Dual-network hydrogel wound dressing with synergistic antibacterial property and glucose responsiveness and preparation method of dual-network hydrogel wound dressing

Abstract

The invention discloses a dual-network hydrogel wound dressing with synergistic antibiosis and glucose responsiveness and a preparation method of the dual-network hydrogel wound dressing. Fucoidin (FU) and broad-spectrum chemical sterilization polylysine (EPL) are self-assembled to form a first layer of glycopeptidyl network simulating an extracellular matrix, 4-formyl phenylboronic acid (FPBA) is added to be grafted with EPL through a Schiff base reaction, and the hydrogel is endowed with the glucose response characteristic; meanwhile, polyacrylamide (PAAM) is used as a second-layer network to enhance the toughness so as to improve the mechanical property, so that double-network hydrogel is formed, and puerarin (PUE)-loaded mesoporous polydopamine (MPDA) drug-loaded nano-particles with low hydrophilicity, which have the functions of promoting wound granulation tissue formation and collagenous fiber deposition, are wrapped in the double-network hydrogel; therefore, the obtained dual-network hydrogel can play roles in promoting wound healing and synergistically resisting bacteria in a high-glucose environment, and on-demand delivery and controllable release of drugs in the high-glucose environment can be realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of composite material preparation, and particularly relates to a double-network hydrogel wound dressing with high antibacterial activity, glucose responsiveness and wound healing promotion and a preparation method thereof. BACKGROUND

[0002] Due to the long-term high blood glucose state of diabetic patients, foot nerve and blood vessel lesions occur, resulting in foot ulceration and delayed healing symptoms. The clinical treatment of diabetic foot ulcers has become a difficult problem to be solved. Traditional methods for treating diabetic foot ulcers include controlling the patient's blood glucose level, debridement and other surgery (removing necrotic tissue to improve healing), negative pressure therapy (removing exudates in the wound, promoting healing and regeneration through vacuum), and applying traditional wound dressings, which can achieve certain positive effects, but are still not satisfactory. Hydrogels have better effects on treating diabetic foot ulcers than traditional dressings due to their good water retention and biocompatibility.

[0003] Traditional glycopeptide hydrogels rely on non-covalent bonds such as hydrogen bonds, hydrophobic interactions, etc. to form a cross-linked network to self-assemble into hydrogels. Although this hydrogel has certain bactericidal effect, it has problems such as poor antibacterial durability and weak antibacterial efficacy due to the influence of various factors in the in vivo or in vitro environment. The introduction of mesoporous polydopamine nanospheres can solve the problem of insufficient antibacterial force, and can load drugs to achieve the effect of targeted drug release for treatment. However, even if the two are simply combined, the hydrogel structure is still easy to be destroyed, and the mechanical properties are poor, which may lead to the fact that the hydrogel cannot effectively fit the wound site, affecting drug delivery and wound protection, and even may cause secondary damage to the wound when changing the dressing. And because it does not have responsiveness, the drug release cannot be precisely controlled, which may cause the local drug concentration of the patient to be too high to cause adverse reactions, or the concentration to be too low to achieve the treatment effect, prolonging the wound healing time and increasing the pain and medical costs of the patient.

[0004] To address these issues, the present invention has constructed a dual-network composite hydrogel with glucose responsiveness, combined chemical and photothermal sterilization, and healing-promoting properties. This hydrogel utilizes fucoidan and polylysine (EPL), a broad-spectrum chemical bactericidal agent, to self-assemble in a weakly alkaline environment through electrophilic reactions to form a first-layer glycopeptide-based network that mimics the extracellular matrix. This network compensates for the loss of extracellular matrix-like properties in difficult-to-heal wounds. This network is then combined with a polyacrylamide network to form a dual-network structure, significantly enhancing mechanical properties such as toughness. Furthermore, unlike traditional dual-network hydrogels, which rely on simple blending (e.g., physically stirring and mixing two network precursors), the present invention utilizes step-by-step infiltration and simultaneous cross-linking techniques to achieve uniform interpenetration of the two network precursor solutions at the molecular scale. Chemical bonding is then achieved through controlled polymerization to avoid phase separation and ensure close interfacial bonding between the two networks, significantly enhancing the mechanical stability and functional synergy of the dual network. Building on this foundation, they innovatively utilized the acyl groups of 4-formylphenylboronic acid to graft amino groups from polylysine via a Schiff base reaction. This dynamic cleavage and reformation of Schiff base bonds under certain conditions maintains the integrity of the hydrogel structure, effectively preventing structural damage. This not only increases the grafting efficiency but also enables stable and specific drug release in high-glucose environments. A template-based method was also used to prepare mesoporous dopamine, which was then mixed with puerarin in the dark to complete the drug loading process. 4-Formylphenylboronic acid forms a boronate ester bond with mesoporous dopamine (MPDA) nanoparticles encapsulated with puerarin (PUE), enabling responsive release of poorly soluble drugs and broadening the application range of responsive drugs. This allows the hydrogel to not only possess photothermal antibacterial properties but also release puerarin in high-glucose environments, which promotes wound granulation tissue formation and collagen fiber deposition, thereby promoting the healing of diabetic foot ulcers. Summary of the Invention

[0005] The purpose of the present invention is to provide a multifunctional composite hydrogel with glucose responsiveness, hypoglycemic properties and antibacterial properties. The biomimetic glycopeptide hydrogel is formed by self-assembly of polylysine grafted with 4-formylphenylboronic acid and fucoidan, which gives it bactericidal and glucose responsive properties; mesoporous polydopamine (MPDA) nanoparticles encapsulating puerarin (PUE) are introduced to give it hypoglycemic, synergistic bactericidal and healing-promoting properties; polyacrylamide (PAAM) is used as the second layer network to enhance its toughness and improve its mechanical properties. The resulting double-network hydrogel wound dressing with synergistic antibacterial and glucose responsiveness can be used for wound healing of diabetic foot ulcers and is a new type of medical material.

[0006] To achieve the above object, the present invention adopts the following technical solutions: A double-network hydrogel wound dressing with synergistic antibacterial and glucose-responsive properties is prepared by grafting polylysine (EPL) with 4-formylphenylboronic acid (FPBA). The 4-formylphenylboronic acid-grafted polylysine then self-assembles with fucoidan (FU) to form a first-layer glycopeptide-based network that mimics the extracellular matrix and has glucose-responsive properties. Meanwhile, mesoporous polydopamine (MPDA) drug-loaded nanoparticles loaded with puerarin (PUE) are encapsulated in polyacrylamide (PAAM) as the second-layer network, and tetramethylethylenediamine and potassium persulfate are used as cross-linkers to form a double-network hydrogel.

[0007] The preparation method of the double-network hydrogel with synergistic antibacterial and glucose responsiveness comprises the following steps: 1) Stirring the polylysine solution and the 4-formylphenylboronic acid solution, adding sodium cyanoborohydride, and continuing to stir and mix until uniform, dialyzing, and freeze-drying to obtain a 4-formylphenylboronic acid-grafted polylysine powder; 2) dissolving poloxamer in an ethanol solution by stirring, then adding 3,3',5,5'-tetramethylbenzidine, dopamine hydrochloride, and ammonia to the resulting mixture, continuing to stir and mix, and then centrifuging, washing, and freeze-drying to obtain nano-mesoporous polydopamine powder; 3) adding the nano-mesoporous polydopamine powder prepared in step 2) to the puerarin solution, stirring and mixing, and then freeze-drying to obtain puerarin-loaded mesoporous polydopamine drug-loaded nanoparticles; 4) Dissolving the puerarin-loaded mesoporous polydopamine drug-loaded nanoparticles obtained in step 3) and acrylamide in a PBS solution to obtain solution A. Dissolving the 4-formylphenylboronic acid-grafted polylysine powder and fucoidan obtained in step 1) in another PBS solution to obtain solution B. Then, an equal volume of solution A was added to solution B, and N,N-methylenebisacrylamide was added and stirred to mix thoroughly. 5) Add tetramethylethylenediamine and potassium persulfate to the mixed system obtained in step 4), and stir rapidly to obtain a double-network glucose-responsive hydrogel.

[0008] Furthermore, the mass ratio of polylysine, 4-formylphenylboronic acid and sodium cyanoborohydride used in step 1) is 1:1:(0.18-0.5).

[0009] Furthermore, the stirring speed in step 1) is 200-300 rpm, and the stirring time is 18-24 hours.

[0010] Furthermore, the dialysis in step 1) uses a dialysis bag with a molecular weight cutoff of 2 kDa, and the total dialysis time is 72 hours, during which the water is changed every 8 hours.

[0011] Furthermore, the volume concentration of the ethanol solution in step 2) is 50%.

[0012] Furthermore, the stirring time in step 2) is 5-10 min and the rotation speed is 600-650 rpm.

[0013] Furthermore, the mass fraction of poloxamer in the mixed solution obtained in step 2) is 1 wt%.

[0014] Furthermore, in step 2), the volume ratio of 3,3',5,5'-tetramethylbenzidine added to the mixed solution is 1:100-1:120, the amount of dopamine hydrochloride added is 12-20 mg / mL, and the volume ratio of ammonia water added to the mixed solution is 1:125.

[0015] Furthermore, in step 2), the stirring time is continued for 3 to 4 hours, and the rotation speed is 600 to 650 rpm.

[0016] Furthermore, the centrifugation speed in step 2) is 8000 rpm and the time is 10-20 min.

[0017] Furthermore, the washing in step 2) uses a mixed solution of ethanol and acetone with a volume ratio of 2:1 as a washing agent.

[0018] Furthermore, the puerarin solution in step 3) is an ethanol solution of puerarin, and its concentration is 125-250 mg / L.

[0019] Furthermore, the amount of puerarin solution added in step 3) is converted based on the amount of puerarin being 25-30 wt% of the mass of the nano-mesoporous polydopamine powder.

[0020] Furthermore, the stirring in step 3) is carried out in the dark, with a rotation speed of 300-350 rpm and a stirring time of 36-48 h.

[0021] Furthermore, in step 4), the pH of the PBS solution is 7.0-7.4.

[0022] Furthermore, in the mixed system obtained in step 4), the mass concentration of puerarin-loaded mesoporous polydopamine drug-loaded nanoparticles is 0-0.6 wt %, the mass concentration of acrylamide is 10 wt %, the mass concentration of 4-formylphenylboronic acid grafted polylysine is 0.5 wt %, the mass concentration of fucoidan is 0.25-1 wt %, and the concentration of N,N-methylenebisacrylamide is 0.75 mg / mL.

[0023] Furthermore, the stirring speed in step 4) is 300-350 rpm, and the stirring time is 40-60 min.

[0024] Furthermore, in step 5), the volume ratio of the added amount of tetramethylethylenediamine to the mixed system is 1:400 to 1:200.

[0025] Furthermore, in step 5), the amount of potassium persulfate added to the mixed system is 2.5 mg / mL.

[0026] Furthermore, the stirring speed in step 5) is 300-350 rpm.

[0027] The present invention is based on nano-mesoporous polydopamine (MPDA), puerarin (PUE), 4-formylphenylboronic acid (FPBA), fucoidan (FU) and polylysine (EPL), and through reasonable material design, a double-network multifunctional composite hydrogel with glucose responsiveness, hypoglycemic properties and synergistic antibacterial properties is prepared. Specifically, fucoidan and broad-spectrum chemically sterilized polylysine are self-assembled to form a glycopeptide-based hydrogel that mimics the extracellular matrix, which helps to make up for the loss of the extracellular matrix in difficult-to-heal wounds and reduce excessive inflammation, impaired angiogenesis and bacterial infection; the introduction of polyacrylamide (PAAM) constitutes the second layer network of the hydrogel, giving the hydrogel higher toughness, providing a more stable support environment, and prolonging the antibacterial effect time; in addition, the present invention covalently grafts 4-formylphenylboronic acid (FPBA) to polylysine (EPL) through a Schiff base reaction, and utilizes the dynamic cleavage and recombination characteristics of the imine bond to achieve glucose responsiveness, that is, when the glucose level is high, the boron atom in the 4-formylphenylboronic acid grafted on the polylysine easily reacts with the hydroxyl group in the glucose molecule, changing its own structure, thereby triggering the associated mesoporous dopamine drug release mechanism; in addition, the mesoporous Porous polydopamine nanoparticles have photothermal effect and antioxidant properties, and can reduce inflammatory response. Their unique mesoporous structure can also efficiently load weakly hydrophilic drugs to form a stable drug-carrying system. Under different physiological environments, the physicochemical properties of mesoporous polydopamine nanoparticles will change accordingly, thereby achieving controlled release of drugs and significantly improving the stability and effectiveness of drug release. Puerarin has the function of regulating blood sugar levels and can also promote the formation of granulation tissue and collagen fiber deposition on the wound surface, showing significant wound healing activity. Therefore, mesoporous polydopamine nanoparticles coated with puerarin can controllably adjust the release rate of puerarin while synergistically antibacterial, giving the product high bactericidal properties, and improving the wound healing speed and antioxidant properties. The double-network composite hydrogel prepared based on the above mechanism can therefore have multiple functions of glucose responsiveness, hypoglycemic properties and synergistic antibacterial properties.

[0028] Furthermore, the dual network structure of the present invention is not simply a physical mixture; rather, it achieves chemical bond-level synergy through step-by-step crosslinking and permeation control. First, in a weakly alkaline PBS solution, fucoidan (FU) and polylysine (EPL) self-assemble through electrophilic interactions to form a glycopeptide-based primary network that mimics the extracellular matrix. The system is stirred at a low speed (300-350 rpm) to avoid excessive shear that disrupts network formation. Subsequently, a polyacrylamide (PAAM) monomer solution is slowly mixed with the primary network solution in equal volume ratios and stirred at the same speed for 40-60 minutes to ensure sufficient interpenetration of the two phases without inducing premature crosslinking. Finally, tetramethylethylenediamine and potassium persulfate are added for free radical polymerization to form a secondary network in situ, allowing the two networks to interpenetrate and form a strong interfacial bond. This step-by-step permeation and simultaneous crosslinking process effectively avoids interfacial delamination caused by physical mixing, achieving mechanical synergy and functional complementarity between the dual networks. Compared with traditional glycopeptide hydrogels and polyacrylamide hydrogels, or even hydrogels prepared by simply mixing them, the present invention has many advantages and solves the problem of weak interfacial bonding existing in traditional methods or their simple combination; the glycopeptide-based network and the polyacrylamide network are only combined by physical adsorption, which is prone to stratification and failure due to mechanical stress or body fluid erosion. The resulting dual network structure can also ensure the good swelling performance of the hydrogel, enabling it to better maintain its morphology and function in wound environments with different humidity.

[0029] In summary, the present invention realizes the intelligent linkage of functional modules through the deep integration of dual network structure design, dynamic grafting of chemical bonds and mesoporous drug delivery system, and upgrades functional modules such as antibacterial, drug release, and mechanical properties from physical mixing to chemical bond-level synergy. In the face of the complex pathological environment of diabetic foot ulcers (such as the coexistence of hyperglycemia and bacterial infection), compared with other single or simple combination treatment methods, the present invention can effectively exert its responsiveness, hypoglycemic properties and synergistic antibacterial properties, thereby improving the treatment efficiency. And from a clinical point of view, because the present invention has good antibacterial properties and promotes wound healing, it can reduce the number of dressing changes for patients, shorten hospitalization time, reduce the risk of amputation, and thus improve the quality of life of patients.

[0030] The significant advantages of the present invention are: (1) The present invention introduces puerarin, which has weak hydrophilicity, into the hydrogel system to endow the hydrogel with the characteristics of promoting the formation of granulation tissue on the wound surface, cell proliferation and significant wound healing activity; and uses the drug-carrying structure to carry it to the wound, which greatly overcomes its shortcomings such as low hydrophilicity, poor bioavailability and low permeability.

[0031] (2) The mesoporous polydopamine is used as a drug loading material to load drugs and endow the hydrogel with photo-thermal antibacterial properties, thereby providing a new antibacterial strategy for the enhanced drug resistance of pathogenic bacteria caused by antibiotic abuse.

[0032] (3) The phenylboric acid is added to the hydrogel to make the hydrogel glucose-responsive, and the loaded drugs can be released according to the ulcer environment of a patient.

[0033] (4) The polylysine used in the application is an FDA-approved natural antibacterial peptide, and has good biocompatibility and broad-spectrum antibacterial properties.

[0034] (5) The fucoidan of marine origin is used as a main raw material, and has strong antioxidant, anticoagulant and antibacterial effects.

[0035] (6) The drug efficiency is maximized through the synergistic effect of various components.

[0036] (7) The application is a double-network hydrogel, and has good antibacterial properties, biocompatibility, moisturizing properties and adhesion, and also has excellent mechanical properties.

[0037] (8) The application has low synthesis cost, simple method, outstanding effect, and can be mass-produced. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 The process flow chart for preparing the double-network hydrogel of Example 1.

[0039] Figure 2 The infrared spectrum of the 4-formyl phenylboronic acid grafted polylysine prepared in Example 1.

[0040] Figure 3 The nuclear magnetic resonance hydrogen spectrum of the 4-formyl phenylboronic acid grafted polylysine prepared in Example 1.

[0041] Figure 4 The TEM comparison chart of the nano-mesoporous polydopamine and the nano-mesoporous polydopamine loaded with puerarin prepared in Example 1.

[0042] Figure 5 The infrared comparison chart of the nano-mesoporous polydopamine and the nano-mesoporous polydopamine loaded with puerarin prepared in Example 1. Figure 6 The drug release effect comparison chart of the double-network hydrogel prepared in Example 1 under different glucose concentrations.

[0043] Figure 7A comparison chart of optical photos of the double network hydrogels prepared for Example 1 and Comparative Example 2.

[0044] Figure 8 A comparison chart of results of the double network hydrogels prepared for Example 1, 2 and Comparative Example 1 in the Cell Counting Kit-8 cell activity experiment.

[0045] Figure 9 A comparison chart of results of the double network hydrogels prepared for Example 1, 2 in the anti-S. aureus test under light and dark conditions.

[0046] Figure 10 A comparison chart of results of the tensile property test of the double network hydrogels prepared for Example 1, 2 and Comparative Example 1, 2. DETAILED DESCRIPTION

[0047] As Figure 1 A double network hydrogel with synergistic antibacterial and glucose responsiveness, the preparation of which comprises the following steps: 1) mixing the polylysine solution and the 4-formylphenylboronic acid solution under stirring at a stirring speed of 200-300 rpm, adding sodium cyanoborohydride, continuously stirring and mixing for 18-24 h, then dialyzing in a dialysis bag with a molecular weight cut-off of 2 kDa for 72 h (with water replacement every 8 h during the dialysis), and then freeze-drying to obtain 4-formylphenylboronic acid grafted polylysine powder; 2) dissolving the poloxamer in a 50 vol% ethanol solution under stirring at a speed of 600-650 rpm for 5-10 min to obtain a mixture with a concentration of 1 wt%, then adding 3,3',5,5'-tetramethylbenzidine, dopamine hydrochloride and ammonia water into the obtained mixture, continuously stirring and mixing for 3-4 h, then centrifuging at 8000 rpm for 10-20 min, washing with a mixed solution of ethanol and acetone in a volume ratio of 2:1, and freeze-drying to obtain nano-mesoporous polydopamine powder; 3) adding the nano-mesoporous polydopamine powder prepared in step 2) into a 125-250 mg / L puerarin ethanol solution, stirring in the dark at a speed of 300-350 rpm for 36-48 h, and then freeze-drying to obtain mesoporous polydopamine drug-loaded nanoparticles loaded with puerarin; 4) The puerarin-loaded mesoporous polydopamine drug-loaded nanoparticles obtained in step 3) and acrylamide were stirred and dissolved in a PBS solution (pH 7.0-7.4) at 300-350 rpm to obtain solution A. The 4-formylphenylboronic acid-grafted polylysine powder and fucoidan obtained in step 1) were stirred and dissolved in another PBS solution (pH 7.0-7.4) at 300-350 rpm to obtain solution B. Then, an equal volume of solution A was added to solution B, and N,N-methylenebisacrylamide was added, and the mixture was stirred and mixed at 300-350 rpm for 40-60 min to obtain a mixed system, wherein the mass concentration of the puerarin-loaded mesoporous polydopamine drug-loaded nanoparticles was 0-0.6 wt %, the mass concentration of acrylamide was 10 wt %, the mass concentration of the 4-formylphenylboronic acid-grafted polylysine was 0.5 wt %, and the mass concentration of the fucoidan was 0.25-1 wt%, and the concentration of N,N-methylenebisacrylamide was 0.75 mg / mL; 5) Tetramethylethylenediamine and potassium persulfate were added to the mixed system obtained in step 4), and the mixture was stirred at 300-350 rpm to obtain a double-network glucose-responsive hydrogel.

[0048] Wherein, the mass ratio of polylysine, 4-formylphenylboronic acid and sodium cyanoborohydride used in step 1) is 1:1:(0.18-0.5).

[0049] The volume ratio of the amount of 3,3',5,5'-tetramethylbenzidine added in step 2) to the mixed solution is 1:100-1:120, the amount of dopamine hydrochloride added is 12-20 mg / mL, and the volume ratio of the amount of ammonia water added to the mixed solution is 1:125.

[0050] The amount of puerarin solution added in step 3) is calculated based on the amount of puerarin used being 25-30 wt% of the mass of the nano-mesoporous polydopamine powder.

[0051] In step 5), the volume ratio of tetramethylethylenediamine added to the mixed system is 1:400 to 1:200, and the amount of potassium persulfate added to the mixed system is 2.5 mg / mL.

[0052] In order to make the contents of the present invention easier to understand, the technical solutions of the present invention are further described below in conjunction with specific implementation methods. However, the following examples are merely examples of the present invention and do not represent the scope of protection of the rights defined by the present invention. The scope of protection of the rights of the present invention shall be subject to the claims.

[0053] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0054] Example 1 (FU:EPL=2:1, MPDA=30 mg) (1) 1 g of ε-polylysine and 1 g of 4-formylphenylboronic acid were added to deionized water respectively and the two solutions were mixed evenly. Then 0.189 g of sodium cyanoborohydride was added and stirred for 18 h to completely dissolve it. The solution was then placed in a dialysis bag with a molecular weight cutoff of 2 kDa and dialyzed for 72 h (the water was changed every 8 h). The solution was then freeze-dried to obtain 4-formylphenylboronic acid grafted polylysine powder.

[0055] (2) Dissolve 0.5 g of poloxamer and 0.5 mL of 3,3',5,5'-tetramethylbenzidine in a mixed solution of 25 mL of ethanol and 25 mL of deionized water, stir at 600-650 rpm, add 0.6 g of dopamine hydrochloride and 0.4 mL of ammonia water, continue stirring for 3 h, then centrifuge the obtained solution in a centrifuge with a centrifugal parameter of 8000 rpm for 15 min, wash it with a mixed solution of ethanol and acetone with a volume ratio of 2:1, and freeze-dry it to obtain nano-mesoporous polydopamine powder. Then, take 0.3 g of the powder and add it to 300 mL of 250 mg / L puerarin solution, stir it at 300-350 rpm for 48 h in the dark, and then freeze-dry it to obtain mesoporous polydopamine drug-loaded nanoparticles loaded with puerarin.

[0056] (3) 30 mg of the puerarin-loaded mesoporous polydopamine drug-loaded nanoparticles obtained in step (2), 1 g of acrylamide, 50 mg of the 4-formylphenylboronic acid grafted polylysine powder obtained in step (1), and 100 mg of fucoidan were dissolved in two 10 mL portions of independently prepared PBS solutions (pH = 7.4). Then, the obtained drug-loaded nanoparticle-polyacrylamide mixed solution was added dropwise to the polylysine-fucoidan mixed solution at a rotation speed of 300-350 rpm and stirred for 60 minutes to ensure that the molecular chains of the two phases were fully entangled but not cross-linked. Subsequently, 0.015 g of the cross-linking agent N,N-methylenebisacrylamide was added to further stabilize the first network structure.

[0057] (4) While maintaining a rotation speed of 300-350 rpm, 0.05 mL of tetramethylethylenediamine and 0.05 g of potassium persulfate were added to initiate free radical polymerization of acrylamide, causing the second network to grow in situ within the already formed glycopeptide network, forming an interpenetrating double network structure, labeled FPPM3. During this process, speed control is crucial: too high a speed will cause the network to break, while too low a speed will lead to uneven mixing, affecting the synergistic properties of the double network.

[0058] Example 2 (FU:EPL=2:1, MPDA=60 mg) (1) 1 g of ε-polylysine and 1 g of 4-formylphenylboronic acid were added to deionized water respectively and the two solutions were mixed evenly. Then 0.189 g of sodium cyanoborohydride was added and stirred for 18 h to completely dissolve it. The solution was then placed in a dialysis bag with a molecular weight cutoff of 2 kDa and dialyzed for 72 h (the water was changed every 8 h). The solution was then freeze-dried to obtain 4-formylphenylboronic acid grafted polylysine powder.

[0059] (2) Dissolve 0.5 g of poloxamer and 0.5 mL of 3,3',5,5'-tetramethylbenzidine in a mixed solution of 25 mL of ethanol and 25 mL of deionized water, stir at 600-650 rpm, add 0.6 g of dopamine hydrochloride and 0.4 mL of ammonia water, continue stirring for 3 h, then centrifuge the obtained solution in a centrifuge with a centrifugal parameter of 8000 rpm for 15 min, wash it in a mixed solution of ethanol and acetone with a volume ratio of 2:1, and freeze-dry it to obtain nano-mesoporous polydopamine powder. Then, take 0.3 g of the powder and add it to 300 mL of 250 mg / L puerarin solution, stir it at 300-350 rpm for 48 h in the dark, and then freeze-dry it to obtain mesoporous polydopamine drug-loaded nanoparticles loaded with puerarin.

[0060] (3) 60 mg of the puerarin-loaded mesoporous polydopamine drug-loaded nanoparticles obtained in step (2), 1 g of acrylamide, 50 mg of the 4-formylphenylboronic acid grafted polylysine powder obtained in step (1), and 100 mg of fucoidan were dissolved in two 10 mL portions of independently prepared PBS solutions (pH = 7.4). Then, the obtained drug-loaded nanoparticle-polyacrylamide mixed solution was added dropwise to the polylysine-fucoidan mixed solution at a rotation speed of 300-350 rpm. The mixture was stirred for 60 minutes to ensure that the molecular chains of the two phases were fully entangled but not cross-linked. Then, 0.015 g of the cross-linking agent N,N-methylenebisacrylamide was added.

[0061] (4) While maintaining a rotation speed of 300-350 rpm, 0.05 mL of tetramethylethylenediamine and 0.05 g of potassium persulfate were added to initiate free radical polymerization of acrylamide, causing the second network to grow in situ within the formed glycopeptide network, forming an interpenetrating double network structure, which was labeled FPPM6.

[0062] Comparative Example 1 (FU:EPL=2:1, MPDA=0) (1) 1 g of ε-polylysine and 1 g of 4-formylphenylboronic acid were added to deionized water respectively and the two solutions were mixed evenly. Then, 0.189 g of sodium cyanoborohydride was added and stirred for 18 h to completely dissolve the solution. The solution was then placed in a dialysis bag with a molecular weight cutoff of 2 kDa and dialyzed for 72 h (the water was changed every 8 h). The solution was then freeze-dried to obtain 4-formylphenylboronic acid grafted polylysine powder.

[0063] (2) Dissolve 1 g of acrylamide, 50 mg of 4-formylphenylboronic acid grafted polylysine powder, and 100 mg of fucoidan in two 10 mL portions of independently prepared PBS solution (pH = 7.4). Then, add the obtained polyacrylamide solution dropwise to the polylysine-fucoidan mixed solution at a rotation speed of 300-350 rpm and continue stirring for 60 minutes to ensure that the molecular chains of the two phases are fully entangled but not cross-linked. Then, add 0.015 g of the cross-linking agent N,N-methylenebisacrylamide.

[0064] (3) While maintaining a rotation speed of 300-350 rpm, 0.05 mL of tetramethylethylenediamine and 0.05 g of potassium persulfate were added to initiate free radical polymerization of acrylamide, causing the second network to grow in situ within the formed glycopeptide network, forming an interpenetrating double network structure, labeled FPP.

[0065] Comparative Example 2 (FU:EPL=2:1, MPDA=30 mg, simple mixing) (1) 1 g of ε-polylysine and 1 g of 4-formylphenylboronic acid were added to deionized water respectively and the two solutions were mixed evenly. Then, 0.189 g of sodium cyanoborohydride was added and stirred for 18 h to completely dissolve the solution. The solution was then placed in a dialysis bag with a molecular weight cutoff of 2 kDa and dialyzed for 72 h (the water was changed every 8 h). The solution was then freeze-dried to obtain 4-formylphenylboronic acid grafted polylysine powder.

[0066] (2) Dissolve 0.5 g of poloxamer and 0.5 mL of 3,3',5,5'-tetramethylbenzidine in a mixed solution of 25 mL of ethanol and 25 mL of deionized water, stir at 600-650 rpm, add 0.6 g of dopamine hydrochloride and 0.4 mL of ammonia water, continue stirring for 3 h, then centrifuge the obtained solution in a centrifuge with a centrifugal parameter of 8000 rpm for 15 min, wash it in a mixed solution of ethanol and acetone with a volume ratio of 2:1, and freeze-dry it to obtain nano-mesoporous polydopamine powder. Then, take 0.3 g of the powder and add it to 300 mL of 250 mg / L puerarin solution, stir it at 300-350 rpm for 48 h in the dark, and then freeze-dry it to obtain mesoporous polydopamine drug-loaded nanoparticles loaded with puerarin.

[0067] (3) 30 mg of the puerarin-loaded mesoporous polydopamine drug-loaded nanoparticles obtained in step (2) and 1 g of acrylamide were dissolved in 10 mL of PBS solution (pH = 7.4), and then 0.015 g of the cross-linking agent N,N-methylenebisacrylamide was added. At the same time, 50 mg of the 4-formylphenylboronic acid-grafted polylysine powder obtained in step (1) and 100 mg of fucoidan were dissolved in 10 mL of PBS solution (pH = 7.4). Then, 0.05 mL of tetramethylethylenediamine and 0.05 g of potassium persulfate were added to the drug-loaded nanoparticle-acrylamide mixed solution at a speed of 300-350 rpm to initiate free radical polymerization of acrylamide. At the same time, the obtained polylysine-fucoidan mixed solution was quickly added to mix the two, and the mixture was labeled as SFPPM3.

[0068] Figure 2 This is the infrared image of polylysine (PPL) grafted with ε-polylysine (EPL), 4-formylphenylboronic acid (PBA), and 4-formylphenylboronic acid prepared in Example 1. The image shows that the imine group C=N generated by the Schiff base reaction of 4-formylphenylboronic acid and polylysine in PPL is 1652 cm -1 A stretching vibration peak is generated at 820 cm -1 A stretching vibration peak was generated at , which proved that 4-formylphenylboronic acid was successfully grafted onto ε-polylysine.

[0069] Figure 3 This is a comparison of the H NMR spectra of ε-polylysine and the polylysine grafted with 4-formylphenylboronic acid prepared in Example 1. As can be seen from the figure, the a and b peaks that appear in PPL are not present in the raw ε-polylysine, which also proves that 4-formylphenylboronic acid is successfully grafted onto ε-polylysine.

[0070] Figure 4 This is a TEM comparison image of the nano-mesoporous polydopamine prepared in Example 1 and the nano-mesoporous polydopamine loaded with puerarin. The image shows that the internal pores of the nano-mesoporous polydopamine after drug loading are filled, confirming the success of drug loading.

[0071] Figure 5 This is an infrared comparison of puerarin (PUE), nano-mesoporous polydopamine (MPDA) prepared in Example 1, and nano-mesoporous polydopamine loaded with puerarin (MPDA@PUE). In the figure, the ether bond -O- group on MPDA@PUE appears at 1103 cm -1 A stretching vibration peak is generated at , which does not exist in mesoporous polydopamine and belongs to the group of puerarin. It can be seen that mesoporous polydopamine is successfully loaded with puerarin.

[0072] Take 0.5 mg of the double network hydrogel prepared in Example 1, disperse it in 10 ml of PBS (pH = 7.4) solution, then dialyze it in 100 ml of PBS, PBS + glucose solution (pH = 7.4) respectively, and measure the concentration of puerarin in the dialysis bag at 0, 1, 2, 4, 6, 8, 10, 12, 24 h respectively to illustrate its glucose responsiveness, and the results are shown in Figure 6 . Figure 6 It is proved that 4-formylphenyl boronic acid (FPBA) is grafted with ε-polylysine, which endows the hydrogel with glucose responsiveness.

[0073] Figure 7 The optical photograph of the double network hydrogel prepared in Example 1 and Comparative Example 2 is shown in the comparison chart. As can be seen from the chart, the double network hydrogel prepared in Comparative Example 2 by simply mixing the glycopeptide network and the polyacrylamide network through physical stirring has obvious phase separation phenomenon and poor forming effect due to the failure to achieve molecular scale interpenetration and synergistic crosslinking, while the double network hydrogel prepared in Example 1 is uniform and continuous in structure and has good integrity. It is proved that the double network hydrogel prepared in the present application has advantages in structural stability and morphological uniformity.

[0074] 1 g of the hydrogels prepared in Examples 1, 2 and Comparative Example 1 were respectively placed in 15 mL centrifuge tubes, 10 mL of basal medium and 200 μL of penicillin streptomycin were added, and soaked for 12 h to obtain hydrogel extracts. Another 10 mL of basal medium and 200 μL of penicillin streptomycin were added to 15 mL centrifuge tubes as control solution. Human umbilical vein endothelial cells were inoculated in a 24-well plate containing complete medium and cultured until the cells formed a single cell layer without gaps, then the medium was discarded, and a 200 μL pipette tip was vertically drawn on the surface of the well plate. After gently washing away the cell debris, 500 μL of hydrogel extract or control solution was added to the residual cells for incubation, and the scratch area was observed using an inverted microscope at different time points (12 h, 24 h, 36 h) and quantitatively processed using ImageJ software. The results are shown in Figure 8 . As can be seen from Figure 8 , after 36 h of culture, the cell viability of the experimental group added with the double network hydrogel of Example 2 is higher, indicating that it has good cell proliferation effect and is more helpful for wound healing.

[0075] The antibacterial rate of the obtained hydrogel against Escherichia coli by photothermal inhibition was qualitatively analyzed by the coating plate method, and the bacterial growth was evaluated by measuring the optical density of bacteria at 600 nm (OD600) with a spectrophotometer to comprehensively evaluate the in vitro antibacterial properties of the hydrogel. The specific operation is to place the same volume of hydrogel samples in a 12-well plate, inoculate Escherichia coli on the surface of the hydrogel after ultraviolet sterilization, and incubate the hydrogel samples for 12 hours without light or with near-infrared (808 nm, 2.5 w / cm²) for 10 minutes, and then resuspend the surviving bacteria in saline. Take about 200 μL of the above suspension and activate it directly in 3 mL of liquid culture medium, incubate it in a shaker at 120 rpm / min for 6-8 hours, and measure the optical density value of the culture medium at 600 nm. At the same time, take 100 μL of the bacterial suspension and drop it onto the LB plate and incubate it for 12 hours to observe the bacterial growth. The results are shown in Figure 9 . Figure 9 The results showed that the double-network hydrogels prepared in Example 1 and Example 2 exhibited excellent antibacterial effects both under illumination and in the absence of illumination.

[0076] A universal material testing machine was used to perform a tensile test at a rate of 50 mm / min to measure the elastic modulus of the double network hydrogels prepared in Examples 1 and 2 and Comparative Examples 1 and 2. The results are shown in FIG. Figure 10 . Figure 10 The results showed that the double-network hydrogel prepared in Example 1 exhibited more excellent tensile mechanical properties.

[0077] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A double-network hydrogel wound dressing with synergistic antibacterial and glucose-responsive properties, characterized by: It uses 4-formylphenylboronic acid to graft polylysine, and then uses the 4-formylphenylboronic acid grafted polylysine to self-assemble with fucoidan to form a first-layer glycopeptide-based network that mimics the extracellular matrix and has glucose-responsive properties. At the same time, mesoporous polydopamine-loaded nanoparticles loaded with puerarin are wrapped in polyacrylamide as the second-layer network, combined with tetramethylethylenediamine and potassium persulfate as cross-linking agents to form a double-network hydrogel.

2. A method for preparing the double-network hydrogel with synergistic antibacterial and glucose-responsive properties as claimed in claim 1, characterized in that: The following steps are involved: 1) Mixing a polylysine solution and a 4-formylphenylboronic acid solution, adding sodium cyanoborohydride, stirring continuously, dialyzing, and freeze-drying to obtain a 4-formylphenylboronic acid-grafted polylysine powder; 2) dissolving poloxamer in an ethanol solution by stirring, then adding 3,3',5,5'-tetramethylbenzidine, dopamine hydrochloride, and ammonia to the resulting mixture, continuing to stir and mix, and then centrifuging, washing, and freeze-drying to obtain nano-mesoporous polydopamine powder; 3) adding the nano-mesoporous polydopamine powder prepared in step 2) to the puerarin solution, stirring and mixing, and then freeze-drying to obtain puerarin-loaded mesoporous polydopamine drug-loaded nanoparticles; 4) Dissolving the puerarin-loaded mesoporous polydopamine drug-loaded nanoparticles obtained in step 3) and acrylamide in a PBS solution to obtain solution A. Dissolving the 4-acylphenylboronic acid-grafted polylysine powder and fucoidan obtained in step 1) in another PBS solution to obtain solution B. Then, an equal volume of solution A was added to solution B, and N,N-methylenebisacrylamide was added and stirred to mix thoroughly. 5) Add tetramethylethylenediamine and potassium persulfate to the mixed system obtained in step 4), and stir rapidly to obtain a double-network glucose-responsive hydrogel.

3. The method for preparing the double-network hydrogel with synergistic antibacterial and glucose-responsive properties according to claim 2, characterized in that: The mass ratio of polylysine, 4-formylphenylboronic acid and sodium cyanoborohydride used in step 1) is 1:1:(0.18~0.5).

4. The method for preparing the double-network hydrogel with synergistic antibacterial and glucose-responsive properties according to claim 2, wherein: The dialysis described in step 1) uses a dialysis bag with a molecular weight cutoff of 2 kDa. The total dialysis time is 72 hours, during which the water is changed every 8 hours.

5. The method for preparing the double-network hydrogel with synergistic antibacterial and glucose-responsive properties according to claim 2, wherein: The volume concentration of the ethanol solution in step 2) is 50%, the mass fraction of poloxamer in the resulting mixed solution is 1 wt%, the volume ratio of the amount of 3,3',5,5'-tetramethylbenzidine added to the mixed solution is 1:100-1:120, the amount of dopamine hydrochloride added is 12-20 mg / mL, and the volume ratio of the amount of ammonia added to the mixed solution is 1:

125.

6. The method for preparing the double-network hydrogel with synergistic antibacterial and glucose-responsive properties according to claim 2, wherein: The centrifugal speed in step 2) is 8000 rpm and the time is 10-20 min; the washing is performed using a mixed solution of ethanol and acetone in a volume ratio of 2:1 as a detergent.

7. The method for preparing the double-network hydrogel with synergistic antibacterial and glucose-responsive properties according to claim 2, wherein: The puerarin solution in step 3) is an ethanol solution of puerarin with a concentration of 125-250 mg / L. The amount of puerarin added is calculated based on the amount of puerarin being 25-30 wt% of the mass of the nano-mesoporous polydopamine powder.

8. The method for preparing the double-network hydrogel with synergistic antibacterial and glucose-responsive properties according to claim 2, wherein: The stirring in step 3) is carried out in the dark, with a rotation speed of 300-350 rpm and a stirring time of 36-48 h.

9. The method for preparing the double-network hydrogel with synergistic antibacterial and glucose-responsive properties according to claim 2, wherein: Step 4) In the resulting mixed system, the mass concentration of puerarin-loaded mesoporous polydopamine drug-loaded nanoparticles is 0-0.6 wt %, the mass concentration of acrylamide is 10 wt %, the mass concentration of 4-formylphenylboronic acid-grafted polylysine is 0.5 wt %, the mass concentration of fucoidan is 0.25-1 wt %, and the concentration of N,N-methylenebisacrylamide is 0.75 mg / mL; the pH of the PBS solution is 7.0-7.

4.

10. The method for preparing the double-network hydrogel with synergistic antibacterial and glucose-responsive properties according to claim 2, wherein: In step 5), the volume ratio of tetramethylethylenediamine added to the mixed system is 1:400-1:200, and the amount of potassium persulfate added is 2.5 mg / mL.

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