Hydrogel capable of controllably releasing antibacterial and anti-inflammatory drugs as well as preparation method and application of hydrogel

Abstract

The invention provides hydrogel capable of controllably releasing antibacterial and anti-inflammatory drugs as well as a preparation method and application of the hydrogel, solves the problems that photoinduced imine cross-linked hydrogel is complex in preparation process and lacks antibacterial and anti-inflammatory functions, and provides an effective way for treating chronic refractory wounds. The photo-crosslinking hydrogel designed by the invention takes an amino modified polymer derivative as a crosslinking matrix and a double-branched small molecule compound as a crosslinking agent, and the crosslinking system is simple to prepare and can quickly form gel under the irradiation of 395nm ultraviolet light. By further introducing a double-branched photo-crosslinking agent-antibacterial drug co-carrier and anti-inflammatory drug sustained-release particles into the cross-linking system, the photo-crosslinking hydrogel disclosed by the invention shows two drug release mechanisms of sudden release of the antibacterial drug and sustained release of the anti-inflammatory drug; therefore, the problems of early-stage bacterial infection and middle-stage chronic inflammation in the chronic refractory wound treatment process are effectively solved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and specifically relates to a hydrogel capable of controlling the release of antibacterial and anti-inflammatory drugs, and a preparation method and application thereof. Background Art

[0002] Diabetes is a common disease worldwide and has become the fourth leading cause of death. Diabetic foot is one of the common and serious complications of diabetes, mainly manifested as foot ulcers 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 suffer from distal nerve abnormalities and peripheral vascular lesions in the lower limbs, making the foot skin susceptible to infection. At the same time, the presence of chronic inflammation hinders skin healing, causing skin trauma to lose its normal repair ability, and severe cases even face the risk of amputation.

[0003] Hydrogel dressing is a new type of wound dressing developed in recent years. Compared with traditional dressings, it has the characteristics of high water content, good biocompatibility, and adjustable chemical and mechanical properties. It 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 conducive to cell adhesion and proliferation, providing an ideal environment for tissue treatment and repair. In order to achieve rapid adhesion to irregular wounds, photoimide cross-linked hydrogels have received widespread attention. This type of hydrogel is usually based on hyaluronic acid grafted with o-nitrobenzyl alcohol (ONB) photoresponsive groups, supplemented by amino-modified polymer derivatives (chitosan, gelatin, hyaluronic acid, etc.). After ultraviolet light irradiation, the aldehyde group generated by ONB forms an imine bond with the surrounding amino-modified materials and the amino groups on the tissue surface, and the hydrogel is constructed in situ while achieving high adhesion between the hydrogel and the tissue.

[0004] However, photoimide cross-linked hydrogels have certain disadvantages, which limit their application in the treatment of diabetic foot, diabetic wounds, and even other chronic and refractory wounds. First, the preparation process of the hydrogel is relatively complicated, including the synthesis of ONB, covalent grafting of hyaluronic acid to ONB, and chemical modification of auxiliary cross-linking materials; second, the current photoimide cross-linked hydrogels have relatively single functions and can only provide the lowest level of wound healing environment. No relevant drugs are added to deal with the complex conditions of chronic and refractory wounds, such as bacterial infection and chronic inflammation.

[0005] In summary, the present invention believes that it is necessary to develop a hydrogel with both antibacterial and anti-inflammatory effects. Summary of the invention

[0006] The purpose of the present invention is to solve the shortcomings of the current photoimide cross-linked hydrogel, which has a complex preparation process and lacks antibacterial and anti-inflammatory functions, and to provide a hydrogel with controllable release of antibacterial and anti-inflammatory drugs and a preparation method and application thereof; the scheme has strong operability and simple process, and can prepare photoimide cross-linked hydrogel with both antibacterial and anti-inflammatory effects.

[0007] The concept of the present invention:

[0008] In view of the problem of the complicated preparation process of the existing photo-induced imine cross-linked hydrogel, the present invention intends to simplify the early two-component cross-linked matrix into a single-component cross-linked matrix, simplifying the cross-linking system while still ensuring the cross-linking density between molecules and within molecules. Therefore, the present invention adopts the same cross-linking mechanism and introduces a bi-branched small molecule photo-crosslinker with ONB as a structural unit. After the photo-crosslinker is irradiated with ultraviolet light, the aldehyde groups generated at both ends of the small molecule photo-crosslinker can form dynamic imine bonds with amino-modified polymer derivatives and amino groups on the tissue surface, thereby constructing a hydrogel with stable structure and good tissue adhesion. At the same time, the bi-branched small molecule photo-crosslinker is further modified to obtain a bi-branched small molecule photo-crosslinker capped with a secondary amino antibiotic (such as ciprofloxacin). The bi-branched photo-crosslinker-antibacterial drug co-carrier can be rapidly cleaved and release the antibiotic under ultraviolet light, such as ciprofloxacin, which has a broad-spectrum antibacterial activity and can achieve a rapid sterilization effect. Finally, polylactic acid-polyglycolic acid copolymer with good biocompatibility and low toxicity is used to encapsulate 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. In the cross-linked network formed by hyaluronic acid and photocrosslinker, the nanoparticles can achieve sustained release of anti-inflammatory drugs. The latter has good anti-inflammatory effects and can promote the regeneration and repair of wound epidermis.

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

[0010] A double-branched small molecule compound, which is special in that it 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 is:

[0011]

[0012] The preparation method of the above-mentioned double-branched small molecule compound is special in that it comprises the following steps:

[0013] S1. Dissolve 4-hydroxy-5-methoxy-2-nitrobenzaldehyde in N,N-dimethylformamide, and add ((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) and potassium carbonate in sequence, then heat to 60-80°C for reaction, and detect by thin layer chromatography until the reaction is completed or water is added to quench the reaction; extract with ethyl acetate for multiple times, combine the organic layers, dry, filter, and concentrate under reduced pressure to obtain a yellow crude product; further purify the crude product on a neutral alumina column to elute 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. 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 the process, then add a tetrahydrofuran solution dissolved with sodium borohydride, and stir the reaction at room temperature. Detect by thin layer chromatography until the reaction is completed, neutralize the reaction solution with hydrochloric acid, extract it with ethyl acetate for multiple times, combine the organic layers, dry, filter, and concentrate 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 photocrosslinker.

[0015] Wherein, the process for synthesizing 4-hydroxy-5-methoxy-2-nitrobenzaldehyde can be:

[0016] ①, dissolve vanillin in N,N-dimethylformamide, then add benzyl bromide (raw material) and potassium carbonate (catalyst), react at room temperature, and add water to quench the reaction after the reaction is completed by thin layer chromatography; extract the reaction product with ether, wash and dry the organic phase, filter and concentrate 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 2h;

[0017] ②, dissolving the 4-(benzyloxy)-3-methoxybenzaldehyde obtained in ① in dichloromethane, and cooling the solution to 0°C; slowly dropping cooled nitric acid, stirring for 30 minutes, continuing to drop cooled nitric acid, and then heating to room temperature for reaction; 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 to nitric acid is 1:50, and the reaction time is 12h;

[0018] ③. At 0°C, slowly add trifluoroacetic acid to the 4-(benzyloxy)-5-methoxy-2-nitrobenzaldehyde obtained in ②; then raise the temperature to 60°C and stir for 8-12 hours. After the reaction is completed by thin layer chromatography, the reaction solution is concentrated under reduced pressure to remove trifluoroacetic acid, and the residue is purified by flash column chromatography to obtain 4-hydroxy-5-methoxy-2-nitrobenzaldehyde; wherein the equivalent ratio of 4-hydroxy-5-methoxy-2-nitrobenzaldehyde to trifluoroacetic acid is 1:20, and the reaction time is 12 hours.

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

[0020] Add p-toluenesulfonyl chloride to a dichloromethane solution containing tetraethylene glycol, and cool the solution to 0°C; slowly add potassium hydroxide to keep the temperature below 5°C, and react at 0°C. After the reaction is completed by thin layer chromatography, add ice water to quench the reaction; extract the reaction product with dichloromethane, wash and dry the organic phase, filter and concentrate to obtain ((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate); wherein the equivalent ratio of tetraethylene glycol, p-toluenesulfonyl chloride and potassium hydroxide is 1:2:8; and the reaction time is 3h.

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

[0022] Furthermore, 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 hours;

[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) and sodium borohydride is 1:2, and the reaction time is 0.5 h.

[0024] At the same time, the present invention provides the use of the above-mentioned double-branched small molecule compound as a photocrosslinking agent in the preparation of a hydrogel precursor solution.

[0025] Based on the above application, the present invention provides a hydrogel precursor solution for controllable release of antibacterial and anti-inflammatory drugs, which is special in that its main components are: a double-branched photocrosslinker, an amino-modified polymer derivative, a double-branched photocrosslinker-antibacterial drug co-carrier and anti-inflammatory drug sustained-release particles;

[0026] Among them, the double-branched photocrosslinker adopts the above-mentioned double-branched small molecule compound, which can induce intramolecular and intermolecular crosslinking of the polymer derivative by forming imine bonds under 395nm light wave irradiation, and at the same time construct a hydrogel-tissue interface;

[0027] Amino-modified polymer derivatives are used as cross-linking matrices;

[0028] The double-branched photocrosslinker-antimicrobial drug co-carrier is prepared by a coupling reaction between the above-mentioned double-branched small molecule compound and an antimicrobial drug containing a secondary amino group;

[0029] The amount of the amino-modified polymer derivative is not more than 0.01 times the mass of the double-branched photocrosslinker;

[0030] The amount of the double-branched photocrosslinker-antimicrobial drug co-carrier is not more than 0.01 times the mass of the double-branched photocrosslinker;

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

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

[0033]

[0034] The double-branched cross-linking agent-antibacterial drug co-carrier is a double-branched cross-linking 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(piperazine-4,1-diyl))bis(1-cyclopropyl-7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid), and the molecular structure is:

[0035]

[0036] ; Under 395nm light wave irradiation, it can quickly break down and release antibacterial drugs, and the entire release process is completed within 10 minutes;

[0037] The anti-inflammatory drug sustained-release particles are polylactic acid-polyglycolic acid copolymer-anti-inflammatory drug nanoparticles, that is, polylactic acid-polyglycolic acid copolymer nanoparticles loaded with metformin, which can slowly release the loaded metformin in a hydrogel environment for up to 72 hours; wherein the anti-inflammatory drug is itaconic acid, metformin, caffeic acid or tannic acid;

[0038] Thus, the hydrogel designed by the present invention can realize the orderly release of antibacterial drugs and anti-inflammatory drugs, and can play a synergistic role in the treatment of chronic refractory wounds (such as diabetic wounds), with early antibacterial and mid-term anti-inflammatory effects.

[0039] Further, the preparation method of the double-branched photocrosslinker-ciprofloxacin co-carrier is as follows:

[0040] 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)

[0041] The double-branched photocrosslinker is dissolved in dry tetrahydrofuran, and the solution is added dropwise to a tetrahydrofuran solution in which p-nitrophenyl chloroformate and pyridine are dissolved, and the reaction is carried out in the dark at room temperature under the protection of an inert gas, and after the reaction is completed by thin layer chromatography, the reaction solution is neutralized with hydrochloric acid and extracted with dichloromethane, and 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);

[0042] Ⅱ. Synthesis of double-branched photocrosslinker-ciprofloxacin co-carrier

[0043] Under the protection of inert gas, the 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) obtained in 1) is dissolved in dry N,N-dimethylformamide, and ciprofloxacin and triethylamine are added in sequence. The reaction is carried out at room temperature in the dark and detected by thin layer chromatography. After the reaction is completed, the reaction solution is neutralized with hydrochloric acid and extracted with ethyl acetate for multiple times. The organic layers are combined and dried. The reaction mixture was filtered and concentrated under reduced pressure to obtain a pale yellow crude product; the crude product was recrystallized from 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(piperazine-4,1-diyl))bis(1-cyclopropyl-7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid), i.e., a double-branched photocrosslinker-ciprofloxacin co-carrier;

[0044] The preparation method of amino-modified hyaluronic acid is as follows:

[0045] Dissolve hyaluronic acid in deionized water at room temperature, add carbohydrazide and 1-hydroxybenzotriazole in sequence, 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 10-20 hours; 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 amino-modified hyaluronic acid;

[0046] The polylactic acid-polyglycolic acid copolymer-anti-inflammatory drug nanoparticles are polylactic acid-polyglycolic acid copolymer-metformin nanoparticles, and the preparation method thereof is as follows:

[0047] Step 1, dissolving metformin in deionized water as the inner water phase;

[0048] Poly(lactic acid)-poly(glycolic acid) copolymer was dissolved in dichloromethane as the oil phase;

[0049] Step 2: Mix the inner water phase and the oil phase, and ultrasonically emulsify to form W / O colostrum;

[0050] Step 3, transferring the W / O colostrum to the external water phase, and ultrasonically emulsifying to form a W / O / W emulsion;

[0051] The external aqueous phase is a polyvinyl alcohol solution with a mass fraction of 1%;

[0052] Step 4: Add the W / O / W emulsion dropwise into deionized water, stir, and age. After the organic solvent is completely evaporated, the resulting nanoparticle solution is centrifuged and washed with ultrapure water to obtain polylactic acid-polyglycolic acid copolymer nanoparticles loaded with metformin, i.e., polylactic acid-polyglycolic acid copolymer-metformin nanoparticles.

[0053] Further, in the process of preparing the double-branched photocrosslinker-ciprofloxacin co-carrier:

[0054] 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;

[0055] 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;

[0056] In the process of preparing amino-modified hyaluronic acid:

[0057] 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 hours, and the dialysis time is 24-48 hours;

[0058] In the process of preparing polylactic acid-polyglycolic acid copolymer-metformin nanoparticles:

[0059] The mass ratio of polylactic acid-polyglycolic acid copolymer to metformin is 1:3.3;

[0060] The ultrasonic emulsification power is 500-600W, the ultrasonic emulsification time is 30s, the aging time is 12h, the centrifugal speed is 15000rpm, and the centrifugal time is 15-20min.

[0061] The method for preparing the hydrogel precursor solution for controlled release of antibacterial and anti-inflammatory drugs is special in that it comprises the following steps:

[0062] 1) dissolving a double-branched photocrosslinker and a double-branched photocrosslinker-antimicrobial drug co-carrier in dimethyl sulfoxide, respectively, under light-proof conditions to obtain a photocrosslinker solution and a photocrosslinker drug solution;

[0063] Dissolving the amino-modified polymer derivative in deionized water, and stirring evenly at room temperature to obtain a polymer derivative solution;

[0064] 2) Under light-proof conditions, the photocrosslinker solution, the photocrosslinker drug solution and the polymer derivative solution are uniformly mixed in proportion, and then the polylactic acid-polyglycolic acid copolymer-metformin nanoparticles are fully dispersed in the mixed solution to obtain a hydrogel precursor solution for controllable release of antibacterial and anti-inflammatory drugs.

[0065] The application of the above-mentioned hydrogel with controllable release of antibacterial and anti-inflammatory drugs in the preparation of chronic refractory wound dressings. Chronic refractory wounds such as diabetic foot, diabetic wounds, venous ulcers of the lower limbs, bedsores, etc., face roughly the same difficulties and treatment ideas in the healing process. Starting from the local wound, it is mainly to deal with the early bacterial infection of the wound and the mid-term inflammation, while starting from the overall situation, it is to actively find and eliminate or control the adverse factors that affect wound healing. Therefore, the hydrogel with controllable release of antibacterial and anti-inflammatory drugs developed based on the present invention can fully cope with most of the current chronic refractory wounds.

[0066] Based on the above application, the present invention also provides a chronic refractory wound dressing, which is special in that its active ingredient is a hydrogel precursor solution of the above antibacterial and anti-inflammatory drugs that can be released in a controlled manner.

[0067] The method for using the above-mentioned chronic refractory wound dressing is special in that the operation is as follows:

[0068] After applying the dressing, ultraviolet light is used to irradiate the dressing to initiate a cross-linking reaction between the double-branched photocrosslinker and the amino-modified polymer derivative, causing them to cross-link into a gel. This use process is also the process of converting the hydrogel precursor solution into a hydrogel. It only requires the active ingredients to be mixed in the dark and cross-linked into a gel under ultraviolet light without the need for covalent grafting.

[0069] Furthermore, the wavelength of the ultraviolet light is 395nm and the power of the ultraviolet light is 30mW / cm 2 , the illumination time is 2-5min.

[0070] Mechanism of the present invention:

[0071] In the present invention, after the bi-branched photocrosslinker, the bi-branched photocrosslinker-antibacterial drug co-carrier, the anti-inflammatory drug sustained-release particles (such as polylactic acid-polyglycolic acid copolymer-anti-inflammatory drug nanoparticles) are fully mixed with the amino-modified polymer derivative solution, the bi-branched photocrosslinker forms a chemical bond with the polymer derivative (such as hyaluronic acid) and the tissue surface through an aldehyde-amine condensation reaction under ultraviolet light irradiation; the bi-branched photocrosslinker-antibacterial drug co-carrier is cleaved simultaneously, and the rapid release of the antibacterial drug is ensured while the gel is formed; after the stable hydrogel is formed, the anti-inflammatory drug sustained-release particles (such as polylactic acid-polyglycolic acid copolymer-anti-inflammatory drug nanoparticles) encapsulated therein can be degraded due to the degradation effect The anti-inflammatory drugs are released continuously and slowly to achieve long-term anti-inflammatory effects, and ultimately provide a key basis for the treatment of chronic refractory wounds (such as diabetic wounds). That is, the present invention designs a photo-crosslinked hydrogel with both antibacterial and anti-inflammatory effects, and induces rapid gelation of polymer derivatives (hyaluronic acid, chitosan, gelatin, etc.) through a double-branched small molecule photo-crosslinking agent. At the same time, by introducing two fast-slow drug release mechanisms, the problems of early bacterial infection and mid-term chronic inflammation in the treatment of chronic refractory wounds are effectively solved; compared with the current photoinduced imine cross-linked hydrogel, its preparation process is simpler (no covalent grafting is required, only ultraviolet light exposure is required), and its functions are richer.

[0072] The advantages of the present invention are:

[0073] 1. The bibranched small molecule compound synthesized by the present invention and capable of being used as a photocrosslinking agent has readily available raw materials, low cost, and simple synthesis steps. In addition, due to the tetraethylene glycol connecting group, the bibranched photocrosslinking agent has good solubility and can be better miscible with the amino-modified polymer derivative, thereby ensuring the crosslinking density of the crosslinking system.

[0074] 2. The double-branched photocrosslinker of the present invention can simultaneously realize crosslinking and rapid release of antibacterial drugs (such as ciprofloxacin) under light stimulation, which solves the problem of single function of current photoimide crosslinked hydrogels. It introduces antibacterial function into photoimide crosslinked hydrogels for the first time, which can effectively solve the problem of rapid bacterial proliferation on the wound surface in the early stage of diabetic foot.

[0075] 3. The present invention utilizes polylactic acid-polyglycolic acid copolymer nanoparticles to coat and slowly 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, and have good application prospects in the treatment of diabetic foot.

[0076] 4. The hydrogel preparation process of the present invention simplifies the preparation of hyaluronic acid, increases the function of controlled drug release, and solves the problem that the current light-controlled hydrogel only focuses on wound closure but not on wound antibacterial and wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 The schematic diagram of the preparation process of the photocrosslinked hydrogel of the present invention and the related synthesis route, wherein A is a schematic diagram of the preparation process of the hydrogel; B is a synthesis route of a double-branched photocrosslinker and a double-branched photocrosslinker-ciprofloxacin co-carrier;

[0078] Figure 2 The double-branched photocrosslinking agent of the present invention is 1 H NMR;

[0079] Figure 3 The double-branched photocrosslinking agent of the present invention is 13 C NMR;

[0080] Figure 4 The double-branched photocrosslinker-ciprofloxacin co-carrier of the present invention 1 H NMR;

[0081] Figure 5 The double-branched photocrosslinker-ciprofloxacin co-carrier of the present invention 13 C NMR;

[0082] Figure 6 The UV-visible absorption spectra of the double-branched photocrosslinker of the present invention under different light irradiation times;

[0083] Figure 7 The double-branched photocrosslinker-ciprofloxacin co-carrier of the present invention releases ciprofloxacin under different light irradiation times;

[0084] Figure 8 The cumulative release curve of ciprofloxacin of the double-branched photocrosslinker-ciprofloxacin co-carrier of the present invention under light stimulation;

[0085] Fig. 9 is a transmission electron microscope image of the polylactic acid-polyglycolic acid copolymer nanoparticles of the present invention;

[0086] Fig.10 is the drug release curve of the polylactic acid-polyglycolic acid copolymer nanoparticles of the present invention in an acidic environment;

[0087] Fig.11 The appearance pictures of the photo-crosslinked hydrogel of the present invention before and after illumination;

[0088] Fig.12 is the X-ray photoelectron spectrum of the photo-crosslinked hydrogel of the present invention before and after illumination;

[0089] Fig.13 is a scanning electron micrograph of the photo-crosslinked hydrogel of the present invention (lyophilized state);

[0090] Fig.14 The rheological study results of the photo-crosslinked hydrogel of the present invention;

[0091] Fig.15 The in vitro swelling test results of the photo-crosslinked hydrogel of the present invention are shown;

[0092] Fig.16 The in vitro degradation test results of the photo-crosslinked hydrogel of the present invention are shown;

[0093] Fig.17 The cytotoxicity test results of the photo-crosslinked hydrogel of the present invention on HUVEC cells;

[0094] Fig.18 The wound model of type 2 diabetic mice was established to show the wound healing conditions of the treatment group and the control group at 0d, 3d, 7d, and 14d. The pictures are photos of representative mouse wounds in each group.

[0095] Fig.19 The figure is a statistical chart of wound healing rates from the day of modeling to 14 days after modeling for the treatment group and the control group; it can be seen from the figure that during the wound healing process, the photo-crosslinked hydrogel group of the present invention heals faster than the control group.

[0096] Fig. 20 HE staining results of wound surface from the day of modeling to 7 days after modeling in the treatment group and the control group;

[0097] Fig.21 The MT staining results of the wound surface from the day of modeling to 7 days after modeling in the treatment group and the control group;

[0098] Fig. 22 These are the collagen scoring results of the wound surface in the treatment group and the control group from the day of modeling to 14 days after modeling. DETAILED DESCRIPTION

[0099] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments:

[0100] 1. Synthesis of bibranched small molecule compounds, i.e. bibranched photocrosslinkers

[0101] 1) Synthesis of ((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate)

[0102] Add p-toluenesulfonyl chloride (5.72 g, 30 mmol) to a dichloromethane (15 mL) solution of tetraethylene glycol (2.91 g, 15 mmol), and cool the solution to 0°C. Slowly add potassium hydroxide (6.72 g, 120 mmol) to keep the temperature below 5°C. Stir the reaction at 0°C for 3 h and perform TLC until the reaction is complete. Add ice water to quench the reaction, extract the reaction product with dichloromethane, wash and dry the organic phase, filter and concentrate to obtain ((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (6.95 g, 92.3%).

[0103] ((Oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate): 1 HNMR (500MHz, DMSO-d6), δ7.78(d,J=8.3Hz,4H),7.47(d,J=8.1Hz,4H),4.11(t,J=4.4Hz,4H),3.60-3.54(m,4H),3.42(d,J=1.0Hz,8H),2.41(s,6H). 13 C NMR(126MHz, 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.

[0104] 2) Synthesis of 4-(Benzyloxy)-3-methoxybenzaldehyde

[0105] Dissolve vanillin (5 g, 32.9 mmol) in 15 mL of N,N-dimethylformamide, add benzyl bromide (5.9 g, 34.5 mmol) and potassium carbonate (9.08 g, 65.7 mmol), react at room temperature for 2 h, and detect by thin layer chromatography until the reaction is complete; add water to quench the reaction, extract with ether, wash and dry the organic phase, filter and concentrate to obtain 4-(benzyloxy)-3-methoxybenzaldehyde (7.91 g, 99%).

[0106] 4-(Benzyloxy)-3-methoxybenzaldehyde: 1 H NMR (500MHz, CDCl3), δ9.82 (s, 1H), 7.44-7.29 (m, 7H), 6.98 (d, J = 8.2Hz, 1H), 5.23 (s, 2H), 3.93 (s, 3H). 13C NMR (126MHz, CDCl3), 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.

[0107] 3) Synthesis of 4-(Benzyloxy)-5-methoxy-2-nitrobenzaldehyde

[0108] Dissolve 4-(Benzyloxy)-3-methoxybenzaldehyde (1.21 g, 5 mmol) in 12 mL of dichloromethane, cool the solution to 0°C, and slowly add cooled nitric acid (70%, 6 mL) dropwise. After stirring for 30 minutes, add another portion of cooled nitric acid (70%, 6 mL), then warm to room temperature for 12 hours. After the reaction is completed, dilute the reaction solution with ethyl acetate, wash and dry the organic phase, filter, and concentrate to obtain a crude product. The crude product is recrystallized 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 (500MHz, CDCl3), δ10.44(s,1H),7.67(s,1H),7.47-7.34(m,6H),5.27(s,2H),4.02(s,3H). 13 C NMR (126MHz, CDCl3), δ (ppm): δ187.91, 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.

[0109] 4) Synthesis of 4-hydroxy-5-methoxy-2-nitrobenzaldehyde

[0110] At 0°C, trifluoroacetic acid (10 ml) was slowly added to 4-(benzyloxy)-5-methoxy-2-nitrobenzaldehyde (900 mg, 3.13 mmol); then the temperature was raised to 60°C and stirred for 12 h, and the reaction was detected by thin layer chromatography until it was complete; the reaction solution was concentrated under reduced pressure to remove trifluoroacetic acid, and the residue was purified by flash column chromatography (petroleum ether: ethyl acetate = 1:1, v / v) to obtain 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 C NMR (126MHz, DMSO-d6), δ (ppm): δ188.34,151.82,151.01,143.80,123.40,111.05,110.66,56.36.LC-MS(ESI):m / z:Calcd.for[M+Na] + :220.03.Found:220.72.

[0111] 5) Synthesis of 4,4'-((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitrobenzaldehyde)

[0112] Dissolve 4-hydroxy-5-methoxy-2-nitrobenzaldehyde (395 mg, 2 mmol) in 20 mL of N,N-dimethylformamide, add ((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) in sequence, then heat to 80 °C for 6 h, and detect with thin layer chromatography until the reaction is complete; add water to quench The reaction was carried out and extracted with ethyl acetate for several times. 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, v / v / v) to obtain 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): 1H NMR(500MHz,DMSO-d6),δ10.18(s,2H),7.71(s,2H),7.34(s,2H),4.32-4.28(m,4H),3.94(s ,6H),3.79(dd,J=5.1,3.7Hz,4H),3.59(dd,J=5.9,3.2Hz,4H),3.54(dd,J=5.8,3.1Hz,4H). 13 C NMR(126MHz,DMSO-d6),δ(ppm):δ188.49,152.63,151.17,143.47,124.71,110.05,108.41,69.88,68.98,68.56,56.40.LC-MS(ESI):m / z:Calcd.for[M+H] + :553.16.Found:553.31.

[0113] 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))dimethanol

[0114] 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, and keep it away from light. Then add a tetrahydrofuran solution (15 mL) containing sodium borohydride (42 mg, 1.1 mmol) and stir at room temperature for 30 min. 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 organic layers are combined, and dried, filtered, and concentrated under reduced pressure to obtain a white crude product; the crude product is purified 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: 1H NMR (500MHz, DMSO-d6), δ7.68(s,2H),7.37(s,2H),5.56(t,J=5.4Hz,2H),4.81(d,J=5.4Hz ,4H),4.19-4.14(m,4H),3.90(s,6H),3.78-3.73(m,4H),3.57(ddd,J=8.8,6.1,3.4Hz,8H). 13 C NMR(126MHz,DMSO-d6),δ(ppm):δ153.65,146.07,138.29,134.26,109.68,109.06,69.86,68.75,68.41,60.08,56.01.LC-MS(ESI):m / z:Calcd.for[M+H] + :557.19.Found:557.34.

[0115] 2. Synthesis of double-branched photocrosslinker-ciprofloxacin co-carrier

[0116] 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)

[0117] (((((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))dimethanol (250 mg, 0.45 mmol) was dissolved in 10 mL of dry tetrahydrofuran, and the solution was added dropwise to tetrahydrofuran (10 mL) containing p-nitrophenyl chloroformate (1.36 g, 6.75 mmol) and pyridine (0.5 mL, 6.75 mmol). 5mL) solution, react in the dark at room temperature under the protection of inert gas for 1h, and detect the reaction by thin layer chromatography until 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).

[0118] 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(piperazine-4,1-diyl))bis(1-cyclopropyl-7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid)

[0119] Under the protection of inert gas, 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) was dissolved in 10 mL of dry N,N-dimethylformamide, and ciprofloxacin (225 mg, 0.68 mmol) and triethylamine (1.2 mL, 8.5 mmol) were added in sequence. The mixture was reacted at room temperature in the dark for 1 h and detected by thin layer chromatography until the reaction was completed. The mixture was neutralized with hydrochloric acid. The reaction solution was extracted with ethyl acetate for several times, the organic layers were combined, dried, filtered, and concentrated under reduced pressure to obtain a pale yellow crude product; the crude product was recrystallized from 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(piperazine-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(piperazine-4,1-diyl))bis(1-cyclopropyl-7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid): 1 H NMR(500MHz, DMSO-d6), δ8.59(s,2H),7.87(d,J=13.3Hz,2H),7.71(d,J=2.9Hz,2H),7.53(d,J=7.4Hz,2H),7.16(s,2H),5.41(s,4H),4.1 9(t,J=4.5Hz,4H),3.91(s,6H),3.76(dd,J=9.7,5.1Hz,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 (126MHz, 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.

[0120] Structural characterization

[0121] use 1 H NMR and 13 C NMR was used to characterize the bibranched photocrosslinker and bibranched photocrosslinker-ciprofloxacin co-carrier obtained above. The results are as follows: Figure 2-5 As shown. NMR spectrum analysis confirmed that the structures of the double-branched photocrosslinker and the double-branched photocrosslinker-ciprofloxacin co-carrier are similar to Figure 1 The chemical structure shown is consistent and the purity is over 99%.

[0122] UV-vis absorption spectroscopy was used to analyze the photolysis reaction of the double-branched photocrosslinker under 395 nm ultraviolet light. Figure 6 As shown in the figure, with the increase of illumination time, the characteristic absorption peak signal at 345nm (attributed to nitro group) of the UV-visible absorption spectrum of the double-branched photocrosslinker gradually decayed, accompanied by the enhancement of the characteristic absorption peak signal at 310nm (attributed to aldehyde group), which proved the photolysis reaction of the double-branched photocrosslinker under UV light.

[0123] High performance liquid chromatography (HPLC) was used to analyze the cleavage of the double-branched photocrosslinker-ciprofloxacin co-carrier under 395 nm ultraviolet light. Figure 7-8 After 10 minutes of UV irradiation, the ciprofloxacin release rate of the double-branched photocrosslinker-ciprofloxacin co-carrier reached 83%.

[0124] 3. Synthesis of amino-modified hyaluronic acid

[0125] Hyaluronic acid (408 mg, 340 KDa) was dissolved in 50 mL of deionized water at room temperature, carbohydrazide (90 mg, 1 mmol) and 1-hydroxybenzotriazole (153 mg, 1 mmol) were added in sequence, and the pH of the solution was adjusted to 4.8 with a 1 M aqueous hydrochloric acid solution. 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, first dialyzed with a dilute aqueous hydrochloric acid solution containing sodium chloride (0.1 M) for 48 h, then dialyzed with deionized water for 24 h, and finally the solution was freeze-dried to obtain amino-modified hyaluronic acid.

[0126] 4. Preparation of poly(lactic acid-co-glycolic acid)-metformin nanoparticles

[0127] 1) Metformin was dissolved in deionized water (272 mg / mL) as the internal aqueous phase;

[0128] 2) dissolving poly(lactic acid-poly(glycolic acid) copolymer in dichloromethane (8.33 mg / mL) as the oil phase;

[0129] 3) 0.6 mL of the internal water phase was mixed with 6 mL of the oil phase and ultrasonically emulsified for 1 min to form W / O colostrum;

[0130] 4) Dissolve 1% polyvinyl alcohol as the external water phase;

[0131] 5) Transfer the W / O colostrum to 6 mL of the external aqueous phase and perform ultrasonic emulsification for 1 min to form a W / O / W emulsion;

[0132] 6) The W / O / W emulsion was added dropwise to 8 mL of deionized water, stirred, and aged for 12 h. After the organic solvent was completely evaporated, the resulting nanoparticle solution was centrifuged (15,000 rpm) with ultrapure water and washed to obtain metformin-loaded polylactic acid-polyglycolic acid copolymer nanoparticles.

[0133] Structural characterization

[0134] The microscopic morphology of the poly(lactic acid-poly(glycolic acid) copolymer)-metformin nanoparticles prepared above was observed using a transmission electron microscope (TEM). Fig. 9 The SEM image shows that the prepared polylactic acid-polyglycolic acid copolymer-metformin nanoparticles are spherical nanoparticles with uniform particle size distribution and an average particle size of 65 nm.

[0135] The drug release of the polylactic acid-polyglycolic acid copolymer-metformin nanoparticles prepared above in an acidic environment was detected by ultraviolet spectrophotometry. Fig.10In an acidic environment, 52% of metformin was released after 12 hours, and the release rate of metformin reached a maximum (about 73%) after 72 hours.

[0136] 5. Preparation of Photocrosslinked Hydrogel

[0137] 1) Under light-proof conditions, dissolving a double-branched photocrosslinker and a double-branched photocrosslinker-ciprofloxacin co-carrier in dimethyl sulfoxide, respectively, to obtain a photocrosslinker solution (10 mM) and a photocrosslinker drug solution (10 mM);

[0138] 2) dissolving the amino-modified hyaluronic acid in deionized water and stirring evenly at room temperature to obtain a hyaluronic acid solution (1-2 wt %);

[0139] 3) Under light-proof conditions, the photocrosslinker solution, the photocrosslinker drug solution and the hyaluronic acid solution are uniformly mixed in different proportions (the amount of the photocrosslinker-ciprofloxacin co-carrier is not higher than 0.01 times the mass of the double-branched photocrosslinker, and the amount of the amino-modified hyaluronic acid is not higher than 0.01 times the mass of the double-branched photocrosslinker), and then 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 photocrosslinker) are fully dispersed in the mixed solution to obtain a hydrogel precursor solution;

[0140] 4) Under ultraviolet light (30mW / cm 2 , 2-5min), initiating the cross-linking reaction between the photocrosslinker and the amino-modified hyaluronic acid to obtain a photocross-linked hydrogel.

[0141] Structural characterization

[0142] The photocrosslinked hydrogels prepared with different proportions of amino-modified hyaluronic acid, bibranched photocrosslinker, bibranched photocrosslinker-ciprofloxacin co-carrier and polylactic acid-polyglycolic acid copolymer-metformin nanoparticles have similar properties. The performance of the photocrosslinked hydrogel prepared with a mass ratio of 1:0.005:0.01:0.05 is described below as an example.

[0143] The precursor solution of the photo-crosslinked hydrogel and the pictures after gelation are as follows Fig.11 The photo-crosslinked hydrogel and its precursor solution of the present invention were analyzed using X-ray photoelectron spectroscopy (XPS), and the results are shown in Fig.12As shown in the figure, the N1s of the -NO2 bond in the photocrosslinked hydrogel sample completely disappears at a binding energy of ~404.5 eV, accompanied by the appearance of the N1s of the -C=N bond at a binding energy of ~399.8 eV, proving that the bibranched molecular compound of the present invention can be used as a photocrosslinker to successfully catalyze the formation of photocrosslinked hydrogels.

[0144] The microscopic morphology of the photocrosslinked hydrogel (freeze-dried state) was observed using a scanning electron microscope (SEM). Fig.13 SEM results show that the photo-crosslinked hydrogel in the freeze-dried state has an interconnected porous structure, which is conducive to the transmission and release of drugs in the hydrogel.

[0145] The gelation process of photocrosslinked hydrogel was monitored by photorheometer. Fig.14 The dynamic time scan results show that the storage modulus (G') exceeds the loss modulus (G") at 40s, confirming irreversible gelation; and as the illumination time increases, the G' and G" of the hydrogel increase, proving that the hydrogel of the present invention has good viscoelasticity.

[0146] The results of in vitro swelling experiments are as follows Fig.15 As shown, after being immersed in water for 72 h, the swelling rate of the photo-crosslinked hydrogel reached 222%, which is beneficial for the absorption of wound exudate.

[0147] The results of in vitro degradation experiments were as follows Fig.16 As shown, after 72 h of incubation with hyaluronidase, more than 70% of the photo-crosslinked hydrogel had been degraded, demonstrating the good degradability of the hydrogel.

[0148] VI. Biocompatibility of photocrosslinked hydrogels

[0149] L929 cells were obtained from the ATCC cell bank;

[0150] Fetal bovine serum (FBS) was purchased from Camus Biotech, brand: HyClone, catalog number: SH30071.03;

[0151] DMEM high-glucose medium was purchased from Camus Biotech, brand: HyClone;

[0152] 1) L929 cells were expanded in a culture flask using a DMEM high-glucose medium containing 10% FBS by volume, and cells in the logarithmic growth phase were selected for later use;

[0153] 2) Add the photo-crosslinked hydrogel prepared above into a 96-well plate at a volume of 100 μL per well, add DMEM high-glucose medium and let it stand for 12 hours until it is fully swollen and set aside;

[0154] 3) L929 cells in logarithmic growth phase were seeded on the photo-crosslinked hydrogel in 2) at a density of 5000 cells per well;

[0155] 4) L929 cells and hydrogels were co-cultured for 24 hours, and CCK-8 test was performed to evaluate the damage of photo-crosslinked hydrogels to cells. The group without hydrogel was used as the control group, and the cell viability was measured at 450nm on a microplate reader and calculated by the following formula: Cell viability = (ODn-ODc) / (ODs-ODc)*100%, where ODn is the UV absorbance of the sample (hydrogel group), ODc is the UV absorbance of the blank group, and ODs is the UV absorbance of the control group; the blank group was CCK-8 and serum-free DMEM high-glucose culture medium; the control group was L929 cells in the proliferation period without hydrogels.

[0156] like Fig.17 As shown, both the photocross-linked hydrogel group and the control group showed a good growth trend (P<0.05), demonstrating its excellent biocompatibility.

[0157] VII. Effect of photocrosslinked hydrogels in promoting wound healing in diabetic mice

[0158] 1) 6-week-old (20 g ± 5 g) SPF-grade BALB / c male mice were purchased and raised for 1 week. After 5 days of intraperitoneal injection of streptozotocin (STZ, 50 mg / kg), a diabetic mouse model was established. One week after the injection, blood glucose was tested, and the model was considered successful when blood glucose was ≥16.7 mmol / L;

[0159] 2) Take the successfully established diabetic mice and induce anesthesia with 2% isoflurane until the mice are completely still, the corneal reflex disappears, and there is no pain response when the tail is clamped. Then create a 10 mm diameter full-thickness circular skin wound on the back;

[0160] 3) The hydrogel precursor solution prepared above was dissolved in sterile water for injection, injected into the wound site, and gelled under light (30 mW / cm 2 ), the dosage was 200 μL per mouse. The wounds of mice were photographed at 0d, 3d, 7d and 14d after hydrogel treatment, and samples were collected at 7d and 14d. In order to determine the degree of healing and healing process of wound repair, the morphology of wound skin tissue was detected. The wound tissue of mice was taken, fixed with 0.4% paraformaldehyde, dehydrated, and then embedded in paraffin. The paraffin-embedded skin tissue was cut into 5 μm thick tissue sections, stained with hematoxylin-eosin (HE) and Masson's trichrome (MT), and the stained sections were observed with a biological microscope.

[0161] like Fig.18As shown in the figure, with the extension of healing time, the wound area of ​​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. Fig.19 As shown, after 3 days of treatment, the photo-crosslinked hydrogel showed a faster wound healing speed 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 photo-crosslinked hydrogel group was reduced to 23.3±9.26%, which was in sharp contrast to the control group, indicating that photo-crosslinked hydrogel can significantly promote diabetic wound healing.

[0162] Through HE( Fig. 20 ) and MT staining ( Fig.21 Quantitative data analysis showed that the photocrosslinked hydrogel had a good wound repair effect at all time points of wound healing; at the same time, the wound collagen deposition effect of the photocrosslinked hydrogel group was more obvious than that of the control group. Fig. 22 As shown in the figure, after 14 days of treatment, the wound collagen score in the photocross-linked hydrogel group was the highest (4.33±0.82), while that in the control group was only 1.17±1.17%, indicating that the photocross-linked hydrogel can effectively promote collagen deposition.

[0163] In summary, the present invention successfully prepared a photocross-linked hydrogel with both antibacterial and anti-inflammatory effects, induced the rapid gelation of polymer derivatives through a double-branched small molecule photocross-linker, and introduced two fast and slow drug release mechanisms, thereby effectively solving the problems of bacterial infection and chronic inflammation in the treatment of diabetic wounds and other chronic and refractory wounds.

Claims

1. A double-branched small molecule compound, characterized in that: It 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 its molecular structure is: 。 2. The method for preparing the double-branched small molecule compound according to claim 1, characterized in that: The following steps are involved: S1. Dissolve 4-hydroxy-5-methoxy-2-nitrobenzaldehyde in N,N-dimethylformamide, and add ((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) and potassium carbonate in sequence, then heat to 60-80°C for reaction, and add water to quench the reaction after the reaction is completed; extract with ethyl acetate for multiple times, combine the organic layers, and dry, filter, and concentrate under reduced pressure to obtain a yellow crude product; further purify the crude product on a neutral alumina column 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 the process, then add a tetrahydrofuran solution dissolved with sodium borohydride, and stir the reaction at room temperature. After the reaction is completed, neutralize the reaction solution with hydrochloric acid and extract it with ethyl acetate for multiple times, combine the organic layers, dry, filter, and concentrate 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, that is, a dibranched light small molecule compound.

3. Use of the double-branched small molecule compound according to claim 1 as a photocrosslinking agent.

4. A hydrogel precursor solution for controlled release of antibacterial and anti-inflammatory drugs, characterized in that: Its main components are: double-branched photocrosslinking agent, amino-modified polymer derivative, double-branched photocrosslinking agent-antibacterial drug co-carrier and anti-inflammatory drug sustained-release particles; Wherein, the double-branched photocrosslinking agent adopts the double-branched small molecule compound described in claim 1; The double-branched photocrosslinker-antibacterial drug co-carrier is prepared by a coupling reaction between the double-branched small molecule compound described in claim 1 and an antibacterial drug containing a secondary amino group; The amount of the amino-modified polymer derivative is not more than 0.01 times the mass of the double-branched photocrosslinking agent; The amount of the double-branched photocrosslinker-antibacterial drug co-carrier is not more than 0.01 times the mass of the double-branched photocrosslinker; 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.

5. The hydrogel for controlled release of antibacterial and anti-inflammatory drugs according to claim 4, characterized in that: The amino-modified polymer derivative is amino-modified hyaluronic acid, which has a molecular weight of 20-40 kDa and a structural formula of: The double-branched cross-linking agent-antibacterial drug co-carrier is a double-branched cross-linking 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(piperazine-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.

6. The hydrogel precursor solution for controlled release of antibacterial and anti-inflammatory drugs according to claim 5, characterized in that: The preparation method of the double-branched photocrosslinker-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) The double-branched photocrosslinker is dissolved in dry tetrahydrofuran, and 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 and in the protection of an inert gas in the dark. After the reaction is completed, the reaction solution is neutralized with hydrochloric acid and extracted with dichloromethane, and 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); Ⅱ. Synthesis of double-branched photocrosslinker-ciprofloxacin co-carrier Under the protection of inert gas, the 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) obtained in 1) is dissolved in dry N,N-dimethylformamide, and ciprofloxacin and triethylamine are added in sequence. 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 multiple times. The organic layers are combined, dried, filtered, reduced, and purified by filtration. The reaction mixture was concentrated under reduced pressure to obtain a pale yellow crude product; the crude product was recrystallized from 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(piperazine-4,1-diyl))bis(1-cyclopropyl-7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid), i.e., a double-branched photocrosslinker-ciprofloxacin co-carrier; The preparation method of amino-modified hyaluronic acid is as follows: Dissolve hyaluronic acid in deionized water at room temperature, add carbohydrazide and 1-hydroxybenzotriazole in sequence, adjust the pH of the solution to 4.8, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and stir for 10-20 hours; 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 amino-modified hyaluronic acid; The polylactic acid-polyglycolic acid copolymer-anti-inflammatory drug nanoparticles are polylactic acid-polyglycolic acid copolymer-metformin nanoparticles, and the preparation method thereof is as follows: Step 1, dissolving metformin in deionized water as the inner water phase; Poly(lactic acid)-poly(glycolic acid) copolymer was dissolved in dichloromethane as the oil phase; Step 2: Mix the inner water phase and the oil phase, and ultrasonically emulsify to form W / O colostrum; Step 3, transferring the W / O colostrum to the external water phase, and ultrasonically emulsifying to form a W / O / W emulsion; The external aqueous phase is a polyvinyl alcohol solution with a mass fraction of 1%; Step 4: Add the W / O / W emulsion dropwise into deionized water, stir, and age. After the organic solvent is completely evaporated, the resulting nanoparticle solution is centrifuged and washed with ultrapure water to obtain polylactic acid-polyglycolic acid copolymer nanoparticles loaded with metformin, i.e., polylactic acid-polyglycolic acid copolymer-metformin nanoparticles.

7. The method for preparing the hydrogel precursor solution for controlled release of antibacterial and anti-inflammatory drugs according to any one of claims 4 to 6, characterized in that: The following steps are involved: 1) Under light-proof conditions, dissolving a double-branched photocrosslinker and a double-branched photocrosslinker-antimicrobial drug co-carrier in dimethyl sulfoxide to obtain a photocrosslinker solution and a photocrosslinker drug solution; Dissolving the amino-modified polymer derivative in deionized water, and stirring evenly at room temperature to obtain a polymer derivative solution; 2) Under light-proof conditions, the photocrosslinker solution, the photocrosslinker drug solution and the polymer derivative solution are uniformly mixed in proportion, and then the polylactic acid-polyglycolic acid copolymer-metformin nanoparticles are fully dispersed in the mixed solution to obtain a hydrogel precursor solution for controllable release of antibacterial and anti-inflammatory drugs.

8. Use of the hydrogel precursor solution with controllable release of antibacterial and anti-inflammatory drugs according to any one of claims 4 to 6 in the preparation of chronic and refractory wound dressings.

9. A chronic and refractory wound dressing, characterized in that: The active ingredient is the hydrogel precursor solution of any one of claims 4-6 for the controllable release of antibacterial and anti-inflammatory drugs.

10. The method for using the chronic non-healing wound dressing according to claim 9, characterized in that: The operation is as follows: After applying the dressing, it is irradiated with ultraviolet light to cross-link it into glue.

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