Silk fibroin drug-loaded hydrogel, preparation method and application thereof

Abstract

This invention relates to a silk fibroin drug-loaded hydrogel, its preparation method, and its application. The silk fibroin drug-loaded hydrogel comprises a hydrogel matrix and nanoparticles, with the nanoparticles loaded onto the hydrogel matrix. The hydrogel matrix comprises phenylboronic acid-grafted methacrylamide silk fibroin and caffeic acid-grafted chitosan. The nanoparticles comprise nanozymes and polydopamine, with the polydopamine coating the nanozymes. The nanozymes comprise epigallocatechin gallate and honeycomb manganese dioxide, with the epigallocatechin gallate and honeycomb manganese dioxide forming the nanozymes through coordination. This invention employs a dual-sequential crosslinking strategy of "dynamic adaptation followed by permanent fixation," simultaneously incorporating EGCG interface-modified MnO2 nanozymes. The resulting hydrogel exhibits superior mechanical strength and unique pH / ROS dual-response drug release characteristics, while avoiding potential damage to the wound surface from explosive gas generation, thus providing a long-lasting controlled-release effect.

Description

Technical Field

[0001] This invention relates to the field of hydrogel technology, and in particular to a silk fibroin drug-loaded hydrogel, its preparation method, and its application. Background Technology

[0002] The skin serves as the body's primary barrier against exogenous pathogens. The repair of infected chronic wounds is a major clinical challenge worldwide, with persistent bacterial infection, oxidative stress damage, inflammatory imbalance, and impaired angiogenesis creating a vicious cycle. Invasion by microbial pathogens such as *Escherichia coli*, *Staphylococcus aureus*, and *Pseudomonas aeruginosa* is a leading cause of infection in infected skin wounds. These pathogens hinder the healing process, leading to unnecessary inflammatory responses, complicating treatment, prolonging treatment cycles, and increasing the healthcare burden. Therefore, accelerating the repair of infected wounds has significant clinical value in improving patient health outcomes and conserving medical resources.

[0003] The core reason why these types of wounds are difficult to heal lies in the formation of a malignant pathological loop in the wound area: bacterial infection, oxidative stress, inflammatory dysregulation, and impaired angiogenesis. Currently, various wound dressings are widely used for repairing infected wounds, among which hydrogels have attracted much attention due to their excellent physicochemical properties and biocompatibility. However, traditional hydrogels lack antibacterial capabilities, making it difficult to meet the treatment needs of infected wounds. Researchers have introduced antibacterial components into traditional hydrogels, such as antibacterial drugs, metal nanoparticles (e.g., silver nanoparticles), antimicrobial peptides, and natural plant extracts. However, current antibacterial hydrogels still have the following problems: low drug loading capacity, poor stability, and most release antibacterial agents through physical diffusion, leading to initial burst release and insufficient drug concentration in the later stages.

[0004] First, current research on antibacterial hydrogels, such as those containing antibiotics, metal nanoparticles, antimicrobial peptides, and cationic polymers, suffers from low drug loading and poor stability. Antibacterial hydrogels formed by covalently modifying antibacterial components into the hydrogel system often involve complex synthesis methods, high costs, poor reproducibility, and difficulty in large-scale production. Furthermore, the modification process can easily impair the activity of the antibacterial agent. Second, the lack of selectivity for the unique physiological environment of wounds (low pH and high reactive oxygen species, ROS) and normal skin also affects therapeutic efficacy. Finally, controlling drug release is challenging, making it difficult to maintain continuous wound closure. Therefore, patients often require frequent medication, impacting treatment adherence and increasing the likelihood of drug toxicity and side effects.

[0005] Silk fibroin (SF) is a structural protein and an excellent natural biomaterial that can be processed into various medical products. SF hydrogels possess excellent biocompatibility, tissue regeneration capacity, minimal immunogenicity, high oxygen and water permeability, high stability, and sustainable release capability. However, pure silk fibroin hydrogels lack antibacterial activity, making them unsuitable for treating infected wounds. Natural antibacterial peptides with good safety profiles and broad antibacterial spectrums, effectively inhibiting bacteria, fungi, and viruses, have shown significant potential in constructing composite antibacterial hydrogels. However, due to the large amount of exudate in infected wounds, the combined use of SF and antibacterial agents in hydrogels alone does not appear to be effective for long-term wound closure and drug delivery.

[0006] In recent years, the prognosis and treatment of infected wounds have received widespread attention. However, conventional drugs cannot simultaneously meet multiple treatment needs and have low utilization rates. Therefore, the development of an anti-inflammatory, antibacterial, antioxidant, and sustained-release material remains a challenge. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention aims to develop a novel therapeutic material. Unlike the traditional strategy of "physically embedding drugs," this invention constructs a novel MnO2-based nanozyme through EGCG interface coordination engineering and loads it into a dual-network hydrogel characterized by "dynamic borate ester bond pre-crosslinking + photocuring permanent crosslinking." This material overcomes the technical bottlenecks of traditional wound antibacterial repair materials, which rely on single antibacterial agents such as antibiotics, antimicrobial peptides, and cationic polymers, leading to bacterial resistance, poor biocompatibility, short duration of action, and high risk of side effects. It constructs a programmable antibacterial, anti-inflammatory, and antioxidant mechanism, achieving dual-response release of the drug based on both pH and ROS.

[0008] The specific technical solution of the present invention is as follows: A silk fibroin drug-loaded hydrogel (SilMA-PBA / CS-CA / P-EMN), characterized in that the silk fibroin drug-loaded hydrogel comprises a hydrogel matrix and nanoparticles, wherein the nanoparticles are loaded on the hydrogel matrix. The hydrogel matrix comprises phenylboronic acid-grafted methacrylamide silk fibroin (SilMA-PBA) and caffeic acid-grafted chitosan (CS-CA). The nanoparticles (P-EMN) include nanozymes (EGCG-Mn) and polydopamine (PDA), wherein the polydopamine coats the nanozymes; The nanozyme comprises epigallocatechin gallate (EGCG) and honeycomb manganese dioxide (HM), which form the nanozyme through coordination.

[0009] In one embodiment, the mass ratio of the phenylboronic acid-grafted methacrylamide silk fibroin to the caffeic acid-grafted chitosan to the nanoparticles is (100~400):(10~20):1.

[0010] A second aspect of the present invention also provides a method for preparing the above-mentioned silk fibroin drug-loaded hydrogel, comprising the following steps: Preparation of nanoparticles: honeycomb manganese dioxide was prepared by adding honeycomb manganese dioxide solution to epigallocatechin gallate solution, stirring at room temperature, centrifuging to collect the precipitate, washing to obtain nanozyme, dispersing nanozyme in buffer solution, adding dopamine, stirring to react, centrifuging to collect the product, and washing to obtain nanoparticles. Preparation of methacrylamide-grafted silk fibroin: Silk fibroin (SF) was extracted, and the SF was added to lithium bromide solution. The mixture was stirred, concentrated hydrochloric acid was added, centrifuged, filtered, and then glycidyl methacrylate (GMA) was added and mixed. The mixture was stirred, centrifuged, filtered, dialyzed, and lyophilized to obtain methacrylamide-grafted silk fibroin. 3-Aminophenylboronic acid (3-APBA), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were sequentially added to dimethyl sulfoxide (DMSO) and stirred to obtain a 3-aminophenylboronic acid solution. Methacrylamide-grafted silk fibroin was dissolved in water to obtain a methacrylamide-grafted silk fibroin solution. The 3-aminophenylboronic acid solution was added to the methacrylamide-grafted silk fibroin solution, and the mixture was stirred at room temperature. The pH was adjusted, dialyzed, and lyophilized to obtain the final product. Preparation of caffeic acid-grafted chitosan: Chitosan (CS) was dissolved in water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added, the pH was adjusted, caffeic acid was added, the pH was adjusted again, the reaction was carried out in the dark, dialyzed, and lyophilized to obtain the product. Preparation of drug-loaded silk fibroin hydrogel: Benzylboronic acid-grafted methacrylamide silk fibroin was dissolved in a photoinitiator solution to obtain a Benzylboronic acid-grafted methacrylamide silk fibroin solution. Caffeic acid-grafted chitosan was dissolved in a buffer solution containing nanoparticles to obtain a caffeic acid-grafted chitosan solution. The Benzylboronic acid-grafted methacrylamide silk fibroin solution was added to the caffeic acid-grafted chitosan solution, sodium hydroxide was added, the mixture was shaken, and then irradiated with light to obtain the drug-loaded silk fibroin hydrogel.

[0011] In one embodiment, the method for preparing the honeycomb-shaped manganese dioxide includes the following steps: Dissolve potassium permanganate in water, add oleic acid, react, centrifuge, and wash to obtain the product. According to the dosage ratio, the potassium permanganate : oleic acid is (0.1~0.3) g : 1 mL; The reaction time is 4-8 hours.

[0012] In one embodiment, during the preparation of the nanoparticles, the ratio of the honeycomb manganese dioxide to the epigallocatechin gallate is 1:1 by mass. The ratio of the nanozyme to the dopamine is 1:1 by mass. The stirring time at room temperature is 1-3 hours, and the stirring reaction time is 4-8 hours.

[0013] In one embodiment, the buffer solution used in the preparation of the nanoparticles is tris-hydroxymethylaminomethane hydrochloride (Tris-HCl).

[0014] In one embodiment, in the preparation of phenylboronic acid-grafted methacrylamide silk fibroin, the ratio of silk fibroin to lithium bromide to concentrated hydrochloric acid to glycidyl methacrylate is (10~30) g: (0.5~1.5) mol: (0.5~2) mL: (10~15) mL. According to the mass ratio, the 3-aminophenylboronic acid : the methacrylamide silk fibroin is (0.004~0.008 : (0.1~0.5). The stirring reaction time is 6-10 hours, and the room temperature stirring reaction time is 40-50 hours.

[0015] In one embodiment, in the preparation of phenylboronic acid-grafted methacrylamide silk fibroin, the pH adjustment is performed by adjusting the pH to 7.3-7.5 using NaOH solution.

[0016] In one embodiment, in the preparation of caffeic acid-grafted chitosan, the chitosan to caffeic acid ratio is (1~2):(1~2) by mass. The light-protected reaction time is 20~26h.

[0017] In one embodiment, in the preparation of the silk fibroin drug-loaded hydrogel, the mass ratio of the phenylboronic acid-grafted methacrylamide silk fibroin to the caffeic acid-grafted chitosan to the nanoparticles is (100~400):(10~20):1.

[0018] In one embodiment, the photoinitiator in the preparation of the silk fibroin drug-loaded hydrogel is LAP.

[0019] A third aspect of the present invention also provides the application of the above-described silk fibroin drug-loaded hydrogel or the silk fibroin drug-loaded hydrogel obtained by the above preparation method in the preparation of drugs for repairing infected wounds.

[0020] In a fourth aspect, the present invention also provides a medicament for repairing infected wounds, comprising the above-described silk fibroin drug-loaded hydrogel or the silk fibroin drug-loaded hydrogel prepared by the above-described preparation method.

[0021] This invention discloses a novel multifunctional intelligent hydrogel drug delivery system. The core of this hydrogel is constructed by grafting caffeic acid onto chitosan (CS-CA) and combining it with phenylboronic acid-modified methacrylamide silk fibroin (SilMA-PBA). Simultaneously, it loads a honeycomb-like manganese dioxide nanozyme (P-EMN) coated with polydopamine (PDA) and in-situ coordinated with EGCG, ultimately creating a SilMA-PBA / CS-CA / P-EMN (SPCC / P-EMN) hydrogel dressing with dual reactive oxygen species (ROS) and pH responsiveness. This invention, by introducing the photocuring properties of methacrylamide silk fibroin (SilMA), innovatively employs a dual-sequential crosslinking strategy of "dynamic adaptation followed by permanent fixation," solving problems such as low mechanical strength, easy detachment due to wound exudate, rapid degradation, and easy drug burst release associated with hydrogels with single dynamic borate ester crosslinking.

[0022] The four core pathological features of difficult-to-heal infected wounds are: (1) bacteria are easily colonized, and the infection is difficult to control repeatedly; (2) excessive reactive oxygen species (ROS) cause oxidative stress to damage tissues; (3) macrophages remain in a pro-inflammatory state, and inflammation continues; (4) local ischemia and hypoxia cause slow regeneration of new blood vessels and granulation tissue.

[0023] To address the aforementioned pathological challenges, epigallocatechin-3-gallate (EGCG) was selected as the core active repair factor. EGCG is the most abundant polyphenol in green tea, possessing antibacterial, anti-inflammatory, antioxidant, and angiogenic effects. However, EGCG suffers from poor physicochemical stability and low bioavailability. Therefore, this invention abandons the traditional physical adsorption drug delivery strategy and adopts an in-situ interfacial coordination strategy: utilizing the galloyl and catechol groups abundant in the EGCG molecule, Mn-OC coordination bonds are formed with the Mn(IV) sites on the honeycomb MnO2 surface to construct EGCG-Mn nanozymes (EMN). This coordination not only improves the stability of EGCG but also reconstructs the electronic structure of the MnO2 surface, giving it mild enzyme-like catalytic activity. Based on this, polydopamine (PDA) was used to coat the surface of EMN. PDA nanoparticles can further remove ROS from the wound and reduce oxidative damage to cells, ultimately achieving a synergistic effect of efficient removal of ROS from the wound and precise relief of oxidative stress.

[0024] Silk fibroin (SF), derived from natural silkworm silk, is a high-molecular-weight polypeptide material with a unique segmental structure and tunable conformation. It exhibits excellent mechanical properties, good biocompatibility, and biodegradability, and is FDA-approved, making it a highly promising biomaterial. However, SF suffers from poor water solubility and spontaneous gelation. Methacrylation modification yields photocurable silk fibroin (SilMA), which significantly improves its water solubility and stability, enabling rapid photocuring in an aqueous environment. Phenylboronic acid (PBA) is a typical smart responsive functional group that can form reversible dynamic borate ester bonds with cis-diol structures in aqueous solution. This dynamic covalent bond not only endows the hydrogel with excellent self-healing properties, but PBA also produces borate ester bonds in weakly alkaline solutions. These borate ester bonds are sensitive to key microenvironmental signals in the wound, such as pH, ROS, and glucose, acting as a molecular responsive switch for the smart hydrogel to adapt to the wound environment. By covalently grafting PBA onto the surface of SilMA, a three-dimensional hydrogel network with tunable structure and dynamic stability can be constructed without the addition of complex crosslinking agents, resulting in SilMA-PBA, a smart hydrogel material that combines reversible crosslinking properties with multiple environmental responsiveness after modification.

[0025] Caffeic acid (CA) is a natural phenolic acid compound widely found in plants. Its molecular structure contains bifunctional groups of catechol and acrylic acid, giving it excellent cell affinity and adhesion to wet wounds. Furthermore, the dynamic bonds of CA catechol are easily broken and can be reversibly reconstructed upon damage, endowing CA hydrogels with rapid self-healing capabilities. Chitosan (CS) is a natural cationic polysaccharide with inherent antibacterial, hemostatic, and immunomodulatory properties. As a natural polymer, CS offers significant advantages in treating infected chronic wounds, overcoming the limitations of traditional antibacterial drugs in addressing bacterial resistance. Moreover, its molecular chain contains active amino and hydroxyl groups, making it easily chemically modified, and its mucosal adhesion can prolong the retention time of the hydrogel on the wound surface. Grafting caffeic acid onto chitosan (CS-CA) allows for covalent grafting of amide bonds between chitosan and caffeic acid under activation. Its self-healing properties and ability to dynamically adhere to irregular wounds provide a natural material for drug-resistant infected wounds.

[0026] The borate ester bond of PBA can bind to the catechol group of caffeic acid (CA). Therefore, this invention combines CS-CA with SilMA-PBA and loads P-EMN functional nanoparticles to finally construct a SilMA-PBA / CS-CA / P-EMN hydrogel dressing with dual reactive oxygen species (ROS) and pH responsiveness. This effectively solves the major limitations of traditional antibacterial materials in terms of drug resistance, mechanical properties, biocompatibility, and long-term antibacterial effect.

[0027] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention combines nanotechnology and smart responsive materials to, for the first time, modify CS by grafting CA onto CS, then mixes it with phenylboronic acid-modified SF, and simultaneously incorporates MnO2 nanozymes with EGCG interface coordination modification to construct a ROS- and pH-responsive SilMA-PBA / CS-CA / P-EMN hydrogel dressing, which can effectively overcome the shortcomings of existing treatment methods.

[0028] 2. The novel SilMA-PBA / CS-CA / P-EMN hydrogel dressing uses silk fibroin (SF) to replace the antibacterial effects of traditional antibiotics, antimicrobial peptides, cationic polymers and other antibacterial agents, solving the problems of low drug loading, poor stability and short duration of action of antibacterial hydrogels.

[0029] 3. The borate groups in the novel SilMA-PBA / CS-CA / P-EMN hydrogel can form borate ester bonds with substances with 1,2 or 1,3-diol structures in weakly alkaline solutions, and these borate ester bonds exhibit temperature, pH, and glucose-responsive properties. Polydopamine (PDA) and EGCG-Mn nanozymes demonstrate synergistic ROS scavenging capabilities. Notably, because EGCG is chemically bonded to the MnO2 surface, not only is drug explosive release avoided, but the hydrogel network also gains additional physical cross-linking points, resulting in the constructed SilMA-PBA / CS-CA / P-EMN hydrogel exhibiting superior mechanical strength and unique pH / ROS dual-responsive drug release characteristics.

[0030] 4. The EGCG-Mn nanozyme (P-EMN) constructed in this invention is different from the violent catalysis of pure MnO2. Due to the coordination modification of EGCG, it achieves a mild and controllable oxygen production capacity, avoids the potential damage to the wound caused by explosive gas production, improves the problem of insufficient oxygen partial pressure in the wound, and has a long-term controlled release effect.

[0031] 5. The introduction of CA into the SilMA-PBA / CS-CA / P-EMN hydrogel endows the hydrogel with excellent antioxidant, adhesion and self-healing properties, enabling it to closely adhere to the wound and maintain a long-term closed microenvironment when dealing with highly exudative infected wounds, thereby achieving stable and efficient drug delivery. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the hydrogel matrix preparation process; Figure 2 TEM images of HM, EMN, and P-EMN; Figure 3 Mapping image of P-EMN nanoparticles; Figure 4This is a particle size distribution diagram of HM nanoparticles; Figure 5 This is a particle size distribution diagram of EMN nanoparticles; Figure 6 This is a particle size distribution diagram of P-EMN nanoparticles; Figure 7 Particle size distribution of P-EMN nanoparticles under different environments; Figure 8 Zeta potential diagrams for three types of nanoparticles: HM, EMN, and P-EMN. Figure 9 NMR spectra of CS and CS-CA; Figure 10 NMR spectra of SF, SilMA, and SilMA-PBA; Figure 11 This is a full-wavelength ultraviolet scan curve of EGCG; Figure 12 This is the standard curve for EGCG. Figure 13 DPPH scavenging rate data for hydrogels with different nanoparticles and nanoparticle-loaded particles; Figure 14 The oxygen production capacity curves of HM and P-EMN nanoparticles under different environments are shown. Figure 15 SEM images of two hydrogels, SilMA-PBA / CS-CA and SilMA-PBA / CS-CA / P-EMN. Figure 16 The strain rheological diagram of the hydrogel is shown. Figure 17 Frequency rheological plot of the hydrogel; Figure 18 This is a step rheological diagram of the hydrogel; Figure 19 This is a shear steady-state diagram of the hydrogel; Figure 20 The time-rheological curve of the hydrogel; Figure 21 This is a graph showing the swelling ratio of the hydrogel. Figure 22 The graph shows the degradation rate of the hydrogel under two different environments. Figure 23 Drug release curves of nanoparticles and nanoparticle-loaded hydrogels under different environments; Figure 24 The appearance of hemolysis after incubation with hydrogels for 8 hours, from left to right: 0.1% Triton X-100, 0.9% NaCl, SPCC, and SPCC / P-EMN; Figure 25 A graph showing the hemolysis rate of the hydrogel; Figure 26 The graphs show the relationship between the adhesion force and displacement of the two groups of hydrogels to pigskin. Figure 27 This is an image of the hydrogel's self-healing mechanism. Detailed Implementation

[0033] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0035] Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all test methods are conventional test methods in this field.

[0036] Example 1 This embodiment provides a method for preparing a silk fibroin drug-loaded hydrogel, including the following steps: 1. Preparation of manganese dioxide oleic acid (HM) nanocarriers 0.1 g of potassium permanganate was completely dissolved in distilled water and stirred continuously for 30 min. Then, 1.0 mL of oleic acid was added to the solution. The emulsion was reacted for 6 h until a brownish-black precipitate was observed. The HM product was centrifuged at 2000 rpm for 20 min, washed repeatedly with deionized water and methanol to remove residual reactants, and then redispersed in deionized water for subsequent experiments.

[0037] 2. Preparation of EGCG-Mn nanozyme (EMN) and PDA coating (P-EMN) 5 mL of HM solution (4 mg / mL) was dispersed in 5 mL of EGCG aqueous solution (4 mg / mL) and stirred at room temperature for 2 h. During this process, the phenolic hydroxyl groups of EGCG undergo an in-situ coordination reaction with Mn ions on the honeycomb MnO2 surface, and EGCG acts as a surface ligand anchored on the MnO2 substrate, forming an EGCG-Mn interfacial active center (denoted as EMN).

[0038] The solution was centrifuged at 12,000 rpm for 10 min and washed three times with water to remove uncoordinated free EGCG. Subsequently, 10 mg of the collected EMN precipitate was dispersed in 10 mL of Tris-HCl (pH=8.5, 10 mM), and 10 mg of dopamine was added, followed by stirring for 6 h. Dopamine underwent oxidative self-polymerization on the EMN surface, forming a polydopamine (PDA) coating layer. The product was then collected by centrifugation, washed twice, and finally dispersed in deionized water and stored at 4 °C for subsequent experiments; this product was designated P-EMN.

[0039] 3. Preparation of phenylboronic acid-grafted methacrylic anhydride-modified silk fibroin (SilMA-PBA) (1) Preparation of methacrylated silk fibroin (SilMA) Weigh 50g of silkworm cocoons (with pupae removed), boil them twice for 30min each time in 1L of 0.05M Na2CO3 (5.3g) solution at 100°C, and wash them several times with distilled water. Then, dry the degummed silk fibroin (SF) in a 60°C oven. Dissolve 20g of degummed SF in 100mL of 9.3M lithium bromide (LiBr) solution (with 0.48g NaOH added), stir at 60°C for 1 hour, and after the degummed SF is completely dissolved, add 1mL of concentrated hydrochloric acid, centrifuge the solution at 10000rpm for 5min, then filter through a miracloth, add 12mL of glycidyl methacrylate, mix, and stir at 1000rpm for 8h at 60°C. After the reaction was complete, the solution was centrifuged at 10,000 rpm for 5 minutes, then filtered through a miracloth and dialyzed with distilled water using a dialysis bag (8-14 kDa) for 3-5 days to remove salts. The solution was then freeze-dried at -80°C to obtain the product SilMA.

[0040] (2) Preparation of phenylboronic acid-grafted methacrylic anhydride-modified silk fibroin (SilMA-PBA) 3-APBA (0.006 g, 0.28 mmol), EDC (0.0546 g, 0.28 mmol), and NHS (0.033 g, 0.28 mmol) were sequentially added to 3 mL of DMSO solution and stirred for 2 h. 0.3 g of SilMA was dissolved in 40 mL of pure water. Then, the activated 3-APBA solution was added dropwise to the SilMA solution using a 1 mL syringe. The mixture was stirred at room temperature for 48 h. After the reaction was complete, the pH was adjusted to approximately 7.4 with 0.1 M NaOH solution. The resulting solution was then placed in a 14000 DA dialysis bag and dialyzed for three days, with the water changed every 4 h. After dialysis, the solution was freeze-dried to obtain the product SilMA-PBA, which was stored at 4 °C for later use.

[0041] 4. Preparation of porous sponge-like chitosan-caffeic acid material (CS-CA) Weigh 5g of CS and dissolve it in 200mL of deionized water, stirring until homogeneous. Then add 6.75g of EDC and 6g of NHS, adjust the pH to 4.7 using 1M NaOH, add 5g of caffeic acid (CA), and continue adjusting the pH to 4.7. React at room temperature in the dark for 24 hours. After the reaction is complete, dialyze the solution through a dialysis bag (14000Da) for three days. Lyophilize the product to obtain the CS-CA sponge.

[0042] 5. Preparation of SilMA-PBA / CS-CA / P-EMN hydrogel First, SilMA-PBA / CS-CA hydrogels were prepared.

[0043] Weigh 0.3g of SilMA-PBA and dissolve it in 1mL of deionized water containing 0.25% LAP. Dissolve at 4℃ to obtain a 30% SilMA-PBA solution. Weigh 0.03g of CS-CA and dissolve it in 1mL of PBS solution. Dissolve by sonication and a 50℃ water bath to obtain a 3% CS-CA solution. Add 200μL of 3% CS-CA to a 2mL centrifuge tube, and add 400μL of 30% SilMA-PBA solution while shaking. Then add 20μL of 1M NaOH solution and continue shaking for 30s to form a microgel. Place the hydrogel under a UV lamp (365nm, 6.9mW cm⁻¹). -2 Irradiation for 30 seconds yields a cured and cross-linked SilMA-PBA / CS-CA (SPCC) hydrogel.

[0044] Finally, the SilMA-PBA / CS-CA / P-EMN hydrogel was prepared.

[0045] Weigh 0.3g of SilMA-PBA and dissolve it in 1mL of deionized water containing 0.25% LAP. Dissolve at 4℃ to obtain a 30% SilMA-PBA solution. Weigh 0.03g of CS-CA and dissolve it in 1mL of PBS containing 2mg P-EMN. Dissolve by sonication and a 50℃ water bath to obtain a 3% CS-CA solution. Add 200μL of 3% CS-CA to a 2mL centrifuge tube, and add 400μL of 30% SilMA-PBA solution while shaking. Then add 20μL of 1M NaOH solution and continue shaking for 30 seconds to form a microgel. Place the hydrogel under a UV lamp (365nm, 6.9mW cm⁻¹). -2 Irradiation for 30 seconds yields a cured and cross-linked SilMA-PBA / CS-CA / P-EMN hydrogel.

[0046] Figure 1This is a schematic diagram of the hydrogel matrix preparation process in this embodiment.

[0047] Implementation effect evaluation 1. Nuclear Magnetic Resonance (NMR) Test Samples: SF, SilMA, SilMA-PBA, CS, CS-CA Test method: 15 mg of sample was weighed and dissolved in an appropriate amount of deuterated reagent (deuterated water D2O or deuterated chloroform CDCl3), and then placed into a clean NMR tube. The NMR structure was determined by an NMR spectrometer at room temperature, and the spectrum was analyzed using MestReNova software.

[0048] Test results: Figure 9 The NMR spectra of CS and CS-CA are shown in the figure. As can be seen from the figure, both curves have dense and complex signals in the δ=3.0~4.0ppm region, which is characteristic of hydrogen on the sugar ring of the CS skeleton. The peak shape of CS-CA in this region is different from that of CS. This is due to the change in the surrounding chemical environment after grafting CA. Therefore, it can be determined that CA modification of CS is successful.

[0049] from Figure 10 It can be seen that, compared to SF, the proton peaks at chemical shifts of SilMA at 5.61 ppm and 6.04 ppm are characteristic peaks of the double bonds on the grafted glycidyl methacrylate, indicating that GMA successfully modified SF. Meanwhile, the chemical shifts of SilMA-PBA... 1 The presence of proton peaks on the benzene ring in the ¹H NMR range of δ=7.0~8.0ppm indicates that SilMA has been successfully grafted with phenylboronic acid groups.

[0050] 2. Transmission electron microscopy (TEM) testing Samples: HM, EMN, P-EMN Test method: The sample solution was diluted to a certain concentration, ultrasonically dispersed, and the solution was dropped onto a copper grid using a 20 μL pipette. Then, 2% phosphotungstic acid was used for negative staining and dropped onto the copper grid. After natural evaporation, the internal morphology of the nanoparticles was observed using a transmission electron microscope.

[0051] Test results: Figure 2 The images show TEM images of three groups of nanoparticles, revealing a typical honeycomb satellite structure. Transmission electron microscopy (TEM) analysis shows that the three groups of nanoparticles are uniformly sized, centrally distributed, and approximately 160 nm in diameter. The images demonstrate that after EGCG coordination modification and PDA coating, the nanoparticles maintained their intact honeycomb morphology, proving that the coordination process did not disrupt the carrier structure. Figure 3It can be seen that C, N, O, and Mn elements are all distributed on P-EMN, and the presence of these elements verifies the successful synthesis of P-EMN nanoparticles.

[0052] 3. Dynamic Light Scattering (DLS) Test Samples: HM, EMN, P-EMN Test method: The solution was diluted to 100 μg / mL, ultrasonically dispersed, and the average particle size and zeta potential of the nanoparticles were tested using a Malvern particle size analyzer.

[0053] Test results: Figure 4-7 DLS results showed that the average particle sizes of HM, EMN, and P-EMN nanoparticles were 169.65 nm, 168.59 nm, and 164.03 nm, respectively, with PDI values ​​of 0.147, 0.113, and 0.085. After loading EGCG and incorporating PDA, the average particle size of the nanoparticles decreased compared to the blank HM nanoparticles. This clearly indicates that EGCG molecules are not simply physically adsorbed, but rather tightly anchored to the nanoparticle surface through coordination, resulting in a densification change in particle size.

[0054] Figure 8 The Zeta potential results showed that the potentials of HM, EMN, and P-EMN nanoparticles were -17.77 mV, -16.09 mV, and -15.33 mV, respectively. After loading EGCG, the absolute value of the negative charge on the nanoparticle surface decreased (from -17.77 mV to -16.09 mV). This is because the phenolic hydroxyl groups of EGCG replaced the negatively charged -OH groups on the MnO2 surface, forming Mn-OC bonds, thereby shielding part of the surface negative charge, directly confirming the occurrence of the coordination reaction. After the PDA was added, the absolute value of the negative charge on the nanoparticle surface further decreased (from -16.09 mV to -15.33 mV), indicating that the loading of the highly electronegative PDA partially neutralized or shielded the original negative charge on the nanoparticle surface.

[0055] 4. Plotting the EGCG standard curve Accurately weigh 1 mg of EGCG powder and dissolve it in 1 mL of deionized water to prepare a 1 mg / mL EGCG stock solution. Then, prepare a series of EGCG solutions with concentration gradients of 5, 10, 15, 20, and 25 μg / mL using appropriate amounts of the stock solution. Measure the absorbance of EGCG at 274 nm using a UV-Vis absorption spectrometer. Combine the measured absorbance values ​​of EGCG with their corresponding concentrations to create a scatter plot, and calculate the linear regression equations for each. The goodness of fit R is required to be... 2 Greater than 0.999.

[0056] result: Figure 11This is a full-wavelength UV scan of EGCG. The data in the image show that the absorbance of EGCG at 274 nm increases significantly with increasing solution concentration. Figure 12 The standard curve shows that the correlation coefficient between the absorbance value of EGCG and the corresponding 0.21 concentration curve is 0.9999, indicating that the correlation meets the requirements.

[0057] 5. Determination of drug loading rate and encapsulation efficiency of nanoparticles Calculate the encapsulation efficiency and drug loading rate using the following formulas: (1) Encapsulation efficiency (%) = (Amount of EGCG added during drug loading - Content of free EGCG) / Amount of EGCG added during drug loading × 100% (2) Drug loading rate (%) = EGCG content in nanoparticles / total mass of nanoparticles × 100% The drug loading and encapsulation efficiency results of EMN and P-EMN nanoparticles are shown in Table 1.

[0058] Table 1

[0059] The data in the table show that the encapsulation efficiencies of EMN and P-EMN nanoparticles are 45.28±0.21 and 40.32±0.41, respectively, and their drug loading efficiencies are 82.76±0.7 and 67.58±1.14, respectively.

[0060] 6. Scanning Electron Microscopy (SEM) Testing Samples: SilMA-PBA / CS-CA (SPCC), SilMA-PBA / CS-CA / P-EMN (SPCC / P-EMN) Test method: The prepared 400 μL hydrogel was pre-frozen in a -20℃ freezer, then freeze-dried in a freeze dryer, and gold was sprayed onto the surface of the hydrogel for 30 seconds. The surface morphology of the hydrogel was observed using a scanning electron microscope.

[0061] Test results: Figure 15The images show SEM microscopic images of SilMA-PBA / CS-CA and SilMA-PBA / CS-CA / P-EMN hydrogels. As can be observed, the SilMA-PBA / CS-CA hydrogel exhibits a typical, relatively loose, porous three-dimensional network structure. Compared to the SilMA-PBA / CS-CA hydrogel, the SilMA-PBA / CS-CA / P-EMN hydrogel shows a significant change in microstructure, with a highly dense network structure, more interlacing points, and smaller pores. This is because the EGCG ligands on the surface of the P-EMN nanoparticles contain abundant phenolic hydroxyl groups, which form additional hydrogen bonds or coordination interactions with groups (such as carboxyl and hydroxyl groups) on the SilMA-PBA / CS-CA polymer chains, increasing the number of physical cross-linking points and thus restricting the movement of the polymer chains.

[0062] 7. Swelling performance test Samples: SilMA-PBA / CS-CA (SPCC), SilMA-PBA / CS-CA / P-EMN (SPCC / P-EMN) Test method: Weigh the hydrogel initially (W0), then immerse it in PBS buffer (pH=7.4) at 37℃. Remove the hydrogel at different time points, wipe off surface moisture with filter paper, and weigh the hydrogel to obtain its weight (W). t The swelling ratio of the hydrogel can be calculated as follows: Swelling rate = (W t -W0) / W0×100% Test results: Figure 21 The swelling rate data of the two hydrogels showed that both hydrogels reached swelling equilibrium after soaking in PBS for approximately 8 hours. The equilibrium swelling rates of SilMA-PBA / CS-CA and SilMA-PBA / CS-CA / P-EMN hydrogels after 8 hours were 11.79% and 9.42%, respectively. Compared with the blank hydrogel, the swelling rate of the P-EMN nanoparticle-loaded hydrogel was reduced, indicating that the nanoparticles may act as physical cross-linking points or interact with the polymer chains, enhancing the cross-linking density of the network structure. The tighter cross-linking network restricts the movement and unfolding of the polymer chains, reducing the penetration and diffusion rate of water molecules, thus leading to a decrease in the swelling rate.

[0063] 8. Self-healing performance test Sample: SilMA-PBA / CS-CA / P-EMN (SPCC / P-EMN) Test Method: The self-healing performance of hydrogels was tested on a macroscopic scale using a fracture-healing experiment. Two hydrogels were prepared, one stained with methylene blue, and cut into two pieces. The fracture surfaces of the hydrogels of different colors were brought into close contact and placed in a petri dish. The dish was covered to prevent moisture evaporation and left at room temperature without external stimuli for 1 hour. The healing phenomenon at the fracture surface of the hydrogel was then observed to evaluate the self-healing behavior of the hydrogel.

[0064] Test results: From Figure 27 As can be seen, after shearing, when the two cut surfaces of the SilMA-PBA / CS-CA / P-EMN hydrogel are placed together, the structure recovers completely after 3 minutes of contact without any obvious cracks, demonstrating the excellent self-healing properties of the hydrogel.

[0065] 9. Viscosity test Samples: SilMA-PBA / CS-CA, SilMA-PBA / CS-CA / P-EMN The hydrogel was injected between two pieces of porcine skin tissue, each 30 mm long and 10 mm wide. An additional 200 g weight was applied to the specimens for 5 minutes to enhance adhesion. Subsequently, the specimens were tested at a constant tensile speed of 1 mm / min until complete separation. Three parallel samples were tested in each group (n=3). The adhesive force was calculated using the following formula: Adhesion force (Pa) = F / S Test results: From Figure 26 It can be seen that the SPCC hydrogel without P-EMN exhibits stronger initial adhesion performance under small displacements, with a peak adhesion stress of approximately 4 kPa. While the initial peak stress of the SPCC / P-EMN system with P-EMN is slightly reduced by about 3.6 kPa, it demonstrates superior adhesion retention and toughness during large displacement tensile stages, with a residual adhesion stress (~2.0 kPa) at a displacement of 10 mm higher than that of the pure SPCC system (~1.8 kPa). This difference stems from the fact that P-EMN doping may interfere with rapid interfacial bonding in the initial stage to some extent, but it significantly improves the material's anti-slip and fatigue resistance, enabling it to maintain a more stable interfacial bond through mechanical interlocking or secondary action under long-term or large deformation.

[0066] 10. Degradation performance test Sample: SilMA-PBA / CS-CA / P-EMN Test method: First, freeze-dry the hydrogel and accurately weigh it as W0. Then, immerse the test sample in PBS solution containing 0 or 1000 U / mL lysozyme and place it in a constant temperature shaker (37°C, 70 rpm). At the measurement time point, wash the hydrogel with ultrapure water, freeze-dry it, and weigh it as W. tThe weight percentage of the hydrogel is calculated using the following formula: Weight loss rate (%) = W t / W0×100% Test results: Figure 22 The graph shows the mass retention rates of the SilMA-PBA / CS-CA / P-EMN hydrogel under two different environments. The graph indicates that the hydrogel degrades faster in the environment with lysozyme, and its mass retention rate is consistently lower than that in the enzyme-free group. In the presence of lysozyme, the hydrogel is completely degraded in 10 days, while under enzyme-free conditions, it reaches complete degradation in 14 days. This comparison suggests that the presence of lysozyme may induce enzymatic hydrolysis of the hydrogel, disrupting its cross-linked structure and leading to rapid degradation. These data demonstrate that the SilMA-PBA / CS-CA / P-EMN hydrogel exhibits good biodegradability.

[0067] 11. In vitro drug release test Samples: P-EMN, SilMA-PBA / CS-CA / P-EMN (SPCC / P-EMN) Test method: Place 1 mL of sample in 5 mL of PBS solution and incubate in a constant temperature shaker (37℃, 70 rpm). Collect 500 μL of PBS supernatant at different time points, and then add 500 μL of fresh PBS solution. Determine the concentration of EGCG in the collected supernatant using a spectrophotometer according to a pre-established standard curve, and calculate the cumulative release rate using the formula: Cumulative release rate (%) = Cumulative drug release / Total drug content × 100% Test Results: The EGCG release behavior of P-EMN nanoparticles and SilMA-PBA / CS-CA / P-EMN hydrogels was evaluated under different conditions. Figure 23 As shown, a small amount of EGCG release was detected in P-EMN nanoparticles and hydrogels in a PBS environment (pH=7.4) without H2O2. After adding 100 μM H2O2 to PBS (pH=5.0), P-EMN nanoparticles cumulatively released 82.75% of EGCG after 48 hours. However, during the same 48-hour incubation period, the cumulative EGCG release from SilMA-PBA / CS-CA / P-EMN hydrogel decreased to 59.16% after adding 100 μM H2O2 to PBS (pH=5.0). This is attributed to the chemical bond (Mn-OC) between EGCG and MnO2, which allows the drug to be released not through simple physical diffusion, but through microenvironmental response (pH / ROS) triggering the breaking of chemical bonds, thus achieving a more precise programmed release.

[0068] 12. Determination of DPPH free radical scavenging rate of nanoparticles and hydrogels DPPH· is a stable N-centered chromogenic free radical that produces a characteristic absorption peak at 517 nm. Antioxidants in the sample reduce DPPH· until it disappears; the change in absorbance is linearly related to the content of antioxidants within a certain range. It can be used to test the antioxidant capacity of nanoparticles and nanoparticle-loaded hydrogels.

[0069] Samples: HM, P-EMN, SilMA-PBA / CS-CA / HM (SPCC / HM), SilMA-PBA / CS-CA / P-EMN (SPCC / P-EMN) Test methods: For the nanoparticle group, two groups of nanoparticles at specific concentrations were mixed with DPPH solution at a 1:1 ratio. For the hydrogel group, the nanoparticle-loaded hydrogel was soaked in deionized water, and the supernatant was extracted and mixed with DPPH solution at a 1:1 ratio. After incubation at 37°C for 30 minutes, the absorbance change at 517 nm was measured. The blank group consisted of 250 μL of ethanol and 250 μL of DPPH solution; the control group consisted of 250 μL of ethanol and 250 μL of sample solution; the experimental group consisted of a mixed solution containing 250 μL of sample solution and 250 μL of DPPH solution. The absorbance values ​​of these groups were recorded as A for the blank group. blank A control group control and experimental group A experiment .

[0070] Clearance rate calculation formula: Clearance rate (%) = 1 - (A) experiment -A control ) / A blank ×100%.

[0071] Test results: Figure 13 The graph shows the DPPH· radical scavenging rates of HM and P-EMN nanoparticles and hydrogels loaded with HM and P-EMN nanoparticles. As can be seen from the graph, the DPPH· radical scavenging rate of the nanoparticles increases after the addition of EGCG. This is because EGCG is the most abundant catechin compound in green tea and has a significant DPPH· radical scavenging ability. The addition of EGCG increases the number of reactive sites in the nanoparticle system. More reactive sites can more effectively capture and scavenge DPPH· radicals, thus improving the radical scavenging rate. Compared with the nanoparticle group, the DPPH· radical scavenging rate of the hydrogel group is lower. This is because the nanoparticles are embedded in the cross-linked network structure of the hydrogel and need to break through the hydrogel's constraints to be released. Since the hydrogel itself does not have a significant DPPH· radical scavenging ability, the DPPH· radical scavenging rate of this group is lower than that of the pure nanoparticle group.

[0072] 13. Determination of the oxygen production capacity of nanoparticles Samples: HM, P-EMN Test method: The oxygen production capacity of the prepared nanoparticles was quantitatively determined using a portable dissolved oxygen meter. Nanoparticles (100 μg / mL) were incubated with 100 μM H₂O₂ for 20 min, and the dissolved oxygen content in the solution was measured at 0, 1, 2, 5, 10, and 20 min. The dissolved oxygen content in distilled water was used as a control.

[0073] Test results: Figure 14 The graphs show the oxygen production capacity of HM and P-EMN nanoparticles under different environments. In the absence of hydrogen peroxide, neither nanoparticle exhibited significant oxygen production capacity. After incubation with H2O2, both HM and P-EMN nanoparticles produced a large amount of O2. The HM group reacted rapidly with H2O2, producing numerous bubbles and exhibiting a more vigorous reaction. The P-EMN nanoparticles produced oxygen at a slower rate. This may be because PDA acts as a protective shell for the nanozyme structure, reducing the nanoparticle's activity and oxygen production capacity.

[0074] 14. Rheological property testing Sample: SilMA-PBA / CS-CA / P-EMN Test method: Steady-state shear, strain scan, frequency scan, and step scan were performed sequentially using a parallel plate (40mm) measuring fixture in a DHR-2 rotational rheometer (TA Instruments, USA).

[0075] Step scan: After time scan, the temperature was set to 25℃, and the self-healing performance of the sample was characterized by scanning step strain test. The test was performed by an amplitude loop between small strain (1%) and large strain (1000%), with a strain interval of 75s.

[0076] Strain scanning: Prepare a gel sample of the corresponding concentration on the rheometer sample stage, quickly lower the parallel plate to make the measurement interval 0.1 mm, set the temperature to 25℃, select the change law as "logarithmic change", set the strain rate to γ=0.1~10000%, angular frequency ω=10rad / s, and the sampling time is determined by the equipment. Observe the changes of sample modulus G' and G” with amplitude.

[0077] Steady-state shear: Viscosity was measured in strain-controlled mode, with shear rates ranging from 0.1 to 100 s⁻¹. -1 At a temperature of 25°C, the apparent viscosity was recorded as a logarithmic function of the shear rate.

[0078] Frequency scanning: After amplitude scanning, set the temperature to 25℃, select a constant strain value, shear strain γ=0.5%, frequency change to "logarithmic change", angular frequency ω=1~100rad / s, set the sampling time control to be set by the equipment, and observe the changes of sample modulus G' and G" with frequency.

[0079] Test results: Figure 16 The results show that as strain increases, the storage modulus (G') is greater than the loss modulus (G' > G") in the early stage, and the hydrogel remains in a gel state. After exceeding the critical point, the loss modulus becomes greater than the storage modulus (G" > G'), and it transforms into a solution state, undergoing a gel-sol transition. This indicates that the hydrogel has certain injectable properties.

[0080] Figure 17 The frequency rheological plot of the hydrogel shows that, under constant oscillation amplitude, the storage modulus (G') is always higher than the loss modulus (G”), indicating that the network structure of the hydrogel is stable (G'>G”).

[0081] from Figure 18 The results show that at the beginning of the first cycle, the value of G' is greater than that of G” at 1% strain. At 1000% strain, G' suddenly decreases and the value of G” exceeds that of G', at which point the hydrogel network structure is completely destroyed due to the breaking of borate ester bonds. When the applied strain is restored to 1%, G' immediately recovers and exceeds G” again. This trend was observed in all three repeated cycles of the hydrogel. This rapid gel-sol-gel transformation under strain cycling conditions demonstrates the excellent self-healing properties of the SilMA-PBA / CS-CA / P-EMN hydrogel.

[0082] The injectability of hydrogels is tested using shear rate, such as... Figure 19 The results show that, due to the correlation between the gel structure and the applied force, the viscosity of the hydrogel continuously decreases with the increase of the shear rate, eventually approaching the liquid state, which proves its shear-thinning property and also shows that the SilMA-PBA / CS-CA / P-EMN hydrogel has excellent injectability.

[0083] 15. Photocurable rheological property testing Sample: SilMA-PBA / CS-CA / P-EMN Test method: The rheological properties of the samples were characterized using a rotational rheometer (TA Instruments, USA, model DISCOVERYHR-2). A 40mm diameter steel plate was used as the rheological fixture with a gap of 0.5mm. A visible light source (wavelength 405nm) was mounted on the instrument to measure the rheological properties.

[0084] Time-oscillation scan: energy storage modulus time total 360s, light intensity 30mW / cm² 2 The ultraviolet irradiation time was 30 s, the constant strain was 1%, and the angular frequency was 5 rad / s.

[0085] Test Results: To observe the changes in hydrogel materials during photocrosslinking and curing, this invention investigated the prepared samples under light intensities of 405 nm and 30 mW / cm². 2 The relationship between modulus change over 30 seconds. Experimental results are as follows: Figure 20 As shown in the figure, the hydrogel exhibits a three-stage change over time. The first stage, from 0 to 115 seconds without UV light, shows no significant change in storage modulus G' and G" over time, with G' being greater than G" indicating a low-strength microgel state. The second stage, from 115 seconds with UV light, sees a dramatic increase in G', while G" shows a smaller increase and remains consistently lower than G'. This dramatic change directly reflects the photoinitiator-induced free radical polymerization of monomers, rapidly constructing a robust three-dimensional cross-linked network, transforming the system from a viscous fluid to an elastic solid. The third stage, from 115 to 360 seconds without UV light, sees the cross-linking reaction enter a slow maturation phase, with the rates of increase for G' and G" gradually slowing and eventually stabilizing, and G' remaining stable at a high modulus level. This indicates further densification of the polymer chains, forming a structurally stable and robust elastic gel system.

[0086] In summary, the evolution of the rheological curves confirms that the rapid cross-linking of SilMA-PBA can be effectively triggered by controlling ultraviolet light irradiation (405 nm), successfully realizing the transformation of the hydrogel from a liquid state to a solid state with high strength and elasticity.

[0087] 16. Blood compatibility test Samples: SilMA-PBA / CS-CA, SilMA-PBA / CS-CA / P-EMN Test method: First, add 2.5 mL of 0.9% sodium chloride injection solution to 2 mL of fresh anticoagulated rabbit blood (ear vein or heart blood), and gently shake up and down at least 10 times to obtain diluted rabbit blood for later use.

[0088] Preparation of sample extract: The sample was extracted with 0.9% sodium chloride injection at a ratio of 0.2 g: 1 mL. The extraction conditions were 37 °C for 30 min.

[0089] The experiment was divided into three groups: a negative control (0.9% sodium chloride injection), a positive control (0.1% Triton-X 100), and a sample group. Three centrifuge tubes were taken from each group, and 1 mL of the corresponding test solution was added to each tube. The tubes were then incubated in a 37°C water bath for 30 min. Subsequently, 0.02 mL of diluted rabbit blood was added to each tube, gently shaken, and incubated in a 37°C water bath for another 1 h. After the reaction was complete, the tubes were centrifuged at 3000 rpm for 20 min. Hemolysis was first observed visually. If the solution in the tube was clear red with no cells or only a small number of red blood cells remaining at the bottom, hemolysis had occurred. If all red blood cells sank and the supernatant was colorless and clear, no hemolysis had occurred. The supernatant was then collected, and the wavelength at 545 nm for each group of solutions was measured using a UV spectrophotometer. The hemolysis rate (HR) was calculated using the following formula.

[0090] HR(%)=(AB) / (CB)×100% Where: A is the absorbance of the sample group; B is the absorbance of the negative control group; C is the absorbance of the positive control group.

[0091] Test Results: The hemolysis test is a common method for evaluating the destructive effect of blood red blood cells on red blood cells when they come into contact with a material. Generally, the better the blood compatibility of the material, the lower the hemolysis rate. Figure 24 As can be seen from the appearance images, neither group of hydrogels showed a hemolytic reaction. Figure 25 The hemolysis test results of the hydrogel samples showed that the hemolysis rate of both hydrogel samples was lower than 2% of the ASTM F756-08 industry standard, indicating that both groups of hydrogels have good blood compatibility.

[0092] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0093] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A silk fibroin drug-loaded hydrogel, characterized in that, The silk fibroin drug-loaded hydrogel comprises a hydrogel matrix and nanoparticles, wherein the nanoparticles are loaded onto the hydrogel matrix. The hydrogel matrix comprises phenylboronic acid-grafted methacrylamide silk fibroin and caffeic acid-grafted chitosan. The nanoparticles include nanoenzymes and polydopamine, wherein the polydopamine coats the nanoenzymes; The nanozyme comprises epigallocatechin gallate and honeycomb manganese dioxide, which form the nanozyme through coordination.

2. The silk fibroin drug-loaded hydrogel according to claim 1, characterized in that, The mass ratio of the phenylboronic acid-grafted methacrylamide silk fibroin to the caffeic acid-grafted chitosan to the nanoparticles is (100~400):(10~20):

1.

3. The method for preparing the silk fibroin drug-loaded hydrogel according to any one of claims 1-2, characterized in that, Includes the following steps: Preparation of nanoparticles: honeycomb manganese dioxide was prepared by adding honeycomb manganese dioxide solution to epigallocatechin gallate solution, stirring at room temperature, centrifuging to collect the precipitate, washing to obtain nanozyme, dispersing nanozyme in buffer, adding dopamine, stirring to react, centrifuging to collect the product, and washing to obtain nanoparticles. Preparation of methacrylamide-grafted silk fibroin: Silk fibroin was extracted, and the silk fibroin was added to lithium bromide solution. The mixture was stirred, concentrated hydrochloric acid was added, centrifuged, filtered, and glycidyl methacrylate was added and mixed. The mixture was stirred, centrifuged, filtered, dialyzed, and lyophilized to obtain methacrylamide-grafted silk fibroin. 3-Aminophenylboronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide were sequentially added to dimethyl sulfoxide and stirred to obtain a 3-aminophenylboronic acid solution. Methacrylamide-grafted silk fibroin was dissolved in water to obtain a methacrylamide-grafted silk fibroin solution. The 3-aminophenylboronic acid solution was added to the methacrylamide-grafted silk fibroin solution, and the mixture was stirred at room temperature. The pH was adjusted, dialyzed, and lyophilized to obtain the final product. Preparation of caffeic acid-grafted chitosan: Chitosan was dissolved in water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added, the pH was adjusted, caffeic acid was added, the pH was adjusted again, the reaction was carried out in the dark, dialyzed, and lyophilized to obtain the product. Preparation of drug-loaded silk fibroin hydrogel: Benzylboronic acid-grafted methacrylamide silk fibroin was dissolved in a photoinitiator solution to obtain a Benzylboronic acid-grafted methacrylamide silk fibroin solution. Caffeic acid-grafted chitosan was dissolved in a buffer solution containing nanoparticles to obtain a caffeic acid-grafted chitosan solution. The Benzylboronic acid-grafted methacrylamide silk fibroin solution was added to the caffeic acid-grafted chitosan solution, sodium hydroxide was added, the mixture was shaken, and then irradiated with light to obtain the drug-loaded silk fibroin hydrogel.

4. The preparation method according to claim 3, characterized in that, The method for preparing the honeycomb-shaped manganese dioxide includes the following steps: Dissolve potassium permanganate in water, add oleic acid, react, centrifuge, and wash to obtain the product. According to the dosage ratio, the potassium permanganate : oleic acid is (0.1~0.3) g : 1 mL; The reaction time is 4-8 hours.

5. The preparation method according to claim 3, characterized in that, In the preparation of nanoparticles, the mass ratio of the honeycomb manganese dioxide to the epigallocatechin gallate is 1:

1. The ratio of the nanozyme to the dopamine is 1:1 by mass. The stirring time at room temperature is 1-3 hours, and the stirring reaction time is 4-8 hours.

6. The preparation method according to claim 3, characterized in that, In the preparation of phenylboronic acid-grafted methacrylamide silk fibroin, the ratio of silk fibroin to lithium bromide to concentrated hydrochloric acid to glycidyl methacrylate is (10~30) g: (0.5~1.5) mol: (0.5~2) mL: (10~15) mL. According to the mass ratio, the 3-aminophenylboronic acid : the methacrylamide silk fibroin is (0.004~0.008 : (0.1~0.5). The stirring reaction time is 6-10 hours, and the room temperature stirring reaction time is 40-50 hours.

7. The preparation method according to claim 3, characterized in that, In the preparation of caffeic acid-grafted chitosan, the chitosan to caffeic acid ratio is (1~2):(1~2) by mass. The light-protected reaction time is 20~26h.

8. The preparation method according to claim 3, characterized in that, In the preparation of the silk fibroin drug-loaded hydrogel, the mass ratio of the phenylboronic acid-grafted methacrylamide silk fibroin to the caffeic acid-grafted chitosan to the nanoparticles is (100~400):(10~20):

1.

9. The use of the silk fibroin drug-loaded hydrogel according to any one of claims 1-2 or the silk fibroin drug-loaded hydrogel obtained by the preparation method according to any one of claims 3-8 in the preparation of drugs for repairing infected wounds.

10. A drug for repairing infected wounds, characterized in that, The invention includes the silk fibroin drug-loaded hydrogel according to any one of claims 1-2 or the silk fibroin drug-loaded hydrogel obtained by the preparation method according to any one of claims 3-8.

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