Glucose-ultrasound dual-responsive intelligent multi-effect hydrogel dressing, preparation method and application thereof

Abstract

The application discloses a glucose-ultrasound double-response intelligent multi-effect hydrogel dressing and a preparation method and application thereof. The gel system takes quaternary ammonium chitosan and a double bond modified product thereof as a macromolecular framework, glucose in a microenvironment of a wound surface as a substrate, generates carbon radicals through a glucose oxidase / glycine ferrous ion cascade catalytic reaction, combines dimethyl acrylamide as a small molecular monomer, and realizes second-level gelation. The hydrogel is coated with a conjugated polymer PFODBT and a raphanusan stabilized by F127. The PFODBT can continuously generate reactive oxygen under the action of ultrasound, and cooperates with the cation of quaternary ammonium chitosan to play an antibacterial role. The SFN has persistent anti-inflammatory and antioxidant effects and promotes angiogenesis. The application constructs an intelligent response hydrogel dressing integrated with'sugar control-antibacterial-promoting repair' through a simple and mild enzymatic gelation method, provides a new treatment strategy for chronic wound repair of diabetes, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the technical field of preparing gel dressings that promote chronic wound repair, and specifically relates to the preparation and application of a glucose-ultrasound dual-response intelligent multi-effect hydrogel dressing. Background Technology

[0002] Diabetes is a common clinical disease, with over 500 million people living with it globally, a number projected to rise to 900 million by 2050. Diabetic foot is one of the major health threats faced by diabetic patients; these wounds are often difficult to heal and can easily lead to serious consequences such as amputation. Notably, the 5-year mortality rate for diabetic foot ulcers is approximately 50%, even higher than the 31% mortality rate for cancer. Given the heavy burden of diabetic wounds, optimizing treatment strategies is crucial.

[0003] Normal wound healing involves four stages: hemostasis, inflammation, proliferation, and remodeling. This process relies on the synergistic effects of various cells and growth factors. However, due to the combined effects of hyperglycemia, bacterial infection, and oxidative stress in diabetic patients, their wounds exhibit a more complex pathological microenvironment compared to traditional wounds, leading to specific damage to blood vessels and the immune system, posing significant challenges to treatment. Specifically, the high-glycemic environment and bacterial infection induce elevated levels of oxidative stress, resulting in excessive superoxide production in the electron transport chain, promoting the generation of advanced glycation end products (AGEs) and downregulating NO levels. Simultaneously, reduced antioxidant enzyme activity leads to extracellular matrix glycosylation, hindering matrix remodeling. Regarding inflammation, macrophages, due to bacterial colonization and metabolic disorders, struggle to complete the M1-M2 phenotypic transition, leading to chronic inflammation. This, coupled with suppressed neutrophil function and excessive protease release, further damages tissue. In terms of angiogenesis, decreased expression of factors such as VEGF results in wound ischemia and hypoxia, delaying healing. Traditional clinical treatments mainly include blood sugar control, surgical debridement, treatment of complications, and local wound care. However, these treatments cannot completely resolve issues such as bacterial infection and impaired angiogenesis, and wound healing is relatively slow. Antibiotic treatment can alleviate these difficulties to some extent, but the rapid spread of drug-resistant bacteria in recent years and the slow progress in the development of new antibiotics urgently necessitate novel strategies for effective wound treatment.

[0004] In recent years, biocompatible hydrogel wound dressings have been widely used. Compared with traditional dressings, hydrogels have advantages such as strong water absorption and a three-dimensional network structure, which can keep the wound moist and promote the healing of diabetic wounds. In addition, the special network structure of hydrogels can introduce functional polymers or bioactive substances, thereby precisely regulating the microenvironment of chronic wounds. Hydrogels can also lower the surface temperature of the wound and play a certain role in analgesia. These excellent properties make them significantly advantageous in the treatment of diabetic chronic wounds. However, most hydrogel wound dressings currently have some limitations. Some hydrogel wound dressings are made of polymers, and although polymers have many potential advantages, they may still have certain toxicity and adverse reactions. On the other hand, the microenvironment of chronic ulcer wounds is complex, involving different molecular pathways, and the cells and growth factors involved in various stages of healing are also dynamically changing. However, some hydrogel wound dressings have only one function and their adaptability to the wound environment needs to be improved, making it difficult to meet the complex needs of the healing process. In view of this, the development of a multi-functional hydrogel that combines antibacterial, hypoglycemic, and repair-promoting properties is urgently needed.

[0005] Sonodynamic therapy (SDT), as an emerging non-invasive treatment, has demonstrated unique advantages in the field of wound healing. Its core principle lies in using ultrasound to excite a sonosensitive agent, generating reactive oxygen species (ROS), thereby effectively killing bacteria and promoting tissue repair. Furthermore, SDT's non-invasive nature allows it to precisely target the affected area in a controlled manner, reducing the risk of infection and accelerating the healing process without damaging normal tissue. SDT's deep penetration, non-invasiveness, and synergistic effects with materials science make it a promising candidate for complex wound management, providing new ideas and tools for clinical treatment.

[0006] Sulforaphane (SFN) offers various health benefits, including improving diabetic complications and cardiovascular disease. Related studies have shown that SFN can activate antioxidant enzymes through the Nrf2-dependent pathway, effectively clearing reactive oxygen species (ROS) at wound sites and reducing oxidative stress. Simultaneously, SFN can inhibit inflammasome activation, reduce the secretion of pro-inflammatory factors such as IL-1β and IL-6, and alleviate wound inflammation. Furthermore, SFN promotes eNOS expression via the Src / PI3K / Akt pathway, releasing nitric oxide (NO) and stimulating angiogenesis, thus improving skeletal muscle injury and dysfunction through a synergistic effect. As a naturally derived compound, SFN exhibits high biocompatibility and low toxicity at doses below anticancer levels, providing a safe and effective treatment for wound healing.

[0007] Glucose oxidase (GOx) is a biological protease that binds glucose and catalyzes its conversion into gluconic acid and hydrogen peroxide (H2O2). By delivering GOx to the wound, glucose levels can be lowered in situ, effectively regulating the hyperglycemic microenvironment of diabetic wounds. Furthermore, the gluconic acid produced by this reaction activates the pH-responsive system by lowering the local pH, and the generated H2O2 can serve as a free substrate for further chemical cross-linking of the gel structure. Therefore, developing GOx-based drug delivery gel systems is of great significance for the repair of diabetic wounds.

[0008] To address the treatment needs of high-glucose wounds in diabetic patients, we designed a multifunctional hydrogel adhesive with excellent biocompatibility. This gel uses quaternized chitosan (QCS) and its double-bond modified products as its macromolecular backbone. Utilizing DMAA as a small molecule and glucose as a substrate, it achieves stable cross-linking through carbon radicals catalyzed by ferrous glycinate and GOx. GOx utilizes the high glucose concentration at the wound site to generate H2O2 and gluconic acid, thereby lowering blood sugar and regulating pH at the wound site. Ferrous glycinate exhibits highly efficient horseradish peroxidase-like activity and can utilize H2O2... Carbon free radicals are generated, enabling in-situ gelation through chemical cross-linking. Simultaneously, the gel carries F127-stabilized conjugated polymers PFODBT and SFN. Under ultrasound, PFODBT generates ROS, which synergistically enhances antibacterial activity with the cationic quaternary ammonium chitosan. In the pH-controlled acidic microenvironment of the wound, the F127 shell softens, and SFN is released slowly, exerting sustained antioxidant and angiogenesis-promoting effects, achieving a long-lasting local effect on the wound. Summary of the Invention

[0009] The technical problem to be solved by this invention is to provide a method for preparing a glucose-ultrasound dual-response intelligent multi-effect hydrogel dressing. This method has mild preparation conditions and is easy to operate. The prepared hydrogel has advantages such as rapid gelation and high stability. The raw materials used are environmentally friendly, the preparation process is simple, and it is feasible for industrialization.

[0010] A glucose-ultrasound dual-response intelligent multi-effect hydrogel dressing uses quaternized chitosan (QCS) and methacrylamide-modified product (QCSMA) as the main carriers, and crosslinks dimethylacrylamide (DMAA). The gel dissolves glucose oxidase (GOx) and ferrous glycine (Fe[Gly]2), and rapidly gels using the carbon free radicals generated during the blood sugar lowering process.

[0011] Preferably, the macromolecular carriers of the present invention are QCS and QCSMA, which regulate blood glucose through the synergistic effect of GOx and Fe[Gly]2.

[0012] Preferably, the coated drug of the present invention further includes PFODBT and SFN, through which PFODBT and QCS synergistically fight bacteria, and SFN provides sustained-release antioxidant and repair-promoting effects.

[0013] A method for preparing a glucose-ultrasound dual-response intelligent multi-effect hydrogel dressing includes the following steps: Step (1) Synthesize QCSMA. First, dissolve QCS in 2% acetic acid solution and stir in a water bath at 60 ℃ for 1.5 h. During the dissolution process, neutralize the pH to 4 with NaOH and slowly add glycidyl methacrylate (GMA). Stir at 60 ℃ for 6 h. Put the viscous fluid into a dialysis bag, dialyze, and freeze dry to obtain white sponge-like solid QCSMA. Step (2) Synthesize PFO nanoparticles. First, PFODBT and F127 are dissolved in tetrahydrofuran. Then, the mixed solution is added to a tetrahydrofuran:water solution of 1:9 under ultrasonic conditions. The mixture is ultrasonicated for 30 min. The tetrahydrofuran is fully volatilized using a shaker and filtered using a PES membrane filtration device. The filtered solution is then placed into an ultrafiltration tube for dialyzing to obtain PFO nanoparticles. Step (3) Synthesize SFN nanoparticles. First, SFN and F127 are dissolved in tetrahydrofuran. Then, the mixed solution is added to a tetrahydrofuran:water solution of 1:9 under ultrasonic conditions. The mixture is ultrasonicated for 30 min. The tetrahydrofuran is fully volatilized using a shaker and filtered using a PES membrane filtration device. The filtered solution is then placed into an ultrafiltration tube for dialyzing to obtain SFN nanoparticles. Step (4) Synthesis of PFO / SFN nanoparticles: First, SFN, PFODBT, and F127 are dissolved in tetrahydrofuran. Then, the mixed solution is added to a tetrahydrofuran:water solution of 1:9 under ultrasonic conditions, and ultrasonicated for 30 min. The tetrahydrofuran is then fully volatilized using a shaker and filtered using a PES membrane filtration device. The filtered solution is then placed into an ultrafiltration tube for dialyzing to obtain PFO / SFN nanoparticles. Step (5) Synthesize smart responsive hydrogel. First, dissolve QCS, QCSMA and DMAA in water and stir at 60 °C for 3 h. After confirming dissolution, add nanoparticles, Fe[Gly]2 and glucose oxidase, and stir in the dark for 30 min to obtain the precursor solution. The precursor solution can be used to quickly obtain hydrogel by contacting glucose.

[0014] An application of a drug-loaded smart responsive hydrogel in the treatment of chronic diabetic wounds.

[0015] The applications of the drug-loaded smart responsive hydrogel of the present invention include effective blood sugar control with GOx, synergistic antibacterial activity of PFODBT with quaternized chitosan, and highly effective antioxidant and angiogenesis-promoting effects with SFN.

[0016] Compared with existing technologies, the drug-loaded smart responsive hydrogel and its preparation method of the present invention have the following advantages: 1. The preparation method of this invention is simple, the main materials are easy to obtain, and the biosafety is good; 2. The PFO / SFN nanoparticles encapsulated in this invention can synergistically fight QCS antibacterial activity and effectively treat bacterial infections in diabetic chronic wounds; 3. The PFO / SFN nanoparticles encapsulated in this invention can be released slowly at the wound site, exhibiting high safety, effectiveness, and utilization rate.

[0017] 4. This invention uses novel materials and enhances the therapeutic effect through a multi-step approach combining blood sugar control, antibacterial action, and repair promotion, thereby maximizing the efficacy of the drug. Attached Figure Description

[0018] Figure 1 This is a flowchart illustrating the preparation process of the drug-loaded intelligent responsive hydrogel of this invention; Figure 2 This is the infrared spectrum of the QCSMA prepared in this invention; Figure 3 This is a rheological analysis diagram of the gel prepared according to the present invention; Figure 4 This is a graph showing the compression modulus of gels prepared in this invention at different QCS concentrations; Figure 5 This is a graph showing the hemolysis rate of PFO / SFN nanoparticles of different concentrations prepared in this invention; Figure 6 The gel system prepared in this invention exhibits singlet oxygen ( ) under ultrasound. 1 O2 generation efficiency assessment over time; Figure 7 These are scanning electron microscope images of the gel system prepared in this invention after lyophilization; Figure 8 The EH (pure gel) and EH prepared in this invention are... SFN (Gel with added SFN nanoparticles), EH PFO (Gel with PFO nanoparticles), EH SFN / PFO Image of coating culture of Escherichia coli and Staphylococcus aureus on gel with PFO / SFN nanoparticles under non-ultrasound and ultrasound conditions; Figure 9 The EH and EH prepared by this invention SFN EH PFO EH SFN / PFO Quantitative graphs of Escherichia coli spread culture under non-ultrasound and ultrasound conditions; Figure 10 The EH and EH prepared by this invention SFN EH PFO EH SFN / PFOQuantitative graphs of Staphylococcus aureus in spread culture under non-ultrasound and ultrasound conditions; Figure 11 The EH and EH prepared by this invention SFN EH PFO EH SFN / PFO HUVEC cell viability assay 24 hours after treatment; Figure 12 The EH and EH prepared by this invention SFN EH PFO EH SFN / PFO Angiogenesis in HUVEC cells after 6 hours of treatment; Figure 13 The EH and EH prepared by this invention SFN EH PFO EH SFN / PFO ROS clearance in L929 cells 24 hours after treatment; Figure 14 This is the glucose consumption curve of the EH gel prepared in this invention before and after ultrasound intervention.

[0019] Figure 15 The EH and EH prepared by this invention SFN EH PFO EH SFN / PFO Quantitative results of IL-10 secretion levels in RAW 264.7 cells 24 hours after treatment.

[0020] Figure 16 This invention relates to the EH prepared by this invention, as well as non-ultrasonic and ultrasonic EH. SFN / PFO Images of wound healing after 14 days of treatment.

[0021] Figure 17 This invention relates to the EH prepared by this invention, as well as non-ultrasonic and ultrasonic EH. SFN / PFO HE staining image after 7 days. Detailed Implementation

[0022] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0023] like Figure 1 As shown, a method for preparing a drug-loaded smart responsive hydrogel includes the following steps: Step (1) Synthesize QCSMA. First, dissolve QCS in 2% acetic acid solution and stir in a water bath at 60 ℃ for 1.5 h. During the dissolution process, neutralize the pH to 4 with NaOH. Slowly add glycidyl methacrylate (GMA) and stir at 60 ℃ for 6 h. Put the viscous fluid into a dialysis bag, take out the liquid from the dialysis bag and freeze dry to obtain white spongy solid QCSMA. Step (2) Synthesize PFO nanoparticles. First, PFODBT and F127 are dissolved in tetrahydrofuran. Then, the mixed solution is added to a tetrahydrofuran:water solution of 1:9 under ultrasonic conditions. The mixture is ultrasonicated for 30 min. The tetrahydrofuran is fully volatilized using a shaker and filtered using a PES membrane filtration device. The filtered solution is then placed into an ultrafiltration tube for dialyzing to obtain PFO nanoparticles. Step (3) Synthesize SFN nanoparticles. First, SFN and F127 are dissolved in tetrahydrofuran. Then, the mixed solution is added to a tetrahydrofuran:water solution of 1:9 under ultrasonic conditions. The mixture is ultrasonicated for 30 min. The tetrahydrofuran is fully volatilized using a shaker and filtered using a PES membrane filtration device. The filtered solution is then placed into an ultrafiltration tube for dialyzing to obtain SFN nanoparticles. Step (4) Synthesis of PFO / SFN nanoparticles: First, SFN, PFODBT, and F127 are dissolved in tetrahydrofuran. Then, the mixed solution is added to a tetrahydrofuran:water solution of 1:9 under ultrasonic conditions, and ultrasonicated for 30 min. The tetrahydrofuran is then fully volatilized using a shaker and filtered using a PES membrane filtration device. The filtered solution is then placed into an ultrafiltration tube for dialyzing to obtain PFO / SFN nanoparticles. Step (5) Synthesize the smart responsive hydrogel. First, dissolve 6% w / v QCS, 4% w / v QCSMA and 2% w / v DMAA in water and stir at 60 ℃ for 3 h. After confirming dissolution, add nanoparticles, Fe[Gly]2 and glucose oxidase. Stir at room temperature in the dark for 30 min to obtain the precursor solution. The precursor solution can quickly obtain the hydrogel by contacting glucose.

[0024] This invention utilizes nuclear magnetic resonance spectroscopy (NMR), Fourier transform infrared spectroscopy (FT-IR), fluorescence spectroscopy, scanning electron microscopy (SEM), and tensile testing to study the prepared drug-loaded smart responsive hydrogels (EH, EH). SFN EH PFO and EH SFN / PFO The structure and physicochemical properties of the compound were systematically characterized. Further biological performance was evaluated through the following experiments: antibacterial activity was determined using an automated colony counter; cell compatibility, in vitro angiogenesis capacity, and ROS scavenging effect were observed using an inverted fluorescence microscope; in vitro anti-inflammatory performance was detected using an ELISA kit; and wound healing maps were used to assess mouse viability and wound repair.

[0025] Example 1: A method for preparing a glucose-ultrasound dual-response intelligent multi-effect hydrogel dressing, comprising the following steps: Dissolve 4 g of QCS in 2% acetic acid solution, stir in a water bath at 60 °C for 1.5 h, neutralize the pH to 4 with NaOH, slowly add 10 ml of GMA, stir at 60 °C for 6 h, dialyze the viscous fluid (molecular weight cutoff of 3000 D), freeze dry to obtain white spongy solid QCSMA; PFODBT (0.5 mg) was dissolved in tetrahydrofuran, and then the mixed solution was added to a tetrahydrofuran:water solution of 1:9. The mixture was sonicated for 30 min, and the tetrahydrofuran was fully volatilized using a shaker. The solution was then filtered using a PES membrane (0.22 μm) filter. The filtered solution was then dialyzed in an ultrafiltration tube (50000D) to obtain PFO nanoparticles. SFN (0.5 mg) was dissolved in tetrahydrofuran, and then the mixed solution was added to a tetrahydrofuran:water solution of 1:9. The mixture was sonicated for 30 min, and the tetrahydrofuran was fully volatilized using a shaker. The solution was then filtered using a PES membrane (0.22 μm) filter. The filtered solution was then dialyzed in an ultrafiltration tube (50000 D) to obtain SFN nanoparticles. SFN (0.5 mg), PFODBT (0.5 mg), and F127 were dissolved in tetrahydrofuran. The mixed solution was then added to a tetrahydrofuran:water solution of 1:9 under ultrasonic conditions and sonicated for 30 min. The tetrahydrofuran was then fully volatilized using a shaker and filtered through a PES membrane (0.22 μm) filter. The filtered solution was then dialyzed through an ultrafiltration tube (50000 D) to obtain PFO / SFN nanoparticles. QCS (6% w / v), QCSMA (4% w / v), and DMAA (2% w / v) were dissolved in water and stirred at 60 °C for 3 h. After confirming dissolution, PFO / SFN nanoparticles (PFODBT concentration of 50 μg / ml and SFN concentration of 10 μM), Fe[Gly]2 (0.1 mg / ml), and glucose oxidase (0.5 U / ml) were added. The mixture was stirred at room temperature in the dark for 30 min to obtain the precursor solution. The precursor solution was then contacted with a glucose solution to obtain the final material EH. SFN / PFO (EH, EH) SFN and EH PFO The synthesis steps are similar to those described above. Test Result 1: Refer to the attached instruction manual. Figure 2 The infrared spectrum of the QCSMA prepared in this invention was measured at 1632.7 cm⁻¹. -1 The appearance of an absorption peak indicates that the double bond modification was successful.

[0026] Test Result 2: Refer to the attached instruction manual. Figure 3 The rheological test results were obtained by using a rotational rheometer to detect the changes in the storage modulus (G′) and loss modulus (G″) of the drug-loaded smart responsive hydrogel prepared in this invention over time (Figure 5). The gelation time was defined as the intersection of the G′ and G″ curves. The drug-loaded smart responsive hydrogel of this invention transforms from liquid to gel solid at an extremely fast speed, which is beneficial for rapid gelation at the wound site.

[0027] Test Result 3: Refer to the attached instruction manual. Figure 4 The compression modulus test results were used to detect the effect of different concentrations of QCS on the strength of the drug-loaded smart responsive hydrogel using a rotational rheometer. Preferably, the drug-loaded smart responsive hydrogel has the highest strength when the concentration of QCS is 6% w / v.

[0028] Test Result 4: Refer to the attached instruction manual. Figure 5 The hemolysis test results showed that the biocompatibility of different concentrations of PFODBT was detected by taking mouse blood and using a multifunctional enzyme-linked immunosorbent assay (ELISA) reader. When the concentration of PFO / SFN nanoparticles reached 100 μg / mL, the hemolysis rate was less than 5%, indicating that the drug encapsulated by the drug-loaded smart responsive hydrogel has high safety.

[0029] Test Result 5: Refer to the attached instruction manual. Figure 6 singlet oxygen ( 1 O2 generation efficiency was evaluated by adding a singlet oxygen green fluorescent probe (SOSG, 2.0 µL) solution to a drug-loaded smart responsive hydrogel solution system and subjecting it to sonication for different time periods. The fluorescence intensity of SOSG was analyzed to determine the efficiency. 1 O2 production was measured. Testing showed that the probe's fluorescence signal gradually increased with ultrasonic time, verifying that the drug-loaded smart responsive hydrogel of this invention possesses excellent acoustic-dynamic oxygen production performance.

[0030] Test Result 6: Refer to the attached instruction manual. Figure 7 The morphology of the drug-loaded smart responsive hydrogel prepared in this invention was observed by scanning electron microscopy (SEM). The SEM images show that the drug-loaded smart responsive hydrogel prepared in this invention has a porous network structure.

[0031] Test Result 7: Refer to the attached instruction manual Figure 8 , 9 Bacterial plating culture images and quantitative results of 10 were used to evaluate EH and EH using Escherichia coli and Staphylococcus aureus. SFN EH PFO and EH SFN / PFO Antibacterial activity before and after ultrasound (US). Escherichia coli and Staphylococcus aureus bacterial suspensions were diluted to 5 × 10⁻⁶ with sterile PBS. 7CFU / mL, bacterial suspension and lyophilized gel powder from each group were prepared into solutions for co-culture. In the non-ultrasonic group, after 10 min of co-culture, the bacterial suspension was diluted tenfold for spread culture. In the ultrasonic group, after 4 min of ultrasonic intervention and 6 min of co-culture, the bacterial suspension was diluted to the same concentration for spread culture. Each group had 3 replicates, and a control group was also set up. The culture dishes were placed in a 37℃, 5% CO2 incubator for 24 h. After culture, the number of colonies in each group was counted using a fully automated colony counter, and the antibacterial ability (e.g., CFU / mL) was calculated based on this value. Figure 9 , 10 (As shown). Compared with the control group, the antibacterial rates against Escherichia coli and Staphylococcus aureus in the non-ultrasound group and the ultrasound group without PFODBT were approximately 60% and 50%, respectively, demonstrating that quaternized chitosan cationic acid has good antibacterial properties. The ultrasound group with PFODBT added had an antibacterial rate exceeding 90% against Escherichia coli and Staphylococcus aureus, which fully demonstrates the excellent antibacterial properties of the drug-loaded smart responsive hydrogel of the present invention. Since the SFN content did not reach the minimum antibacterial concentration, no effect of SFN on the antibacterial properties was observed in the drug-loaded smart responsive hydrogel of the present invention.

[0032] Test Result 8: Refer to the attached instruction manual. Figure 11 Cell viability and death staining results were used to evaluate the effect of the drug-loaded smart responsive hydrogel prepared in this invention on cell viability, using HUVEC cells as a model cell. In this experiment, Calcein-AM / PI double staining was used to label cells, and the cell state after different treatment groups was observed using an inverted fluorescence microscope. By statistically analyzing the number of viable cells (Calcein-AM labeled, green fluorescence) and the ratio of viable to dead cells, the effect of the gel on cell survival and proliferation was analyzed, thereby evaluating its proliferative biological activity. Cells were soaked in cell culture medium at EH and EH... SFN EH PFO EH SFN / PFO The lyophilized powder was sterilized by overnight ultraviolet irradiation. Each group had three replicates and a control group. Cells were resuspended in gel extract at 2×10⁻⁶. 4 Cells were seeded in 48-well plates and cultured in a 37 °C, 5% CO2 incubator for 24 h. After culture, the gel extract was aspirated, and the cells were slowly washed three times with PBS. A culture medium containing Calcein-AM (live cell dye) and Propidium Iodide was added to the wells, and the plates were incubated in a 37 °C, 5% CO2 incubator for another 30 min. The plates were then removed and observed using an inverted fluorescence microscope. Compared with the PBS control group, there were no significant differences in HUVEC cell morphology and survival rate among the gel groups, demonstrating that the synthesized drug-loaded smart responsive hydrogel has good biocompatibility and can be used for therapeutic purposes.

[0033] Test Result 9: Refer to the attached instruction manual. Figure 12 Angiogenesis evaluation was conducted using HUVEC cells as a model cell to evaluate the effect of the drug-loaded smart responsive hydrogel prepared in this invention on angiogenesis. Cell culture medium was used to soak EH and EH cells... SFN EH PFO EH SFN / PFO The lyophilized powder was sterilized by overnight UV irradiation. 10-20 μl of matrix gel was added to each well of a 24-well plate, spread into a circle using a pipette tip, and incubated overnight at 4 °C. Three replicates were set up for each group, along with a control group. Digested HUVEC cells were resuspended in gel extract at 5 × 10⁻⁶ cells / well. 4 Cells were seeded into 24-well plates after overnight incubation, and the plates were incubated at 37 °C in a 5% CO2 incubator for 6 h. The plates were then removed, gently washed twice with PBS, and culture medium containing Calcein-AM / PI solution was added to the wells. The plates were then incubated again at 37 °C in a 5% CO2 incubator for 30 min. Observation was performed using an inverted fluorescence microscope. Compared to the PBS group, the group with SFN-containing gel extract showed stronger cell tube formation activity, which fully demonstrates that the drug-loaded smart responsive hydrogel of this invention has a significant angiogenic effect.

[0034] Test Result 10: Refer to the attached instruction manual. Figure 13 The ability of the drug-loaded smart responsive hydrogel of this invention to scavenge intracellular ROS was evaluated using L929 cells as a model cell. Cells were loaded with 2 × 10⁻⁶ cells... 4 Cells were seeded in 48-well plates and incubated at 37 °C for 24 h in a 5% CO2 incubator. The plates were then removed, and the control group received cell culture medium with 10 µL PBS, the blank group received cell culture medium with 10 µL H2O2, and the experimental group received gel extraction buffer with 10 µL H2O2. All cells were incubated at 37 °C for 2 h in a 5% CO2 incubator. Then, culture medium containing DCFH-DA was prepared and added to the wells, and incubated for 20 min. Observation was performed using an inverted fluorescence microscope. Compared with the control group, the EH group and the EH... PFO Due to the presence of Fe[Gly]2, the green fluorescence produced by ROS in group EH was significantly weakened. SFN EH SFN / PFO The addition of SFN further weakened the green fluorescence, bringing it close to that of the control group. This indicates that the drug-loaded smart responsive hydrogel of this invention can effectively reduce intracellular oxidative stress and provide good biochemical effects.

[0035] Test Result 11: Refer to the attached instruction manual. Figure 14A glucose solution containing glucose oxidase (1 mg / mL) was used as the control group, while a glucose solution containing the gel was used as the experimental group. The solutions were incubated at 37 ℃ in a shaker, with 100 µL samples taken every 30 min. The glucose concentration in the solution was determined using a glucose assay kit. The gel continuously consumed glucose; although glucose oxidase was partially inactivated after ultrasonic intervention, it still maintained high glucose-consuming activity.

[0036] Test Result 12: Refer to the attached instruction manual. Figure 15 The in vitro anti-inflammatory activity of the drug-loaded smart responsive hydrogel of this invention was evaluated using RAW 264.7 cells. EH and EH were used. SFN EH PFO EH SFN / PFO Lyophilized samples were extracted with cell culture medium and sterilized overnight by UV irradiation. RAW 264.7 cells were then cultured at a concentration of 1×10⁻⁶. 4 Cells were seeded per well in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h. The original culture medium was discarded, and medium containing LPS (100 ng / mL) was added to induce M1 polarization. After 24 h, the medium was replaced with the sample extract mentioned above, and cultured for another 24 h. The cell culture supernatant was collected, and IL-10 secretion levels were detected using an ELISA kit. Compared with the control group, the EH group and EH... PFO Because it contains Fe[Gly]2, it can alleviate oxidative stress and reduce inflammatory response; EH SFN Group and EH SFN / PFO The anti-inflammatory effect was further enhanced in the group with the additional introduction of SFN. These results demonstrate that the drug-loaded smart responsive hydrogel of this invention can effectively inhibit inflammatory responses and exert beneficial biological effects.

[0037] Test Result 13: Refer to the attached instruction manual. Figure 16 The mouse wound repair diagram shown demonstrates that, compared to the control group, the drug-loaded smart responsive hydrogel of this invention exhibits excellent skin repair capabilities.

[0038] Test Result 14: Refer to the attached instruction manual. Figure 17 The mouse wound HE staining image shown indicates that, compared with the control group, the drug-loaded smart responsive hydrogel group of this invention showed less inflammatory cell infiltration. SFN / PFO Due to the anti-inflammatory and antioxidant effects of SFN, inflammatory infiltration was further reduced. The above results indicate that the drug-loaded smart responsive hydrogel of the present invention has good skin repair capabilities.

[0039] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural modifications made based on the description and drawings of the present invention, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A glucose-ultrasound dual-response intelligent multi-effect hydrogel dressing, characterized in that, Using quaternized chitosan and methacrylamide modified products as the macromolecular backbone, dimethacrylamide as the small molecule comonomer, a glucose oxidase / ferrous glycine cascade catalytic system is introduced, and F127-stabilized conjugated polymers PFODBT and sulforaphane are supported, and then chemically crosslinked to form the product.

2. The glucose-ultrasound dual-response intelligent multi-effect hydrogel dressing according to claim 1, characterized in that... The rapid gelling ability described is triggered by a cascade effect of glucose oxidase and ferrous glycine.

3. The glucose-ultrasound dual-response intelligent multi-effect hydrogel dressing according to claim 1, characterized in that... include: The quaternized chitosan content was 6% w / v, the methacrylamide modified product content was 4% w / v, the dimethacrylamide content was 2% w / v, the final concentration of glucose oxidase was 0.5 U / ml, and the final concentration of ferrous glycine was 0.1 mg / ml.

4. The glucose-ultrasound dual-response intelligent multi-effect hydrogel dressing according to claim 1, characterized in that... include: The concentration of the conjugated polymer PFODBT was 50 μg / ml, and the concentration of sulforaphane was 10 μM.

5. A method for preparing the glucose-ultrasound dual-response intelligent multi-effect hydrogel dressing according to any one of claims 1-4, characterized in that... Includes the following steps: Step (1) Synthesize the methacrylamide-modified product QCSMA. First, dissolve the quaternized chitosan QCS in 2% acetic acid solution and stir in a water bath at 60 ℃ for 2-4 h. During the dissolution process, neutralize the pH to 4-5 with NaOH. Slowly add glycidyl methacrylate GMA and stir at 60-65 ℃ for 4-6 h. Pack the viscous fluid into a dialysis bag, dialyze, and freeze dry to obtain a white sponge-like solid QCSMA. Step (2) Synthesis of PFO nanoparticles: First, the conjugated polymers PFODBT and F127 are dissolved in tetrahydrofuran. Then, the mixed solution is added to a tetrahydrofuran:water solution of 1:9 under ultrasonic conditions. The mixture is ultrasonicated for 30-45 min, and the tetrahydrofuran is fully volatilized using a shaker. The solution is then filtered using a PES membrane filtration device. The filtered solution is then dialyzed into an ultrafiltration tube to obtain PFO nanoparticles. Step (3) Synthesize SFN nanoparticles. First, dissolve sulforaphane (SFN) and F127 in tetrahydrofuran. Then, add the mixed solution to a tetrahydrofuran:water solution of 1:9 under ultrasonic conditions. Sonicate for 30-45 min, use a shaker to fully volatilize the tetrahydrofuran, and filter with a PES membrane filter. Dialyze the filtered solution into an ultrafiltration tube to obtain SFN nanoparticles. Step (4) Synthesis of PFO / SFN nanoparticles: First, sulforaphane SFN, conjugated polymer PFODBT, and F127 are dissolved in tetrahydrofuran. Then, the mixed solution is added to a tetrahydrofuran:water solution of 1:9 under ultrasonic conditions and ultrasonicated for 30-45 min. The tetrahydrofuran is fully volatilized using a shaker and filtered using a PES membrane filtration device. The filtered solution is then dialyzed into an ultrafiltration tube to obtain PFO / SFN nanoparticles. Step (5) Synthesize the smart responsive hydrogel. First, dissolve QCS, QCSMA, and dimethacrylamide (DMAA) in water and stir at 60-65℃ for 3-5 h. After confirming dissolution, add nanoparticles, ferrous glycine (Fe[Gly]2), and glucose oxidase (GOx). Stir in the dark for 30-45 min to obtain the precursor solution. The precursor solution can rapidly polymerize into EH and EH upon contact with glucose. SFN EH PFO EH SFN / PFO Hydrogel.

6. The preparation method according to claim 5, characterized in that... In step (1), QCS is 4 g, GMA is 10 ml, and the molecular weight cutoff of the ultrafiltration tube is 50,000 D; in step (2), PFODBT is 0.5 mg; and in step (3), SFN is 0.5 mg.

7. The preparation method according to claim 6, characterized in that, The PES membrane filtration device used in steps (2) and (3) has a filter membrane pore size of 0.22 μm.

8. The preparation method according to claim 6, characterized in that... The conditions for membrane dialysis in step (1) are as follows: the molecular weight cutoff of the dialysis bag used is 3000 D; the dialysis time is 72-96 h; and the stirring conditions are: room temperature.

9. The preparation method according to claim 6, characterized in that, In steps (2) and (3), the ultrafiltration tube used for ultrafiltration has a rejection capacity of 50,000 D; the centrifuge parameters are: 1500-3000 rpm, 4 ℃, 1.5 h.

10. The application of the glucose-ultrasound dual-response intelligent multi-effect hydrogel dressing of claim 1, characterized in that, It is applied to the treatment of chronic diabetic wounds through multiple aspects of "blood sugar control, antibacterial and repair promotion".

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