Preparation method of hydrogel for improving microenvironment of chronic wound of diabetes mellitus

Abstract

The invention discloses a preparation method of hydrogel for improving a diabetic chronic wound microenvironment, and relates to the technical field of biomedical materials. Comprising the following steps: dissolving sodium alginate and gallic acid in water to form a mixed solution, and adding a calcium ion solution for crosslinking to prepare antioxidant alginate hydrogel; the dosage of the sodium alginate is 100mg / 5mL of water, and the dosage of the gallic acid is 50mg / 5mL of water; the calcium ion solution is a CaCl2 solution with the concentration of 10 mg / mL, and the volume ratio of the added calcium ion solution to the mixed liquid is 1: 1. The multifunctional hydrogel disclosed by the invention realizes comprehensive treatment on chronic diabetic wounds through a synergistic effect mechanism; compared with traditional dressings, the hydrogel can synchronously solve the two core problems of excessive oxidative stress and biological membrane infection, and the wound microenvironment is fundamentally improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a method for manufacturing a hydrogel for improving the microenvironment of chronic wounds in diabetic patients. Background Technology

[0002] Among the many complications of diabetes, chronic wounds are one of the most destructive and clinically challenging problems, accounting for about 15%-25% of all diabetic patients. Foot ulcers, as the most common manifestation, are a major cause of disability and death in patients. The healing process of chronic wounds in diabetic patients is severely impaired, with the core characteristic being the persistent stagnation of the inflammatory phase in the healing cycle, preventing a smooth transition to the proliferative and remodeling phases. This pathological state primarily stems from two key pathological microenvironmental disturbances: excessive oxidative stress and persistent bacterial colonization and biofilm formation. In the diabetic pathological state, metabolic disorders lead to local vascular and neurological damage at the wound site, resulting in insufficient blood supply, oxygen and nutrient deficiencies. Simultaneously, abnormal activation of immune cells such as neutrophils leads to the release of large amounts of reactive oxygen species (ROS). Excessive ROS not only directly damages normal tissue cells surrounding the wound and disrupts the extracellular matrix but also inhibits key healing processes such as fibroblast proliferation and vascular endothelial cell migration, further exacerbating the persistent inflammatory response. On the other hand, the moist and hypoxic microenvironment at the wound site readily fosters bacterial growth. These bacteria rapidly colonize and secrete extracellular polysaccharide matrix, forming a dense biofilm structure. Biofilms not only provide a physical protective barrier for bacteria, resisting their clearance by the host's immune system, but also significantly reduce the penetration efficiency of antibacterial drugs, leading to increased bacterial resistance and persistent infection. The two factors combined result in delayed wound healing, recurrent ulceration, and ultimately a significant increase in the patient's amputation risk. Data shows that the amputation rate for diabetic foot ulcer patients is as high as 14%-24%, seriously affecting the patient's quality of life and imposing a heavy medical burden on families and society. Currently, traditional clinical treatments for chronic diabetic wounds (such as debridement, negative pressure drainage, and routine topical antibiotics) have many limitations, often failing to simultaneously address the two core issues of oxidative stress imbalance and biofilm infection. In recent years, hydrogels, due to their excellent biocompatibility, hydrophilicity, and three-dimensional porous structure, can mimic the extracellular matrix environment of human cells, providing a moist microenvironment for wound healing. They can also be loaded with drugs for localized sustained release, and have been widely used in the development of wound dressings. However, simple hydrogel therapy systems still have significant shortcomings and cannot meet the complex treatment needs of chronic diabetic wounds: First, ordinary hydrogels lack effective ROS scavenging capabilities, cannot improve the local redox microenvironment of the wound, and are unable to break the vicious cycle of stagnant inflammation; Second, hydrogels have weak inhibitory effects on bacteria, especially unable to penetrate mature biofilm structures, making them ineffective against persistent infections that have already formed biofilms; Third, traditional hydrogels mostly use a passive sustained-release mode after drug loading, and the drug release rate cannot be dynamically adjusted according to the degree of wound infection, which can easily lead to insufficient drug concentration in the early stage to effectively kill bacteria, and excessive drug in the later stage to cause toxic side effects or bacterial resistance; In addition, some hydrogels also have problems such as insufficient mechanical strength and a mismatch between degradation rate and wound healing process, which further limit their clinical application effects. Given the aforementioned clinical treatment challenges and the inadequacy of existing materials, it is urgent to develop a multifunctional treatment platform that can simultaneously address the two core issues of oxidative stress and biofilm infection, and possess intelligent drug delivery capabilities. Summary of the Invention

[0003] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for manufacturing a hydrogel for improving the microenvironment of chronic wounds in diabetic patients.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: A method for manufacturing a hydrogel for improving the microenvironment of chronic wounds in diabetic patients includes the following steps: Sodium alginate and gallic acid were dissolved in water to form a mixture, and calcium ion solution was added to crosslink the mixture to prepare an antioxidant alginate hydrogel.

[0005] Preferably, the amount of sodium alginate used is 100 mg / 5 mL of water, and the amount of gallic acid used is 50 mg / 5 mL of water.

[0006] Preferably, the calcium ion solution is a CaCl2 solution with a concentration of 10 mg / mL, and the volume ratio of the added solution to the volume of the mixed solution is 1:1.

[0007] Preferably, the method further includes the step of preparing ultrasonically responsive PLGA nanoparticles, wherein the PLGA nanoparticles are loaded with vancomycin.

[0008] Preferably, the PLGA nanoparticles are prepared using a double emulsion solvent evaporation method, which includes dissolving PLGA and VCM in an organic solvent, adding perfluoropentane for emulsification, then emulsifying it a second time with a polyvinyl alcohol solution, and finally evaporating the solvent to obtain nanoparticles.

[0009] Preferably, in the PLGA nanoparticles, the amount of PLGA is 50 mg, the amount of VCM is 4 mg, and the amount of PFP is 200 μL.

[0010] Preferably, the PLGA nanoparticles are embedded in the hydrogel of claim 1 to form a composite hydrogel.

[0011] A hydrogel composition manufactured by the above method comprises a sodium alginate substrate, gallic acid functionalization modification, and embedded PLGA-VCM nanoparticles.

[0012] Preferably, the PLGA nanoparticles have an average hydrodynamic diameter of 300-400 nm and a zeta potential of -20 mV to -40 mV.

[0013] The application of the hydrogel composition in the preparation of a medicament for treating chronic wounds of diabetes.

[0014] The beneficial effects of this invention are as follows: 1. The multifunctional hydrogel of the present invention achieves comprehensive treatment of diabetic chronic wounds through a synergistic mechanism; compared with traditional dressings, this hydrogel can simultaneously solve the two core problems of excessive oxidative stress and biofilm infection, fundamentally improving the wound microenvironment.

[0015] 2. Through functionalization modification with gallic acid (GA), the hydrogel of this invention possesses a strong free radical scavenging ability; the DPPH free radical scavenging test shows that its scavenging rate reaches 67.23%, which can effectively reduce the excessive reactive oxygen species (ROS) level in the wound and break the vicious cycle of inflammatory stagnation; DHE staining results show that the red fluorescence (high ROS level) in the treatment group is effectively inhibited, confirming its ability to improve the redox microenvironment.

[0016] 3. This invention integrates PLGA-VCM nanoparticles to achieve precise antibacterial treatment; through remote control by low-intensity focused ultrasound (LIFU), it can achieve on-demand and targeted drug release, solving the problem that traditional dressings cannot dynamically control drug release; in vitro experiments have confirmed that LIFU activation can promote rapid-triggered antibiotic release, avoiding toxic side effects caused by insufficient initial drug concentration or excessive later concentration.

[0017] 4. Rheological analysis in this invention shows that both drug-loaded and unloaded hydrogels form stable viscoelastic networks with good mechanical stability, which can meet the clinical application requirements of wound dressings; this stable cross-linked network ensures that the hydrogel will not collapse during wound treatment. Attached Figure Description

[0018] Figure 1 This is a schematic diagram illustrating the encapsulation efficiency and drug loading of the formulation of the present invention; Figure 2 This is a schematic diagram illustrating the rheological evaluation and verification of the mechanical integrity of the hydrogel scaffold of this invention. Figure 3 This is a porosity diagram observed by scanning electron microscopy in this invention; Figure 4 This is a schematic diagram of the oxidation performance test of the present invention; Figure 5 This is a schematic diagram illustrating the cell compatibility of the hydrogel of the present invention; Figure 6 This is a graph showing the weight changes of mice during the modeling process of this invention; Figure 7 This is a schematic diagram illustrating the wound healing process according to the present invention; Figure 8 This is a diagram showing the bacterial coating results of the present invention; Figure 9 This is the HE detection diagram of the present invention; Figure 10 This is a DHE staining image of the PBS group of the present invention; Figure 11 This is a DHE staining image of the A+ ultrasound group of the present invention; Figure 12 This is a TNF-α staining image from the present invention; Figure 13 This is an immunohistochemical staining image of IL-6 from the present invention. Detailed Implementation

[0019] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0020] Example 1: 1. Materials and Methods 1.1 Materials Sodium alginate (ALG) and streptozotocin (STZ) were purchased from Beijing Solarbio Science & Technology Co., Ltd., Beijing, China. Polylactic acid-glycolic acid copolymer (PLGA, L:G=50:50, average molecular weight 90,000 Da), vancomycin hydrochloride (VCM), and polyvinyl alcohol (PVA, degree of hydrolysis 87-89%) were purchased from Shanghai Maclean Biotechnology Co., Ltd., Shanghai, China. Gallic acid (GA) and perfluoropentane (PFP) were purchased from Aladdin Biotechnology Co., Ltd. (Shanghai). Cell counting kit-8 (CCK-8) was purchased from Invigentech, and MRSA was purchased from Beijing Zhongyuan Heju Biotechnology Co., Ltd. Dichloromethane (CHCl2) and isopropanol (IPA) were purchased from Chongqing Chuandong Chemical (Group) Co., Ltd., Chongqing, China. L929 mouse fibroblasts were purchased from Wuhan Pronosei Life Sciences Co., Ltd. All other chemical reagents were of analytical grade and used directly.

[0021] 1.2. Preparation of antioxidant alginate hydrogels Antioxidant hydrogel scaffolds were prepared using an ionic crosslinking method. Briefly, 100 mg of sodium alginate and 50 mg of gallic acid were dissolved in 5 mL of deionized water. The mixture was magnetically stirred at 65 °C for 30 minutes to ensure complete dissolution and homogeneous mixing. After cooling to room temperature, 5 mL of CaCl2 solution (10 mg / mL) was added dropwise with gentle stirring to initiate the crosslinking reaction. The resulting hydrogel was cured for 1 hour. For structural analysis, the hydrogel samples were freeze-dried for 24 hours, sputter-coated with gold, and observed using a scanning electron microscope (SEM).

[0022] 1.3 Hydrogel performance characterization Rheological Analysis: To evaluate the mechanical stability and viscoelasticity of the hydrogels, their rheological properties were quantitatively analyzed using a rotational rheometer (TA Instruments). A 600-second time-scan test was performed at 25°C with a constant frequency (1 Hz) and strain (1%). Storage modulus (G') and loss modulus (G'') recorded the gel state and structural integrity of the drug-loaded (GA / PLGA-VCM) and unloaded (GA) hydrogels.

[0023] Swelling and Degradation: The swelling behavior of the lyophilized hydrogel was evaluated by immersing it in phosphate-buffered saline (PBS, pH 7.4) at 37°C. Samples were weighed after removing excess surface moisture at predetermined time points (2, 6, and 12 hours). Degradation was monitored by measuring weight loss over 14 days in PBS containing lysozyme.

[0024] Antioxidant activity: Antioxidant activity was determined by a DPPH free radical scavenging assay. The hydrogel extract was mixed with 0.1 mM DPPH ethanol solution, incubated in the dark for 30 minutes, and the absorbance was measured at 517 nm using a microplate reader.

[0025] 1.4. Synthesis of Ultrasonic-Responsive PLGA-VCM Nanoparticles Ultrasonic-responsive nanoparticles were synthesized using a modified two-emulsion (W / O / W) solvent evaporation method. First, 4 mg of VCM was added to the reaction system. 200 μL of PFP was emulsified with 2 mL of dichloromethane containing 50 mg of PLGA and 4 mg of VCM. To impart ultrasonic responsiveness, 200 μL of PFP (perfluoropentane) was added. The mixture was emulsified using an ultrasonic disruptor (40% amplitude, 3 min, 50% duty cycle) under ice bath conditions. Immediately after emulsification, 8 mL of pre-cooled 4% PVA solution was added to the primary emulsion, and emulsification was repeated under the same conditions. The resulting secondary emulsion was added to 10 mL of pre-cooled 2% isopropanol solution and stirred for 4 h under ice bath conditions to evaporate the organic solvent. The nanoparticles were collected by centrifugation (10000 rpm, 8 min, 4 °C), washed three times with cold distilled water, and then freeze-dried.

[0026] Characterization of 1.5 nanoparticles The hydrodynamic dimensions, polydispersity index (PDI), and zeta potential of the nanoparticles were determined using dynamic light scattering (DLS, Malvern Zetasizer Nano ZS). The morphology of the particles was observed by transmission electron microscopy (TEM). To determine the drug loading, the nanoparticles were dissolved in methanol to extract vancomycin (VCM). The concentration of VCM was quantitatively determined at 280 nm using UV-Vis spectrophotometry using a pre-established standard curve (C = 4.820A–8.043). The encapsulation efficiency (EE) and drug loading (DL) were calculated as follows: Encapsulation efficiency = (mass of vancomycin in nanoparticles / amount of vancomycin added) * 100%; Drug loading = (mass of vancomycin in nanoparticles / total mass) * 100% 1.6. In vitro cytotoxicity assay (CCK-8) 1.6.1 Experimental Grouping The experiment was divided into a Control group and a Sample group (including samples 1 and 2). 100 μL of complete culture medium was added to each well in the Control group, and 100 μL of the corresponding concentration of working solution was added to each well in the Sample group. Three replicates were set up for each group to ensure experimental repeatability. GA / PLGA-VCM hydrogel (sample 1), GA hydrogel (sample 2).

[0027] 1.6.2 CCK8 assay for cell viability 1. Cell seeding: Take L929 cells in logarithmic growth phase, digest them with trypsin, count the cells, adjust the cell concentration to 4×10^3 cells / well, seed them in 96-well plates, and incubate them in a 5% CO2, 37℃ incubator.

[0028] 2. Sample intervention: After the cells adhered to the wall, they were treated according to the above grouping and cultured in a 5% CO2, 37℃ constant temperature incubator for 1 day, 3 days and 5 days respectively.

[0029] 3. Detection procedure: After the culture is completed, remove the culture medium from each well, wash three times with PBS buffer, add 100 μL of culture medium containing 10% CCK8 reagent to each well, and incubate again in a 5% CO2, 37℃ constant temperature incubator for 2 hours.

[0030] 4. Absorbance measurement: The absorbance (OD value) of each well was measured at a wavelength of 450 nm using an ELISA reader, and the experimental data were recorded.

[0031] 1.7. In vivo diabetic wound model All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) and strictly followed ethical guidelines. Male Sprague-Dawley rats (200-250g) were acclimatized to the environment for one week. Streptozotocin working solution was prepared using 0.1M citrate / sodium citrate buffer (pH 4.5), taking care to avoid light during preparation. Experimental animals were fasted for 16 hours but allowed free access to water. They were administered a single intraperitoneal injection of 65mg / kg, followed by gavage with 20% glucose solution 4 hours later to prevent hypoglycemia-induced death. On day 7 post-injection, tail clipping was performed, and a blood glucose level >16.7mmol / L was considered a successful modeling. A circular full-thickness wound with a diameter of 8 mm was created on the back of the experimental animal. After cutting the wound, it was measured with a ruler and the diameter was 0.8 ± 0.1 cm. The wound was inoculated with 10 μL of MRSA (concentration of 1.25 × 10^6 CFU / mL), and the wound was sealed with a 3M membrane. The appearance of turbid pus from the wound 24 hours after infection was considered a successful infection model.

[0032] 1.8 Treatment Plan Rats were randomly assigned to five groups (n=6): (1) animal model + PBS (negative control); (2) animal model + LIFU (ultrasound control); (3) animal model + GA / PLGA-VCM hydrogel (passive treatment); (4) animal model + GA / PLGA-VCM hydrogel + LIFU (active treatment); and (5) animal model + GA hydrogel without PLGA-VCM loading + LIFU (material control). Treatment was performed every 3 days by local injection of the hydrogel precursor followed by in-situ crosslinking. For the LIFU group, 5 minutes of ultrasound therapy (1MHz, 4W / cm²) was performed after injection using a portable ultrasound transducer (coupled to the gel). 2 (50% duty cycle).

[0033] 1.9. Healing status and histological assessment Wound healing was digitally monitored on days 0, 3, 7, 10, 14, 17, and 21. Wound area was analyzed using ImageJ software. On day 7, wound tissue was collected, weighed, homogenized in sterile saline, and serially diluted on LB agar plates for colony-forming unit (CFU) counting. For histological analysis, tissue collected on day 21 was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Sections were stained with hematoxylin and eosin (HE) to assess epithelial regeneration and inflammation. Immunohistochemical (IHC) staining was performed using primary antibodies against TNF-α and IL-6. Oxidative stress was observed in frozen sections using dihydroethidium (DHE) fluorescence staining. Sample images were acquired using a fluorescence microscope (Olympus).

[0034] 2. Results 2.1 Physicochemical and Rheological Characterization Constructing a stable and responsive delivery system is crucial for in vivo application. PLGA nanoparticles synthesized using a two-emulsion method exhibit a smooth, spherical morphology. Dynamic light scattering (DLS) analysis revealed that the blank nanoparticles were monodisperse with an average hydrodynamic diameter of 282.4 nm. After encapsulation with VCM, the particle size increased to 383.4 nm, confirming successful drug encapsulation. The zeta potential of the VCM-loaded nanoparticles ranged from -20 mV to -40 mV, indicating strong electrostatic repulsion that prevents aggregation and ensures colloidal stability in biological fluids. High encapsulation efficiency is essential for maintaining therapeutic efficacy; based on standard curve calculations, the formulation achieved an encapsulation efficiency (EE) of 90.5% and a drug loading (DL) of 6.75%.

[0035] like Figure 1 As shown.

[0036] Rheological evaluation was used to verify the mechanical integrity of the hydrogel scaffold, such as... Figure 2 As shown.

[0037] Both the drug-loaded antioxidant hydrogel (GA / PLGA-VCM) and the unloaded antioxidant hydrogel (GA) exhibited typical viscoelastic solid behavior. Specifically, the storage modulus (G'), representing the elastic response, consistently exceeded the loss modulus (G'') throughout the testing period. Both hydrogels maintained a stable storage modulus, and this sustained elastic advantage (G'>G'') indicates that the hydrogel matrix has a stable cross-linked network that does not undergo structural collapse or sol-gel transition under the testing conditions, thus ensuring its effective use as a dressing.

[0038] The alginate hydrogel scaffold exhibits a highly interconnected porous structure with pore sizes ranging from 100 to 200 micrometers. Scanning electron microscopy (SEM) revealed a high porosity. This porosity facilitates the drainage of exudate and the permeation of oxygen.

[0039] like Figure 3 As shown.

[0040] Furthermore, the addition of gallic acid (GA) endows the hydrogel with significant antioxidant activity. The hydrogel extract effectively scavenges DPPH free radicals, achieving a scavenging rate of 67.23%, which can be observed through the decolorization of the DPPH solution.

[0041] like Figure 4 As shown.

[0042] 2.2. In vitro biocompatibility (cytotoxicity) Ensuring the biocompatibility of wound dressings is a prerequisite for clinical application. This study used the CCK-8 assay to evaluate the cytotoxicity of functionalized hydrogels on L929 fibroblasts. Results showed that both GA / PLGA-VCM hydrogel (sample 1) and GA hydrogel (sample 2) exhibited good cell compatibility. On day 1, cell viability for both samples was close to 100% at all concentrations (1%–100%), with no significant difference compared to the control group. Cells continued to proliferate actively on days 3 and 5. Although a slight decrease in cell viability was observed at the highest concentration (100%) on day 5 in a dose-dependent manner, the relative viability of both groups was well above 80%. This is significantly higher than the toxicity threshold (70%) defined by ISO 10993-5, confirming that the leachate products of the hydrogels (including gallic acid and trace PLGA degradation products) are non-toxic and support fibroblast proliferation.

[0043] like Figure 5 As shown.

[0044] 2.3. Kinetics of accelerated wound healing in diabetic rats The therapeutic effect was rigorously evaluated in a rat model of diabetes. During the 21-day study, the rats experienced a slight decrease in body weight.

[0045] like Figure 6 As shown.

[0046] Consistent with the streptozotocin (STZ)-induced diabetic phenotype, but no significant differences were observed between the treatment and control groups, indicating good systemic biocompatibility of the biomaterial. Macroscopic assessment of wound healing revealed significant differences. Wound healing was delayed in the PBS group and the control group receiving only ultrasound therapy, with persistent signs of inflammation and open wound beds even in later stages. In contrast, wound healing was accelerated in the group receiving functionalized hydrogel therapy. The “A+Ultrasound” (GA / PLGA-VCM+LIFU) group and the “B+Ultrasound” (GA+LIFU) group showed a statistically significant therapeutic advantage compared to the control group as early as day 3. This early accelerated healing has important clinical significance for preventing chronic transformation. By day 21, the wound healing in the “A+Ultrasound” group was the most complete, significantly better than that in the PBS control group (whose wounds were essentially unhealed).

[0047] like Figure 7 As shown.

[0048] 2.4. In vivo antibacterial effects and biofilm clearance The main obstacle to healing of diabetic foot ulcers is bacterial bioburden. Bacterial colony counts on day 7 showed that the infection burden remained high in the PBS control group at 7.26 ((CFU / mL / mg) × 10^5). After treatment with low-intensity focused ultrasound (LIFU) alone, the colony-forming unit (CFU) count moderately decreased to 4.46 ((CFU / mL / mg) × 10^5). This decrease ((CFU / mL / mg) × 10^5) is likely due to biofilm thickening caused by mechanical disturbance. The synergistic effect of the drug-loaded hydrogel and ultrasound was significant. The bacterial count in the passive treatment group (Group A, without LIFU treatment) decreased to 2.31 ((CFU / mL / mg) × 10^5). However, the aggressive “A+Ultrasound” treatment significantly reduced CFU / mg to 0.63 ((CFU / mL / mg) × 10^5). This reduction of more than one log unit compared to the control group highlights the necessity of an active triggering mechanism, with the A+Ultrasound group showing a more significant effect compared to the control group. Scanning electron microscopy analysis of wound tissue on day 21 further confirmed these findings: the control group wounds showed disordered fibers colonized by bacterial clusters, while the wounds in the "A+ Ultrasound" treatment group had smooth, sterile surfaces and orderly arranged collagen fibers.

[0049] like Figure 8 As shown.

[0050] 2.5 Tissue regeneration and microenvironment regulation Histological evaluation (H&E staining) on ​​day 21 confirmed the quality of tissue repair. The PBS group showed incomplete epithelial regeneration and extensive inflammatory cell infiltration. In contrast, the "A+ ultrasound" group showed intact layered epidermis and orderly dermal structure with minimal inflammatory cell infiltration.

[0051] like Figure 9 As shown.

[0052] Mechanistically, this regeneration is related to the regulation of the oxidative and inflammatory microenvironment. DHE staining showed strong red fluorescence (high ROS levels) in the PBS group.

[0053] like Figure 10 As shown.

[0054] The fluorescence in the "A+ ultrasound" group was effectively suppressed.

[0055] like Figure 11 As shown.

[0056] Immunohistochemistry showed widespread expression of the pro-inflammatory cytokine TNF-α.

[0057] like Figure 12 As shown.

[0058] The level of IL-6 in the wound was elevated in the control group.

[0059] like Figure 13 As shown.

[0060] “A+ ultrasound” treatment significantly reduced these markers to near baseline levels, indicating a successful transition from a chronic inflammatory state to a pro-regenerative phenotype.

[0061] in conclusion In summary, this study developed a multifunctional ultrasound-responsive antioxidant hydrogel that effectively creates a healing-promoting microenvironment for diabetic foot ulcers. Rheological and CCK-8 assays validated the material's physical stability and excellent biocompatibility. Through synergistic GA-mediated ROS scavenging and LIFU-triggered antibiotic release, this system significantly reduced bacterial load, alleviated inflammation, and accelerated wound healing compared to the passive control group. This "on-demand" treatment strategy provides an effective solution for overcoming the barriers of biofilm resistance and oxidative stress in chronic diabetic foot ulcers.

[0062] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for manufacturing a hydrogel for improving the microenvironment of chronic wounds in diabetic patients, characterized in that, Includes the following steps: Sodium alginate and gallic acid were dissolved in water to form a mixture, and calcium ion solution was added to crosslink the mixture to prepare an antioxidant alginate hydrogel.

2. The method for manufacturing a hydrogel for improving the microenvironment of chronic diabetic wounds according to claim 1, characterized in that, The dosage of sodium alginate is 100 mg / 5 mL water, and the dosage of gallic acid is 50 mg / 5 mL water.

3. A method for manufacturing a hydrogel for improving the microenvironment of chronic diabetic wounds according to claim 2, characterized in that, The calcium ion solution is a CaCl2 solution with a concentration of 10 mg / mL, and the volume ratio of the added solution to the volume of the mixed solution is 1:

1.

4. A method for manufacturing a hydrogel for improving the microenvironment of chronic diabetic wounds according to claim 1, characterized in that, It also includes the step of preparing ultrasonically responsive PLGA nanoparticles, wherein the PLGA nanoparticles are loaded with vancomycin.

5. A method for manufacturing a hydrogel for improving the microenvironment of chronic diabetic wounds according to claim 4, characterized in that, The PLGA nanoparticles were prepared using a double emulsion solvent evaporation method, which included dissolving PLGA and VCM in an organic solvent, adding perfluoropentane for emulsification, then emulsifying it a second time with a polyvinyl alcohol solution, and finally evaporating the solvent to obtain nanoparticles.

6. A method for manufacturing a hydrogel for improving the microenvironment of chronic diabetic wounds according to claim 5, characterized in that, In the PLGA nanoparticles, the amount of PLGA is 50 mg, the amount of VCM is 4 mg, and the amount of PFP is 200 μL.

7. A method for manufacturing a hydrogel for improving the microenvironment of chronic diabetic wounds according to claim 4, characterized in that, The PLGA nanoparticles are embedded in the hydrogel of claim 1 to form a composite hydrogel.

8. A hydrogel composition manufactured by any one of claims 1-7, characterized in that: It contains sodium alginate substrate, gallic acid functionalization modification, and encapsulated PLGA-VCM nanoparticles.

9. A method for manufacturing a hydrogel for improving the microenvironment of chronic diabetic wounds according to claim 8, characterized in that, The PLGA nanoparticles have an average hydrodynamic diameter of 300-400 nm and a zeta potential of -20 mV to -40 mV.

10. The use of the hydrogel composition according to claim 8 in the preparation of a medicament for treating chronic diabetic wounds.

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