An antibacterial hydrogel, its preparation method and application

Abstract

This invention relates to the field of biomedical materials, specifically to an antibacterial hydrogel, its preparation method, and its applications. The method involves first preparing thiolated tannic acid and copper-doped Prussian blue nanozyme (CuPB). Methacrylated gelatin is mixed with the thiolated tannic acid, followed by the sequential addition of CuPB, ε-polylysine, trehalose, horseradish peroxidase, and a photoinitiator. The mixture is stirred until homogeneous to obtain a photosensitive prepolymer solution, which is then photocured under visible light to obtain the antibacterial hydrogel. The antibacterial hydrogel prepared by this invention responds to changes in H2O2 concentration at the wound site: during infection, CuPB and HRP cascade catalyze the generation of reactive oxygen species, synergistically with ε-polylysine and copper ions to achieve multi-mechanism, highly efficient antibacterial action; simultaneously, it triggers oxidative crosslinking of the thiolated tannic acid, enhancing its mechanical properties and accompanied by color changes, thus aiding in monitoring the infection status; possessing excellent biocompatibility, antibacterial properties, and mechanical properties, it can be used in wound dressings, biosensors, and other fields.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, specifically to an antibacterial hydrogel, its preparation method, and its application. Background Technology

[0002] Hydrogels have garnered significant attention in wound dressings due to their excellent biocompatibility, tissue adhesion, and moisture retention capabilities, and are gradually becoming important materials for promoting wound healing. This is especially true in the treatment of infected wounds (such as burns, diabetic foot ulcers, and purulent wounds), where the wound environment is complex and prone to bacterial infection, inflammatory responses, and oxidative stress. Therefore, higher demands are placed on hydrogel materials, requiring not only excellent physical coverage and moisture retention but also multiple functions including intelligent antibacterial properties, mechanical adaptability, and anti-inflammatory and healing-promoting effects.

[0003] However, existing antibacterial hydrogels still have certain limitations. Some antibacterial hydrogels rely on exogenous antibacterial agents such as antibiotics and nano-silver to achieve their antibacterial effect. Long-term use can easily induce bacterial resistance and bring certain biotoxicity. At the same time, their antibacterial effect is usually a fixed release pattern, making it difficult to dynamically regulate according to the degree of wound infection. Other responsive hydrogels, although possessing certain stimulus-responsive characteristics, mostly rely on a single cross-linking or a single antibacterial triggering mechanism, lacking multi-level response capabilities: in the early stage of infection, their mechanical strength is insufficient, making it difficult to form a stable antibacterial barrier; while in the repair stage, an overly dense network structure can affect cell migration and tissue regeneration, hindering wound healing.

[0004] Furthermore, existing hydrogel systems often rely on exogenous enzymes, specific substrates, or harsh reaction conditions, leading to complex preparation processes and limited applications. Simultaneously, facing oxidative stress caused by excessive reactive oxygen species (ROS) in the infection microenvironment, traditional hydrogels struggle to simultaneously meet the synergistic requirements of efficient antibacterial activity, anti-inflammatory clearance, and tissue repair. Moreover, the functional components are mostly simple physical mixtures lacking systematic synergistic design, causing nano-antibacterial agents to easily aggregate and precipitate, severely affecting the stability of material properties and the uniformity of therapeutic efficacy.

[0005] Based on the above statements, there is an urgent need to provide an antibacterial hydrogel, its preparation method, and its application. Summary of the Invention

[0006] To address the problems of existing antibacterial hydrogels, such as reliance on exogenous antibacterial agents leading to drug resistance and cytotoxicity, limited functionality, inability to dynamically adjust according to the degree of infection, difficulty in achieving both high-efficiency bactericidal effect and removal of oxidative stress damage, complex preparation process, and easy aggregation of functional components, this invention provides an antibacterial hydrogel, its preparation method, and its application.

[0007] In a first aspect, the present invention provides a method for preparing an antibacterial hydrogel, employing the following technical solution: A method for preparing an antibacterial hydrogel includes the following steps: S1. Under light-protected conditions, methacrylamide gelatin (GelMA) and mercapto-tannic acid were added to PBS buffer and stirred to dissolve, resulting in mixed solution A. S2. Under light-protected conditions, copper-doped Prussian blue nanozyme (CuPB) was added to mixed solution A and stirred to react. Then, ε-polylysine was added and stirred to dissolve, resulting in mixed solution B. S3. Under light-protected conditions, add trehalose to mixed solution B, stir to dissolve, then add horseradish peroxidase (HRP), stir evenly, and obtain mixed solution C; S4. Under light-protected conditions, add the photoinitiator to the mixed solution C, stir evenly, centrifuge to remove air bubbles, and obtain the photosensitive prepolymer solution; S5. Inject the photosensitive prepolymer into the mold and place it under visible light irradiation to cure it, thus obtaining the antibacterial hydrogel.

[0008] Preferably, the method for preparing thiolized tannic acid in step S1 is as follows: Tannic acid and a mercapto-containing compound were dissolved in deionized water, the pH was adjusted, and the reaction was carried out at room temperature in the dark. After the reaction was completed, the reaction product was purified by dialysis and freeze-dried to obtain mercapto-tannic acid.

[0009] Preferably, the specific preparation method of the thiolized tannic acid in step S1 is as follows: Tannic acid was dissolved in deionized water to prepare a tannic acid solution with a mass concentration of 5-15 mg / mL. Then, a thiol-containing compound was added to the tannic acid solution, making the molar ratio of tannic acid to the thiol-containing compound 1:3-5. The mixture was thoroughly mixed to obtain a homogeneous solution. The pH of the system was adjusted to 6.0-8.0 using 0.01-0.03 mol / L hydrochloric acid solution and 0.01-0.03 mol / L sodium hydroxide solution. The reaction was carried out at room temperature in the dark for 18-24 hours. After the reaction was completed, the reaction mixture was... The product was transferred into a dialysis bag with a molecular weight cutoff of 3000-5000 Da, and dialyzed at room temperature for 48-72 hours using deionized water as the dialysis medium. During this period, the deionized water was replaced every 6-12 hours to remove unreacted mercapto-containing compounds and small molecule impurities. After dialysis, the product in the dialysis bag was pre-frozen at -80℃ to -70℃ for 1-3 hours, and then transferred to a freeze dryer and freeze-dried at -55℃ to -45℃ and a vacuum degree ≤15Pa for 24-48 hours to obtain mercapto-tannic acid.

[0010] Preferably, the thiol-containing compound is selected from one or more of cysteine ​​hydrochloride, thioglycolic acid, glutathione, and thioglycerol.

[0011] Preferably, in step S1, the mass-to-volume ratio of methacrylamide gelatin, thiolated tannic acid, and PBS buffer is 50-150 mg: 10-20 mg: 1 mL; the concentration of the PBS buffer is 30-50 mM, and the pH value is 7.0-7.4.

[0012] Preferably, the preparation method of the copper-doped Prussian blue nanozyme in step S2 is as follows: Potassium ferrocyanide was dissolved in deionized water to obtain a ligand source solution; copper salt was dissolved in a polyol to obtain a metal source solution; the metal source solution was added dropwise to the ligand source solution under stirring, and the reaction was carried out at a higher temperature; the reaction product was purified by dialysis and freeze-dried to obtain copper-doped Prussian blue nanozyme.

[0013] Preferably, the specific preparation method of the copper-doped Prussian blue nanozyme in step S2 is as follows: Potassium ferrocyanide was dissolved in deionized water to prepare a ligand source solution with a concentration of 10-20 mmol / L; copper salt was dissolved in polyol to prepare a metal source solution with a concentration of 20-30 mmol / L; the metal source solution was added dropwise to the ligand source solution under magnetic stirring at 300-500 rpm for 1-2 h; after the addition was complete, the temperature was raised to 60-80 °C and the reaction was carried out for 2-4 h; after the reaction was completed, the reaction product was cooled to room temperature and transferred to a filtration membrane. Dialysis was performed in dialysis bags with a molecular weight of 8000-12000 Da, using deionized water as the dialysis medium, at room temperature for 48-72 hours. During this period, the deionized water was replaced every 8-12 hours to remove unreacted raw materials and small molecule byproducts. After dialysis, the product in the dialysis bag was pre-frozen at -80℃ to -70℃ for 2-4 hours, and then transferred to a freeze dryer for freeze drying at -55℃ to -40℃ and a vacuum degree ≤15Pa for 24-48 hours to obtain copper-doped Prussian blue nanozyme.

[0014] Preferably, the copper salt is selected from one or more of copper nitrate, copper chloride, and copper acetate; the polyol solvent is selected from one or more of ethylene glycol, propylene glycol, and glycerol.

[0015] Preferably, in step S2, the mass-to-volume ratio of copper-doped Prussian blue nanozyme, ε-polylysine, and mixed solution A is 3-5 mg: 6-10 mg: 1 mL.

[0016] Preferably, in step S3, the mass-to-volume ratio of trehalose, horseradish peroxidase, and mixed solution B is 80-100 mg: 0.3-0.5 mg: 1 mL.

[0017] Preferably, the photoinitiator in step S4 is one or more of lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), Irgacure 2959, and Irgacure 127; the mass-to-volume ratio of the photoinitiator to the mixed solution C is 2-4 mg: 1 mL.

[0018] Preferably, the visible light irradiation conditions in step S5 include: a visible light wavelength range of 400-500nm, a light intensity of 30-50mW / cm², and an irradiation time of 2-5min.

[0019] Secondly, the present invention provides an antibacterial hydrogel prepared by the method described above.

[0020] Thirdly, the present invention provides an application of the antibacterial hydrogel as described above in the preparation of wound dressings, biosensors, or materials for monitoring inflammatory microenvironments.

[0021] The antibacterial hydrogel in this invention is constructed based on a "trigger-dual response" mechanism of "infection microenvironment triggering": During the preparation process, tannic acid is modified by thiolation to obtain thiolated tannic acid with redox activity, while CuPB with good dispersibility and peroxidase-like activity is prepared simultaneously. Subsequently, trehalose is used to protect the native conformation of HRP, and ε-polylysine, CuPB, and HRP are introduced into the GelMA and thiolated tannic acid system through electrostatic interaction, forming a covalent cross-linked network through visible light-initiated polymerization; at the same time, thiolated tannic acid forms a metal-polyphenol coordination structure with copper ions through its polyphenolic hydroxyl groups, thereby constructing a dual-network hydrogel composed of a covalent network and a coordination structure.

[0022] During application, this hydrogel can regulate its functional state in response to changes in H2O2 concentration in the wound microenvironment. When the wound is in the normal healing stage, the local H2O2 level is low, and the activity of CupPb and HRP is weak. The hydrogel mainly relies on the photocrosslinking network and metal-polyphenol coordination structure to maintain stability, exhibiting a soft and elastic state that can adhere to the wound and provide moist protection. When the wound becomes infected, the local H2O2 concentration increases. CupPb and HRP synergistically catalyze the generation of various reactive oxygen species, causing oxidative damage to bacteria. At the same time, ε-polylysine disrupts the bacterial cell membrane, and copper ions inhibit bacterial metabolism, with multiple mechanisms synergistically enhancing the antibacterial effect. In addition, under oxidative conditions, the thiol groups in thiolated tannic acid can be oxidized to form disulfide crosslinks, while the phenolic hydroxyl groups on the tannic acid backbone are oxidized to quinone structures. These two factors synergistically enhance the crosslinking density and mechanical strength of the gel network, thereby further inhibiting bacterial spread, accompanied by a darkening of color to reflect changes in the oxidative environment of the wound.

[0023] In summary, compared with the prior art, the present invention has the following beneficial effects: (1) Adaptive response to infection microenvironment, achieving on-demand antibacterial treatment: The "infection microenvironment trigger-dual response" mechanism constructed in this invention enables the hydrogel to automatically switch its working mode according to the local hydrogen peroxide concentration at the wound site: In the absence of infection or in the presence of low concentrations of hydrogen peroxide, the hydrogel remains soft and elastic, mainly playing the role of physical barrier and moisturizing function, without interfering with the normal tissue healing process; when infection causes an increase in hydrogen peroxide concentration, the hydrogel immediately switches to enhanced defense mode and initiates a powerful sterilization program. This "on-demand response" characteristic avoids the cytotoxicity and bacterial resistance problems caused by the continuous release of drugs in traditional antibacterial dressings, achieving precise and safe infection management.

[0024] (2) Synergistic antibacterial action with multiple mechanisms, high bactericidal efficiency and low risk of drug resistance: The hydrogel in this invention integrates three synergistic antibacterial mechanisms: the hydroxyl radicals generated by the cascade catalysis of copper-doped Prussian blue nanozyme and horseradish peroxidase exert a strong oxidative bactericidal effect; ε-polylysine disrupts the integrity of bacterial cell membranes; and copper ions themselves have metal toxicity. The synergistic effect of multiple mechanisms not only significantly improves the bactericidal efficiency, but more importantly, because it acts on different targets of bacteria, it greatly reduces the risk of bacteria developing drug resistance, making it particularly suitable for the treatment of wounds infected by drug-resistant bacteria.

[0025] (3) Dynamic enhancement of mechanical properties to construct an adaptive physical barrier during infection: This invention achieves dynamic regulation of the mechanical properties of hydrogels through the in-situ cross-linking reaction of thiolized tannic acid in an oxidizing environment. Under infection conditions, hydrogen peroxide triggers multiple cross-linking, increasing the density and modulus of the gel network and forming a dense physical barrier to inhibit bacterial spread. This "on-demand enhancement" mechanical response characteristic allows the hydrogel to rapidly increase its structural strength during infection to meet barrier requirements, while maintaining a relatively loose network structure during non-infection periods to facilitate material exchange and tissue adaptation, better adapting to the differentiated needs of different stages of wound healing.

[0026] (4) Visual infection monitoring for integrated diagnosis and treatment: This invention utilizes the color change during the oxidation of thiolated tannic acid (from light amber to brownish-red) to give the hydrogel a real-time visual monitoring function. Medical staff or patients can directly observe the color of the gel with the naked eye to help determine the status and severity of wound infection and adjust the treatment plan in a timely manner.

[0027] (5) Dual protection strategy for enzyme activity to ensure stable and reliable response function: This invention adopts a dual protection strategy combining the "water substitution" effect of trehalose with visible light curing, which effectively maintains the natural conformation and bioactivity of horseradish peroxidase during material preparation. Trehalose forms a protective layer before photocuring, reducing the damage of the enzyme to the free radical environment; 400-500nm visible light causes less damage to enzyme proteins than traditional ultraviolet light. This design solves the key technical problem of easy inactivation of bioactive substances during the preparation of photocrosslinked hydrogels, ensuring the stability and reliability of the material's response function. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0029] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0030] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0031] The methacrylamide gelatin (GelMA) was purchased from Beijing Solarbio Technology Co., Ltd., product model: G7002; ε-polylysine was purchased from Shandong Pingju Biotechnology Co., Ltd. Horseradish peroxidase (HRP) was purchased from Beijing Bio-Sens Biotechnology Co., Ltd., product model: C-0001, RZ>3.1, activity ≥300U / mg; Lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) was purchased from Jiangxi Lote Chemical Co., Ltd. Tannic acid was purchased from Zhangjiajie Jiurui Biotechnology Co., Ltd. Cysteine ​​hydrochloride was purchased from Shandong Suihua Biotechnology Co., Ltd.

[0032] Example 1 A method for preparing an antibacterial hydrogel includes the following steps: S1. Under light-protected conditions, according to the mass-to-volume ratio of methacrylamide gelatin (GelMA), mercapto-tannic acid, and PBS buffer (50 mg: 10 mg: 1 mL), GelMA and mercapto-tannic acid were added to 30 mM PBS buffer with a pH of 7.0 and stirred at 40 °C and 300 rpm for 2 h to dissolve, thus obtaining mixed solution A. S2. Under light-protected conditions, CuPB was added to mixed solution A and stirred at 400 rpm for 40 min. Then, ε-polylysine was added and stirred at 400 rpm for 20 min to dissolve, resulting in mixed solution B. The mass-volume ratio of CuPB, ε-polylysine, and mixed solution A was 3 mg: 6 mg: 1 mL. S3. Under light-protected conditions, trehalose was added to mixed solution B and stirred at 200 rpm for 20 min to dissolve. Then HRP was added and stirred at 200 rpm for 10 min to obtain mixed solution C. The mass-volume ratio of trehalose, HRP and mixed solution B was 80 mg: 0.3 mg: 1 mL. S4. Under light-protected conditions, LAP was added to mixed solution C at a mass-to-volume ratio of 2 mg to 1 mL. The mixture was stirred at 200 rpm until the solid was completely dissolved. Then, it was placed in a centrifuge and centrifuged at 3000 rpm for 3 min to remove air bubbles, thus obtaining the photosensitive prepolymer solution. S5. Inject the photosensitive prepolymer into a glass mold to a depth of 2mm, and place it at 405nm, 30mW / cm². 2 The antibacterial hydrogel was obtained by photocuring under visible light for 2 minutes.

[0033] The specific preparation method of thiolized tannic acid is as follows: Tannic acid was dissolved in deionized water to prepare a tannic acid solution with a mass concentration of 5 mg / mL. Then, a thiol-containing compound was added to the tannic acid solution, making the molar ratio of tannic acid to the thiol-containing compound 1:3. The mixture was thoroughly mixed to obtain a homogeneous solution. The pH of the system was adjusted to 6.0 using 0.01 mol / L hydrochloric acid solution and 0.01 mol / L sodium hydroxide solution. The reaction was carried out at room temperature in the dark for 18 hours. After the reaction, the reaction product was transferred to a dialysis bag with a molecular weight cutoff of 3000 Da. Dialysis was performed using deionized water as the dialysis medium at room temperature for 48 hours, with the deionized water replaced every 6 hours to remove unreacted thiol-containing compounds and small molecule impurities. After dialysis, the product in the dialysis bag was pre-frozen at -80℃ for 1 hour, and then transferred to a freeze dryer. The product was freeze-dried at -55℃ and a vacuum of 15 Pa for 24 hours to obtain thiolized tannic acid.

[0034] The specific preparation method of CuPB is as follows: Potassium ferrocyanide was dissolved in deionized water to prepare a ligand source solution with a concentration of 10 mmol / L; copper nitrate was dissolved in ethylene glycol to prepare a metal source solution with a concentration of 20 mmol / L; the metal source solution was added dropwise to the ligand source solution under magnetic stirring at 300 rpm for 1 h; after the addition was completed, the temperature was raised to 60 °C and reacted for 2 h; after the reaction was completed, the reaction product was cooled to room temperature and transferred to a dialysis bag with a molecular weight cutoff of 8000 Da. Dialysis was performed at room temperature for 48 h using deionized water as the dialysis medium, with the deionized water being replaced every 8 h to remove unreacted raw materials and small molecule byproducts. After dialysis, the product in the dialysis bag was pre-frozen at -80 °C for 2 h and then transferred to a freeze dryer for freeze drying at -55 °C and a vacuum of 15 Pa for 24 h to obtain CupP.

[0035] Example 2 A method for preparing an antibacterial hydrogel includes the following steps: S1. Under light-protected conditions, according to the mass-to-volume ratio of methacrylamide gelatin (GelMA), mercapto-tannic acid, and PBS buffer (100 mg: 15 mg: 1 mL), GelMA and mercapto-tannic acid were added to 40 mM PBS buffer with a pH of 7.2 and stirred at 40 °C and 500 rpm for 2.5 h to dissolve, thus obtaining mixed solution A. S2. Under light-protected conditions, CuPB was added to mixed solution A and stirred at 600 rpm for 50 min. Then, ε-polylysine was added and stirred at 600 rpm for 25 min to dissolve, resulting in mixed solution B. The mass-volume ratio of CuPB, ε-polylysine, and mixed solution A was 4 mg: 8 mg: 1 mL. S3. Under light-protected conditions, trehalose was added to mixed solution B and stirred at 300 rpm for 25 min to dissolve. Then HRP was added and stirred at 300 rpm for 15 min to obtain mixed solution C. The mass-volume ratio of trehalose, HRP and mixed solution B was 90 mg: 0.4 mg: 1 mL. S4. Under light-protected conditions, LAP was added to mixed solution C at a mass-to-volume ratio of 3 mg to 1 mL. The mixture was stirred at 300 rpm until the solid was completely dissolved. Then, it was placed in a centrifuge and centrifuged at 4000 rpm for 4 min to remove air bubbles, thus obtaining the photosensitive prepolymer solution. S5. Inject the photosensitive prepolymer into a glass mold to a depth of 3mm, and place it at 405nm, 40mW / cm². 2 The antibacterial hydrogel was obtained by photocuring under visible light for 3 minutes.

[0036] The specific preparation method of thiolized tannic acid is as follows: Tannic acid was dissolved in deionized water to prepare a tannic acid solution with a mass concentration of 10 mg / mL. Then, a thiol-containing compound was added to the tannic acid solution, making the molar ratio of tannic acid to the thiol-containing compound 1:4. The mixture was thoroughly mixed to obtain a homogeneous solution. The pH of the system was adjusted to 7.0 using 0.02 mol / L hydrochloric acid solution and 0.02 mol / L sodium hydroxide solution. The reaction was carried out at room temperature in the dark for 20 h. After the reaction, the reaction product was transferred to a dialysis bag with a molecular weight cutoff of 4000 Da. Dialysis was performed using deionized water as the dialysis medium at room temperature for 60 h, with the deionized water replaced every 10 h to remove unreacted thiol-containing compounds and small molecule impurities. After dialysis, the product in the dialysis bag was pre-frozen at -75℃ for 2 h, and then transferred to a freeze dryer and freeze-dried at -50℃ and a vacuum of 15 Pa for 36 h to obtain thiolized tannic acid.

[0037] The specific preparation method of CuPB is as follows: Potassium ferrocyanide was dissolved in deionized water to prepare a ligand source solution with a concentration of 15 mmol / L; copper nitrate was dissolved in ethylene glycol to prepare a metal source solution with a concentration of 25 mmol / L; the metal source solution was added dropwise to the ligand source solution under magnetic stirring at 400 rpm for 1.5 h; after the addition was completed, the temperature was raised to 70 °C and reacted for 3 h; after the reaction was completed, the reaction product was cooled to room temperature and transferred to a dialysis bag with a molecular weight cutoff of 10000 Da. Dialysis was performed at room temperature for 60 h using deionized water as the dialysis medium, with the deionized water being replaced every 10 h to remove unreacted raw materials and small molecule byproducts. After dialysis, the product in the dialysis bag was pre-frozen at -75 °C for 3 h and then transferred to a freeze dryer for freeze drying at -50 °C and a vacuum of 15 Pa for 36 h to obtain CupP.

[0038] Example 3 A method for preparing an antibacterial hydrogel includes the following steps: S1. Under light-protected conditions, according to the mass-to-volume ratio of methacrylamide gelatin (GelMA), mercapto-tannic acid, and PBS buffer (150 mg: 20 mg: 1 mL), GelMA and mercapto-tannic acid were added to 50 mM PBS buffer with a pH of 7.4. The mixture was stirred at 40 °C and 600 rpm for 3 h to dissolve the mixture, thus obtaining mixed solution A. S2. Under light-protected conditions, CuPB was added to mixed solution A and stirred at 800 rpm for 60 min. Then, ε-polylysine was added and stirred at 800 rpm for 30 min to dissolve, resulting in mixed solution B. The mass-volume ratio of CuPB, ε-polylysine, and mixed solution A was 5 mg: 10 mg: 1 mL. S3. Under light-protected conditions, trehalose was added to mixed solution B and stirred at 400 rpm for 30 min to dissolve. Then HRP was added and stirred at 400 rpm for 20 min to obtain mixed solution C. The mass-volume ratio of trehalose, HRP and mixed solution B was 100 mg: 0.5 mg: 1 mL. S4. Under light-protected conditions, LAP was added to mixed solution C at a mass-to-volume ratio of 4 mg to 1 mL. The mixture was stirred at 400 rpm until the solid was completely dissolved. Then, it was placed in a centrifuge and centrifuged at 5000 rpm for 5 min to remove air bubbles, thus obtaining the photosensitive prepolymer solution. S5. Inject the photosensitive prepolymer into a glass mold to a depth of 4 mm, and place it at 405 nm and 50 mW / cm². 2 The antibacterial hydrogel was obtained by photocuring under visible light for 5 minutes.

[0039] The specific preparation method of thiolized tannic acid is as follows: Tannic acid was dissolved in deionized water to prepare a tannic acid solution with a mass concentration of 15 mg / mL. Then, a thiol-containing compound was added to the tannic acid solution, making the molar ratio of tannic acid to the thiol-containing compound 1:5. The mixture was thoroughly mixed to obtain a homogeneous solution. The pH of the system was adjusted to 8.0 using 0.03 mol / L hydrochloric acid solution and 0.03 mol / L sodium hydroxide solution. The reaction was carried out at room temperature in the dark for 24 hours. After the reaction, the reaction product was transferred to a dialysis bag with a molecular weight cutoff of 5000 Da. Dialysis was performed using deionized water as the dialysis medium at room temperature for 72 hours, with the deionized water replaced every 12 hours to remove unreacted thiol-containing compounds and small molecule impurities. After dialysis, the product in the dialysis bag was pre-frozen at -70℃ for 3 hours, and then transferred to a freeze dryer. The product was freeze-dried at -45℃ and a vacuum of 15 Pa for 48 hours to obtain thiolized tannic acid.

[0040] The specific preparation method of CuPB is as follows: Potassium ferrocyanide was dissolved in deionized water to prepare a ligand source solution with a concentration of 20 mmol / L; copper nitrate was dissolved in ethylene glycol to prepare a metal source solution with a concentration of 30 mmol / L; the metal source solution was added dropwise to the ligand source solution under magnetic stirring at 500 rpm for 2 h; after the addition was completed, the temperature was raised to 80 °C and reacted for 4 h; after the reaction was completed, the reaction product was cooled to room temperature and transferred to a dialysis bag with a molecular weight cutoff of 12000 Da. Dialysis was performed at room temperature for 72 h using deionized water as the dialysis medium, with the deionized water being replaced every 12 h to remove unreacted raw materials and small molecule byproducts. After dialysis, the product in the dialysis bag was pre-frozen at -70 °C for 4 h and then transferred to a freeze dryer for freeze drying at -40 °C and a vacuum of 15 Pa for 48 h to obtain CupP.

[0041] Comparative Example 1 This comparative example provides a method for preparing an antibacterial hydrogel. The only difference from Example 2 is that mercapto-tannic acid is not added in step S1, while the other raw material types, amounts, and preparation process parameters are completely consistent with Example 2.

[0042] Comparative Example 2 This comparative example provides a method for preparing an antibacterial hydrogel. The only difference from Example 2 is that in step S1, tannic acid of equal mass is used to replace thiolated tannic acid. The other raw material types, amounts, and preparation process parameters are completely consistent with those of Example 2.

[0043] Comparative Example 3 This comparative example provides a method for preparing an antibacterial hydrogel. The only difference from Example 2 is that CupB is not added in step S2, while the other raw material types, amounts, and preparation process parameters are completely consistent with Example 2.

[0044] Comparative Example 4 This comparative example provides a method for preparing an antibacterial hydrogel. The only difference from Example 2 is that in step S2, Prussian blue nanoparticles (PBNPs) of equal mass are used to replace Cubat. All other raw material types, amounts, and preparation process parameters are completely consistent with Example 2.

[0045] Comparative Example 5 This comparative example provides a method for preparing an antibacterial hydrogel. The only difference from Example 2 is that HRP is not added in step S3, while the other raw material types, amounts, and preparation process parameters are completely consistent with Example 2.

[0046] Comparative Example 6 This comparative example provides a method for preparing an antibacterial hydrogel. The only difference from Example 2 is that trehalose is replaced with an equal mass of sucrose in step S3. All other raw material types, amounts, and preparation process parameters are completely consistent with Example 2.

[0047] Performance testing I. Rheological property testing 1. Testing Method The antibacterial hydrogels prepared in Examples 1-3 and Comparative Examples 1-6 were cut into circular samples with a diameter of 20 mm and a thickness of 2 mm, with 3 parallel samples per group. A rotational rheometer (model: TA DHR-2) equipped with a 20 mm parallel plate clamp was used. The test temperature was set to 37℃, the clamp gap to 1000 μm, and silicone oil was applied to the edges of the samples to prevent moisture evaporation. The following two tests were performed: Basic network stability test: First, dynamic strain scanning was performed with a strain range of 0.1-100% and a fixed frequency of 1Hz to determine the linear viscoelastic region of the sample; then, a fixed strain of 0.5% was selected in the linear region, and dynamic frequency scanning was performed to record the storage modulus (G′) and loss modulus (G′′) at an angular frequency of 10 rad / s to evaluate the basic network stability of the hydrogel.

[0048] Oxidative response test: Using time-scan mode, with a fixed strain of 0.5% and a fixed frequency of 1Hz, the initial modulus G0′ of the hydrogel was measured. Then, 100μL of PBS solution containing 500μM H2O2 (simulating a wound infection environment) was dropped onto the sample surface. The storage modulus Gt′ was immediately and continuously monitored at 0h, 1h, 2h, and 4h after the drop. The modulus growth factor R was calculated, R=G t ′ / G0′ is used to characterize the dynamic response of hydrogels to oxidative environments.

[0049] 2. Test Results and Analysis The specific test results are shown in Table 1.

[0050] Table 1. Rheological property test results of antibacterial hydrogel

[0051] Table 1 shows that the G′ values ​​of Examples 1-3 are all higher than those of the comparative examples, and G′ is much larger than G′′, indicating that the antibacterial hydrogel has excellent basic network stability. Under simulated infection conditions, the modulus growth factor of Examples 1-3 continues to increase over time, with Example 2 showing the best performance, increasing its modulus to 2.5 times its initial value in 4 hours, demonstrating a sensitive response to oxidative environments. Due to the absence or substitution of components, the performance of the comparative examples is weaker than that of Examples 1-3: Comparative Example 1 lacks thiolized tannic acid, and Comparative Example 2 uses unmodified tannic acid instead of thiolized tannic acid, both of which cannot effectively trigger oxidative crosslinking, resulting in almost no modulus growth; Comparative Example 3 lacks CupP, lacking enzyme-like activity and exhibiting weak oxidative responsiveness; Comparative Example 4 uses PBNPs to replace CupP, and Comparative Example 5 lacks HRP, both of which lead to a decrease in cascade catalytic efficiency and a reduced modulus growth rate; Comparative Example 6 uses sucrose to replace trehalose, and the HRP activity is not effectively protected, resulting in a slightly lower oxidative response than the examples.

[0052] II. Mechanical Property Testing 1. Testing Method The antibacterial hydrogels prepared in Examples 1-3 and Comparative Examples 1-6 were cut into strips 20 mm long, 5 mm wide, and 2 mm thick, with 5 parallel samples in each group. Using an electronic universal testing machine at a temperature of 37°C and a tensile rate of 10 mm / min, the following two tests were performed: Basic mechanical property testing: Tensile tests were performed on the hydrogel samples to measure and record the tensile strength at break (MPa) and elongation at break (%), in order to evaluate the basic mechanical compatibility of the hydrogel.

[0053] Mechanical response test in oxidative environment: After immersing the hydrogel sample in PBS solution containing 500 μM H2O2 (simulating wound infection environment) for 4 h, a tensile test was performed under the same test conditions. The tensile strength at break (MPa) and elongation at break (%) of the sample were measured and recorded to characterize the mechanical response of the hydrogel to the oxidative environment.

[0054] 2. Test Results and Analysis The specific test results are shown in Table 2.

[0055] Table 2. Test results of mechanical properties of antibacterial hydrogel

[0056] Table 2 shows that the initial tensile strength and initial elongation at break of Examples 1-3 are higher than those of the comparative examples, exhibiting both good tensile strength and high flexibility. After oxidation treatment, the tensile strength of the examples is significantly improved, while the elongation at break shows a reasonable downward trend, with Example 2 showing the best performance. Due to the absence or substitution of components, the performance of each comparative example was weaker than that of Examples 1-3: Comparative Example 1 lacked thiolized tannic acid, thus failing to form a double network structure, resulting in the worst basic mechanical properties and almost no change after oxidation; Comparative Example 2 used ordinary tannic acid to replace thiolized tannic acid, failing to form chemical cross-links, achieving only a slight increase in strength through weak physical action, with weak responsiveness; Comparative Example 3 lacked CupP, lacking enzyme-like activity and unable to trigger effective oxidative cross-linking, resulting in almost no improvement in mechanical properties; Comparative Example 4 used ordinary PBNPs to replace CupP, significantly reducing catalytic activity, and the increase in strength after oxidation was far lower than that of Examples 1-3; Comparative Example 5 lacked HRP, resulting in the absence of a cascade catalytic system, insufficient oxidative cross-linking, limited strength improvement, and a small decrease in elongation; Comparative Example 6 used sucrose to replace trehalose, failing to effectively protect HRP activity, reducing catalytic efficiency, and the increase in strength after oxidation was weaker than that of Examples 1-3.

[0057] III. Reactive Oxygen Species (ROS) Generation Capacity Test 1. Testing Method The antibacterial hydrogels prepared in Examples 1-3 and Comparative Examples 1-6 were cut into circular samples with a diameter of 4 mm and a thickness of 3 mm. Three parallel samples were set up for each group. After being sterilized by ultraviolet irradiation, they were ready for use. The amount of ROS generated was detected by the DCFH-DA fluorescent probe method. The specific steps are as follows: DCFH-DA was diluted with PBS buffer to a final concentration of 10 μM working solution. The antibacterial hydrogel sample was immersed in 1 mL of the working solution and incubated at 37°C in the dark for 30 min to allow the probe to fully penetrate into the gel network. The sample was taken out and washed twice with PBS buffer to remove the free probes on the surface that had not entered the gel. Then, 100 μL of PBS solution containing 500 μM H2O2 (simulating the wound infection environment) was dropped onto the sample surface. Simultaneously, a negative control group (100 μL of H2O2-free PBS buffer was added to the sample surface) and a blank group (no hydrogel, 1 mL of DCFH-DA working solution + 100 μL of PBS solution containing 500 μM H2O2 were added). All samples were placed in a 37℃, 5% CO2 humidified incubator and reacted in the dark for 4 h. The fluorescence intensity of each group was measured using a fluorescence microplate reader (excitation wavelength 488 nm, emission wavelength 525 nm). The relative ROS generation was calculated using the formula: Relative ROS generation = [(Fluorescence intensity of sample group - fluorescence intensity of negative control group) / fluorescence intensity of blank group] × 100%.

[0058] 2. Test Results and Analysis The specific test results are shown in Table 3.

[0059] Table 3 Results of ROS generation capacity test for antibacterial hydrogel

[0060] Table 3 shows that the relative ROS generation in Examples 1-3 all reached over 880%, with Example 2 reaching nearly 980%. This indicates that the hydrogel prepared by the present invention can efficiently catalyze the generation of ROS under simulated infection conditions. Because CuPB and HRP form a highly efficient cascade catalytic system, they can fully utilize H2O2 to generate active oxygen, providing the core material basis for oxidative sterilization and oxidative crosslinking of thiolized tannins. The ROS generation capacity of each comparative example was significantly weaker than that of Examples 1-3: Comparative Example 1 lacked thiolized tannic acid, and Comparative Example 3 lacked CupP, so an effective catalytic system could not be formed. The ROS generation was basically the same as that of the blank group, with almost no catalytic generation effect; Comparative Example 2 used ordinary tannic acid to replace thiolized tannic acid, but there was no thiol-mediated catalytic enhancement effect, and only a small amount of ROS was generated; Comparative Example 4 used ordinary PBNPs to replace CupP, and the catalytic activity was greatly reduced, and the ROS generation was significantly reduced; Comparative Example 5 lacked HRP, and the cascade catalytic system was missing. Only the enzyme-like activity of CupP generated a small amount of ROS; Comparative Example 6 used sucrose to replace trehalose, and the HRP activity was not effectively protected, the catalytic efficiency decreased, and the ROS generation was slightly lower but still better than the other comparative examples.

[0061] IV. Antibacterial Performance Test 1. Testing Method The antibacterial hydrogels prepared in Examples 1-3 and Comparative Examples 1-6 were cut into circular samples with a diameter of 4 mm and a thickness of 3 mm. Three parallel samples were prepared for each group. After sterilization by ultraviolet irradiation, they were used for later use. Staphylococcus aureus (S. aureus) was selected. Staphylococcus aureus ATCC 25923), Escherichia coli ( Escherichia coli ATCC 25922 was used as the test strain. Both strains were inoculated into culture medium and incubated at 37°C for 12 hours, then diluted to 2×10⁻⁶. 5 A bacterial suspension was prepared at CFU / mL.

[0062] Sterilized hydrogel samples were placed in 96-well plates, and 200 μL of the above bacterial suspension was added to each well. After incubation at 37°C for 24 h, the absorbance of the liquid in each well was measured at a wavelength of 600 nm using an ELISA reader. The bacterial suspension without added hydrogel was used as the blank group. The sterilization rate was calculated as follows: sterilization rate = (absorbance of blank group - absorbance of sample group) / absorbance of blank group × 100%.

[0063] 2. Test Results and Analysis The specific test results are shown in Table 4.

[0064] Table 4. Test results of antibacterial properties of antibacterial hydrogel

[0065] Table 4 shows that the bactericidal rates of Examples 1-3 against Staphylococcus aureus and Escherichia coli all exceeded 97%, with Example 2 exhibiting the best bactericidal effect, achieving a bactericidal rate of over 99% against both bacteria. This indicates that the hydrogel prepared by this invention possesses excellent and broad-spectrum antibacterial properties. The antibacterial properties of each comparative example were weaker than those of Examples 1-3: Comparative Example 1 lacked thiolated tannins, resulting in a loose network structure that led to rapid loss of the loaded ε-polylysine and the loss of the synergistic antibacterial effect of tannin-Cu coordination, resulting in a significantly lower bactericidal rate; Comparative Example 2 used ordinary tannins instead of thiolated tannins, leading to insufficient oxidative cross-linking and reactive oxygen species generation, resulting in a significant decrease in antibacterial effect; Comparative Examples 3-6 lacked efficient catalytic systems (no CuPB, ordinary PBNPs, or no HRP), failing to effectively catalyze the generation of sufficient reactive oxygen species from H2O2, resulting in a significant weakening of the chemodynamic antibacterial effect; Comparative Example 6 used sucrose instead of trehalose, resulting in partial loss of HRP activity, decreased catalytic efficiency, and a slightly lower bactericidal rate than Examples 1-3.

[0066] V. Cytotoxicity Test 1. Testing Method ① Sample preparation: Take the antibacterial hydrogels prepared in Examples 1-3 and Comparative Examples 1-6, cut them into circular samples with a diameter of 4 mm and a thickness of 3 mm, and set up 3 parallel samples in each group. After sterilization by ultraviolet irradiation, they are ready for use.

[0067] ② Preparation of extraction solution: Place the sterilized hydrogel sample in a sterile container, add complete culture medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) at a sample surface area to culture medium volume ratio of 3 cm² / mL, and extract at 37℃ for 24 h. Collect the supernatant as the hydrogel extraction solution and store at 4℃ for later use.

[0068] ③ Cell Culture and Seeding: Mouse fibroblasts (L929) were used as test cells and cultured in a 37℃, 5% CO2 incubator with complete culture medium, changing the medium every 2 days. Logarithmic growth phase cells were seeded into 48-well plates at a density of 20,000 cells per well, with 500 μL of complete culture medium added to each well, and cultured for 12 h to allow cell adhesion.

[0069] ④ Experimental grouping and treatment: Discard the original culture medium in the wells and treat according to the following groups (3 replicates per group): Sample group: Add 500 μL of hydrogel extraction buffer to each well; Negative control group: Add 500 μL of fresh complete culture medium to each well; Blank group: No cells are seeded, and 500 μL of fresh complete culture medium is added to each well (to subtract background absorbance). Incubate the culture plates at 37℃ in a 5% CO2 incubator for 24 h.

[0070] ⑤ Cell viability assay (MTT method): After culture, add 20 μL of MTT solution (5 mg / mL) to each well and incubate for another 4 h. Discard the supernatant, add 150 μL of DMSO to each well, and shake for 10 min to completely dissolve the formazan crystals. Measure the absorbance (OD value) at 490 nm using a microplate reader.

[0071] ⑥ Result calculation: Calculate the relative cell activity using the following formula: Relative cell activity = (Absorbance of sample group - Absorbance of blank group) / (Absorbance of negative control group - Absorbance of blank group) × 100%.

[0072] 2. Test Results and Analysis The specific test results are shown in Table 5.

[0073] Table 5 Results of antibacterial hydrogel cytotoxicity test

[0074] Table 5 shows that the relative activity of Examples 1-3 against L929 cells all exceeded 98%, indicating that the antibacterial hydrogel prepared by this invention has no significant cytotoxicity, excellent biocompatibility, and that the functional components in the gel system work synergistically with release concentrations within the biosafety range. The relative cell activity of each comparative example was also at a high level, with no significant cytotoxicity, meeting the safety requirements for biomedical materials.

[0075] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A method for preparing an antibacterial hydrogel, characterized in that, Includes the following steps: S1. Under light-protected conditions, methacrylamide gelatin and mercapto-tannic acid were added to PBS buffer and stirred to dissolve, resulting in mixed solution A. S2. Under light-protected conditions, copper-doped Prussian blue nanozyme was added to mixed solution A, stirred and reacted, then ε-polylysine was added and stirred to dissolve, resulting in mixed solution B. S3. Under light-protected conditions, add trehalose to mixed solution B, stir to dissolve, then add horseradish peroxidase, stir evenly, and obtain mixed solution C; S4. Under light-protected conditions, add the photoinitiator to the mixed solution C, stir evenly, centrifuge to remove air bubbles, and obtain the photosensitive prepolymer solution; S5. Inject the photosensitive prepolymer into the mold and place it under visible light irradiation to cure it, thus obtaining the antibacterial hydrogel.

2. The method for preparing the antibacterial hydrogel according to claim 1, characterized in that, The method for preparing thiolized tannic acid in step S1 is as follows: Tannic acid and a mercapto-containing compound were dissolved in deionized water, the pH was adjusted, and the reaction was carried out at room temperature in the dark. After the reaction was completed, the reaction product was purified by dialysis and freeze-dried to obtain mercapto-tannic acid.

3. The method for preparing the antibacterial hydrogel according to claim 1, characterized in that, In step S1, the mass-to-volume ratio of methacrylamide gelatin, thiolated tannic acid, and PBS buffer is 50-150 mg: 10-20 mg: 1 mL; the concentration of the PBS buffer is 30-50 mM, and the pH value is 7.0-7.

4.

4. The method for preparing the antibacterial hydrogel according to claim 1, characterized in that, The preparation method of copper-doped Prussian blue nanozyme in step S2 is as follows: Potassium ferrocyanide was dissolved in deionized water to obtain a ligand source solution; copper salt was dissolved in a polyol to obtain a metal source solution; the metal source solution was added dropwise to the ligand source solution under stirring, and the reaction was carried out at a higher temperature. The reaction product was purified by dialysis and freeze-dried to obtain copper-doped Prussian blue nanozyme.

5. The method for preparing the antibacterial hydrogel according to claim 1, characterized in that, In step S2, the mass-to-volume ratio of copper-doped Prussian blue nanozyme, ε-polylysine, and mixed solution A is 3-5 mg: 6-10 mg: 1 mL.

6. The method for preparing the antibacterial hydrogel according to claim 1, characterized in that, In step S3, the mass-to-volume ratio of trehalose, horseradish peroxidase, and mixed solution B is 80-100 mg: 0.3-0.5 mg: 1 mL.

7. The method for preparing the antibacterial hydrogel according to claim 1, characterized in that, In step S4, the photoinitiator is one or more of lithium phenyl-2,4,6-trimethylbenzoylphosphonate, Irgacure 2959, and Irgacure 127; the mass-to-volume ratio of the photoinitiator to the mixed solution C is 2-4 mg: 1 mL.

8. The method for preparing the antibacterial hydrogel according to claim 1, characterized in that, The visible light irradiation conditions in step S5 include: a visible light wavelength range of 400-500nm, a light intensity of 30-50mW / cm², and an irradiation time of 2-5min.

9. An antibacterial hydrogel prepared by the method of any one of claims 1-8.

10. The use of the antibacterial hydrogel as described in claim 9 in the preparation of wound dressings, biosensors, or materials for monitoring inflammatory microenvironments.

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