and the experimental group containing only H2O2. This phenomenon indicates that PDA-MnO2NPs-loaded hydrogel has a rapid scavenging effect on H2O2, which can effectively reduce the accumulation of reactive oxygen species. Figure 15 ·OH radical is a highly active ROS that can cause direct oxidative damage to cells. Therefore, the scavenging effect of hydrogel on ·OH is crucial for protecting cells from oxidative stress. 1 mL of 2 mM H2O2 was reacted with 150 µL of 2 mM FeSO4 for 3 min to generate ·OH. Then the hydrogel was incubated with the above mixture at a mass / volume ratio of 1:10 at 37°C for 30 min. Then, 900 µL of the solution was mixed with 100 µL of 15 mM SA for 30 min. After centrifugation, the clear supernatant of the mixture was obtained, and finally, the ultraviolet absorption intensity of the solution in the wavelength range of 300 nm to 800 nm was recorded using a UV-visible spectrophotometer (MAPUDA, China).

[0072] (b) of FIG. 6 shows that the ultraviolet absorption of the PDA-MnO2NPs-loaded hydrogel group decreased significantly compared with the control group, which was much lower than the control group, indicating that the hydrogel had a significant scavenging effect on ·OH radicals. Figure 15

[0073] ​The antioxidant activity was evaluated by measuring the decrease in absorbance of DPPH radicals at the maximum wavelength of light absorption. The hydrogels showed significant scavenging effect on DPPH radicals, 10 mg of prepared hydrogels were immersed in 10 mL of DPPH ethanol (0.1 mM) solution (n = 3). Then the mixture was incubated at 37 °C for 40 min. Finally, the absorbance of each sample was recorded at 517 nm wavelength using a UV-Vis spectrophotometer (Cary 60, Agilent, USA) to calculate the DPPH radical scavenging capacity. The results are shown in Fig. 1 (c), and the clearance rate reached 42.91%. Figure 15

[0074] The ABTS+radical experiment evaluated the antioxidant activity of radical scavengers by measuring the change in absorbance of radicals at the maximum wavelength of light absorption. 7 mM ABTS+stock solution (10 mL) was reacted with 2.45 mM potassium persulfate solution (10 mL) overnight in the dark to generate ABTS+radical cations. The mixture was diluted to prepare a stock solution with absorbance at 734 nm wavelength. The next steps were the same as the DPPH scavenging test (n = 3), and the ABTS+radical scavenging activity was calculated. The experimental results are shown in Fig. 1 (d), and the hydrogel also has a significant scavenging effect on ABTS+radicals, with a clearance rate of 16.81%, further confirming its antioxidant activity. Figure 15

[0075] To confirm that the obtained hydrogel can scavenge ROS at the cellular level, we co-cultured L929 cells with the hydrogel and H2O2. L929 cells were seeded in 96-well plates at a density of 1 x 10 4 cells per well, and after 12 h of culture, the cell culture medium was replaced with fresh medium containing H2O2 and hydrogel of the same volume. Cells cultured with H2O2 (0.5 mM) and PBS (equivalent weight of hydrogel) were set as negative control groups. After 6 h of culture, the cell culture medium was removed, and CCK-8 was added to evaluate cell viability (n = 3). As shown in Fig. 2, in the H2O2 positive control group with a concentration of 500 mM, only 40% of the cells survived. At the same time, the cell survival rate of the PP and PM gel groups without embedding PDA-MnO2NPs did not show significant improvement compared with the positive control group. In contrast, the relative cell survival rate of the PMM and PMMR hydrogel groups loaded with PDA-MnO2NPs increased significantly to about 80%. These results indicate that the prepared hydrogel can effectively scavenge ROS in an in vitro environment and protect cells from oxidative damage, which is of great significance for improving the oxidative stress environment existing in diabetic wounds and the like. Figure 16

[0076] ​​​To investigate whether the embedding of hydrogel would affect the oxygen generation ability of PDA-MnO2nanoparticles, the oxygen generation ability of PDA-MnO2nanoparticle-loaded hydrogel and blank gel was studied. First, blank and PDA-MnO2nanoparticle-loaded hydrogels were prepared and placed in sample bottles (1 mL). After the gel was formed, 30% H2O2solution (500 μL) was added to the sample bottles, and the oxygen generation was observed, and the phenomenon in the sample bottle was recorded by taking pictures at different time points. We observed that a large number of colorless bubbles rapidly formed on the surface of the gel within just 3 minutes Figure 17 ). Based on the known theory that MnO2can catalyze the decomposition of H2O2to generate oxygen, this phenomenon indicates that the PDA-MnO2NP-containing hydrogel can react with H2O2to generate oxygen. This not only qualitatively confirms that the hydrogel has CAT-like nanoscale enzyme activity, but also can provide oxygen while scavenging reactive oxygen species (ROS). This oxygen supply capacity is crucial for improving the hypoxic condition in the diabetic wound environment and helps to promote the healing process of diabetic wounds.

[0077] Long-term stability test. By comparing the performance indicators of the samples at different time points (1 month, 2 months, and 3 months) with the initial state (0 days), it can be observed that the P407 / P188 / Sal temperature-sensitive gel maintains stability in clarity and uniformity, and no obvious changes are observed. At the same time, the key parameters of the gel system such as gelation temperature, gelation time, viscosity, and pH value also show good stability and no significant fluctuations occur. Although the drug content decreases slightly after 3 months of storage under different temperature conditions, the decrease is small, indicating that the drug has high stability. In summary, the P407 / P188 / Sal temperature-sensitive gel exhibits excellent long-term stability within the temperature range investigated, which is of great significance for its practical application in the field of drug delivery.

[0078] Example 4 Biological evaluation of the multifunctional thermoresponsive hydrogel Cell compatibility assay. In application, the cytotoxicity of hydrogel is also an important indicator for its use as a wound dressing. 25 mg of freeze-dried composite hydrogel was weighed and sterilized under ultraviolet light for 30 min. Then, the sterilized hydrogel was added to 1 mL of DMEM medium and incubated at 37°C for 24 h to extract the soluble components in the hydrogel. After that, the obtained extract was sterilized by 0.22 μm filter membrane (n = 3). The cytotoxicity of the four hydrogels was evaluated by CCK8 method, and the hydrogel extract was co-cultured with L929 cells to calculate the cell survival rate. As Figure 18As shown, the cell viability of L929 cells co-incubated with SOP hydrogels for 24 and 48 hours was all above 80%, indicating that the PHMB-loaded hydrogels had good cytocompatibility with L929 cells. Figure 19 The results of the live / dead cell staining assay were consistent with the results of the CCK8 experiment. Calcein-AM / PI was used to stain the cells, which produced green and red fluorescence in live and dead cells, respectively. After L929 cells were co-incubated with the SOP hydrogels and the control group for 24 hours, most of the L929 cells were green live cells that maintained a spindle shape and were uniformly distributed, and there were basically no red cells and dead cells, indicating that the hydrogels had no cytotoxicity to L929 cells. At the same time, the treatment of the hydrogels had no effect on the growth and proliferation of the cells. This would allow the hydrogels to be used as potential wound dressings.

[0079] Hemolysis rate analysis. To evaluate the compatibility of the hydrogel wound dressings with human blood, we tested the hemolytic properties of the four hydrogels using sheep whole blood. The sheep whole blood was centrifuged at a speed of 1000 rpm for 10 min at room temperature. After centrifugation, the supernatant was removed, and the red blood cell precipitate was washed with sterile PBS buffer for 3 times until the supernatant was completely transparent. Finally, the washed red blood cells were resuspended in PBS buffer and adjusted to a cell concentration of 5% (v / v). 0.3 mL of different hydrogel samples were mixed with 0.7 mL of red blood cell suspension, and negative and positive controls were set up (PBS + red blood cells and pure water + red blood cells, respectively). The mixture was incubated in a 37°C constant temperature shaker at a speed of 100 rpm for 1 h. After centrifugation at 1000 rpm for 10 min, 150 μL of supernatant was accurately transferred to a 96-well plate, and the absorbance was detected at a wavelength of 540 nm using a microplate reader (n = 3), and the hemolysis rate was calculated. Figure 20 Figure 6 shows the results of the hemolysis experiment of the P407 / P188 / Sal hydrogel, in which PBS solution was used as a blank control and deionized water was used as a positive control. The results showed that the supernatant of the four hydrogels was yellowish compared with the blank control group, while the positive control group was red, indicating that all the tested hydrogels did not cause hemolysis. Further data showed that, as shown in Figure 6(b), the hemolysis rates of the four hydrogels were 2.61%, 3.64%, 3.96% and 4.45%, respectively, all of which were lower than 5%, meeting the standard of hemolytic performance of medical device biomaterials. These results confirmed that the four hydrogels all exhibited good blood compatibility and had no toxic effects on blood. Figure 20

[0080] ​The pro-angiogenic effect of hydrogel. Studies have shown that NO gas can promote the angiogenesis of human umbilical vein endothelial cells. NO, as a key angiogenic factor, participates in the angiogenesis process together with other cytokines such as vascular endothelial growth factor (VEGF). In order to further verify the effect of hydrogel on angiogenesis, human umbilical vein endothelial cells were selected to verify the influence of hydrogel on angiogenesis. Matrigel matrix glue was pre-cooled at 4°C overnight to fully liquefy. The 24-well culture plate was pre-cooled before the experiment, and the whole operation process was carried out in an ice bath environment. 30 μL of Matrigel matrix glue solution was added to each well and shaken evenly. A circle of PBS was added around the matrix glue to prevent the matrix glue from drying out. The well plate with matrix glue was placed in a 4°C refrigerator overnight, and then the well plate with flat matrix glue was placed in a 37°C incubator for 30 min. HUVEC was recovered and placed in DMEM medium containing 10% serum and 1% double antibody. When the cells reached 80-90% of the logarithmic growth phase, the HUVEC cells were digested with trypsin containing EDTA, and then the cell suspension was diluted to a final concentration of 7 x 106 / mL. 100 μL of uniform cell suspension was inoculated into each well of the cell culture plate with matrix glue, ensuring that the cell density of each well was 1 x 105 cells. The culture plate was placed in the incubator for incubation. Finally, at 2 h, 4 h, and 6 h, the growth and tube formation of the cells were observed and photographed using a fluorescence inverted microscope. The experimental results are shown in Figure 21 It was found that the PMMR hydrogel group showed more significant effect in promoting angiogenesis. This phenomenon is mainly due to the PES-RSNO particles embedded in the PMMR hydrogel, which can release NO gas under the culture conditions of 37°C. As an important messenger of cell signal transmission, NO gas plays a key role in angiogenesis. It can promote the proliferation and migration of vascular endothelial cells, and thus promote the formation of new blood vessels.

[0081] Example 5 Evaluation of the repair efficiency of the multifunctional thermoresponsive hydrogel on diabetic wounds Establishment of diabetic rat model. First, male SD rats (6-8 weeks) were adaptively fed for one week, and then the SD rats were randomly divided into 4 groups, respectively, (1) control group (PBS), (2) commercial dressing group (3M dressing), (3) blank gel group (PP) and (4) drug-loaded gel group+NIR (PMMR+NIR), and then the modeling of type I diabetes was performed. The SD rats after overnight fasting for 12 h were injected with streptozotocin (STZ) 65 mg / kg intraperitoneally, and the rats were fasted for 2 hours but not watered, and then the normal diet of the rats was restored. The random blood glucose of the rats was measured in sections on the 3rd and 7th day and recorded, and the blood glucose >16.7 mmol / L was considered to be successful modeling.

[0082] Hydrogel promotes diabetic wound healing experiment. After the successful modeling of type I diabetic rats, the modeling of infected wounds was performed. The experimental scheme first anesthetized the rats with urethane (20%, 5 mL / kg), shaved the hair on the back of the rats, and then created a full-thickness skin wound with a diameter of 1.2 cm on the back of the rats with surgical scissors, and then injected 50 μL of E. coli bacterial suspension (10 8 CFU / mL) into the wound surface of each rat to promote the infected environment of the wound, and then covered the wounds on the back of the rats with commercial dressing and hydrogel dressing, respectively, while the PPMR group used 808 nm near-infrared light treatment for 10 minutes, and the control group used PBS to treat the wounds on the back of the rats. The wound surfaces on the 0th, 3rd, 7th, 10th, and 14th days were photographed and recorded, and then the wound area was calculated using Image J software to determine the healing of the wound. The calculation formula of the wound healing rate (%) is: wound healing rate (%)=(A0-A t ) / A0x100; where A0 is the area of the wound on the 0th day, and A t is the area of the wound on the tth day.

[0083] The wound conditions were statistically analyzed using Image J software. By comparing the wound healing of each group on the 0th, 3rd, 7th, 10th, and 14th days, it can be found that on the 0th day of the wound, the sizes of all the wounds were similar, and the wounds of the control group and the commercial dressing group were slightly smaller than the blank gel group and the drug-loaded gel group+NIR. Figure 22The wound size of the PPMR gel group was reduced compared to the other three groups on day 3, with a healing rate of 36.39%; on day 7, the wound size of the PPMR gel group was significantly reduced, with a healing rate of 76.04%, while the healing rate of the commercial 3M dressing group was 52.75%. On day 10, the wound area of the four groups of rats was significantly reduced, among which the wound healing rate of the control group was 49.52%, the wound healing rate of the 3M commercial dressing group was 63.72%, and the wound healing rate of the PPMR gel group was 85.15%. On day 14, the PPMR gel group + NIR had the best healing, which was significantly better than the commercial dressing group and the PPMR blank gel group, with a wound healing rate of 93.62%, and new hair began to cover the wound around the wound, with good wound healing.

[0084] Tissue staining. On days 7 and 14, tissues were taken from the wound site, fixed in 4% paraformaldehyde after simple treatment, and then embedded in paraffin after washing the fixed tissue samples at different time points. Then, 5 μm thick sections were cut, stained with hematoxylin and eosin (H&E), Masson, immunohistochemistry (IL-6, TNF-α) and immunofluorescence (CD31, α-SMA) according to standard requirements, and observed and photographed under a microscope.

[0085] HE staining analysis as Figure 23 As shown in FIG. 7, H&E staining results showed that only a small amount of new epidermis was observed in the blank control group and the blank gel group on day 7, while the new epidermis of the 3M commercial dressing group and the PPMR group was separated from the dermal tissue. The epidermis of the other groups was new and adhered tightly to the dermal tissue. The wound gap of the PPMR group was the smallest, at 5213 μm, indicating the fastest wound healing rate. In addition, a small amount of sebaceous glands and hair follicles were observed in the magnified area of the PPMR, and the wound healing process was faster. On day 14, the blank control group, the 3M hydrogel group and the PP gel group were still in the re-epithelialization process, and the epithelial thickness of the 3M commercial dressing group was 32.85 μm. The epithelial thickness of the wound treated with the 3M commercial dressing was 3798 μm, about 2600 μm more than that of the control group, indicating that it was in the rapid proliferation stage of granulation tissue. In contrast, the PPMR gel group had already completed this process, with an epidermal thickness of 61.44 μm, a large number of hair follicles, and a high degree of skin maturity, showing excellent healing effect. In addition, the dermal-epidermal junction density of the blank control group, the 3M hydrogel group and the PP gel group was very low, and obvious inflammatory cell infiltration (neutrophils, lymphocytes) was also observed. In summary, the PPMR gel can significantly promote the healing of diabetic wounds, with reduced inflammatory infiltration, promotion of epithelialization and generation of skin appendages such as hair follicles.

[0086] Masson staining reveals collagen fibers as blue and muscle fibers as red, allowing for differentiation between the two. The intensity of the blue stain indicates the collagen fiber content; a darker blue indicates a higher collagen fiber content. Figure 24 As shown, on day 7, the collagen in the control group, the 3M commercial dressing group, and the PP gel group exhibited a disordered and immature state, while the collagen fibers in the PPMR group were more tightly packed. The collagen deposition density in the 3M dressing group was 24.91%, while that in the PPMR group reached 46.28%. Furthermore, compared with other groups, the collagen deposition level in the PPMR group was significantly higher. On day 14, the collagen deposition density in the PPMR group was the highest, reaching 80.82%, which was more mature than other groups and 2.24 times that of the control group. The suppressed collagen expression in other groups may be due to bacterial infection. Therefore, the use of PPMR gel can accelerate the healing of diabetic wounds by promoting collagen deposition.

[0087] Hydrogel wound dressings can suppress inflammatory responses caused by bacteria and other microorganisms. The severity of the inflammatory stage can be characterized by immunohistochemistry. To study the expression of inflammation-related factors during wound healing, immunohistochemical analysis was performed on wound tissue from diabetic rats. The degree of wound inflammation can be assessed by characterizing common pro-inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). The expression level of IL-6 was mainly determined by the intensity of the staining on the immunohistochemical sections; the darker the color, the higher the expression level. On day 7, obvious brown areas were observed in the blank control group, the 3M hydrogel group, and the blank gel group, indicating elevated IL-6 expression levels and severe inflammatory response. In contrast, the PPMR group showed a very light brown color, and the expression of the pro-inflammatory factor IL-6 was significantly reduced, with a relative expression level of 28.67%. Figure 25 The results showed that an inflammatory response occurred in the untreated group, while the PPMR hydrogel group exhibited a lower degree of inflammation. Similarly, compared to the control group, the TNF-α expression level was decreased in the 3M commercial dressing group (relative expression 52.88%), while the PPMR group also showed a relatively lower TNF-α level (relative expression 24.43%). This can be attributed to the antioxidant and anti-inflammatory properties of the PPMR gel, and the ability of the antibacterial agent PES-RSNO to further reduce bacterial stimulation and inflammation of the wound. Therefore, the prepared drug-loaded gel helps to suppress the inflammatory response at the wound site, thereby promoting wound healing.

[0088] CD31 is a marker of neovascularization, mainly involved in the formation of neovascularization and the proliferation of endothelial cells, and is usually expressed on vascular endothelial cells. The expression level may be related to angiogenesis and tumor development. Immunofluorescence staining of CD31 was performed on the skin wound tissue of 7 days to determine the angiogenesis. Red fluorescence represents angiogenesis. As can be seen from the figure, the CD31 index of the PPMR hydrogel group is more, and the blood vessel diameter is larger. It shows that the hydrogel can effectively promote the vascular regeneration of the skin tissue of the wound site, and further promote the wound repair of the skin. α-SMA is closely related to the differentiation and function of vascular smooth muscle cells, and expression usually occurs in the late stage of vascular development, indicating the maturation and stability of blood vessels. α-SMA and CD31 both maintain the stability and function of blood vessels. Red fluorescence represents angiogenesis, and as can be seen from the figure, the PPMR gel group has more angiogenesis. In summary, PPMR gel has a good effect of promoting angiogenesis. Figure 26

[0089] In order to study the tissue compatibility of PMMR hydrogel in vivo, the main organs of rats, heart, liver, spleen, lung and kidney, treated with hydrogel for 14 days were subjected to HE staining. It can be found by HE staining that after treatment with hydrogel, the main organs of rats have no obvious damage and abnormality, and still maintain the integrity of the complete tissue structure. Figure 27

[0090] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.​​

Claims

1. A dual mode antimicrobial pro-rep air ing injectable hydrogel, characterized in that, The dual-mode antibacterial and repair-promoting injectable hydrogel hydrogel is that PES-RSNO microparticles are encapsulated in a hydrogel matrix doped with PDA-MnO2 NPs and CVM microspheres, and the hydrogel matrix is composed of P407, P188 and Sal.

2. The method of producing a dual-responsive antibacterial pro-repair injectable hydrogel according to claim 1, characterized in that, It comprises the following steps: S1, respectively, P407, P188, Sal is dissolved in pre-cooled deionized water, stirring until completely dissolved, obtain transparent micellar solution: S2, PES-RSNO microparticles, PDA-MnO2 NPs, CVM microspheres are added to the transparent micellar solution and vortexed to disperse, and then obtained.

3. The production method according to claim 2, wherein The step S1 specifically comprises: Sal is weighed and placed in a centrifuge tube, pre-cooled distilled water is added and stirred until completely dissolved, then pre-weighed P407 and P188 powder is added to the Sal solution, and the solution is placed at 4±1℃ for 24-36h until a transparent micellar solution is obtained; In the transparent micellar solution, the amount of Sal is 0.5%-1%, w / v; the amount of P407 is 22%-24%, w / v; the amount of P188 is 2%-3%, w / v; Preferably, the amount of Sal is 0.7±0.1%, w / v; the amount of P407 is 23±0.5%, w / v; the amount of P188 is 2.5±0.1%, w / v.

4. The production method according to claim 2, wherein In step S2, the preparation method of PES-RSNO microparticles comprises: collecting Lactobacillus rhamnosus culture, adding deionized water to the culture, treating at 80±5℃ for 24±5h, centrifuging to separate orange transparent liquid, and obtaining PES mother liquor; dispersing RSNO powder in PES mother liquor, stirring to obtain PES-RSNO solution, and freeze-drying the obtained solution to obtain PES-RSNO microparticles.

5. The production method according to claim 2, wherein In step S2, the preparation method of PDA-MnO2 NPs comprises: adding MnO2 nanoparticles to Tris / HCl buffer solution and ultrasonic dispersion treatment, then adding hydrochloric acid dopamine, ultrasonic stirring at room temperature in the dark, centrifuging to collect the nanoparticle precipitate, and washing the nanoparticle precipitate with anhydrous ethanol and pure water respectively, and drying to obtain; Wherein, the pH of Tris / HCl buffer solution is 8.5±0.5, and the mass-volume ratio of MnO2 nanoparticles to Tris / HCl buffer solution is 1:

1.

6. The production method according to claim 2, wherein In step S2, the preparation method of CVM microspheres comprises: Dissolve chitosan in 1% acetic acid solution to obtain chitosan solution, then dissolve metformin hydrochloride in chitosan solution to obtain a drug-loaded aqueous phase solution; add emulsifier Span-80 to soybean oil to obtain an oil phase solution; add the drug-loaded aqueous phase solution to the stirred oil phase solution for emulsification; then add vanillin crosslinker solution for reaction to obtain microsphere precipitate, which is washed and dried.

7. A pharmaceutical composition, characterized by, The active ingredient comprises the dual-mode antibacterial and repair-promoting injectable hydrogel of claim 1.

8. The pharmaceutical composition of claim 7, wherein The pharmaceutical composition further comprises at least one non-active pharmaceutical ingredient.

9. The pharmaceutical composition according to claim 7 or 8, wherein The non-active pharmaceutical ingredient is at least one of the commonly used carriers, excipients and diluents in pharmacy.

10. Use of the dual-mode antimicrobial pro-rep air injectable hydrogel of claim 1 or the pharmaceutical composition of any one of claims 7-9 for the preparation of a diabetic wound repair product.

Images