Endogenous signal response closed-loop regulation hydrogel wound dressing and preparation method and application thereof

Abstract

This application relates to the field of biomedical materials technology, specifically disclosing a closed-loop regulated hydrogel wound dressing with endogenous signal response, its preparation method, and its application. The closed-loop regulated hydrogel wound dressing provided in this application is constructed from metal polyphenol nanoparticles (MPNs), a GSNO gas donor, oxidized sodium alginate grafted with phenylboronic acid groups, and a multi-hydroxyl-modified soybean protein derivative, forming a reconfigurable hydrogel network through multi-level dynamic cross-linking. The closed-loop regulated hydrogel wound dressing obtained in this application can recognize endogenous pathological signals such as glucose levels and pH values ​​in diabetic wounds, and based on these signals, achieve adaptive regulation of structure and function, thereby playing a synergistic role in anti-infection, anti-inflammatory regulation, and tissue repair. The closed-loop regulated hydrogel wound dressing provided in this application has good biocompatibility, environmental responsiveness, and regulatory ability, and can be used for the treatment of chronic diabetic wounds.

Description

Technical Field

[0001] This application relates to the field of biomedical materials technology, and in particular to a closed-loop regulated hydrogel wound dressing with endogenous signal response, its preparation method and application. Background Technology

[0002] Chronic diabetic wounds are one of the most common and difficult-to-treat complications of metabolic diseases, and their healing process is often influenced by multiple pathological factors. Compared with normal wounds, diabetic wounds typically exhibit prolonged inflammatory response, impaired angiogenesis, and decreased tissue regeneration capacity, leading to chronic wound healing difficulties. Under this pathological condition, the wound microenvironment is generally characterized by high glucose levels, excessive accumulation of reactive oxygen species (ROS), and recurrent bacterial infections. These abnormal factors interact and severely inhibit the migration and proliferation of fibroblasts and endothelial cells, thereby hindering the wound repair process.

[0003] Currently, most wound dressings used in clinical practice and research primarily function by providing a physical barrier, maintaining a moist environment, or passively loading active ingredients. Their functions are mainly limited to isolating external stimuli or improving local conditions, and they are unlikely to actively participate in the regulation of the wound microenvironment. In the highly dynamic and complex pathological environment of diabetic chronic wounds, a single, passive dressing method is insufficient to meet the actual needs for synergistic regulation of inflammation control, infection prevention, and tissue regeneration.

[0004] In recent years, hydrogel materials have attracted widespread attention in the field of wound repair due to their excellent biocompatibility, tunable mechanical properties, and high water content. However, most existing hydrogel systems employ static cross-linked structures, meaning their physicochemical properties and functional release behavior are fixed during the preparation stage, primarily relying on predetermined composition ratios and cross-linking densities to function. These hydrogels have limited interaction with the wound microenvironment, typically lacking the ability to recognize and respond to changes in in vivo biochemical signals. They struggle to adaptively adjust to dynamic changes in glucose levels and pH during wound healing, resulting in a mismatch between their functional effects and the actual healing process.

[0005] Therefore, how to construct a functional hydrogel system that can sense the endogenous pathological signals of diabetic wounds and transform these signals into changes in material structure and function, thereby achieving dynamic regulation of the wound microenvironment, remains a key technical problem that urgently needs to be solved in the field of wound repair. Summary of the Invention

[0006] The purpose of this application is to overcome the shortcomings of the prior art and provide a closed-loop regulated hydrogel wound dressing with endogenous signal response, its preparation method, and its application. The closed-loop regulated hydrogel wound dressing provided in this application achieves adaptive intervention of the wound microenvironment by recognizing pathological signal changes in the microenvironment of diabetic wounds and triggering dynamic regulation of material structure and function, thereby promoting efficient repair of chronic diabetic wounds.

[0007] To achieve the above objectives, the technical solution adopted in this application is as follows: This application provides a method for preparing a closed-loop regulated hydrogel wound dressing based on endogenous signal response, comprising the following steps: S1. Dissolve plant polyphenols in an aqueous solvent and adjust the solution to alkaline conditions. Then add metal salts to form metal polyphenol nanoparticles. After separation, purification and drying, metal polyphenol nanoparticles (MPNs) are obtained. S2. Dissolve glutathione and sodium nitrite in deionized water and adjust the solution to acidic conditions. Then add a precipitant to terminate the reaction, and then separate, purify and dry to obtain GSNO gas donor. S3. Dissolve sodium alginate in an aqueous solvent, add an oxidant to carry out an oxidation reaction, then add a precipitant to terminate the reaction, and after purification and drying, obtain oxidized sodium alginate; dissolve the oxidized sodium alginate in an aqueous solvent, add a coupling agent and a phenylboronic acid compound to carry out a grafting reaction, and after purification and drying, obtain oxidized sodium alginate grafted with phenylboronic acid groups. S4. Soy protein is dissolved in an aqueous solvent, and a coupling agent, activator and amino compound containing a polyhydroxy structure are added to carry out a coupling reaction. After purification and drying, a polyhydroxy modified soybean protein derivative is obtained. S5. The metal polyphenol nanoparticles (MPNs) obtained in step S1, the GSNO gas donor obtained in step S2, the sodium alginate (BOSA) grafted with phenylboronic acid groups obtained in step S3, and the polyhydroxy-modified soybean protein derivative (SPI-Tri) obtained in step S4 are added to an aqueous solution and mixed. Calcium salt is introduced into the system to make Ca... 2+ It forms ionic crosslinks with sodium alginate to create a closed-loop regulated hydrogel wound dressing.

[0008] The closed-loop regulated hydrogel wound dressing provided in this application is constructed from metal polyphenol nanoparticles (MPNs), a GSNO gas donor, oxidized sodium alginate grafted with phenylboronic acid groups, and a multi-hydroxyl-modified soybean protein derivative. Through multi-level dynamic cross-linking, a reconfigurable hydrogel network is formed. The closed-loop regulated hydrogel wound dressing obtained in this application can recognize endogenous pathological signals such as glucose levels and pH values ​​in diabetic wounds, and based on these signals, achieve adaptive regulation of structure and function, thereby playing a synergistic role in anti-infection, anti-inflammatory regulation, and tissue repair. The closed-loop regulated hydrogel wound dressing provided in this application has good biocompatibility, environmental responsiveness, and regulatory capacity, and can be used for the treatment of chronic diabetic wounds.

[0009] In step S1, plant polyphenols are dissolved in an aqueous solvent and the solution is adjusted to alkaline conditions to partially dissociate the phenolic hydroxyl groups in the plant polyphenols into phenolic acid ions, thereby enhancing their coordination ability with metal ions; then metal salts are added to drive the polyphenol molecules to self-assemble in the solution to form metal polyphenol nanoparticles.

[0010] In step S2, glutathione and sodium nitrite are dissolved in deionized water and the solution is adjusted to acidity. Under acidic conditions, the thiol groups in the glutathione molecule undergo a thiothionitrosylation reaction with sodium nitrite to generate S-nitrosoglutathione (GSNO). After the reaction is complete, a precipitant is added to terminate the reaction and precipitate the target product. After separation, purification, and drying, the GSNO gas donor is obtained. GSNO can release NO in a controlled manner under metal ion conditions, exerting antibacterial, angiogenesis-promoting, and inflammation-regulating biological functions.

[0011] In step S3, sodium alginate is dissolved in an aqueous solvent, and an oxidizing agent is added to selectively oxidize the vicinal diol structure on the sodium alginate molecular chain to an aldehyde group, thereby obtaining oxidized sodium alginate (OSA). Based on this, OSA is dissolved in an aqueous solvent, a coupling agent is added to activate its carboxyl group, and then an amide bond coupling reaction is performed with the amino group of a phenylboronic acid compound to obtain oxidized sodium alginate (BOSA) grafted with phenylboronic acid groups. The phenylboronic acid groups can form reversible borate ester bonds with polyhydroxy structures, thereby endowing the material with glucose responsiveness and pH responsiveness.

[0012] In step S4, soybean protein is dissolved in an aqueous solution, and an amino compound containing a polyhydroxy structure is added under the action of a coupling agent and an activator. The polyhydroxy groups are introduced into the soybean protein molecular chain through amide bond coupling to obtain a polyhydroxy modified soybean protein derivative (SPI-Tris).

[0013] As a preferred embodiment of the method for preparing the closed-loop regulated hydrogel wound dressing of endogenous signal response described in this application, in step S1, the plant polyphenols include at least one of tannic acid, catechin, and anthocyanin. And / or, in step S1, the metal salt includes at least one of magnesium sulfate, ferrous sulfate, and copper chloride.

[0014] The closed-loop regulated hydrogel wound dressing of this application introduces a network of interactions such as metal polyphenol coordination, ionic cross-linking and dynamic covalent bonds to construct a closed-loop regulated hydrogel wound dressing. It can identify endogenous pathological signals such as high glucose levels and abnormal pH in the microenvironment of diabetic chronic wounds, and use these as triggering conditions to induce changes in the material's structure and function. This breaks through the limitation of traditional wound dressings and static hydrogels that can only be passively applied to the wound, enabling the material to actively participate in the process of regulating the wound microenvironment.

[0015] Among them, plant polyphenols and metal salts are used, and the two undergo chelation and self-assembly to form stable metal polyphenol nanoparticles (MPNs). This structure not only endows the material with good antioxidant and antibacterial properties, but also promotes the decomposition of GSNO in the hydrogel environment and enhances the release efficiency of NO.

[0016] In a preferred embodiment of the method for preparing the closed-loop regulated hydrogel wound dressing of endogenous signal response described in this application, in step S1, the mass ratio of plant polyphenols to metal salts is (10~30):1. In step S1, adjusting the solution to alkaline conditions involves adjusting the pH value of the solution to 8-10.

[0017] The plant polyphenols and metal salts used in this application, with the aforementioned mass ratio, can effectively encapsulate metal ions and stably form uniformly sized metal polyphenol nanoparticles, provided that the polyphenol hydroxyl groups fully participate in coordination. When the amount of polyphenols is too low, metal ions are difficult to fully coordinate, which can easily lead to problems such as uneven particle size, agglomeration, or residual free metal ions, thereby reducing the material's antioxidant properties and biocompatibility.

[0018] The purpose of adjusting the solution to alkaline conditions in this application is to promote the deprotonation of the phenolic hydroxyl groups in plant polyphenol molecules, transforming them from the –OH form into more coordinating phenolic acid anions. Polyphenols have weak coordination ability under neutral or acidic conditions, making it difficult to fully form stable polydentate coordination structures with metal ions. Under alkaline conditions, the number of phenolic acid anions increases significantly, thereby enabling the rapid, uniform, and self-assembly of metal polyphenol nanoparticles.

[0019] In a preferred embodiment of the preparation method of the closed-loop regulated hydrogel wound dressing with endogenous signal response described in this application, in step S2, the mass ratio of glutathione to sodium nitrite is (2~10):1.

[0020] Using the aforementioned mass ratio range for glutathione and sodium nitrite ensures that the thiomercaptonitrosation reaction proceeds efficiently and stably. Within this mass ratio range, GSH is in a relative excess, effectively avoiding side reactions, NO loss, or the generation of various unstable nitration byproducts caused by excess nitrite, thereby guaranteeing the purity and yield of GSNO.

[0021] In a preferred embodiment of the method for preparing the closed-loop regulated hydrogel wound dressing with endogenous signal response described in this application, in step S2, the precipitant includes at least one of acetone, ethanol, and isopropanol. In step S2, adjusting the solution to acidic conditions involves adjusting the pH value of the solution to 4-6.

[0022] The purpose of adjusting the solution to acidic conditions in this application is to promote the formation of nitrous acid with nitrosylation activity from sodium nitrite, thereby enabling the thiol group in the glutathione molecule to undergo a thiothionitrosylation reaction to generate stable S-nitrosoglutathione.

[0023] In a preferred embodiment of the method for preparing the closed-loop regulated hydrogel wound dressing of the endogenous signal response described in this application, the oxidant in step S3 is a periodate oxidant. The precipitant includes at least one of acetone, ethanol, and isopropanol; The coupling agent includes at least one of EDC, EDC·HCl, DCC, and DIC; And / or, the phenylboronic acid compound includes at least one of aminophenylboronic acid, hydroxyphenylboronic acid, and substituted phenylboronic acid.

[0024] In a preferred embodiment of the method for preparing the closed-loop regulated hydrogel wound dressing of the endogenous signal response described in this application, in step S4, the activator includes at least one of N-hydroxysuccinimide and sulfonated N-hydroxysuccinimide; and the polyhydroxyamino compound is at least one of trihydroxymethylaminomethane and trihydroxymethylglycine.

[0025] In a preferred embodiment of the preparation method of the closed-loop regulated hydrogel wound dressing with endogenous signal response described in this application, in step S3, the mass ratio of sodium alginate to oxidant is 1:(0.05~1.0).

[0026] The mass ratio range of sodium alginate and oxidant used in this application is intended to enable the vicinal diol structure of sodium alginate to be selectively oxidized in a moderate and uniform manner, thereby obtaining oxidized sodium alginate containing an appropriate amount of aldehyde group.

[0027] The mass ratio of the oxidized sodium alginate, coupling agent, and phenylboronic acid compound is 1:(0.8~1.2):(0.6~1.0).

[0028] Using the above-mentioned mass ratio range of oxidized sodium alginate, coupling agent, and phenylboronic acid compound, it is possible to efficiently graft phenylboronic acid groups onto the sodium alginate molecular chain while ensuring effective activation of carboxyl groups.

[0029] In step S4, the mass ratio of soybean protein, coupling agent, activator and polyhydroxyamino compound is 1:(1.0~3.0):(1.0~3.0):(1.0~3.0).

[0030] The soybean protein, coupling agent, activator, and polyhydroxy amino compound in this application are used in the above-mentioned mass ratio range to ensure that the carboxyl groups on the soybean protein molecular chain are fully activated and undergo efficient amidation reaction with the amino groups of the polyhydroxy amino compound, thereby obtaining a suitable degree of polyhydroxy modification.

[0031] In step S5, the mass ratio of metal polyphenol nanoparticles (MPNs), GSNO gas donor, oxidized sodium alginate grafted with phenylboronic acid groups, polyhydroxy modified soybean protein derivative, and calcium salt is 1:(50~80):(50~80):(1~5):(0.1~1).

[0032] The above-mentioned mass ratio range enables the optimal matching of gelation rate, mechanical properties, environmental responsiveness, and NO controlled release function, ensuring that the hydrogel has a good steady-state structure, agile pathological signal response capability, and ideal therapeutic effect in the wound environment.

[0033] In some preferred embodiments, in step S5, the metal polyphenol nanoparticles (MPNs) obtained in step S1, the GSNO gas donor obtained in step S2, the sodium alginate grafted with phenylboronic acid groups obtained in step S3, and the soybean protein derivative modified with polyhydroxyl groups are dissolved in an aqueous solution, and the pH of the resulting reaction solution is 5-12.

[0034] In some preferred embodiments, in steps S4 and S5, the purification process involves transferring the reaction product to a dialysis bag with a molecular weight cutoff of 8-12 kDa, dialyzing it in deionized water for 3-5 days, and changing the dialysis solution every 12 hours.

[0035] This application also provides a method for preparing the above-mentioned closed-loop regulated hydrogel wound dressing based on the intrinsic signal response, resulting in a closed-loop regulated hydrogel wound dressing.

[0036] This application also provides the application of the above-mentioned closed-loop regulated hydrogel wound dressing in the preparation of products for the healing of chronic diabetic wounds.

[0037] Compared with the prior art, this application has the following beneficial effects: 1. Achieve active perception and response to endogenous pathological signals in diabetic wounds: This application introduces multiple interactions such as metal polyphenol coordination, ionic crosslinking and dynamic covalent bonds to construct a hydrogel network, which can recognize endogenous pathological signals such as high glucose levels and abnormal pH in the microenvironment of diabetic chronic wounds, and use these as triggering conditions to induce changes in the material's structure and function. This breaks through the limitation of traditional wound dressings and static hydrogels that can only be passively applied to the wound, enabling the material to actively participate in the process of regulating the wound microenvironment.

[0038] 2. Construct a closed-loop regulation mechanism to achieve adaptive treatment based on "perception-response-feedback": This application utilizes the synergistic effects of multiple components, including metal-polyphenol coordination, reversible borate ester and Schiff base dynamic covalent bonds, and nitric oxide release triggered by pathological signals. The metal-polyphenol nanoparticles provide antioxidant and antibacterial functions through metal-polyphenol coordination; S-nitrosoglutathione provides nitric oxide release triggered by pathological signals; the reversible borate ester and Schiff base dynamic covalent bonds formed between phenylboronic acid-grafted sodium alginate and polyhydroxy-modified soybean protein provide glucose and pH responsiveness; and the matrix network constructed by ionic crosslinking between calcium ions and sodium alginate enables the closed-loop regulated hydrogel wound dressing to regulate the release behavior of gaseous functional factors after recognizing pathological signals, further improving the wound microenvironment and forming a self-regulating closed-loop control process. This closed-loop mechanism matches the material function with the wound healing stage, improving the stability and sustainability of the treatment process.

[0039] 3. The material system has good biocompatibility and is suitable for use in wound dressings: The biomass materials used in this application, such as polysaccharides, proteins, and natural polyphenols, have good biocompatibility and processability. The resulting closed-loop regulated hydrogel wound dressing system has high water content and good softness, which can closely adhere to the wound surface, reduce mechanical stimulation to newly formed tissues, and is suitable for long-term coverage of chronic wounds.

[0040] This application constructs a closed-loop regulatory system for hydrogel wound dressings based on endogenous signal response, achieving active sensing and adaptive regulation of the pathological microenvironment of diabetic chronic wounds. This overcomes the shortcomings of existing wound dressings and static hydrogels, which are passive and lack sufficient response. The system possesses excellent structural stability, biocompatibility, and multifunctional synergistic effects, effectively improving the wound microenvironment and promoting efficient repair of diabetic chronic wounds, demonstrating promising application prospects. Attached Figure Description

[0041] Figure 1 This is the 1H NMR spectrum of BOSA; Figure 2 Infrared spectrum of SPI-Tris; Figure 3 The 1H NMR spectrum of GSNO; Figure 4 Infrared spectra of hydrogel wound dressings under different pH conditions; Figure 5 The diagram shows the mechanical properties of hydrogel wound dressings under different pH conditions; Figure 6 The graph shows the NO release capacity test of hydrogel wound dressings under different glucose contents. Figure 7 Antibacterial test results for different hydrogel wound dressings; Figure 8 Graphs showing liver hemostasis tests in SD rats with different hydrogel wound dressings; Figure 9 Figure 1 shows the wound healing ability of SD rats with different hydrogel wound dressings. Detailed Implementation

[0042] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0043] In the following examples and comparative examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified. Furthermore, the raw materials used in each parallel experiment are the same.

[0044] Example 1: A closed-loop modulated hydrogel wound dressing based on endogenous signal response and its preparation method. This embodiment provides a closed-loop modulated hydrogel wound dressing based on endogenous signal response and its preparation method. The preparation method includes the following steps: S1. 7 mg of anthocyanin was added to 7 mL of deionized water and dissolved completely under ultrasonication at 50 kHz. Then, 10 μL of 0.05 M sodium hydroxide solution was added to the resulting solution under low-speed stirring to adjust the pH to 8. Next, 15 μL of 2.5 wt% copper chloride dihydrate aqueous solution was added under ultrasonication at 50 kHz to induce a coordination reaction between anthocyanin and metal ions, forming metal polyphenol nanoparticles (MPNs). After the reaction was complete, the reaction mixture was centrifuged at 3000 rpm for 10 min, and the resulting product was freeze-dried to obtain MPNs. S2. Dissolve 3.07 g of glutathione and 0.69 g of sodium nitrite in a mixed solution consisting of 15 mL of ultrapure water and 8 mL of 0.1 mol / L hydrochloric acid (pH=4). Stir the mixture thoroughly in an ice bath at 0 °C for 1 h. After the reaction is complete, add 20 mL of acetone to terminate the reaction and continue stirring for 10 min. Filter the reaction mixture, collect the precipitate, and wash it three times each with acetone and diethyl ether. Freeze-dry the washed product under vacuum to obtain the GSNO gas donor. S3. Dissolve 5.0 g of sodium alginate in 500 mL of buffer solution and stir until completely dissolved at room temperature. Then add 1.5 g of sodium periodate and react under light-protected conditions for 5 h. After the reaction is complete, add 5.0 mL of ethylene glycol and continue the reaction under light-protected conditions for 1 h to terminate the oxidation. The resulting solution is dialyzed against deionized water for 4 days using a dialysis bag with a molecular weight cutoff of 8-14 kDa, and then freeze-dried to obtain oxidized sodium alginate (OSA). Dissolve 1.5 g of oxidized sodium alginate (OSA) in 100 mL of buffer solution to prepare a 1.5 wt% solution. Add 1.452 g of EDC·HCl and activate for 15 min, then add 1.245 g of 3-aminophenylboronic acid and stir at room temperature for 24 h. After the reaction is complete, dialyze the mixture against a dialysis bag with a molecular weight cutoff of 8-12 kDa for 5 days, and then freeze-dry to obtain oxidized sodium alginate grafted with phenylboronic acid groups (BOSA). S4. Dissolve 2 g of soy protein in 200 mL of deionized water and stir at room temperature until completely dissolved. Then add 2.70 g of EDC and 1.46 g of NHS and react for 30 min. Then add 3.86 g of trihydroxymethylglycine and react at room temperature for 48 h. After the reaction is complete, dialyze the resulting solution through a dialysis bag with a molecular weight cutoff of 3500 Da for 4 days and freeze-dry to obtain a polyhydroxy modified soy protein derivative (SPI-Tris). S5. Add 5 g of SPI-Tris to 95 g of deionized water, heat to 80 ℃ and stir for 30 min. After cooling to room temperature, adjust the pH of the system to 9 with sodium hydroxide solution to obtain a 5 wt% SPI-Tris solution. At the same time, dissolve 1 g of BOSA in 19 g of deionized water to obtain a 5 wt% BOSA solution. Then take 5 g of BOSA solution and 5 g of SPI-Tris solution, add 0.1 g of MPNs, 0.1 g of GSNO and 0.05 g of CaCO3, and mix them evenly with magnetic stirring at room temperature. Pour the resulting mixture into a mold. The pH of the mixture is 8, and a closed-loop regulated hydrogel wound dressing with endogenous signal response is obtained, which is named SSA / MPNs / NO.

[0045] Example 2: A closed-loop modulated hydrogel wound dressing based on endogenous signal response and its preparation method. This embodiment provides a closed-loop modulated hydrogel wound dressing based on endogenous signal response and its preparation method. The preparation method includes the following steps: S1. 3.8 mg of catechin was added to 7 mL of deionized water and dissolved completely under ultrasonication at 50 kHz. Then, 10 μL of 0.05 M sodium hydroxide solution was added to the resulting solution under low-speed stirring to adjust the pH of the system to 8. 15 μL of 2.5 wt% magnesium sulfate aqueous solution was added under ultrasonication at 50 kHz to induce a coordination reaction between anthocyanins and metal ions to form metal polyphenol nanoparticles. After the reaction was complete, the reaction mixture was centrifuged at 3000 rpm for 10 min, and the resulting product was freeze-dried to obtain metal polyphenol nanoparticles (MPNs). S2. Dissolve 1 g of glutathione and 0.5 g of sodium nitrite in a mixed solution consisting of 15 mL of ultrapure water and 8 mL of 0.1 mol / L hydrochloric acid (pH=4). Stir the mixture thoroughly in an ice bath at 0°C for 1 h. After the reaction is complete, add 20 mL of isopropanol to terminate the reaction and continue stirring for 10 min. Filter the reaction mixture, collect the precipitate, and wash it three times each with acetone and diethyl ether. Freeze-dry the washed product under vacuum to obtain the GSNO gas donor. S3. Dissolve 5 g of sodium alginate in 500 mL of buffer solution and stir until completely dissolved at room temperature. Then add 0.25 g of sodium periodate and react under light-protected conditions for 5 h. After the reaction is complete, add 5.0 mL of ethylene glycol and continue the reaction under light-protected conditions for 1 h to terminate the oxidation. The resulting solution is dialyzed against deionized water for 4 days using a dialysis bag with a molecular weight cutoff of 8-14 kDa, and then freeze-dried to obtain oxidized sodium alginate (OSA). Dissolve 1.5 g of oxidized sodium alginate (OSA) in 100 mL of buffer solution to prepare a 1.5 wt% solution. Add 1.452 g of DCC and activate for 15 min, then add 0.9 g of 3-aminophenylboronic acid and stir at room temperature for 24 h. After the reaction is complete, dialyze the mixture against a dialysis bag with a molecular weight cutoff of 8-12 kDa for 5 days, and then freeze-dry to obtain oxidized sodium alginate grafted with phenylboronic acid groups (BOSA). S4. Dissolve 2 g of soy protein in 200 mL of deionized water and stir at room temperature until completely dissolved. Then add 2 g of DIC and 2 g of NHS and react for 30 min. Next, add 2 g of trihydroxymethylglycine and react at room temperature for 48 h. After the reaction is complete, dialyze the resulting solution through a dialysis bag with a molecular weight cutoff of 3500 Da for 4 days and freeze-dry to obtain a polyhydroxy modified soy protein derivative (SPI-Tris). S5. Add 5 g of SPI-Tris to 95 g of deionized water, heat to 80 ℃ and stir for 30 min. After cooling to room temperature, adjust the pH of the system to 9 with sodium hydroxide solution to obtain a 5 wt% SPI-Tris solution. At the same time, dissolve 1 g of BOSA in 19 g of deionized water to obtain a 5 wt% BOSA solution. Then take 5 g of BOSA solution and 5 g of SPI-Tris solution, add 0.1 g of MPNs, 0.1 g of GSNO and 0.01 g of CaCO3, and mix them evenly with magnetic stirring at room temperature. Pour the resulting mixture into a mold. The pH of the mixture is 8, and a closed-loop regulated hydrogel wound dressing with endogenous signal response is obtained, which is named SSA / MPNs / NO.

[0046] Example 3: A closed-loop modulated hydrogel wound dressing based on endogenous signal response and its preparation method. This embodiment provides a closed-loop modulated hydrogel wound dressing based on endogenous signal response and its preparation method. The preparation method includes the following steps: S1. 11.3 mg of tannic acid was added to 7 mL of deionized water and dissolved completely under ultrasonication at 50 kHz. Then, 10 μL of 0.05 M sodium hydroxide solution was added to the resulting solution under low-speed stirring to adjust the pH of the system to 10. Then, 15 μL of 2.5 wt% ferrous sulfate aqueous solution was added under ultrasonication at 50 kHz to induce a coordination reaction between anthocyanins and metal ions to form metal polyphenol nanoparticles. After the reaction was complete, the reaction mixture was centrifuged at 3000 rpm for 10 min, and the resulting product was freeze-dried to obtain metal polyphenol nanoparticles (MPNs). S2. Dissolve 5 g of glutathione and 0.5 g of sodium nitrite in a mixed solution consisting of 15 mL of ultrapure water and 8 mL of 0.1 mol / L hydrochloric acid (pH=6). Stir the mixture thoroughly in an ice bath at 0 °C for 1 h. After the reaction is complete, add 20 mL of acetone to terminate the reaction and continue stirring for 10 min. Filter the reaction mixture, collect the precipitate, and wash it three times each with acetone and diethyl ether. Freeze-dry the washed product under vacuum to obtain the GSNO gas donor. S3. Dissolve 5 g of sodium alginate in 500 mL of buffer solution and stir until completely dissolved at room temperature. Then add 5 g of sodium periodate and react for 5 h in the dark. After the reaction is complete, add 5.0 mL of ethylene glycol and continue the reaction in the dark for 1 h to terminate the oxidation. Dialyze the resulting solution to deionized water for 4 days using a dialysis bag with a molecular weight cutoff of 8-14 kDa, and then freeze-dry to obtain oxidized sodium alginate (OSA). Dissolve 1.5 g of oxidized sodium alginate (OSA) in 100 mL of buffer solution to prepare a 1.5 wt% solution. Add 1.8 g of DIC and activate for 15 min, then add 1.5 g of 3-aminophenylboronic acid and stir for 24 h at room temperature. After the reaction is complete, dialyze the mixture to 8-12 kDa using a dialysis bag for 5 days, and then freeze-dry to obtain oxidized sodium alginate grafted with phenylboronic acid groups (BOSA). S4. Dissolve 2.00 g of soy protein in 200 mL of deionized water and stir at room temperature until completely dissolved. Then add 6 g of DCC and 6 g of NHS and react for 30 min. Next, add 6 g of trihydroxymethylglycine and react at room temperature for 48 h. After the reaction is complete, dialyze the resulting solution through a dialysis bag with a molecular weight cutoff of 3500 Da for 4 days and freeze-dry to obtain a polyhydroxy modified soy protein derivative (SPI-Tris). S5. Add 5 g of SPI-Tris to 95 g of deionized water, heat to 80 ℃ and stir for 30 min. After cooling to room temperature, adjust the pH of the system to 9 with sodium hydroxide solution to obtain a 5 wt% SPI-Tris solution. At the same time, dissolve 1 g of BOSA in 19 g of deionized water to obtain a 5 wt% BOSA solution. Then take 8 g of BOSA solution and 8 g of SPI-Tris solution, add 0.1 g of MPNs, 0.5 g of GSNO and 0.1 g of CaCO3, and mix them evenly with magnetic stirring at room temperature. Pour the resulting mixture into a mold. The pH of the mixture is 8. A closed-loop regulated hydrogel wound dressing with endogenous signal response is obtained, which is named SSA / MPNs / NO.

[0047] Example 4: A closed-loop modulated hydrogel wound dressing based on endogenous signal response and its preparation method. Unlike Example 1, Example 4 adjusted the amount of GSNO added in step S5 from 0.1g to 0.05g, while keeping the other steps the same. The resulting closed-loop regulated hydrogel wound dressing was named SSA / MPNs / NO1.

[0048] Example 5: A closed-loop modulated hydrogel wound dressing based on endogenous signal response and its preparation method. Unlike Example 1, Example 5 changed the amount of GSNO added in step S5 from 0.1g to 1.5g, while keeping the other steps the same. The resulting closed-loop regulated hydrogel wound dressing was named SSA / MPNs / NO2.

[0049] Comparative Example 1 Unlike Example 1, Comparative Example 1 did not add the metal polyphenol nanoparticles (MPNs) and GSNO gas donor synthesized in steps S1 and S2; it did not use the method in step S3 to activate and modify sodium alginate, but directly added unmodified sodium alginate in step S5. The other steps were the same, and the resulting hydrogel wound dressing was named SSA.

[0050] Comparative Example 2 Unlike Example 1, Comparative Example 2 did not add the GSNO gas donor synthesized in step S2, but the other steps were the same. The resulting hydrogel wound dressing was named SSA / MPNs.

[0051] Comparative Example 3 Unlike Example 1, in Comparative Example 3, the pH of the hydrogel was adjusted to 5 with HCl (1 mol / L) in step S5, so that the hydrogel was in an acidic state.

[0052] Step S5: Add 5 g of SPI-Tris to 95 g of deionized water, heat to 80 ℃ and stir for 30 min. After cooling to room temperature, adjust the pH of the system to 9 with sodium hydroxide solution to obtain a 5 wt% SPI-Tris solution. Simultaneously, dissolve 1 g of BOSA in 19 g of deionized water to obtain a 5 wt% BOSA solution. Then, take 8 g of BOSA solution and 8 g of SPI-Tris solution, add 0.1 g of MPNs, 0.5 g of GSNO and 0.1 g of CaCO3, and mix thoroughly with magnetic stirring at room temperature. Adjust the pH of the hydrogel to 5 with HCl (1 mol / L) to make the hydrogel acidic.

[0053] Comparative Example 4 Unlike Example 1, in Comparative Example 4, the pH of the hydrogel was adjusted to 12 with NaOH (1 mol / L) in step S5, so that the hydrogel was in an alkaline state.

[0054] Step S5: Add 5 g of SPI-Tris to 95 g of deionized water, heat to 80 ℃ and stir for 30 min. After cooling to room temperature, adjust the pH of the system to 9 with sodium hydroxide solution to obtain a 5 wt% SPI-Tris solution. Simultaneously, dissolve 1 g of BOSA in 19 g of deionized water to obtain a 5 wt% BOSA solution. Then take 8 g of BOSA solution and 8 g of SPI-Tris solution, add 0.1 g of MPNs, 0.5 g of GSNO and 0.1 g of CaCO3, and mix thoroughly with magnetic stirring at room temperature. Adjust the pH of the hydrogel to 12 with NaOH (1 mol / L) to make the hydrogel alkaline.

[0055] Test cases, performance tests The 1H NMR spectrum analysis of the BOSA synthesized in Example 1 above confirmed the successful synthesis of BOSA. Figure 1 As shown.

[0056] Infrared spectroscopy analysis of the SPI-Tris synthesized in Example 1 above confirmed the successful synthesis of SPI-Tris. Figure 2 As shown.

[0057] The 1H NMR spectrum analysis of the GSNO synthesized in Example 1 above confirmed the successful synthesis of GSNO. Figure 3 As shown.

[0058] Infrared spectroscopy analysis of the hydrogels synthesized in Examples 1, 3, and 4 revealed that in a neutral environment, the hydrogel exhibited a significant C=N stretching vibration peak at 1640 cm⁻¹, indicating the effective formation of Schiff base bonds. Simultaneously, the hydroxyl absorption peak at 3500 cm⁻¹ narrowed significantly, reflecting an increase in hydrogen bond content and a denser network structure. Under acidic conditions, however, Schiff base bonds hydrolyzed, and the coordination of Ca²⁺-carboxyl groups and the metal-polyphenol complexes successively failed, leading to the rupture of the cross-linked structure and a loosening of the gel network. In a strongly alkaline environment, some dynamic cross-linking bonds were deactivated, triggering network slippage and rearrangement. The infrared spectra of the hydrogel wound dressings under different pH conditions are shown below. Figure 4 As shown.

[0059] Mechanical property tests were conducted on the hydrogels synthesized in Example 1, Comparative Example 3, and Comparative Example 4. Under acidic or strongly alkaline conditions, some cross-linking bonds were destroyed, the integrity of the cross-linking network decreased, and the mechanical properties weakened accordingly. The mechanical properties of the hydrogel wound dressings under different pH conditions are shown in the figure below. Figure 5 As shown.

[0060] NO release behavior tests were conducted on the above Example 1 under different glucose concentrations. NO release kinetics were tested under simulated physiological conditions (6 mM), mild hyperglycemia (8 mM), and typical hyperglycemia conditions of diabetic wounds (10 mM). The results showed that NO release exhibited a significant concentration-dependent effect. With increasing environmental glucose concentration, borate ester bonds gradually broke, leading to the continuous release of metal-polyphenol complexes, which in turn promoted the decomposition of -SNO bonds in GSNO, thus increasing the NO release rate. The NO release capacity test results of the hydrogel wound dressing under different glucose contents are shown in the figure below. Figure 6 As shown.

[0061] The hydrogel constructed in this application can adaptively enhance NO release under high glucose stimulation, achieving responsive compensation to the pathological environment. This avoids the risk of excessive NO exposure in normal tissues and can accurately meet the gas signal level required for the repair of diabetic wounds, providing important theoretical basis and technical support for the design of blood glucose-responsive smart dressings.

[0062] Antibacterial performance tests were conducted on Examples 1, 1, 2, 4, and 5. The results showed that the hydrogel constructed in this application exhibited a significant synergistic antibacterial effect. Its antibacterial mechanism can be attributed to a multi-layered synergistic effect of "NO gas molecule attack + metal ion killing (Cu²⁺ / Cu⁺) + polyphenol synergistic antibacterial effect + network controlled release support." Specifically, copper ions can bind to bacterial membrane proteins and DNA, disrupting cell membrane integrity and interfering with metabolism; the phenolic hydroxyl groups of polyphenols interact with membrane proteins, further weakening membrane permeability; simultaneously, NO molecules are small and highly lipophilic, allowing them to freely penetrate the bacterial outer membrane, inducing membrane lipid peroxidation, protein nitration, and DNA breakage, ultimately leading to cell death. Antibacterial test results for different hydrogel wound dressings are shown in the figure below. Figure 7 As shown.

[0063] Hemostasis tests were performed on the livers of SD rats in Examples 1, 1, and 2. The results showed that, compared with the blank control group and commercially available hemostatic sponges, the hydrogel constructed in this application significantly shortened the hemostasis time and reduced bleeding. Figures show the hemostasis test results of different hydrogel wound dressings on the livers of SD rats. Figure 8 As shown.

[0064] SD rat full-thickness skin defect models were established for Examples 1, 1, and 2 to evaluate the wound repair effect of hydrogel dressings. The wounds were dynamically observed and recorded for two consecutive weeks post-surgery, with images taken every two days to monitor the healing process. Results showed that the hydrogel prepared in this application can achieve efficient healing of diabetic wounds by maintaining a moist environment, regulating inflammation, and promoting angiogenesis and tissue regeneration. The wound healing ability test results of different hydrogel wound dressings in SD rats are shown in the figure below. Figure 9 As shown.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A method for preparing a closed-loop regulated hydrogel wound dressing based on endogenous signal response, characterized in that, Includes the following steps: S1. Dissolve plant polyphenols in an aqueous solvent and adjust the solution to alkaline conditions. Then add metal salts to form metal polyphenol nanoparticles. After separation, purification and drying, metal polyphenol nanoparticles (MPNs) are obtained. S2. Dissolve glutathione and sodium nitrite in deionized water and adjust the solution to acidic conditions. Then add a precipitant to terminate the reaction, and then separate, purify and dry to obtain GSNO gas donor. S3. Dissolve sodium alginate in an aqueous solvent, add an oxidant to carry out an oxidation reaction, then add a precipitant to terminate the reaction, and after purification and drying, obtain oxidized sodium alginate; dissolve the oxidized sodium alginate in an aqueous solvent, add a coupling agent and a phenylboronic acid compound to carry out a grafting reaction, and after purification and drying, obtain oxidized sodium alginate grafted with phenylboronic acid groups. S4. Soy protein is dissolved in an aqueous solvent, and a coupling agent, activator and amino compound containing a polyhydroxy structure are added to carry out a coupling reaction. After purification and drying, a polyhydroxy modified soybean protein derivative is obtained. S5. The metal polyphenol nanoparticles (MPNs) obtained in step S1, the GSNO gas donor obtained in step S2, the sodium alginate grafted with phenylboronic acid groups obtained in step S3, and the soybean protein derivative modified with polyhydroxyl groups obtained in step S4 are added to an aqueous solution and mixed. Calcium salt is introduced into the system to allow Ca²⁺ to form ionic crosslinks with sodium alginate, thereby forming a closed-loop regulated hydrogel wound dressing.

2. The method for preparing the closed-loop regulated hydrogel wound dressing based on endogenous signal response as described in claim 1, characterized in that, In step S1, plant polyphenols include at least one of tannic acid, catechin, and anthocyanin. And / or, in step S1, the metal salt includes at least one of magnesium sulfate, ferrous sulfate, and copper chloride.

3. The method for preparing the closed-loop regulated hydrogel wound dressing based on endogenous signal response as described in claim 1, characterized in that, In step S1, the mass ratio of plant polyphenols to metal salts is (10~30):1; And / or, in step S1, adjusting the solution to alkaline conditions means adjusting the pH of the solution to 8~10.

4. The method for preparing the closed-loop regulated hydrogel wound dressing based on endogenous signal response as described in claim 1, characterized in that, In step S2, the mass ratio of glutathione to sodium nitrite is (2~10):

1.

5. The method for preparing the closed-loop regulated hydrogel wound dressing based on endogenous signal response as described in claim 1, characterized in that, In step S2, the precipitant includes at least one of acetone, ethanol, and isopropanol; And / or, in step S2, adjusting the solution to acidic conditions means adjusting the pH of the solution to 4~6.

6. The method for preparing the closed-loop regulated hydrogel wound dressing based on endogenous signal response as described in claim 1, characterized in that, In step S3, the oxidant is a periodate oxidant; And / or, the precipitant includes at least one of acetone, ethanol, and isopropanol; And / or, the coupling agent includes at least one of EDC, EDC·HCl, DCC, and DIC; And / or, the phenylboronic acid compound includes at least one of aminophenylboronic acid, hydroxyphenylboronic acid, and substituted phenylboronic acid.

7. The method for preparing the closed-loop regulated hydrogel wound dressing based on endogenous signal response as described in claim 1, characterized in that, In step S4, the activator includes at least one of N-hydroxysuccinimide and sulfonated N-hydroxysuccinimide; the polyhydroxyamino compound is at least one of trihydroxymethylaminomethane and trihydroxymethylglycine.

8. The method for preparing the closed-loop regulated hydrogel wound dressing based on endogenous signal response as described in claim 1, characterized in that, In step S3, the mass ratio of sodium alginate to oxidant is 1:(0.05~1.0); And / or, the mass ratio of the oxidized sodium alginate, coupling agent, and phenylboronic acid compound is 1:(0.8~1.2):(0.6~1.0); And / or, in step S4, the mass ratio of the soybean protein, coupling agent, activator, and polyhydroxyamino compound is 1:(1.0~3.0):(1.0~3.0):(1.0~3.0); And / or, in step S5, the mass ratio of metal polyphenol nanoparticles (MPNs), GSNO gas donor, oxidized sodium alginate grafted with phenylboronic acid groups, polyhydroxy modified soybean protein derivative and calcium salt is 1:(50~80):(50~80):(1~5):(0.1~1).

9. The closed-loop regulated hydrogel wound dressing prepared by the method for preparing the closed-loop regulated hydrogel wound dressing based on any one of claims 1 to 8.

10. The application of the closed-loop regulated hydrogel wound dressing as described in claim 9 in the preparation of a product for the healing of chronic diabetic wounds.

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