An antibacterial and anti-inflammatory drug controlled-release hydrogel and a preparation method and application thereof

Abstract

The application provides an antibacterial and anti-inflammatory drug controllable release hydrogel and a preparation method and application thereof, solves the problems of complex preparation process of photo-induced imine crosslinking hydrogel and lack of antibacterial and anti-inflammatory functions, and provides an effective way for treating chronic refractory wounds. The photo-crosslinking hydrogel designed in the application uses an amino-modified polymer derivative as a crosslinking matrix, and uses a double-branched small molecule compound as a crosslinking agent. The crosslinking system is simple to prepare, and can be quickly gelled under 395nm ultraviolet light. By further introducing a double-branched photo-crosslinking agent-antibacterial drug co-carrier and an anti-inflammatory drug slow-release particle into the above crosslinking system, the photo-crosslinking hydrogel of the application exhibits two drug release mechanisms of burst release of antibacterial drugs and slow release of anti-inflammatory drugs, thereby effectively solving the problems of bacterial infection in the early stage and chronic inflammation in the middle stage in the treatment process of chronic refractory wounds.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to an antibacterial and anti-inflammatory drug controllable release hydrogel as well as a preparation method and application thereof. BACKGROUND

[0002] Diabetes is a high-incidence disease globally, and has become the fourth disease that is easy to cause human death. Diabetic foot is one of common and serious complications of diabetes, and mainly manifests as foot ulcer and gangrene. According to statistics, the incidence of diabetic foot is as high as 15% in diabetic patients. Due to long-term high blood sugar, diabetic patients cause abnormality of distal lower extremity nerves and peripheral vascular diseases, so that the foot skin is easy to be infected, and the existence of chronic inflammation hinders skin healing, so that the skin wound loses normal repair ability, and severe patients even face the risk of amputation.

[0003] Hydrogel dressing is a new type of wound dressing developed in recent years, and compared with traditional dressings, it has the characteristics of high water content, good biocompatibility and adjustable chemical and mechanical properties, and can maintain its unique volume and shape to cope with physical pressure from surrounding tissues. At the same time, the three-dimensional network structure inside the hydrogel is beneficial to cell adhesion and proliferation, and provides an ideal environment for tissue treatment and repair. In order to realize rapid fitting of irregular wounds, photo-induced imine cross-linked hydrogel has attracted widespread attention. This type of hydrogel usually takes hyaluronic acid grafted with ortho-nitrobenzyl alcohol (ONB) photoresponsive groups as the main component, and amino-modified polymer derivatives (chitosan, gelatin, hyaluronic acid, etc.) as the auxiliary component. After ultraviolet irradiation, the aldehyde group generated by ONB forms an imine bond with the amino-modified materials around and the amino group on the tissue surface, thereby constructing the hydrogel in situ and realizing high adhesion of the hydrogel to the tissue.

[0004] However, the photo-induced imine cross-linked hydrogel has certain defects, which limits its application in the treatment of diabetic foot, diabetic wounds and even other chronic refractory wounds. First, the preparation process of the hydrogel is relatively complicated, including synthesis of ONB, covalent grafting of ONB on hyaluronic acid and chemical modification of auxiliary cross-linking materials; second, the current photo-induced imine cross-linked hydrogel has a single function, and can only provide a minimum level of wound healing environment, and no related drugs are added to cope with the complex situation of chronic refractory wounds, such as bacterial infection and chronic inflammation.

[0005] In summary, the present application considers it necessary to develop a hydrogel with antibacterial and anti-inflammatory effects. SUMMARY

[0006] The present application aims to solve the problems of the current photo-induced imine cross-linked hydrogel preparation process being complex, lacking antibacterial and anti-inflammatory functions, and provides an antibacterial and anti-inflammatory drug controlled release hydrogel and its preparation method and application; the scheme has strong operability and simple process, and can prepare photo-induced imine cross-linked hydrogel with antibacterial and anti-inflammatory effects.

[0007] The concept of the present application is:

[0008] In view of the problem of the complex preparation process of the existing photo-induced imine cross-linked hydrogel, the present application intends to simplify the early two-component cross-linked matrix into a single-component cross-linked matrix, while simplifying the cross-linking system, still ensuring the cross-linking density between molecules and within molecules, so the present application adopts the same cross-linking mechanism, introduces a double-branched small molecule photo-crosslinking agent with ONB as a structural unit, the aldehyde groups generated at both ends of the small molecule photo-crosslinking agent after ultraviolet irradiation can form dynamic imine bonds with the amino-modified polymer derivatives and the amino groups on the tissue surface, thereby constructing a hydrogel with stable structure and good tissue adhesion. At the same time, the double-branched small molecule photo-crosslinking agent is further modified to obtain a double-branched small molecule photo-crosslinking agent capped with a secondary amino antibacterial drug (such as ciprofloxacin), which can quickly cleave and release the antibacterial drug, such as ciprofloxacin, under ultraviolet light, which has broad-spectrum antibacterial activity and can achieve rapid sterilization effect. Finally, the biocompatible and low-toxicity polylactic acid-polyglycolic acid copolymer is used to coat the anti-inflammatory drugs (such as itaconic acid, metformin, caffeic acid or tannic acid, etc.) to form polylactic acid-polyglycolic acid copolymer-anti-inflammatory drug nanoparticles, which can achieve the slow release of anti-inflammatory drugs in the cross-linking network formed by hyaluronic acid and photo-crosslinking agent, and the latter has good anti-inflammatory effect and can promote the regeneration and repair of the wound epidermis.

[0009] Based on the above-mentioned inventive concept, in order to achieve the above-mentioned purpose, the technical solution provided by the present application is:

[0010] A double-branched small molecule compound, which is specifically (((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))dimethanol, and the molecular structure formula is:

[0011] .

[0012] The preparation method of the above-mentioned double-branched small molecule compound, which is specifically characterized by comprising the following steps:

[0013] S1, 4-hydroxy-5-methoxy-2-nitrobenzaldehyde is dissolved in N,N-dimethylformamide, and ((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl) bis(4-methylbenzenesulfonate) and potassium carbonate are added in turn, followed by heating to 60-80°C, and thin layer chromatography is used to detect the completion of the reaction or the reaction is quenched by adding water; the reaction product is extracted with ethyl acetate, the organic phase is washed and dried, filtered, and concentrated under reduced pressure to obtain a yellow crude product; the crude product is further purified on a neutral alumina chromatographic column, and elution is performed to obtain 4,4'-((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitrobenzaldehyde);

[0014] S2, 4,4'-((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitrobenzaldehyde) obtained in S1 is dissolved in tetrahydrofuran, and the whole process is kept away from light, then sodium borohydride dissolved in tetrahydrofuran is added, and the reaction is stirred at room temperature; thin layer chromatography is used to detect the completion of the reaction, the reaction solution is neutralized with hydrochloric acid, and the reaction product is extracted with ethyl acetate; the organic phase is combined, dried, filtered, and concentrated under reduced pressure to obtain a white crude product; the crude product is purified by flash column chromatography to obtain (((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))dimethanol, i.e., a double-branched photocrosslinking agent.

[0015] The process for synthesizing 4-hydroxy-5-methoxy-2-nitrobenzaldehyde can be as follows:

[0016] ①, vanillin is dissolved in N,N-dimethylformamide, then benzyl bromide (raw material) and potassium carbonate (catalyst) are added, and the reaction is carried out at room temperature; thin layer chromatography is used to detect the completion of the reaction, and water is added to quench the reaction; the reaction product is extracted with diethyl ether, the organic phase is washed and dried, filtered, and concentrated to obtain 4-(benzyloxy)-3-methoxybenzaldehyde; wherein the equivalent ratio of vanillin, benzyl bromide, and potassium carbonate is 1:1.05:2, and the reaction time is 2 h;

[0017] ii. dissolving 4-(benzyloxy)-3-methoxybenzaldehyde obtained in i. in dichloromethane, and cooling the solution to 0°C; slowly adding cooled nitric acid, stirring for 30 minutes, and then continuously adding cooled nitric acid, followed by warming to room temperature to react; after the reaction is completed, diluting the reaction solution with ethyl acetate, washing and drying the organic phase, filtering, and concentrating to obtain a crude product; recrystallizing the crude product in an ethyl acetate / n-heptane system to obtain 4-(benzyloxy)-5-methoxy-2-nitrobenzaldehyde; wherein the equivalent ratio of 4-(benzyloxy)-3-methoxybenzaldehyde and nitric acid is 1:50, and the reaction time is 12 h;

[0018] iii. slowly adding trifluoroacetic acid to 4-(benzyloxy)-5-methoxy-2-nitrobenzaldehyde obtained in ii. at 0°C; then warming to 60°C and stirring for 8-12 h, detecting the completion of the reaction by thin layer chromatography, and then concentrating the reaction solution under reduced pressure to remove trifluoroacetic acid, and purifying the residue by flash column chromatography to obtain 4-hydroxy-5-methoxy-2-nitrobenzaldehyde; wherein the equivalent ratio of 4-hydroxy-5-methoxy-2-nitrobenzaldehyde and trifluoroacetic acid is 1:20, and the reaction time is 12 h.

[0019] The process for synthesizing ((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl) bis(4-methylbenzenesulfonate) can be as follows:

[0020] adding p-toluenesulfonyl chloride to a dichloromethane solution in which triethylene glycol is dissolved, and cooling the solution to 0°C; slowly adding potassium hydroxide to keep the temperature below 5°C, and reacting at 0°C, detecting the completion of the reaction by thin layer chromatography, and then adding ice water to quench the reaction; extracting the reaction product with dichloromethane, washing and drying the organic phase, filtering, and concentrating to obtain ((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl) bis(4-methylbenzenesulfonate); wherein the equivalent ratio of triethylene glycol, p-toluenesulfonyl chloride, and potassium hydroxide is 1:2:8; and the reaction time is 3 h.

[0021] In the above reaction, the solvent is preferably added in an amount such that the concentration of the reactants in the solvent is 0.3-0.5 M.

[0022] Further, in S1, the equivalent ratio of 4-hydroxy-5-methoxy-2-nitrobenzaldehyde, ((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl) bis(4-methylbenzenesulfonate), and potassium carbonate is 1:2:2.2, and the reaction time is 6 h;

[0023] In S2, the equivalent ratio of 4,4'-((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitrobenzaldehyde) to sodium borohydride is 1:2, and the reaction time is 0.5 h.

[0024] Meanwhile, the application provides application of the above-mentioned double-branched small molecule compound as a photocrosslinking agent in preparation of a hydrogel precursor solution.

[0025] Based on the above-mentioned application, the application provides a hydrogel precursor solution for controlled release of antibacterial and anti-inflammatory drugs, which is characterized in that the main components are: a double-branched photocrosslinking agent, an amino-modified polymer derivative, a double-branched photocrosslinking agent-antibacterial drug co-carrier and anti-inflammatory drug sustained-release particles.

[0026] The double-branched photocrosslinking agent is the above-mentioned double-branched small molecule compound, which can induce intramolecular and intermolecular crosslinking of the polymer derivative by forming an imine bond under irradiation of a 395 nm light wave, and simultaneously construct a hydrogel-tissue interface.

[0027] The amino-modified polymer derivative is used as a crosslinking matrix.

[0028] The double-branched photocrosslinking agent-antibacterial drug co-carrier is prepared by coupling the above-mentioned double-branched small molecule compound and an antibacterial drug containing a secondary amino group.

[0029] The amount of the amino-modified polymer derivative is not higher than 0.01 times the mass of the double-branched photocrosslinking agent.

[0030] The amount of the double-branched photocrosslinking agent-antibacterial drug co-carrier is not higher than 0.01 times the mass of the double-branched photocrosslinking agent.

[0031] The amount of the anti-inflammatory drug sustained-release particles is not higher than 0.05 times the mass of the double-branched photocrosslinking agent.

[0032] Further, the amino-modified polymer derivative is an amino-modified hyaluronic acid, the molecular weight of which is 20-40 kDa, the amino grafting rate of which is 4%, and the mass concentration of the hyaluronic acid is 1-2%, and the structural formula of the amino-modified hyaluronic acid is as follows:

[0033]

[0034] The double-branched photocrosslinking agent-antibacterial drug co-carrier is a double-branched photocrosslinking agent-ciprofloxacin co-carrier, which is 6,6'-((((((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(methylene))bis(oxy))bis(carbonyl))bis(piperazin-4,1-diyl))bis(1-cyclopropyl-7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid), and the molecular structure is:

[0035] The antibacterial drug can be quickly cleaved and released under irradiation of 395 nm light waves, and the whole release process is completed within 10 minutes;

[0036] The anti-inflammatory drug slow-release particles are poly-lactic acid-poly-glycolic acid copolymer-anti-inflammatory drug nanoparticles, namely, poly-lactic acid-poly-glycolic acid copolymer nanoparticles loaded with metformin, which can slowly release the loaded metformin in a hydrogel environment, and the release time is as long as 72 hours; wherein the anti-inflammatory drug is itaconic acid, metformin, caffeic acid or tannic acid;

[0037] In this way, the hydrogel designed in the application can realize the ordered release of the antibacterial drug and the anti-inflammatory drug, and the treatment of chronic refractory wounds (such as diabetic wounds) can play a synergistic role, antibacterial in the early stage and anti-inflammatory in the middle stage.

[0038] Further, the preparation method of the double-branched photocrosslinking agent-ciprofloxacin co-carrier is as follows:

[0039] I. Synthesis of bis(4-nitrophenyl)(((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(methylene))bis(carbonate)

[0040] The double-branched photocrosslinking agent is dissolved in dry tetrahydrofuran, the solution is added dropwise to a tetrahydrofuran solution in which p-nitrophenyl chloroformate and pyridine are dissolved, and the reaction is carried out at room temperature under the protection of inert gas and in the dark. After thin layer chromatography detection until the reaction is completed, the reaction solution is neutralized with hydrochloric acid and extracted with dichloromethane. The organic phase is washed and dried, filtered, and concentrated to obtain bis(4-nitrophenyl)(((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(methylene))bis(carbonate);

[0041] II. Synthesis of double-branched photocrosslinking agent-ciprofloxacin co-carrier

[0042] Dissolve the obtained bis(4-nitrophenyl)(((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(methylene))bis(carbonate) in dry N,N-dimethylformamide under inert gas protection, and sequentially add ciprofloxacin and triethylamine, react at room temperature in the dark, and detect by thin layer chromatography until the reaction is completed. The reaction solution is neutralized with hydrochloric acid and extracted with ethyl acetate for several times. The combined organic layers are dried, filtered, and concentrated under reduced pressure to obtain a light yellow crude product. The crude product is recrystallized in dichloromethane to obtain 6,6'-((((((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(methylene))bis(oxy))bis(carbonyl))bis(piperazin-4,1-diyl))bis(1-cyclopropyl-7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid), i.e., the double-branched photocrosslinking agent-ciprofloxacin co-carrier;

[0043] The preparation method of the amino-modified hyaluronic acid is as follows:

[0044] Dissolve the hyaluronic acid in deionized water at room temperature, sequentially add carbonyl hydrazine and 1-hydroxybenzotriazole, and adjust the pH of the solution to 4.8 (for example, use 0.5-2M hydrochloric acid aqueous solution to adjust the pH), add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and stir for 10-20h; transfer the reaction solution to a dialysis bag, first dialyze with a dilute hydrochloric acid aqueous solution containing sodium chloride, then dialyze with deionized water, and finally freeze-dry the solution to obtain the amino-modified hyaluronic acid;

[0045] The polylactic acid-polyglycolic acid copolymer-anti-inflammatory drug nanoparticles are polylactic acid-polyglycolic acid copolymer-metformin nanoparticles, and the preparation method is as follows:

[0046] Step one, dissolve metformin in deionized water as an internal water phase;

[0047] Dissolve the polylactic acid-polyglycolic acid copolymer in dichloromethane as an oil phase;

[0048] Step two, mix the internal water phase with the oil phase, ultrasonic emulsify to form a W / O primary emulsion;

[0049] Step three, transfer the W / O primary emulsion to an external water phase, ultrasonic emulsify to form a W / O / W multiple emulsion;

[0050] The external water phase uses a polyvinyl alcohol solution with a mass fraction of 1%;

[0051] Step four, the W / O / W re-emulsified droplets are added into deionized water, stirred, aged, and after the organic solvent is completely volatilized, the obtained nanoparticle solution is centrifuged and washed with ultrapure water to obtain metformin-loaded polylactic acid-polyglycolic acid copolymer nanoparticles, i.e. polylactic acid-polyglycolic acid copolymer-metformin nanoparticles.

[0052] Further, in the process of preparing the double-branched photocrosslinking agent-ciprofloxacin co-carrier:

[0053] In I, the equivalent ratio of (((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))dimethanol, p-nitrophenyl chloroformate and pyridine is 1:15:15, and the reaction time is 1 h;

[0054] In II, the equivalent ratio of bis(4-nitrophenyl)(((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(methylene))bis(carbonate), ciprofloxacin and triethylamine is 1:2:25, and the reaction time is 1 h;

[0055] In the process of preparing the amino-modified hyaluronic acid:

[0056] The equivalent ratio of hyaluronic acid, carbohydrazide, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 1:1:1:0.5, the stirring reaction time is 24 h, and the dialysis time is 24-48 h;

[0057] In the process of preparing the polylactic acid-polyglycolic acid copolymer-metformin nanoparticle:

[0058] The mass ratio of polylactic acid-polyglycolic acid copolymer and metformin is 1:3.3;

[0059] The ultrasonic emulsification power is 500-600 W, the ultrasonic emulsification time is 30 s, the aging time is 12 h, the centrifugal speed is 15000 rpm, and the centrifugal time is 15-20 min.

[0060] The preparation method of the above-mentioned antibacterial and anti-inflammatory drug controlled release hydrogel precursor solution has the special feature that it comprises the following steps:

[0061] 1) Under light shielding conditions, the double-branched photocrosslinking agent and the double-branched photocrosslinking agent-antibacterial drug co-carrier are respectively dissolved in dimethyl sulfoxide to obtain a photocrosslinking agent solution and a photocrosslinking agent drug solution;

[0062] Dissolve the amino-modified polymer derivative in deionized water, and stir uniformly at room temperature to obtain a polymer derivative solution;

[0063] 2) Under light shielding conditions, the photocrosslinking agent solution, the photocrosslinking agent drug solution and the polymer derivative solution are uniformly mixed in proportion, and the polylactic acid-polyglycolic acid copolymer-metformin nanoparticles are fully dispersed in the mixed solution to obtain an antibacterial and anti-inflammatory drug controlled-release hydrogel precursor solution.

[0064] The antibacterial and anti-inflammatory drug controlled-release hydrogel is applied to the preparation of a chronic non-healing wound dressing. Chronic non-healing wounds such as diabetic foot, diabetic wounds, lower extremity venous ulcers, bedsores and the like have roughly the same difficulties and treatment ideas in the healing process. Starting from the local wound, the main problem is to deal with bacterial infection in the early stage of the wound surface and inflammation in the middle stage, and starting from the whole body, the problem is to actively seek and eliminate or control adverse factors affecting wound healing. Therefore, the antibacterial and anti-inflammatory drug controlled-release hydrogel developed based on the application can completely deal with most chronic non-healing wounds.

[0065] Based on the above application, the application further provides a chronic non-healing wound dressing, and the special feature thereof is that the effective component is the antibacterial and anti-inflammatory drug controlled-release hydrogel precursor solution.

[0066] The use method of the chronic non-healing wound dressing is characterized in that the operation is as follows:

[0067] After the dressing is applied, the dressing is irradiated with ultraviolet light to initiate the crosslinking reaction between the double-branched photocrosslinking agent and the amino-modified polymer derivative, so that the crosslinking agent is crosslinked into glue; the use process is also the process of converting the hydrogel precursor solution into a hydrogel, and only the effective components are mixed in the dark, and the crosslinking glue is obtained by ultraviolet light irradiation, without covalent grafting.

[0068] Further, the wavelength of the ultraviolet light is 395 nm, the power of the ultraviolet light is 30 mW / cm 2 , and the irradiation time is 2-5 min.

[0069] Mechanism of the application:

[0070] The branched light crosslinking agent, the branched light crosslinking agent-antibacterial drug co-carrier, the anti-inflammatory drug sustained-release particles (such as: polylactic acid-polyglycolic acid copolymer-anti-inflammatory drug nanoparticles) and the amino-modified polymer derivative solution are fully mixed, and under the irradiation of ultraviolet light, the branched light crosslinking agent forms chemical bonds with the polymer derivative (such as: hyaluronic acid) and the tissue surface through aldehyde-amine condensation reaction; at the same time, the branched light crosslinking agent-antibacterial drug co-carrier is also cracked, ensuring the rapid release of the antibacterial drug while forming the gel; after the stable hydrogel is formed, the anti-inflammatory drug sustained-release particles (such as: polylactic acid-polyglycolic acid copolymer-anti-inflammatory drug nanoparticles) wrapped therein can continuously and slowly release the anti-inflammatory drug due to the degradation effect, realizing long-term anti-inflammatory effect, and ultimately providing a key basis for the treatment of chronic refractory wounds (such as: diabetic wounds), that is, the present application designs a light crosslinking hydrogel with antibacterial and anti-inflammatory effects, which is induced by a branched small molecule light crosslinking agent to quickly gel the polymer derivative (hyaluronic acid, chitosan, gelatin, etc.), and at the same time, by introducing fast-slow two drug release mechanisms, the problems of early bacterial infection and chronic inflammation in the treatment of chronic refractory wounds are effectively solved; compared with the current light-induced imine crosslinking hydrogel, the preparation process is simpler (without covalent grafting, only ultraviolet irradiation is needed), and the function is more abundant.

[0071] The advantages of the present application are:

[0072] 1. The branched small molecule compound synthesized as a light crosslinking agent in the present application is easy to obtain, low in cost and simple in synthesis steps, and because of the three condensed tetraethylene glycol linking group, the branched light crosslinking agent has good solubility and can be better miscible with the amino-modified polymer derivative, ensuring the crosslinking density of the crosslinking system.

[0073] 2. The branched light crosslinking agent in the present application can realize crosslinking and rapid release of antibacterial drugs (such as: ciprofloxacin) under light stimulation at the same time, solve the problem of single function of the current light-induced imine crosslinking hydrogel, and for the first time introduce the antibacterial function into the light-induced imine crosslinking hydrogel, which can effectively solve the problem of rapid proliferation of bacteria in the early stage of diabetic foot.

[0074] 3. The present application uses polylactic acid-polyglycolic acid copolymer nanoparticles to coat and release anti-inflammatory drugs, which can effectively inhibit chronic inflammation in the middle stage of diabetic foot, further improve the healing efficiency of the wound surface, and has good application prospect in the treatment of diabetic foot.

[0075] 4. The preparation process of the hydrogel of the present application simplifies the preparation of hyaluronic acid, increases the function of drug controlled release, and solves the problem that the current light-controlled hydrogel only focuses on wound sealing and does not pay attention to the antibacterial and healing promotion of the wound surface. BRIEF DESCRIPTION OF DRAWINGS

[0076] Figure 1 Schematic diagram of preparation process of photocrosslinking hydrogel and related synthesis route of the application, wherein A is the schematic diagram of preparation process of hydrogel; B is the synthesis route of double-branched photocrosslinking agent and double-branched photocrosslinking agent-ciprofloxacin co-carrier;

[0077] Figure 2 Synthesis route of double-branched photocrosslinking agent of the application 1 H NMR

[0078] Figure 3 Synthesis route of double-branched photocrosslinking agent of the application 13 C NMR

[0079] Figure 4 Synthesis route of double-branched photocrosslinking agent-ciprofloxacin co-carrier of the application 1 H NMR

[0080] Figure 5 Synthesis route of double-branched photocrosslinking agent-ciprofloxacin co-carrier of the application 13 C NMR

[0081] Figure 6 UV-visible absorption spectrum of double-branched photocrosslinking agent of the application under different light irradiation times

[0082] Figure 7 Release of ciprofloxacin from double-branched photocrosslinking agent-ciprofloxacin co-carrier of the application under different light irradiation times

[0083] Figure 8 Cumulative release curve of ciprofloxacin from double-branched photocrosslinking agent-ciprofloxacin co-carrier of the application under light stimulation

[0084] Figure 9 Transmission electron microscope image of polylactic acid-polyglycolic acid copolymer nanoparticles of the application

[0085] Figure 10 Drug release curve of polylactic acid-polyglycolic acid copolymer nanoparticles of the application in acidic environment

[0086] Figure 11 Appearance pictures of photocrosslinking hydrogel of the application before and after light irradiation

[0087] Figure 12 X-ray photoelectron spectroscopy of photocrosslinking hydrogel of the application before and after light irradiation

[0088] Figure 13 Scanning electron microscope image (lyophilized state) of photocrosslinking hydrogel of the application

[0089] Figure 14 Rheological study results of photocrosslinking hydrogel of the application

[0090] Figure 15 In vitro swelling experiment results of the photo-crosslinked hydrogel of the present application;

[0091] Figure 16 In vitro degradation experiment results of the photo-crosslinked hydrogel of the present application;

[0092] Figure 17 Cell toxicity experiment results of the photo-crosslinked hydrogel of the present application on HUVEC cells;

[0093] Figure 18 The wound healing conditions of the 0d, 3d, 7d and 14d treatment groups and the control group of the type 2 diabetes mouse wound model were established, and the pictures were the photos of the wounds of the representative mice in each group.

[0094] Figure 19 The wound healing rate statistics chart of the treatment group and the control group from the modeling day to 14d after modeling; it can be seen from the chart that in the wound healing process, the healing speed of the photo-crosslinked hydrogel group of the present application is faster than that of the control group.

[0095] Figure 20 The HE staining results of the wound surface of the treatment group and the control group from the modeling day to 7d after modeling;

[0096] Figure 21 The MT staining results of the wound surface of the treatment group and the control group from the modeling day to 7d after modeling;

[0097] Figure 22 The collagen protein score results of the wound surface of the treatment group and the control group from the modeling day to 14d after modeling. DETAILED DESCRIPTION

[0098] The content of the present application is further described in detail below in combination with the drawings and specific embodiments:

[0099] I. Synthesis of the double-branched small molecule compound, i.e. the double-branched photo-crosslinking agent

[0100] 1) Synthesis of ((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl) bis(4-methylbenzenesulfonate)

[0101] p-Toluenesulfonyl chloride (5.72 g, 30 mmol) was added to a solution of triethylene glycol (2.91 g, 15 mmol) in dichloromethane (15 mL) and the solution was cooled to 0 °C, and potassium hydroxide (6.72 g, 120 mmol) was added slowly keeping the temperature below 5 °C; the reaction was stirred at 0 °C for 3 h, thin layer chromatography was used to check the completion of the reaction; the reaction was quenched by the addition of ice water and the reaction product was extracted with dichloromethane, the organic phase was washed and dried, filtered and concentrated to obtain ((oxybis(ethane-2, 1-diyl))bis(oxy))bis(ethane-2, 1-diyl) bis(4-methylbenzenesulfonate) (6.95 g, 92.3 %).

[0102] ((oxybis(ethane-2, 1-diyl))bis(oxy))bis(ethane-2, 1-diyl) bis(4-methylbenzenesulfonate): 1 HNMR (500 MHz, DMSO-d6), δ 7.78 (d, J = 8.3 Hz, 4H), 7.47 (d, J = 8.1 Hz, 4H),4.11 (t, J = 4.4 Hz, 4H), 3.60-3.54 (m, 4H), 3.42 (d, J = 1.0 Hz, 8H), 2.41(s, 6H). 13 C NMR (126 MHz, DMSO-d6), δ (ppm): 144.83, 132.45, 130.03, 127.57,69.90, 69.69, 67.90, 20.98. LC-MS (ESI): m / z: Calcd. for [M+H] + : 503.13.Found: 503.28.

[0103] 2) Synthesis of 4-(benzyloxy)-3-methoxybenzaldehyde

[0104] Vanillin (5 g, 32.9 mmol) was dissolved in 15 mL of N,N-dimethylformamide, and benzyl bromide (5.9 g, 34.5 mmol) and potassium carbonate (9.08 g, 65.7 mmol) were added, the reaction was carried out at room temperature for 2 h, thin layer chromatography was used to check the completion of the reaction; the reaction was quenched by the addition of water and extracted with diethyl ether, the organic phase was washed and dried, filtered and concentrated to obtain 4-(benzyloxy)-3-methoxybenzaldehyde (7.91 g, 99 %).

[0105] 4-(benzyloxy)-3-methoxybenzaldehyde: 1H NMR (500 MHz, CDC13), δ 9.82 (s, 1H), 7.44-7.29 (m, 7H), 6.98 (d, J = 8.2 Hz, 1H), 5.23 (s, 2H), 3.93 (s, 3H). 13 CNMR (126 MHz, CDC13), δ (ppm): 191.00, 153.70, 150.18, 136.12, 130.40, 128.82, 128.31, 127.31, 126.68, 112.50, 109.46, 70.96, 56.15. LC-MS (ESI): m / z: Calcd. for [M+H] + : 243.09. Found: 243.16.

[0106] 3) Synthesis of 4-(benzyloxy)-5-methoxy-2-nitrobenzaldehyde

[0107] Dissolve 4-(benzyloxy)-3-methoxybenzaldehyde (1.21 g, 5 mmol) in 12 mL of dichloromethane, cool the solution to 0 °C and slowly add a cooled nitric acid (70%, 6 mL), after 30 minutes of stirring, add another portion of cooled nitric acid (70%, 6 mL), then warm to room temperature for 12 h; after completion of the reaction, dilute the reaction with ethyl acetate, wash and dry the organic phase, filter, concentrate to obtain the crude product; recrystallize the crude product in ethyl acetate / n-heptane system (1:3, v / v) to obtain 4-(benzyloxy)-5-methoxy-2-nitrobenzaldehyde (0.89 g, 62%). 4-(benzyloxy)-5-methoxy-2-nitrobenzaldehyde: 1 H NMR (500 MHz, CDC13), δ 10.44 (s, 1H), 7.67 (s, 1H), 7.47-7.34 (m, 6H), 5.27 (s, 2H), 4.02 (s, 3H). 13 C NMR (126 MHz, CDC13), δ (ppm): δ 187.91, 153.87, 151.56, 143.77, 134.98, 129.06, 128.86, 127.72, 125.87, 110.17, 109.05, 71.71, 56.87. LC-MS (ESI): m / z: Calcd. for [M+H] + : 288.08. Found:288.19.

[0108] 4) Synthesis of 4-hydroxy-5-methoxy-2-nitrobenzaldehyde

[0109] To 4-(benzyloxy)-5-methoxy-2-nitrobenzaldehyde (900 mg, 3.13 mmol) was added slowly dropwise trifluoroacetic acid (10 ml) at 0 °C; then warmed to 60 °C and stirred for 12 h, TLC monitored until the reaction was completed; the reaction was concentrated under reduced pressure, trifluoroacetic acid was removed, the residue was purified by flash column chromatography (petroleum ether: ethyl acetate = 1:1, v / v) to give 4-hydroxy-5-methoxy-2-nitrobenzaldehyde (550 mg, 89%). 4-hydroxy-5-methoxy-2-nitrobenzaldehyde: 1 H NMR (500MHz, DMSO- d6), 10.16 (s, 1H), 7.51 (s, 1H), 7.36 (s, 1H), 3.95 (s, 3H). 13 CNMR (126 MHz, DMSO-d6), δ (ppm): δ 188.34, 151.82, 151.01, 143.80, 123.40,111.05, 110.66, 56.36. LC-MS (ESI): m / z: Calcd. for [M+Na] + : 220.03. Found:220.72.

[0110] 5) Synthesis of 4,4'-((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitrobenzaldehyde)

[0111] 4-Hydroxy-5-methoxy-2-nitrobenzaldehyde (395 mg, 2 mmol) was dissolved in 20 mL of N,N-dimethylformamide. Then, ((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (502 mg, 1 mmol) and potassium carbonate (305 mg, 2.2 mmol) were added sequentially. The mixture was then heated to 80 °C and reacted for 6 h. Thin-layer chromatography was used to detect the reaction until completion. The reaction was quenched with water and extracted multiple times with ethyl acetate. The organic layers were combined, dried, filtered, and concentrated under reduced pressure to obtain a yellow crude product. The crude product was further purified on neutral alumina (ethanol:petroleum ether:ethyl acetate = 0.2:8:3). The solution was eluted v / v / v to give 4,4'-((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitrobenzaldehyde) (397.5 mg, 72%). 4,4'-((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitrobenzaldehyde): 1 HNMR (500 MHz, DMSO-d6), δ 10.18 (s, 2H), 7.71 (s, 2H), 7.34 (s, 2H), 4.32-4.28 (m, 4H), 3.94 (s, 6H), 3.79 (dd, J = 5.1, 3.7 Hz, 4H), 3.59 (dd, J =5.9, 3.2 Hz, 4H), 3.54 (dd, J = 5.8, 3.1 Hz, 4H). 13 C NMR (126 MHz, DMSO-d6), δ (ppm): δ 188.49, 152.63, 151.17, 143.47, 124.71, 110.05, 108.41, 69.88,68.98, 68.56, 56.40. LC-MS (ESI): m / z: Calcd. for [M+H] + : 553.16. Found: 553.31.

[0112] 6) Synthesis of (((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))diethanol

[0113] Dissolve 4,4'-((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitrobenzaldehyde) (300 mg, 0.55 mmol) in 10 mL of tetrahydrofuran, keep it away from light throughout, then add sodium borohydride (42 mg, 1.1 mmol) dissolved in tetrahydrofuran (15 mL) to it, and stir the reaction at room temperature for 30 min, check the reaction completion by thin layer chromatography; neutralize the reaction with hydrochloric acid and extract with ethyl acetate several times, combine the organic layers, and dry, filter, and concentrate under reduced pressure to obtain a white crude product; purify the crude product by flash column chromatography (dichloromethane:methanol = 80:1, v / v) to obtain (((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))dimethanol (278 mg, 92%). (((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))dimethanol: 1 H NMR (500 MHz, DMSO-d6), δ 7.68 (s, 2H), 7.37 (s, 2H), 5.56 (t,J = 5.4 Hz, 2H), 4.81 (d, J = 5.4 Hz, 4H), 4.19-4.14 (m, 4H), 3.90 (s, 6H),3.78-3.73 (m, 4H), 3.57 (ddd, J = 8.8, 6.1, 3.4 Hz, 8H). 13 C NMR (126 MHz,DMSO-d6), δ (ppm): δ 153.65, 146.07, 138.29, 134.26, 109.68, 109.06, 69.86,68.75, 68.41, 60.08, 56.01. LC-MS (ESI): m / z: Calcd. for [M+H] + : 557.19.Found: 557.34.

[0114] II. Synthesis of the dual branched photocrosslinker-ciprofloxacin co-carrier

[0115] 1) Synthesis of bis(4-nitrophenyl)(((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(methylene))bis(carbonate)

[0116] (((((oxybis(ethane-2, 1 -diyl))bis(oxy))bis(ethane-2, 1 - diyl))bis(oxy))bis(5-methoxy-2-nitro-4, 1 -phenylene))bismethanol (250 mg, 0.45 mmol) was dissolved in 10 mL of dry tetrahydrofuran and the solution was added dropwise to a solution of p-nitrophenyl chloroformate (1.36 g, 6.75 mmol) and pyridine (0.5 mL, 6.75 mmol) in tetrahydrofuran (5 mL) at room temperature under an inert gas atmosphere and protected from light for 1 h, until the reaction was complete by thin layer chromatography; the reaction was neutralized with hydrochloric acid and extracted with dichloromethane, the organic phase was washed and dried, filtered and concentrated to obtain bis(4-nitrophenyl)(((((oxybis(ethane-2, 1 -diyl))bis(oxy))bis(ethane-2, 1 - diyl))bis(oxy))bis(5-methoxy-2-nitro-4, 1 -phenylene))bis(methylene))bis(carbonate).

[0117] 2) Synthesis of 6,6'-((((((((oxybis(ethane-2, 1 -diyl))bis(oxy))bis(ethane-2, 1 - diyl))bis(oxy))bis(5-methoxy-2-nitro-4, 1 -phenylene))bis(methylene))bis(oxy))bis(carbonyl))bis(piperazin-4, 1 -diyl))bis( 1 -cyclopropyl-7-fluoro-4-oxo- 1,4-dihydroquinoline-3-carboxylic acid)

[0118] Dissolve bis(4-nitrophenyl)(((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(methylene))bis(carbonate) (300 mg, 0.34 mmol) in 10 mL of dry N,N-dimethylformamide under inert gas protection, and add cyclofloxacin (225 mg, 0.68 mmol) and triethylamine (1.2 mL, 8.5 mmol) in turn, and react at room temperature for 1 h in the dark, and test by thin layer chromatography until the reaction is complete; neutralize the reaction solution with hydrochloric acid and extract with ethyl acetate several times, combine the organic layers, and dry, filter, and concentrate under reduced pressure to obtain a light yellow crude product; recrystallize the crude product in dichloromethane to obtain 6,6'-((((((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(methylene))bis(oxy))bis(carbonyl))bis(piperazin-4,1-diyl))bis(1-cyclopropyl-7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid) (236 mg, 55%). 6,6'-((((((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(methylene))bis(oxy))bis(carbonyl))bis(piperazin-4,1-diyl))bis(1-cyclopropyl-7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid): 1 H NMR (500 MHz, DMSO-d6), δ 8.59 (s, 2H), 7.87 (d, J = 13.3 Hz, 2H), 7.71 (d, J = 2.9 Hz, 2H), 7.53 (d, J = 7.4 Hz, 2H), 7.16 (s, 2H), 5.41 (s, 4H), 4.19 (t, J = 4.5 Hz, 4H), 3.91 (s, 6H), 3.76 (dd, J = 9.7, 5.1 Hz, 6H), 3.65 (s, 8H), 3.59-3.54 (m, 8H), 3.17 (s, 8H), 1.32-1.28 (m, 4H), 1.13 (s, 4H). 13C NMR (126 MHz, DMSO-d6), δ (ppm): 171.33, 164.21, 153.80, 153.29, 147.64, 147.06, 143.32, 139.63, 137.87, 127.53, 126.68, 121.97, 111.43, 109.79, 109.37, 106.28, 69.79, 69.65, 68.61, 63.30, 56.10, 49.42, 49.19, 34.45, 7.29. HRMS (ESI): m / z: Calcd. for [M+Na] + : 1293.4154. Found: 1293.4010.

[0119] Structural characterization

[0120] Use 1 H NMR and 13 C NMR were used to characterize the double-branched photocrosslinker and the double-branched photocrosslinker-ciprofloxacin co-carrier obtained above, and the results are shown in Figures 2-5 NMR spectrum analysis confirmed that the structure of the double-branched photocrosslinker and the double-branched photocrosslinker-ciprofloxacin co-carrier was consistent with the chemical structure shown in Figure 1 , and the purity was more than 99%.

[0121] UV-vis absorption spectroscopy was used to analyze the photolysis reaction of the double-branched photocrosslinker under 395 nm ultraviolet light irradiation, and the results are shown in Figure 6 With the increase of light irradiation time, the characteristic absorption peak (nitro group attribution) signal at 345 nm on the UV-vis absorption spectrum of the double-branched photocrosslinker gradually attenuated, accompanied by the enhancement of the characteristic absorption peak (aldehyde group attribution) signal at 310 nm, which proved the photolysis reaction of the double-branched photocrosslinker under ultraviolet light irradiation.

[0122] High performance liquid chromatography (HPLC) was used to analyze the cleavage of the double-branched photocrosslinker-ciprofloxacin co-carrier under 395 nm ultraviolet light irradiation, and the results are shown in Figures 7-8 After 10 min of ultraviolet light irradiation, the release rate of ciprofloxacin of the double-branched photocrosslinker-ciprofloxacin co-carrier reached 83%.

[0123] III. Synthesis of amino-modified hyaluronic acid

[0124] Hyaluronic acid (408 mg, 340 KDa) was dissolved in 50 mL of deionized water at room temperature, and hydrazine (90 mg, 1 mmol) and 1-hydroxybenzotriazole (153 mg, 1 mmol) were added successively. The pH of the solution was adjusted to 4.8 with 1 M aqueous hydrochloric acid solution, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (90 mg, 0.47 mmol) was added and stirred for 20 h. The reaction solution was transferred to a dialysis bag and dialyzed first with dilute aqueous hydrochloric acid solution in which sodium chloride (0.1 M) was dissolved for 48 h, and then with deionized water for 24 h. Finally, the solution was lyophilized to obtain the amino-modified hyaluronic acid.

[0125] IV. Preparation of Poly(lactic-co-glycolic acid)-metformin nanoparticles

[0126] 1) Metformin was dissolved in deionized water (272 mg / mL) as the internal aqueous phase;

[0127] 2) Poly(lactic-co-glycolic acid) was dissolved in dichloromethane (8.33 mg / mL) as the oil phase;

[0128] 3) 0.6 mL of the internal aqueous phase was mixed with 6 mL of the oil phase, and ultrasonic emulsification was performed for 1 min to form a W / O primary emulsion;

[0129] 4) 1% polyvinyl alcohol was dissolved as the external aqueous phase;

[0130] 5) The W / O primary emulsion was transferred to 6 mL of the external aqueous phase, and ultrasonic emulsification was performed for 1 min to form a W / O / W multiple emulsion;

[0131] 6) The W / O / W multiple emulsion was added dropwise to 8 mL of deionized water, stirred, and aged for 12 h. After the organic solvent was completely volatilized, the obtained nanoparticle solution was centrifuged (15000 rpm) with ultrapure water, washed, and poly(lactic-co-glycolic acid) nanoparticles loaded with metformin were obtained.

[0132] Structural characterization

[0133] The micro-morphology of the aforementioned prepared poly(lactic-co-glycolic acid)-metformin nanoparticles was observed using a transmission electron microscope (TEM), as shown in Figure 9 The SEM picture shows that the prepared poly(lactic-co-glycolic acid)-metformin nanoparticles are spherical nanoparticles with uniform particle size distribution, and the average particle size is 65 nm.

[0134] The drug release of the aforementioned prepared poly(lactic-co-glycolic acid)-metformin nanoparticles in an acidic environment was detected using ultraviolet spectrophotometry, and the results are shown in Figure 10The release of metformin in acidic environment was 52% after 12 h and reached a maximum (about 73%) after 72 h.

[0135] V. Preparation of photo-crosslinked hydrogel

[0136] 1) Dissolve the double-branched photo-crosslinker and the double-branched photo-crosslinker-ciprofloxacin co-carrier in dimethyl sulfoxide respectively under light shielding condition to obtain a photo-crosslinker solution (10 mM) and a photo-crosslinker-drug solution (10 mM);

[0137] 2) Dissolve the amino-modified hyaluronic acid in deionized water and stir uniformly at room temperature to obtain a hyaluronic acid solution (1-2 wt%);

[0138] 3) Under light shielding condition, uniformly mix the photo-crosslinker solution, the photo-crosslinker-drug solution and the hyaluronic acid solution in different proportions (the amount of the double-branched photo-crosslinker-ciprofloxacin co-carrier is not higher than 0.01 times the mass of the double-branched photo-crosslinker, and the amount of the amino-modified hyaluronic acid is not higher than 0.01 times the mass of the double-branched photo-crosslinker), and then fully disperse the polylactic acid-polyglycolic acid copolymer-metformin nanoparticles (the amount of the polylactic acid-polyglycolic acid copolymer-metformin nanoparticles is not higher than 0.05 times the mass of the double-branched photo-crosslinker) in the mixed solution to obtain a hydrogel precursor solution;

[0139] 4) Under ultraviolet light irradiation (30 mW / cm 2 , 2-5 min), initiate the crosslinking reaction between the photo-crosslinker and the amino-modified hyaluronic acid to obtain a photo-crosslinked hydrogel.

[0140] Structural characterization

[0141] The photo-crosslinked hydrogels prepared from the above different proportions of the amino-modified hyaluronic acid, the double-branched photo-crosslinker, the double-branched photo-crosslinker-ciprofloxacin co-carrier and the polylactic acid-polyglycolic acid copolymer-metformin nanoparticles have similar properties, and only the photo-crosslinked hydrogel prepared from the mass ratio of 1:0.005:0.01:0.05 is taken as an example for performance description.

[0142] The pictures of the precursor solution and the gelled photo-crosslinked hydrogel are shown in Figure 11 . The photo-crosslinked hydrogel and its precursor solution are analyzed by X-ray photoelectron spectroscopy (XPS), and the results are shown in Figure 12The N 1s of -NO2 bond in the photocrosslinked hydrogel sample completely disappeared at the binding energy of ~404.5 eV, accompanied by the appearance of N 1s of -C=N bond at the binding energy of ~399.8 eV, which proved that the double-branched molecular compound of the application could successfully catalyze the formation of photocrosslinked hydrogel as a photocrosslinking agent.

[0143] The microstructure of the photocrosslinked hydrogel (in the freeze-dried state) was observed using a scanning electron microscope (SEM), as shown in Figure 13 The SEM results showed that the photocrosslinked hydrogel in the freeze-dried state had a connected pore structure, which was conducive to the transmission and release of drugs in the hydrogel.

[0144] The photocrosslinked hydrogel formation process was monitored using a photo-rheometer, and the results are shown in Figure 14 The dynamic time scan results showed that the storage modulus (G’) exceeded the loss modulus (G”) at 40 s, confirming irreversible gelation; and as the light exposure time increased, both G’ and G” of the hydrogel increased, proving that the hydrogel had good viscoelasticity.

[0145] The in vitro swelling experiment results are shown in Figure 15 After soaking in water for 72 h, the swelling rate of the photocrosslinked hydrogel reached 222%, which was conducive to the absorption of wound exudate.

[0146] The in vitro degradation experiment results are shown in Figure 16 After co-incubation with hyaluronidase for 72 h, more than 70% of the photocrosslinked hydrogel was degraded, proving the good degradability of the hydrogel.

[0147] Six, biocompatibility of photocrosslinked hydrogel

[0148] L929 cells were obtained from the ATCC cell bank;

[0149] Fetal bovine serum (FBS) was purchased from the Kaymu Biological Technology Company, brand: HyClone, item number: SH30071.03;

[0150] DMEM high-sugar medium was purchased from the Kaymu Biological Technology Company, brand: HyClone;

[0151] 1) L929 cells were expanded in culture bottles with DMEM high-sugar medium containing a volume fraction of 10% FBS, and cells in the logarithmic growth phase were taken for standby;

[0152] 2) The photocrosslinked hydrogel prepared as described above was added to a 96-well plate at a volume of 100 µL per well, and DMEM high-sugar medium was added and left for 12 h to allow it to swell fully, ready for use;

[0153] 3) L929 cells in the logarithmic growth phase were seeded on the photocrosslinked hydrogel in 2) at a density of 5000 cells per well;

[0154] 4) L929 cells and hydrogel were co-cultured for 24 h, and CCK-8 test was performed to evaluate the damage of photocrosslinked hydrogel to cells. The group without hydrogel was used as a control group, and the measurement was performed at 450 nm on a microplate reader and calculated by the following formula: cell survival rate = , wherein ODn is the ultraviolet absorbance of the sample (hydrogel group), ODc is the ultraviolet absorbance of the blank group, and ODs is the ultraviolet absorbance of the control group; wherein the blank group is CCK-8 and serum-free DMEM high-glucose medium; and the control group is L929 cells in the logarithmic growth phase without hydrogel.

[0155] As shown in Figure 17 , both the photocrosslinked hydrogel group and the control group showed good growth trend (P < 0.05), proving excellent biocompatibility.

[0156] Seven, the role of photocrosslinked hydrogel in promoting the healing of diabetic wounds in mice

[0157] 1) SPF level BALB / c male mice aged 6 weeks (20 g ± 5 g) were purchased, and after being raised for 1 week, streptozotocin (STZ, 50 mg / kg) was injected intraperitoneally for 5 days to establish a diabetic mouse model. One week after injection, blood glucose was detected, and a blood glucose of ≥16.7 mmol / L was considered to be successfully established;

[0158] 2) The successfully modeled diabetic mice were anesthetized with 2% isoflurane, and after the mice were completely motionless and the corneal reflex disappeared, the tail was clamped without pain response, a 10 mm diameter circular full-thickness skin wound was created on the back;

[0159] 3) The hydrogel precursor solution prepared in the foregoing was dissolved in sterile water for injection, injected into the wound site, and gelled under light (30 mW / cm 2 ) at a dose of 200 µL per mouse. The mouse wound was photographed at 0 d, 3 d, 7 d, and 14 d after hydrogel treatment, and the tissue was taken at 7 d and 14 d. To determine the healing degree and healing process of the wound repair, the skin tissue morphology of the wound was detected. The mouse wound tissue was fixed with 0.4% paraformaldehyde, dehydrated, and then paraffin-embedded. The paraffin-embedded skin tissue was cut into 5 µm thick tissue sections, stained with hematoxylin-eosin (HE) and Masson trichrome (MT), and the stained sections were observed under a biological microscope.

[0160] As shown in Figure 18As shown, with the extension of healing time, the wound area of ​​both the control group and the photocrosslinked hydrogel group gradually decreased, but the wound area of ​​the photocrosslinked hydrogel group was smaller than that of the control group. Figure 19 As shown, after 3 days of treatment, the photocrosslinked hydrogel exhibited a faster wound healing rate than the control group (the wound areas of the two groups were 94.47±3.76% and 64.25±3.35%, respectively). After 14 days of treatment, the wound area of ​​the photocrosslinked hydrogel group shrank to 23.3±9.26%, which was in stark contrast to the control group. This indicates that the photocrosslinked hydrogel can significantly promote the healing of diabetic wounds.

[0161] Through HE ( Figure 20 (as shown) and MT staining ( Figure 21 As shown in the analysis, the photocrosslinked hydrogel exhibited good wound repair effects at all time points during wound healing; simultaneously, the collagen deposition effect in the wound of the photocrosslinked hydrogel group was more significant compared to the control group. Quantitative data analysis showed ( Figure 22 As shown in the figure, after 14 days of treatment, the wound collagen score of the photocrosslinked hydrogel group was the highest (4.33±0.82), while that of the control group was only 1.17±1.17%, indicating that the photocrosslinked hydrogel can effectively promote collagen deposition.

[0162] In summary, this invention successfully prepared a photocrosslinked hydrogel with both antibacterial and anti-inflammatory effects. By inducing rapid gelation of polymeric derivatives through a doubly branched small molecule photocrosslinking agent, and by introducing both fast and slow drug release mechanisms, it effectively solves the problems of bacterial infection and chronic inflammation in the treatment of diabetic wounds and other chronic refractory wounds.

Claims

1. An antibacterial, anti-inflammatory drug controlled release hydrogel precursor solution, characterized in that, The main components are: double branched photo-crosslinking agent, amino modified polymer derivative, double branched photo-crosslinking agent-antibacterial drug co-carrier and anti-inflammatory drug sustained-release particles. The amino modified polymer derivative is amino modified hyaluronic acid, the molecular weight is 20-40kDa, and the structural formula is: The double branched photo-crosslinking agent is (((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))dimethanol, and the molecular structure formula is: The double branched photo-crosslinking agent-antibacterial drug co-carrier is a double branched photo-crosslinking agent-ciprofloxacin co-carrier, which is 6,6'-((((((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(methylene))bis(oxy))bis(carbonyl))bis(piperazin-4,1-diyl))bis(1-cyclopropyl-7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid), and the molecular structure is: The anti-inflammatory drug sustained-release particles are polylactic acid-polyglycolic acid copolymer-anti-inflammatory drug nanoparticles, wherein the anti-inflammatory drug is itaconic acid, metformin, caffeic acid or tannic acid; The amount of the amino modified polymer derivative is not more than 0.01 times the mass of the double branched photo-crosslinking agent; The amount of the double branched photo-crosslinking agent-antibacterial drug co-carrier is not more than 0.01 times the mass of the double branched photo-crosslinking agent; The amount of the anti-inflammatory drug sustained-release particles is not more than 0.05 times the mass of the double branched photo-crosslinking agent.

2. The antibacterial and anti-inflammatory drug controlled release hydrogel precursor solution according to claim 1, characterized in that: The preparation method of the double branched photo-crosslinking agent comprises the following steps: S1, 4-hydroxy-5-methoxy-2-nitrobenzaldehyde is dissolved in N,N-dimethylformamide, and ((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl) bis(4-methylbenzenesulfonate) and potassium carbonate are added in turn, then the temperature is raised to 60-80℃ for reaction, after the reaction is completed, water is added to quench the reaction; multiple times of ethyl acetate extraction, combined organic layer, and dried, filtered, and reduced pressure concentrated to obtain yellow crude product; further purify the crude product on a neutral alumina chromatographic column, and elute to obtain 4,4'-((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitrobenzaldehyde); S2, dissolve the 4,4'-((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitrobenzaldehyde) obtained in S1 in tetrahydrofuran, keep it away from light throughout, then add sodium borohydride dissolved in tetrahydrofuran solution thereto, and stir the reaction at room temperature, after the reaction is completed, neutralize the reaction solution with hydrochloric acid and extract with ethyl acetate for several times, combine the organic layers, dry, filter, and concentrate under reduced pressure to obtain a white crude product; purify the crude product by flash column chromatography to obtain (((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))dimethanol, i.e. the double-branched photocrosslinking agent.

3. The solution of the hydrogel precursor for controlled release of antibacterial and anti-inflammatory drugs according to claim 2, characterized in that: The preparation method of the double-branched photocrosslinking agent-ciprofloxacin co-carrier is as follows: Ⅰ. Synthesis of bis(4-nitrophenyl)(((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(methylene))bis(carbonate) Dissolve the double-branched photocrosslinking agent in dry tetrahydrofuran, drop the solution into a tetrahydrofuran solution containing p-nitrophenyl chloroformate and pyridine, and carry out the reaction at room temperature under the protection of inert gas and away from light, after the reaction is completed, neutralize the reaction solution with hydrochloric acid and extract with dichloromethane, wash and dry the organic phase, filter, and concentrate to obtain bis(4-nitrophenyl)(((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(methylene))bis(carbonate); Ⅱ. Synthesis of double-branched photocrosslinking agent-ciprofloxacin co-carrier The obtained bis(4-nitrophenyl)(((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(methylene))bis(carbonate) is dissolved in dry N,N-dimethylformamide under inert gas protection, and cyclofloxacin and triethylamine are sequentially added, and the reaction is carried out at room temperature in the dark, after the reaction is completed, the reaction solution is neutralized with hydrochloric acid and extracted with ethyl acetate for several times, the combined organic layer is dried, filtered and concentrated under reduced pressure to obtain a light yellow crude product; the crude product is recrystallized in dichloromethane to obtain 6,6'-((((((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(methylene))bis(oxy))bis(carbonyl))bis(piperazin-4,1-diyl))bis(1-cyclopropyl-7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid), namely the double-branched photocrosslinking agent-cyclofloxacin co-carrier; The preparation method of the amino-modified hyaluronic acid is as follows: The hyaluronic acid is dissolved in deionized water at room temperature, and hydrazine carbohydrazide and 1-hydroxybenzotriazole are sequentially added, and the pH of the solution is adjusted to 4.8, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is added and stirred for 10-20 hours; the reaction solution is transferred to a dialysis bag, first dialyzed with a dilute hydrochloric acid aqueous solution containing sodium chloride, then dialyzed with deionized water, and finally the solution is freeze-dried to obtain the amino-modified hyaluronic acid; The poly(lactic-co-glycolic acid)-anti-inflammatory drug nanoparticles are poly(lactic-co-glycolic acid)-metformin nanoparticles, and the preparation method is as follows: Step one, metformin is dissolved in deionized water as an internal aqueous phase; The poly(lactic-co-glycolic acid) is dissolved in dichloromethane as an oil phase; Step two, the internal aqueous phase and the oil phase are mixed and ultrasonically emulsified to form a W / O primary emulsion; Step three, the W / O primary emulsion is transferred to an external aqueous phase and ultrasonically emulsified to form a W / O / W multiple emulsion; The external aqueous phase is a 1% polyvinyl alcohol solution by mass fraction; Step four, the W / O / W multiple emulsion is added dropwise into deionized water, stirred, aged, and after the organic solvent is completely volatilized, the obtained nanoparticle solution is washed by centrifugation with ultrapure water to obtain poly(lactic-co-glycolic acid) nanoparticles loaded with metformin, i.e. poly(lactic-co-glycolic acid)-metformin nanoparticles.

4. A method for preparing the hydrogel precursor solution for controlled release of antibacterial, anti-inflammatory drugs according to any one of claims 1 to 3, characterized in that, The following steps are included: 1) under light-proof conditions, the double-branched photocrosslinking agent and the double-branched photocrosslinking agent-antibacterial drug co-carrier are respectively dissolved in dimethyl sulfoxide to obtain a photocrosslinking agent solution and a photocrosslinking agent drug solution; The amino-modified polymer derivative is dissolved in deionized water and stirred uniformly at room temperature to obtain a polymer derivative solution; 2) Under the condition of avoiding light, the photo-crosslinking agent solution, the photo-crosslinking agent drug solution and the high polymer derivative solution are mixed uniformly according to the proportion, and then the polylactic acid-polyglycolic acid copolymer-metformin nanoparticles are dispersed in the mixed solution to obtain an antibacterial and anti-inflammatory drug controlled-release hydrogel precursor solution.

5. The use of the antibacterial and anti-inflammatory drug controlled-release hydrogel precursor solution according to any one of claims 1-3 in the preparation of a chronic refractory wound dressing.

6. A chronic difficult to heal wound dressing characterized by: The effective component is the antibacterial and anti-inflammatory drug controlled-release hydrogel precursor solution according to any one of claims 1-3.

7. A method of using a chronic wound dressing as claimed in claim 6, characterised in that, The operation is as follows: After the dressing is applied, the dressing is irradiated with ultraviolet light to crosslink into glue.

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