Metal coordination driven dynamic composite hydrogel and preparation method and application thereof

Abstract

The application discloses a kind of metal coordination driven dynamic composite hydrogel and its preparation method and application.The application is by adding 0.5~1.0 mol / L sodium periodate solution to 1%w / v sodium hyaluronate aqueous solution, under the condition of light shielding, stirring reaction, after reaction is ended, quenching reaction, continue stirring 1~3 h;The reaction liquid is dialyzed, and the obtained dialysate is freeze-dried to obtain oxidized hyaluronic acid OHA;8.0~10% w / v dopamine hydrochloride aqueous solution and 8.0~10% w / v ferric trichloride aqueous solution are mixed, and the pH value of mixed solution is adjusted to 7.0~7.5, to obtain coordination mixed solution;4.0%~6.0%w / v oxidized hyaluronic acid OHA solution is added to the coordination mixed solution, and after being mixed uniformly, reaction is carried out at room temperature to obtain metal coordination driven dynamic composite hydrogel.The dynamic composite hydrogel is used for preparing a drug for treating multiple drug-resistant bacteria infected diabetic wounds, can realize mild photothermal antibacterial, inflammation control and antioxidant effect in cooperation, and finally speeds up the repair of infected diabetic wounds.

Description

Technical Field

[0001] This invention relates to the field of drug development technology, and in particular to a metal coordination-driven dynamic composite hydrogel (OFD composite hydrogel), its preparation method and application. Background Technology

[0002] Chronic skin wounds caused by diabetes typically exhibit persistent inflammation and delayed healing due to impaired microcirculation and local immune function. Simultaneously, these wounds are highly susceptible to multidrug-resistant (MDR) bacterial infections. Once bacteria form a biofilm on the wound surface, it not only weakens the penetration and efficacy of antibiotics but also continuously induces excessive accumulation of reactive oxygen species (ROS). The combined effects of infection and oxidative stress keep the wound in a pro-inflammatory state for a prolonged period, further inhibiting the transformation of macrophages to the M2 type and hindering angiogenesis and tissue regeneration. Therefore, effectively controlling bacterial infection while regulating the local oxidative stress microenvironment is of great significance for promoting the repair of chronic diabetic wounds.

[0003] Near-infrared photothermal therapy (PTT) generates a localized thermal effect through photothermal conversion, thereby disrupting bacterial structure and reducing drug resistance. However, traditional PTT typically relies on high temperatures (often above 55°C) to achieve the desired bactericidal effect. The tissue surrounding diabetic wounds is already in a state of ischemia and hypoxia, making it highly sensitive to thermal stimuli. Excessively high temperatures can easily cause secondary thermal damage to normal tissues and exacerbate local inflammation. In contrast, mild photothermal therapy (Mild-PTT) can achieve antibacterial effects at lower temperatures, making it more suitable for treating chronic wounds. However, Mild-PTT alone has limited ability to regulate the complex infection microenvironment. Summary of the Invention

[0004] To overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide a metal coordination-driven dynamic composite hydrogel, its preparation method, and its application.

[0005] The technical solution provided by this invention is as follows: A method for preparing a metal coordination-driven dynamic composite hydrogel (OFD composite hydrogel), the method comprising the following steps: (1) Add 0.5~1.0 mol / L sodium periodate solution to a 1% w / v sodium hyaluronate aqueous solution, react under light-proof and stirring conditions, quench the reaction after the reaction is completed, and continue stirring for 1~3 h; dialyze the reaction solution, freeze dry the obtained dialysate to obtain oxidized hyaluronic acid (OHA); (2) Mix 8.0~10% w / v dopamine hydrochloride aqueous solution and 8.0~10% w / v ferric chloride aqueous solution, adjust the pH of the mixture to 7.0~7.5, and obtain a complexation mixture; (3) Add 4.0%~6.0% w / v of oxidized hyaluronic acid (OHA) solution to the coordination mixture, mix evenly, and react at room temperature to obtain metal coordination driven dynamic composite hydrogel (OFD composite hydrogel).

[0006] Preferably, in step (1), the relative molecular mass of the sodium hyaluronate is 800,000 to 1,500,000 Da.

[0007] Preferably, in step (1), the volume ratio of the sodium hyaluronate aqueous solution to the sodium periodate solution is 100 mL: 0.5 mL to 1 mL.

[0008] Preferably, in step (1), 0.5~1.0 mL of ethylene glycol is added to quench the reaction after the reaction is completed.

[0009] Preferably, in step (1), the dialysis specifically involves transferring the reaction solution to a dialysis bag for dialysis treatment, with a dialysis time of 3 days and the dialysis fluid being replaced every 8 hours.

[0010] Preferably, in step (2), the molar ratio of dopamine hydrochloride to ferric chloride in the coordination mixture is 3:1.

[0011] Preferably, in step (3), the volume ratio of the coordination mixture to the oxidized hyaluronic acid (OHA) solution is 1:8~17.

[0012] This invention further discloses the metal coordination-driven dynamic composite hydrogel (OFD composite hydrogel) prepared by the above method.

[0013] This invention further discloses the application of the above-mentioned metal coordination-driven dynamic composite hydrogel (OFD composite hydrogel) in the preparation of a drug for treating diabetic wounds infected with multidrug-resistant bacteria.

[0014] This invention overcomes the shortcomings of existing technologies and provides a method based on dynamic covalent bonds and dopamine-ferric chloride (DA-Fe) hydrochloride. 3+This paper discusses the preparation method and applications of metal coordination-driven dynamic composite hydrogels with double cross-linking coordination bonds (OFD composite hydrogels). Hydrogels, due to their high water content and three-dimensional network structure similar to the natural extracellular matrix, can maintain a moist wound environment and adapt to irregular tissue defects, and have been widely used in wound repair. A suitable moist environment is conducive to cell migration, granulation tissue formation, and tissue regeneration, while traditional gauze or pre-formed dressings usually only provide passive coverage, making it difficult to adapt to complex wound morphologies and lacking the ability to effectively regulate the local pathological microenvironment. Based on this, dynamically cross-linked hydrogels can further adhere tightly to the wound through in-situ molding and form a stable physical barrier, reducing external stimuli and bacterial invasion while providing a suitable microenvironment for tissue repair. Therefore, constructing a dynamic hydrogel system with both antibacterial and microenvironment regulation capabilities is of great significance for the treatment of chronically infected wounds. Furthermore, dopamine (DA), as a typical polyphenol molecule, possesses both metal coordination and free radical scavenging capabilities, showing good application potential in the construction of functionalized wound materials. Among them, the catechol groups in the DA structure not only endow the material with good tissue affinity, but also alleviate local oxidative stress by scavenging excess ROS; while DA and Fe 3+ The resulting dynamic coordination structure endows the material with certain photothermal response properties. Based on this, this invention utilizes oxidized hyaluronic acid, dopamine hydrochloride, and ferric chloride to construct a double dynamically crosslinked OFD composite hydrogel via a one-step blending method. This system is linked by Schiff base bonds with dopamine hydrochloride-ferric chloride (DA-Fe) 3+ The coordination bonds form a dynamic network, giving the hydrogel excellent tissue adaptability and stability. Under 808 nm near-infrared light irradiation, the OFD hydrogel can generate a mild photothermal effect to inhibit bacterial growth, while promoting dopamine hydrochloride-ferric chloride (DA-Fe) production. 3+ The release of coordination complexes is facilitated. Furthermore, the polyphenolic structures within the gel network can further scavenge excess ROS, thereby alleviating local oxidative stress and blocking the inflammatory cascade. Based on these characteristics, this invention systematically evaluated the physicochemical properties, photothermal response behavior, and in vitro antibacterial and antioxidant activities of OFD hydrogels. Furthermore, its application potential in regulating the local inflammatory microenvironment and promoting the repair of infected wounds was verified using a live mouse hemorrhage model and a full-thickness skin defect model on the back of diabetic mice infected with MRSA.

[0015] The beneficial effects of this invention after adopting the above technical solution are as follows: The OFD composite hydrogel of this invention is used for the treatment of diabetic wounds infected with multidrug-resistant bacteria. The dynamic network structure in the OFD composite hydrogel endows the hydrogel with good injectability, self-healing ability, and tissue adaptability, and also gives it auxiliary hemostatic properties. Under irradiation with 808 nm near-infrared light, the OFD hydrogel can produce a mild photothermal effect, exhibiting an inhibitory effect on bacteria and biofilms; at the same time, photothermal stimulation can promote the production of dopamine hydrochloride-ferric chloride (DA-Fe) 3+ The OFD hydrogel responsively releases the coordination complex, while the polyphenolic structure within the gel network scavenge excess reactive oxygen species (ROS), effectively alleviating infection-related oxidative stress and excessive inflammatory responses. In vitro and in vivo experiments demonstrate that the OFD hydrogel effectively modulates the inflammatory microenvironment of infected wounds, promotes macrophage M2 polarization and angiogenesis, ultimately accelerating the repair of infectious diabetic wounds. Overall, this OFD composite hydrogel synergistically achieves mild photothermal antibacterial, inflammatory regulation, and antioxidant effects, providing a promising hydrogel strategy for the treatment of drug-resistant bacterial infections of chronic wounds. Attached Figure Description

[0016] Figure 1 This section describes the preparation and characterization of OFD hydrogels; (a) images of inverted vials of OFD4, 6, and 8 hydrogels; (b) representative SEM images of OFD hydrogels; (c) the self-healing properties of OFD hydrogels; and (d) the injectability of OFD hydrogels.

[0017] Figure 2 Characterization of OFD hydrogels includes: (a) strain scan tests of OFD hydrogels from 0.1% to 1000% at 1 Hz; (b) frequency scan tests of OFD hydrogels at 1% strain; (e) step strain tests of OFD hydrogels under low (1%) and high (1000%) strain conditions; and (d) shear viscosity tests of OFD hydrogels at shear rates from 0% to 300%.

[0018] Figure 3 The photothermal properties of OFD hydrogels; among which, (a) Water, Fe 3+ DA, DA-Fe 3+ (a) Temperature changes of OFD4, 6, and 8 hydrogels under 808 nm NIR (0.4 W / cm²) irradiation; (b) Thermal imaging image of temperature changes of OFD6 hydrogel; (c) DA-Fe 3+ (d) Temperature changes of OFD4, 6, and 8 hydrogels during on / off cycles; (e) Temperature changes of OFD6 hydrogel at different power levels; (f) Thermal stability of OFD6 hydrogel during five on / off cycles; (c) Thermal imaging images of temperature changes of OFD6 hydrogel at different power levels.

[0019] Figure 4 The results show the biocompatibility, swelling, and release properties of OFD hydrogels; (a) blood compatibility of OFD hydrogels; (b) swelling of OFD6 hydrogels; (c) in vitro release properties of OFD6 hydrogels; (d) cell viability after co-incubation of OFD hydrogels with mouse fibroblasts (L929) for 24, 48, and 72 h; and (e) live-dead fluorescence microscopy images (scale bar = 200 μm) after co-incubation of OFD hydrogels with mouse fibroblasts (L929) for 24, 48, and 72 h.

[0020] Figure 5 This study investigated the in vivo degradation of OFD hydrogel; (a) representative images of residual OFD6 hydrogel in the subcutaneous tissue of mice on the back, detected by B-mode ultrasound on days 1, 3, 5, 7, and 14 (scale bar = 2 mm); (b) quantitative analysis of residual hydrogel area based on ultrasound images; (c) changes in body weight of mice after subcutaneous implantation of OFD6 hydrogel; (d) representative H&E staining images of major organs (heart, liver, spleen, lung, and kidney) of mice treated with OFD6 hydrogel; (e) concentrations of inflammatory factors IL-1β, IL-6, and TNF-α in mouse skin tissue at the hydrogel injection site determined by qPCR (n=3); (f, g) analysis of complete blood count and biochemical indicators in mice (n=3) (unit: WBC-10). 9 / L, LYMPH-10 9 / L, RBC-10¹² / L, HCT-%, HGB-g / L, PLT-10 9 / L, ALT-U / L, AST-U / L, BUN-mg / dL, CREA-μmol / L); "ns" indicates no significant difference between the two groups.

[0021] Figure 6 shows the in vitro antibacterial, antioxidant, and anti-inflammatory properties of OFD6 hydrogel; (a) representative colony images of Staphylococcus aureus, Escherichia coli, and MRSA after co-incubation of OFD6 hydrogel and OHA with or without near-infrared light (NIR); (b) Staphylococcus aureus; (c) Escherichia coli; (d) Quantitative analysis of MRSA (n=3); (e) H2O2 scavenging rate of OFD6 hydrogel and sodium ascorbate (VcNa); (f) PTIO scavenging rate of OFD6 hydrogel and sodium ascorbate (VcNa); (j) ABTS scavenging rate of OFD6 hydrogel and sodium ascorbate (VcNa); (g~i) relative mRNA expression of IL-6, IL-1β, and TNF-α in LPS-stimulated RAW264.7 macrophages after treatment with OFD6 hydrogel for 24 h (n=3). =4); *p<0.05, **p<0.01, ***p<0.001, ****p <0.0001, “ns” indicates no significant difference between the two groups.

[0022] Figure 7 shows the in vivo hemostatic efficacy evaluation of OFD6 hydrogel; (a, d, g) are representative images of different treatment groups (n=3) in mouse femoral artery, liver, and tail vein bleeding models; (b, e, h) are quantitative analyses of hemostasis time in each treatment group's femoral artery, liver, and tail vein bleeding models (n=3); (c, f, i) are the total blood loss measured in each treatment group in each bleeding model (n=3); *p<0.05, **p<0.01, ***p<0.001, ****p <0.0001, "ns" indicates no significant difference between the two groups.

[0023] Figure 8 This study evaluated the therapeutic effect of OFD6 hydrogel on diabetic wounds infected with MRSA. The results included: (a) representative wound images of each treatment group on Days 0, 3, 5, 7, 10, 14, 18, and 21; (b) representative photographs of MRSA bacterial colonies formed on MH agar plates after 3 days in different treatment groups; (c) temperature changes in the OFD6 group under 808 nm NIR treatment; (d) skin wound healing rates at different time points in each group; (e) MRSA colony counts in the wound skin tissue of mice in different groups after 3 days of treatment (n=8); (f) DHE and DAPI staining of the back skin tissue of mice in different treatment groups after 7 days of treatment (scale bar = 100 μm); (g) relative quantitative analysis of DHE expression in different treatment groups; and (h) H2O2 concentration in the back skin tissue of mice in different groups after 11 days of treatment (n=6). *p < 0.05, **p < 0.01, ***p < 0.001.

[0024] Figure 9 shows the histological assessment of full-thickness skin wound infection in diabetic mice; (a) optical microscopic images of H&E-stained tissue from the back of mice in each treatment group on days 14 and 21 (scale bar = 2000 μm, 500 μm); (b) optical microscopic images of MASSON-stained tissue from the back of mice in each treatment group on days 14 and 21 (scale bar = 2000 μm, 500 μm); (c) quantitative analysis of wound length in each treatment group on day 14 (n = 3); (d) quantitative analysis of wound length in each treatment group on day 21 (n = 3); (e) quantitative analysis of collagen deposition in the wound area in each treatment group on day 14 (n = 3); (f) quantitative analysis of collagen deposition in the wound area in each treatment group on day 21 (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0025] Figure 10 This study evaluated the therapeutic intervention of OFD6 hydrogel in a full-thickness skin wound infection model in diabetic mice. Specifically, (a) the relative expression of IL-6, IL-1β, and TNF-α genes in wound skin tissue of mice in each treatment group was measured by qPCR on day 7 (n=3); (b) the relative mRNA expression levels of HIF-1α, α-SMA, and CD31 in wound skin tissue on day 14 were measured by qPCR. *p<0.05, **p<0.01, ***p<0.001, ****p <0.0001, “ns” indicates no significant difference between the two groups. Detailed Implementation

[0026] The technical solution of the present invention will be further described in detail below with reference to specific embodiments, but this does not constitute any limitation on the present invention.

[0027] I. Implementation Methods 1. Reagents and materials Dopamine hydrochloride was purchased from Anhui Zesheng Technology Co., Ltd. ABTS and PTIO were both purchased from Shanghai Bide Pharmaceutical Co., Ltd. (Shanghai, China). Hyaluronic acid, hydroxylamine hydrochloride solution, Calcein-AM (calcium oxymethyl fluorescein), Hoechst 33342, and ethidium dihydrogen ester (DHE) were all purchased from Maclean's Biochemical Co., Ltd. (Shanghai, China). Propidium iodide (PI) and lipopolysaccharide (LPS) were both purchased from Sol Biosciences Co., Ltd. (Beijing, China).

[0028] 2. Cell lines and strains The L929 cell line (RRID: CVCL_L929) was provided by the Medical Experimental Center of Lanzhou University, while RAW264.7 was purchased from Purcell Biotechnology Co., Ltd. (Wuhan, China). All cell lines were cultured in high-glucose DMEM medium (Servicebio, China) containing 10% fetal bovine serum (FBS; Israel Biosciences). In bacterial experiments, Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922), and methicillin-resistant Staphylococcus aureus (ATCC 43300) were all obtained from the American Type Culture Collection (ATCC).

[0029] 3. Laboratory animals Male Kunming mice (18–22 g) and male Sprague-Dawley (SD) rats (180–220 g) were purchased from the Laboratory Animal Center of Lanzhou University. All animals were housed in a temperature-controlled environment at 22 ± 1 °C with a 12-hour light-dark cycle. Animals had free access to ample food and water. All experimental protocols complied with European Community Council Directive of 24 November 1986 (86 / 609 / EEC) and were approved by the Ethics Committee of the School of Basic Medical Sciences, Lanzhou University.

[0030] 4. Preparation of metal coordination-driven dynamic composite hydrogels (OFD composite hydrogels) (1) Weigh 1.0 g of sodium hyaluronate with a relative molecular mass of 800,000 to 1,500,000 Da, place it in a 250 mL round-bottom flask, add 100 mL of pure water, and stir on a magnetic stirrer until completely dissolved; then slowly add 0.5 mL to 1 mL of sodium periodate solution with a concentration of 0.5 to 1.0 mol / L, and stir the reaction for 30 min under light-protected conditions; after the reaction is completed, add 0.5 to 1.0 mL of ethylene glycol to quench the reaction, and continue stirring for 1 to 3 h; transfer the reaction solution to a dialysis bag for dialysis treatment, and the dialysis time is 3 days, with the dialysis fluid changed every 8 h (i.e., the water is changed 3 times a day); finally, collect the dialysis fluid and freeze-dry it to obtain the oxidized hyaluronic acid (OHA) product.

[0031] (2) Weigh out appropriate amounts of dopamine hydrochloride and ferric chloride respectively, dissolve them in deionized water, and prepare dopamine hydrochloride stock solution and ferric chloride stock solution with a mass-volume concentration of 8.0~10% (w / v) respectively, and keep them away from light for later use.

[0032] In preparing a 1 mL hydrogel system, the required volumes of the above-mentioned dopamine hydrochloride stock solution and ferric chloride stock solution were transferred and mixed. The molar ratio of dopamine hydrochloride to ferric chloride was controlled at 3:1, and a regulating agent was added dropwise to adjust the pH of the mixture to 7.0-7.5, so that the two could fully undergo a metal coordination reaction to obtain a coordination mixture.

[0033] (3) Add 4.0%~6.0% (w / v) of oxidized hyaluronic acid (OHA) solution to the above coordination mixture and make up to 1 mL. The volume ratio of coordination mixture to oxidized hyaluronic acid OHA solution is 1:8~17. After mixing evenly, Schiff base crosslinking reaction occurs rapidly at room temperature to form metal coordination driven dynamic composite hydrogel (OFD composite hydrogel).

[0034] 5. Scanning electron microscopy observation The microstructure of the hydrogel was observed using field emission scanning electron microscopy (FE-SEM). First, the hydrogel was placed on a clean silicon wafer and left at room temperature until all moisture evaporated. Then, gold was sputtered onto its surface, and the microstructure was recorded using FE-SEM. The microstructure of the hydrogel network was observed using FE-SEM. The hydrogel sample was fixed to a silicon substrate, dried at room temperature, and then surface-sputtered with gold to enhance surface conductivity. Finally, its micromorphology was imaged at an accelerating voltage of 5 kV.

[0035] 6. Determination of hydrogel rheological properties The rheological properties of hydrogels were systematically evaluated using a rotational rheometer in oscillatory mode. The viscoelastic behavior of the material was characterized by quantitative analysis of storage modulus (G′) and loss modulus (G″) and their interrelationship. A strain scan range of 1%–1000% (logarithmic mode) was set to determine the linear viscoelastic region (LVR) of the hydrogel, providing suitable strain parameters for subsequent testing. Based on the determined LVR, the strain was fixed at 1% and the frequency at 1 Hz, and the changes in G′ and G″ were continuously monitored over 30 min to assess the structural stability of the material. Maintaining a 1% strain, scans were performed within a frequency range of 1–100 rad / s to examine the frequency dependence and determine the effect of scan frequency on the rheological properties of the hydrogel. A cyclic step strain mode was used: first, a baseline value was measured at 1% strain and 1 Hz for 5 min, then switched to 100% strain for 1 min, and this cycle was repeated 5 times to evaluate the shear thinning effect and structural recovery capability of the material. The thermal stability of the material was characterized by monitoring the temperature dependence of G′ and G″ under the conditions of 1 Hz frequency and 1% strain, from 25°C to 70°C at a constant heating rate (e.g., 2°C / min).

[0036] 7. Determination of photothermal properties of hydrogels To systematically evaluate the in vitro heating effect of the hydrogel, samples of OFD hydrogels containing H2O, FeCl3, DA, DA-Fe³⁺, and different concentrations were irradiated with 808 nm near-infrared light at a power density of 0.4 W / cm², with temperature changes recorded every 10 seconds. Simultaneously, infrared thermography was used to visualize and evaluate the heating process of each group of samples. Based on this, the effect of different laser powers on the heating behavior of OFD6 hydrogels was further investigated, and the heating-cooling curves were recorded through five consecutive on / off irradiations to evaluate the photothermal stability of the hydrogel.

[0037] 8. Swelling behavior of hydrogels The in vitro swelling behavior of the hydrogels was evaluated in phosphate-buffered saline (PBS) at 37°C. Precisely weighed hydrogel samples were immersed in excess PBS. Samples were removed at predetermined time points, and excess free water was gently blotted off with filter paper before weighing until the weight no longer changed (swelling equilibrium was reached). The swelling ratio was calculated using the following formula: . In the formula W t Let Wt be the sample weight at time t, and W0 be the initial weight of the sample.

[0038] 9. In vitro DA-Fe 3+ Release assay To investigate the in vitro release kinetics of DA-Fe³⁺, this invention established a non-photothermal group (PBS, pH 7.4) and a photothermal stimulation group (808 nm near-infrared laser irradiation, power density 0.4 W / cm²) under physiological conditions, and systematically monitored the release using ultraviolet-visible (UV-Vis) spectrophotometry. Before the experiment, a standard curve was plotted by measuring the absorbance of a series of DA-Fe³⁺ standard solutions at 380 nm to ensure a linear correlation coefficient R² > 0.99. In the in vitro release test, precisely weighed hydrogel samples were completely immersed in the release medium and incubated at 37°C with constant temperature shaking. The photothermal stimulation group was subjected to corresponding laser irradiation at preset time points. At the preset time points, quantitative release samples were aspirated, appropriately diluted, and their absorbance was measured. The cumulative release of DA-Fe³⁺ at different time points was then calculated using the standard equation.

[0039] 10. Determination of the biocompatibility of hydrogels This invention comprehensively evaluated the in vitro biosafety of the hydrogel through in vitro hemolysis experiments and cytotoxicity tests. Hemolytic toxicity: First, red blood cells (RBCs) were isolated from rat whole blood by centrifugation (800×g, 5 min), and after repeated washing with PBS, an 8% (v / v) suspension was prepared. Subsequently, the RBC suspension was mixed with an equal volume of hydrogel and incubated at 37°C for 1 h. 1% Triton X-100 and physiological saline were used as positive and negative controls, respectively. After incubation, the sample was centrifuged twice (1200×g, 15 min), and the absorbance (OD) of the supernatant was measured at 490 nm using an ELISA reader. The hemolysis rate was calculated using the following formula: . Cytotoxicity assessment: Cell viability was assessed using the MTT assay and a double staining method for live and dead cells. L929 mouse fibroblasts were cultured in DMEM complete medium containing 10% FBS and 1% penicillin antibiotics at 37°C and 5% CO2.

[0040] MTT assay: Cells were injected at a concentration of 1×10⁻⁶. 4 Cells were seeded per well in 96-well plates and allowed to adhere overnight. The medium was then replaced with fresh medium containing different concentrations of hydrogel extract (three replicates per group). After co-culturing for 24, 48, and 72 h, 20 μL of MTT reagent was added to each well for 4 h of incubation. The supernatant was discarded, and 150 μL of DMSO was added and shaken for 10 min to dissolve the purple formazan. OD values ​​were measured at 570 nm, and cell viability was calculated as follows: . Live / dead staining: L929 cells were co-cultured with the extract at different time points (24, 48, 72 h), then the culture medium was removed, and cells were stained with Calcein-AM (5 μM) and PI (50 μM) in the dark for 30 min and 15 min, respectively. After washing with PBS, fluorescence images were acquired using a laser confocal microscope (CLSM).

[0041] 11. In vivo degradation behavior In vivo degradation and biocompatibility of the hydrogel were evaluated using animal experiments. The back of mice was selected as the injection site, with 500 μL of hydrogel material injected subcutaneously at each site. On days 1, 3, 5, 7, and 14 post-administration, the volume changes of the subcutaneous hydrogel were dynamically monitored using a small animal high-resolution ultrasound imaging system. Animals were sacrificed on day 10 post-surgery, and tissues from the injection sites and major organs (heart, liver, spleen, lung, and kidney) were collected. Total RNA was extracted from the injection site tissues, and the mRNA expression levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α were analyzed by real-time quantitative polymerase chain reaction (qPCR). After fixation and embedding, the skin and organs at the injection sites were stained with hematoxylin and eosin (H&E) to assess histopathological changes. Simultaneously, whole blood samples were collected. One portion was used for whole blood cell analysis (using a fully automated blood analyzer, Mindray BC-2800vet); the other portion, after centrifugation to separate serum, was used to detect the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), and blood urea nitrogen (BUN) using an automated biochemical analyzer (Rayto Chemray 800) to assess liver and kidney function.

[0042] 12. Determination of the in vitro antibacterial activity of hydrogels Collected bacterial suspensions of Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and methicillin-resistant Staphylococcus aureus (MRSA) grown to the logarithmic phase and diluted 20-fold with fresh culture medium. 50 μL of each diluted bacterial suspension was added dropwise to the surface of a hydrogel, with separate groups for no near-infrared light (NIR) and near-infrared light (NIR). The NIR group samples were irradiated with 808 nm near-infrared light at a power density of 0.4 W / cm² for 10 min, while the NIR group samples were treated for the same duration at room temperature in the dark. All samples were then transferred to a 37 ℃ constant temperature and humidity incubator and incubated for 2 h. After incubation, 950 μL of fresh culture medium was added to each tube and mixed thoroughly. 100 μL of the mixture was then evenly spread onto a solid culture medium plate and incubated at 37 ℃ constant temperature and humidity for 18–24 h. Colony-forming units (CFU) were counted on each plate. A saline-treated group was used as a blank control to evaluate the antibacterial effect of the hydrogel under different conditions. The colony count (N) on the plate is converted to the viable bacteria count (CFU / mL) per milliliter of sample using the following formula: CFU / mL = (N × D) / V, where, This is the total dilution factor of the bacterial suspension after incubation. Let be the volume of the coated plate. Therefore, To facilitate statistical analysis, the results are converted into the commonly used logarithmic form: Log 10 (CFU / mL) = Log 10 (N×10).

[0043] 13. Determination of the in vitro antioxidant activity of hydrogels This invention comprehensively evaluates the in vitro antioxidant activity of the hydrogel using ABTS, PTIO, and hydrogen peroxide (H2O2) scavenging experiments. ABTS cationic radical scavenging experiment: First, an ABTS⁺ stock solution was prepared by mixing equal volumes of 2.45 mM ABTS and 2.45 mM potassium persulfate solution and reacting at room temperature in the dark for 12–16 h. Before use, the stock solution was diluted 20 times with ultrapure water to prepare the working solution. Subsequently, the hydrogel was incubated with 1.5 mL of this working solution for 25 minutes, and its absorbance at 734 nm was measured. PTIO radical scavenging experiment: An equal volume of the hydrogel was mixed with 0.2 mM PTIO aqueous solution and reacted at room temperature in the dark for 2 h. The absorbance of the mixture at 557 nm was then measured. H2O2 scavenging experiment: An equal volume of the hydrogel was mixed with 1 mM H2O2 solution and reacted for 2 h. Subsequently, 50 μL of the reaction supernatant was taken and 100 μL of titanium sulfate colorimetric reagent was added (the colorimetric reagent formula was prepared by mixing 1.33 mL of 24% Ti(SO4)2, 8.33 mL of H2SO4, and 50 mL of ultrapure water). After standing for 30 minutes, the absorbance at 405 nm was measured. Sodium ascorbate (VcNa) was used as a positive control in all the above antioxidant evaluation experiments. Each experiment was repeated in triplicate (n = 3). The scavenging rate was calculated using the following formula: . (Note: OD) control The absorbance of the solvent control group is shown in Figure OD. blank (The background absorbance of the sample without colorimetric reagent).

[0044] 14. In vitro anti-inflammatory activity assay of hydrogel To evaluate the in vitro anti-inflammatory effect of the hydrogel, the transcriptional levels of relevant inflammatory factors were detected by real-time quantitative polymerase chain reaction (qPCR). RAW264.7 mouse macrophages were used at a density of 4 × 10⁶ cells per well. 5 Cells were seeded at a density of 2 mL per well in 6-well plates. After 24 h of cell adhesion, 300 ng / mL lipopolysaccharide (LPS) was added to induce an inflammatory state, followed by replacement with hydrogel extraction medium and continued culturing for another 24 h. Total RNA was extracted from the cells using TRIzol reagent and reverse transcribed into cDNA. Using GAPDH as an internal control, the relative mRNA expression levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were analyzed by qPCR. Primer sequences are as follows: GADPH-F: 5'-TGTGTCCGTCGTGGATCTGA-3', GADPH-R: 5'-TTGCTGTTGAAGTCGCAGGAG-3'; IL-1β-F: 5'-TGGTGTGTGACGTTCCCATT-3', IL-1β-R: 5'-TGTCGTTGCTTGGTTCTCCT-3'; IL-6-F: 5'-CGGCCTTCCCTACTTCACAA-3', IL-6-R: 5'-GCAAGTGCATCATCGTTGTTC-3'; TNF-α-F: 5'-ACTCCAGGCGGTGCCTATGT-3', TNF-α-R: 5'-GTGAGGGTCTGGGCCATAGAA-3'.

[0045] 15. In vivo hemostatic activity assay To verify the in vivo hemostatic ability of the material, three animal models were established: liver hemorrhage, tail vein transection, and femoral artery hemorrhage. The in vivo hemostatic efficacy of the hydrogel was systematically evaluated. Healthy KM mice (18-22 g) were randomly divided into a model control group, a hydrogel group, and a medical gauze group, with 3 mice in each group. Animals were fasted for 12 hours before the experiment but had free access to water. After anesthesia, the surgical area was shaved, prepared, and disinfected with povidone-iodine. Liver hemostasis model: The abdominal cavity was opened along the midline, and the liver lobes were gently separated. A pre-weighed sterile filter paper (W0) was placed below the liver lobe. A 26G needle was used to puncture the liver parenchyma to a depth of approximately 2 mm to establish a hemorrhage model. The hydrogel (or gauze) was immediately applied to the wound and gently pressed for 5 seconds. The time to cessation of bleeding (T) was recorded. The filter paper was collected and weighed again (W1), and the blood loss (W1~W0) was calculated based on the increase in the weight of the filter paper. Tail vein hemostasis model: The mouse tail was fixed, and the tail vein was completely transversely severed at the distal 1 / 3. A pre-weighed filter paper (W0) was placed under the severed end. Hydrogel was then applied or gauze was used for pressure to stop the bleeding. The hemostasis time and filter paper weight gain were recorded. Femoral artery hemostasis model: The mouse hind limb was fixed, the femoral skin was incised, and the femoral artery was bluntly dissected and completely transversely severed at the midpoint of the vessel. A pre-weighed filter paper (W0) was placed under the bleeding point, and hydrogel was quickly applied or gauze was used for pressure bandaging. The hemostasis time and blood loss were recorded.

[0046] 16. Effect of hydrogels on wound healing in diabetic mice Male Kunming mice aged 6–8 weeks (20±2 g) were acclimatized for one week before modeling. They were intraperitoneally injected with streptozotocin (STZ, Macklin, China) for 5 consecutive days. The diabetic model was successfully established when the blood glucose level consistently exceeded 16.7 mmol L⁻¹. The day before the experiment, the hair on the backs of the mice was completely removed with depilatory cream. An 8 mm diameter full-thickness wound was created on the back of each mouse using a biopsy punch, and MRSA bacteria diluted 10-fold and in the logarithmic growth phase were injected into the wound (50 μL). Physiological saline, sterile dressings, and OFD6 and OFD6+NIR hydrogels were applied to the wounds sequentially. The OFD6+NIR group was irradiated with 808 nm near-infrared light for 5 min after application. Wound area was observed and photographed on days 0, 3, 5, 7, 10, 14, 18, and 21 post-surgery. On day 3, MRSA-infected skin tissue was homogenized and spread on MH plates to quantify bacterial load and photograph colonies. On day 7, ROS in the wound tissue were determined by DHE staining. Fresh tissue sections were stained with DHE (5 μM) and DAPI at 37°C for 20 minutes in the dark, and fluorescence imaging was performed using CLSM. Semi-quantitative analysis of ROS intensity was performed using ImageJ software. Simultaneously, skin tissue from the wound site was collected, homogenized, and RNA was extracted using the TRIzol method and reverse transcribed. GADPH was used as an internal control gene, and the expression of cytokines in the tissue was measured by real-time quantitative PCR. Primer sequences are as follows: GAPDH-F: 5'-TGTGTCCGTCGTGGATCTGA-3'; GAPDH-R: 5'-TTGCTGTTGAAGTCGCAGGAG-3'; IL-1β-F: 5'-TGGTGTGTGACGTTCCCATT-3'; IL-1β-R: 5'-TGTCGTTGCTTGGTTCTCCT-3'; IL-6-F: 5'-CGGCCTTCCCTACTTCACAA-3'; IL-6-R: 5'-GCAAGTGCATCATCGTTGTTC-3'; TNF-α-F: 5'-ACTCCAGGCGGTGCCTATGT-3'; TNF-α-R: 5'-GTGAGGGTCTGGGCCATAGAA-3'.

[0047] On day 14, all mice were euthanized. Skin tissue from the dorsal wounds was fixed in 4% formaldehyde and embedded in paraffin for sectioning. The paraffin sections were stained with H&E and MASSON for histological examination. Cytokine expression in the tissues was measured by real-time quantitative PCR. GADPH was used as an internal control gene, and cytokine expression in the tissues was measured by real-time quantitative PCR. Primer sequences are as follows: GAPDH-F: 5'-TGTGTCCGTCGTGGATCTGA-3'; GAPDH-R: 5'-TTGCTGTTGAAGTCGCAGGAG-3'; CD31-F: 5'-CAACAGAGCCAGCAGTATGAG-3'; CD31-R: 5'-AATGACAACCACCGCAATGA-3'; α-SMA-F: 5'-CCCTGAAGAGCATCCGACAC-3'; α-SMA-R: 5'-CCAGAGTCCAGCACAATACCCA-3'; HIF-1α-F: 5'-CCCTCCGATTTAGCATGCAGAC-3'; HIF-1α-R: 5'-CACCCTGCAGTAGGTTTCTGCT-3'; CD206-F: 5'-TGGAGGGTGCGGTACACTAA-3'; CD206-R: 5'-CTGTTCTGACTCTGGACACTTGC-3'.

[0048] On day 21, all mice were euthanized. Skin tissue from the wounds on the backs of the mice was fixed in 4% formaldehyde and embedded in paraffin for sectioning. The paraffin sections were stained with H&E and MASSON for histological examination.

[0049] 17. Statistical Analysis Statistical analysis was performed using GraphPad Prism 9.0. All data are expressed as mean ± standard deviation (SD). Student's t-test was used to compare statistically significant differences between two groups, and one-way ANOVA was used for multiple group comparisons. P < 0.05 was considered statistically significant.

[0050] II. Implementation Results 1. Preparation of OFD hydrogels OFD hydrogels were constructed in a one-step process via coordination of dopamine hydrochloride-ferric chloride (DA-Fe³⁺) and a Schiff base reaction with oxidized hyaluronic acid (OHA), resulting in a dual network of both chemical and physical cross-linking. By adjusting the concentrations of dopamine hydrochloride-ferric chloride and the amount of oxidized hyaluronic acid solution added, the final concentrations of dopamine hydrochloride (0.4%–0.8%), ferric chloride (0.11%–0.23%), and oxidized hyaluronic acid (4.0%–6.0%) in the resulting hydrogels were achieved. Three types of hydrogels, OFD4, OFD6, and OFD8, were prepared respectively. Figure 1 a). Scanning electron microscopy revealed that the hydrogel exhibited a uniform porous structure inside. Figure 1 b). For example Figure 1 As shown in Figure c, after the cut OFD gel blocks were placed end-to-end for 30 minutes, the two cut surfaces healed spontaneously, confirming its self-healing properties. Furthermore, the OFD hydrogel could be easily extruded from the syringe, even continuously extruded to form the letter "Fe³⁺" (…). Figure 1 d), demonstrating its good injectability.

[0051] 2. Rheological properties of OFD hydrogels The rheological properties of OFD hydrogels were systematically characterized using a rheometer. Oscillatory shear rheological studies showed that ( Figure 2 a) When the strain is below 501%, G' > G'', exhibiting typical gel behavior; when the strain exceeds 501%, G' < G'', and the system transitions from a gel state to a sol state. Dynamic frequency scanning results show ( Figure 2 (b) Within the angular frequency range of 1–100 rad / s, the storage modulus (G') of the hydrogel is consistently higher than its loss modulus (G''), and both increase slightly with increasing frequency, indicating that the material maintains a stable solid-like state at different frequencies and exhibits good viscoelasticity. The self-healing properties of the hydrogel were investigated by cycling between high and low strain. Figure 2 c). Under high strain (1000%), G' < G'', the gel network structure is disrupted, transforming into a flowable sol; when the strain recovers to 1%, the system rapidly rebuilds into a viscoelastic gel (G' > G''). After multiple cycles, the hydrogel can still stably recover to the gel state, indicating its excellent self-healing ability. Steady-state shear tests show ( Figure 2 d) The viscosity of the hydrogel gradually decreases with increasing shear rate, exhibiting shear-thinning properties.

[0052] 3. Photothermal properties of OFD hydrogels Suitable photothermal temperature and good photothermal stability are key to the effective antibacterial properties of wound dressings. Therefore, before conducting subsequent antibacterial and healing-promoting experiments, this invention first evaluated the photothermal performance of OFD hydrogels under 808 nm near-infrared light. Irradiation was performed at 0.4 W / cm² for 600 s, and the temperature rise of H₂O, FeCl₃ solution, DA solution, DA-Fe³⁺ solution, and OFD₄, (6), and (8) hydrogels was compared. Figure 3 a). The results showed that the temperature of DA-Fe³⁺ and the three OFD hydrogels gradually increased with irradiation time, with endpoint temperatures of 42.1℃, 48.0℃, 50.7℃, and 58.7℃, respectively; after the laser was turned off, the temperatures of all groups naturally decreased. Figure 3 c). Considering both skin tolerance limitations and the temperature range required for photothermal antibacterial effects, this invention selected OFD6 hydrogel for subsequent research. Its temperature changes under thermal imaging are as follows: Figure 3 As shown in b. Next, the photothermal response of OFD6 under different laser powers was evaluated. After irradiation for 600 s at 0.4, 0.6, 1.0, and 1.2 W / cm², the hydrogel temperature rose to 50.5℃, 64.3℃, 78.4℃, and 110.5℃, respectively, exhibiting a significant power dependence. Figure 3 d, g). Furthermore, after five cycles of switching lasers, the hydrogel's heating capacity did not decrease significantly. Figure 3 e), indicating that OFD6 has good photothermal stability.

[0053] Based on the above results, the photothermal temperature of OFD series hydrogels can be effectively controlled by adjusting the content of dopamine-ferric chloride (DA-Fe³⁺) or the power density of near-infrared laser.

[0054] 4. Swelling behavior and photothermal response drug release characteristics The swelling behavior of the OFD hydrogel is shown in Figure 4b. All three formulations (OFD4, 6, and 8) exhibited excellent water absorption properties, with equilibrium swelling rates of approximately 29.5%, 31.2%, and 32.8%, respectively. This indicates that these hydrogels can effectively remove wound exudate, thus preventing exudate from adversely affecting the healing process. Furthermore, the in vitro release curves (Figure 4c) show that the hydrogel exhibits a significant photothermal response release characteristic. Under normal physiological conditions (PBS), the dopamine hydrochloride-ferric chloride (DA-Fe³⁺) complex exhibits a stable sustained-release mode, with a final cumulative release rate of approximately 30%. However, after irradiation with 0.4 W / cm² 808 nm near-infrared light for 5 minutes, the release rate of the complex significantly increased, with the cumulative release rate ultimately reaching approximately 75%. This phenomenon can be attributed to the dopamine hydrochloride-ferric chloride (DA-Fe³⁺) coordination structure in the hydrogel network acting as a photothermal conversion center: photothermal stimulation induces dynamic relaxation of the gel network, thereby accelerating the responsive release of the dopamine hydrochloride-ferric chloride (DA-Fe³⁺) complex. This "on-demand" release mechanism endows the hydrogel with intelligent regulatory capabilities—under photothermal action, the release of active ingredients at the wound site is accelerated, achieving targeted delivery of antibacterial, anti-inflammatory, and antioxidant functions, which not only improves treatment efficiency but also reduces ineffective drug loss in non-target areas.

[0055] 5. Biocompatibility of OFD hydrogels Good biocompatibility is fundamental for the application of biomaterials in the medical field. As a prerequisite for evaluating the biological function of OFD6 hydrogel, this invention first systematically evaluated its in vitro biocompatibility. The blood compatibility of the hydrogel was assessed through a hemolysis experiment. OFD4, 6, and 8 hydrogels were co-incubated with rat erythrocytes for 1 hour, and the supernatant was collected by centrifugation for quantitative analysis. The results showed that the hemolysis rate of all hydrogel groups was extremely low, comparable to the PBS control group (Figure 4a), indicating good blood compatibility. The cytotoxicity of the hydrogel to L929 cells was detected using the MTT assay. After treating cells with different concentrations of hydrogel extract for 24, 48, and 72 hours, the cell viability in each experimental group remained above 80% (Figure 4d). To further visually assess cell viability, this invention used Calcein-AM / PI double staining to distinguish between live and dead cells. The principle is as follows: Calcein-AM can permeate the cell membrane, enter living cells, and be hydrolyzed by intracellular esterases to generate calcein, which emits strong green fluorescence; while PI can only pass through damaged cell membranes to enter dead cells, where it binds to DNA and emits red fluorescence. Staining results showed that after hydrogel treatment, the vast majority of L929 cells exhibited green fluorescence, while the red fluorescence signal was extremely weak, showing no significant difference from the control group (Figure 4e). Based on these results, OFD6 hydrogel demonstrates excellent biocompatibility in vitro, providing important support for its subsequent in vivo application.

[0056] 6. In vivo degradation and safety assessment of OFD hydrogels The degradation characteristics of hydrogels are crucial to the safety and efficacy of their clinical applications. Therefore, before evaluating the in vivo therapeutic potential of OFD6 hydrogel, this invention first assessed its degradation behavior in vivo. By subcutaneously injecting a quantitative amount of hydrogel into mice and dynamically observing it using a small animal ultrasound imaging system, the results showed that the volume of the subcutaneous hydrogel gradually decreased with increasing days (Figure 5a), and by day 14, it was almost completely degraded with no obvious residue (Figure 5b), indicating that the hydrogel can be completely degraded in vivo without the need for secondary surgical removal. During the observation period, the mice showed a slow trend of weight gain (Figure 5c), indicating good overall health. Further H&E staining of major organs (heart, liver, spleen, lungs, and kidneys) showed that the tissue structure of each organ in the hydrogel-treated group was normal, with no obvious inflammatory cell infiltration or tissue damage (Figure 5d). Simultaneously, skin tissue homogenates from the injection site were collected, and the expression levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α were detected by qPCR. The results showed no significant difference between the hydrogel-treated group and the control group (Figure 5e). In addition, mouse blood was collected for routine and biochemical parameter testing. There were no significant differences in hematological parameters (white blood cell count WBC, lymphocyte count LYMPH, red blood cell count RBC, hematocrit HCT, hemoglobin HGB, platelet count PLT) and biochemical parameters (alanine aminotransferase ALT, aspartate aminotransferase AST, creatinine CREA, blood urea nitrogen BUN) between the hydrogel-treated group and the control group (Figures 5f and g). These results indicate that OFD6 hydrogel has good biocompatibility and biosafety in vivo.

[0057] 7. In vitro antibacterial properties of OFD hydrogel Based on literature reports, hydrogels containing dopamine hydrochloride-ferric chloride (DA-Fe³⁺) can convert light energy into heat energy under near-infrared (NIR) irradiation, thereby achieving a photothermal bactericidal effect. Accordingly, this invention hypothesizes that OFD6 hydrogel may possess excellent antibacterial properties, and evaluates its antibacterial activity with and without NIR irradiation using *Staphylococcus aureus*, *Escherichia coli*, and methicillin-resistant *Staphylococcus aureus* (MRSA) as models. As shown in Figures 6a-d, even without NIR irradiation, the number of colonies significantly decreased after co-incubation of OFD6 hydrogel with the three bacteria, demonstrating a certain antibacterial effect. Under NIR irradiation, the bactericidal effect of OFD6 hydrogel on the three bacteria is further enhanced, exhibiting superior antibacterial performance. This is mainly attributed to the photothermal conversion effect of the dopamine hydrochloride-ferric chloride (DA-Fe³⁺) complex. OFD6 hydrogel exhibits excellent antibacterial properties under near-infrared irradiation, indicating its potential as an effective photothermal antibacterial wound dressing.

[0058] 8. In vitro antioxidant properties of OFD hydrogels A complex interaction exists between oxidative stress and diabetic infected wounds. Oxidative stress not only directly damages the skin barrier and cellular function, but also further aggravates the infection state by activating inflammatory signaling pathways and altering the wound microbiota composition. Therefore, antioxidant therapy has become an important strategy for managing diabetic infected wounds. Hydrogen peroxide (H2O2) is a typical reactive oxygen species. The scavenging ability of three hydrogels, OFD4, OFD6, and OFD8, for H2O2 was evaluated by measuring the absorbance at 405 nm using the Ti(SO4)2 colorimetric method. The results showed (Figure 6e) that the scavenging rates of H2O2 by the three hydrogels were 85.36±2.095%, 90.51±2.046%, and 95.26±0.747%, respectively, all of which were superior to the antioxidant VcNa. ABTS and PTIO are commonly used model molecules for evaluating the scavenging ability of materials for reactive nitrogen species (RNS). ABTS is oxidized to generate ABTS⁺ cationic free radicals, which are blue-green and have a characteristic absorption peak at 734 nm. The ABTS scavenging rates of OFD4, (6), and (8) were 73.27±2.426%, 76.98±2.905%, and 80.19±1.8%, respectively (Figure 6j). The PTIO solution was deep blue and had a characteristic absorption peak at 557 nm; the PTIO scavenging ability of the three hydrogels was comparable to that of VcNa (Figure 6f). The above results indicate that the OFD series hydrogels have good scavenging effects on both oxygen and nitrogen free radicals, exhibiting excellent antioxidant activity.

[0059] 9. In vitro anti-inflammatory properties of OFD hydrogel In diabetic wound infections, uncontrolled inflammation is a core driver of delayed healing. The high-glucose environment continuously activates inflammatory pathways such as NF-κB, leading to the excessive release of pro-inflammatory factors such as IL-6, IL-1β, and TNF-α, which in turn causes abnormal macrophage polarization, resulting in prolonged inflammatory stasis in the wound. Therefore, inhibiting the overexpression of pro-inflammatory factors and regulating macrophage polarization towards the M2 pattern are key strategies for promoting wound healing. Based on this, this invention utilizes lipopolysaccharide (LPS) to induce RAW264.7 mouse macrophage cells to establish an in vitro inflammation model, evaluating the anti-inflammatory activity of OFD6 hydrogel at the gene and protein levels. qPCR results showed that, compared with the control group, LPS stimulation significantly upregulated the mRNA expression levels of IL-1β, IL-6, and TNF-α, confirming the successful establishment of the inflammation model; while after OFD6 hydrogel treatment, the expression levels of the above pro-inflammatory factors were significantly reduced (Figure 6g~i). These results indicate that OFD6 hydrogel can effectively inhibit the expression of pro-inflammatory factors in macrophages, exhibiting good anti-inflammatory activity.

[0060] 10. In vivo hemostatic properties of OFD hydrogel Diabetic infections often involve tissue bleeding, and the resulting scabs and fibrin networks not only provide an initial scaffold for bacterial colonization but also hinder subsequent granulation tissue ingrowth. This invention validated the hemostatic properties of the hydrogel under physiological conditions in mouse femoral artery, liver, and tail vein injury models. Results showed that OFD6 hydrogel rapidly closed wounds and significantly reduced blood loss in all models. Particularly in the challenging femoral artery injury model, the hydrogel group achieved hemostasis within approximately 50 seconds, with blood loss of only 27.57 ± 7.70 mg, far lower than the control group's 523.5 ± 138.1 mg (Figures 7a-c). The liver and tail vein injury models also confirmed that the hemostatic effect of OFD6 hydrogel was significantly superior to the gauze control group (Figures 7d-i). The potent hemostatic effect of OFD6 hydrogel stems from a multi-component synergistic mechanism. On one hand, Fe³⁺, after coordinating with dopamine hydrochloride, can interact with proteins and cell membranes in the blood, promoting erythrocyte and platelet aggregation, thereby accelerating the coagulation process. On the other hand, the hydrogel matrix rapidly forms a physical barrier upon contact with blood, locally concentrating clotting factors and blood cells, and promoting stable blood clot formation. This combined effect effectively limits local bleeding and scab formation at the wound site, creating favorable conditions for subsequent infection control and tissue repair.

[0061] 11. The therapeutic effect of OFD hydrogel on wounds in diabetic mice This invention further evaluated the therapeutic effect of OFD6 hydrogel on diabetic infected wounds under near-infrared (NIR) irradiation. Figure 8 c is an infrared thermographic image of the mouse during the treatment process. The experimental design and sampling time points are shown in Figure 8a. Wound photographs were recorded on days 0, 3, 5, 7, 10, 14, 18, and 21, and wound tissue was collected on days 3, 7, 14, and 21 for subsequent testing.

[0062] On day 3 of treatment (Figure 8d), the wound healing rate in the OFD6+NIR group reached 36.20% ± 10.89%, significantly higher than that in the Model group (19.46% ± 7.81%), the Dress group (14.72% ± 9.75%), and the OFD6-only group (17.78% ± 6.41%). As treatment progressed, the OFD6+NIR group maintained its advantage in healing speed. By day 14, the healing rate in this group reached 96.14% ± 2.96%, significantly better than that in the OFD6 group (85.39% ± 4.91%), the Model group (76.70% ± 3.79%), and the Dress group (69.58% ± 9.61%). On day 21, the wounds in the OFD6+NIR group and the OFD6 group were basically closed, with healing rates of 99.66% ± 0.22% and 97.92% ± 0.72%, respectively. New epidermal tissue covered the wounds, and hair regrowth was evident. In contrast, the Model group (96.17% ± 1.32%) and the Dress group (88.76% ± 7.47%) still showed residual dry scabs, and hair growth was lacking around the wounds. These results indicate that OFD6 hydrogel combined with NIR irradiation can significantly accelerate the healing process of diabetic infected wounds. To evaluate the in vivo antibacterial effect of each material, wound skin tissue was collected for bacterial culture on day 3 of treatment. As shown in Figures 8b and 8e, the bacterial load in the OFD6+NIR group was significantly lower than the other three groups, indicating that this treatment strategy has good infection control capabilities in vivo. Impaired wound healing in diabetic patients is closely related to elevated levels of reactive oxygen species (ROS). On day 7 of treatment, DHE staining was used to assess ROS levels in the wound tissue. The results showed that the Model and Dress groups exhibited large areas of red fluorescence, indicating a significant increase in ROS levels; while the fluorescence range of the OFD6 group was reduced, and the OFD6+NIR group had the lowest red fluorescence intensity, significantly lower than the other three groups (Figure 8f). ImageJ semi-quantitative analysis further verified this trend (Figure 8g). Simultaneously, this invention used the Amplex Red kit to detect the H2O2 content in the supernatant of wound tissue homogenate (…). Figure 8 The results showed that the H2O2 levels in the OFD6 group and the OFD6+NIR group were significantly lower than those in the Model group and the Dress group, with the OFD6+NIR group showing the most significant decrease.

[0063] Wound healing was assessed using HE staining (Figure 9a). On day 14, the Model and Dress groups still had significant epithelial defects, with wound lengths of 6454 ± 413.9 μm and 4831 ± 285.0 μm, respectively. Granulation tissue was loose, and obvious crusting was visible. In contrast, the OFD6 and OFD6+NIR groups showed significantly shorter wound lengths, at 3511 ± 255.2 μm and 2476 ± 232.6 μm, respectively. Re-epithelialization was essentially complete, with dense new granulation tissue visible in the dermis and no obvious crusting (Figure 9c). On day 21, the wound length in all groups further shortened, with the OFD6+NIR group still showing the shortest length (1884 ± 409.6 μm), followed by the OFD6 group (2711 ± 196.4 μm), the Dress group (3455 ± 354.0 μm), and the Model group (4073 ± 472.3 μm) (Figure 9d). Masson's trichrome staining was used to assess collagen deposition and remodeling in the wound (Figure 9b). On day 14, the Model, Dress, and OFD6 groups showed less collagen fiber deposition, lighter staining, sparse and disordered arrangement, and crusting in some areas. In contrast, the OFD6+NIR group showed significantly increased collagen deposition, deeper blue staining, and collagen bundles began to arrange themselves in an orderly manner along the skin direction. Quantitative analysis showed (Figure 9e) that the collagen-positive area in the OFD6+NIR group was 2.35 times, 2.03 times, and 1.35 times that of the Model, Dress, and OFD6 groups, respectively. On day 21, the total collagen amount increased in all groups, but the arrangement in the Model, Dress, and OFD6 groups remained disordered; while the OFD6+NIR group had abundant collagen deposition, with a tight and orderly arrangement, exhibiting a parallel bundle structure. Quantitative analysis showed that its collagen area was 1.73 times, 1.61 times, and 1.29 times that of the Model, Dress, and OFD6 groups, respectively. Figure 9 f).

[0064] Successful conversion of macrophages from the M1 to the M2 phenotype is crucial for the transition of wound tissue from the inflammatory phase to the proliferative phase. In this invention, wound tissue was collected on days 7 and 14. On day 7, the expression levels of M1 pro-inflammatory cytokines IL-6, IL-1β, and TNF-α in the wound tissue were detected by qPCR. The results showed that the expression levels of these inflammatory cytokines in the OFD6+NIR group were significantly lower than in other groups, suggesting that this treatment strategy effectively suppressed the local inflammatory response (Figure 10a). Further qPCR was used to detect the mRNA expression levels of HIF-1α, CD31, and α-SPA in the wound tissue on day 14. The results (Figure 10b) showed that the expression of all three genes was significantly upregulated in the OFD6+NIR group. This indicates that the OFD6+NIR group effectively promoted the formation and maturation of a functional vascular network in the wound by promoting angiogenesis and pericyte recruitment.

[0065] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a metal coordination-driven dynamic composite hydrogel, characterized in that, The method includes the following steps: (1) Add 0.5~1.0 mol / L sodium periodate solution to a 1% w / v sodium hyaluronate aqueous solution, react under light-proof and stirring conditions, quench the reaction after the reaction is completed, and continue stirring for 1~3 h; dialyze the reaction solution, freeze dry the obtained dialysate to obtain oxidized hyaluronic acid (OHA); (2) Mix 8.0~10% w / v dopamine hydrochloride aqueous solution and 8.0~10% w / v ferric chloride aqueous solution, adjust the pH of the mixture to 7.0~7.5, and obtain a complexation mixture; (3) Add 4.0%~6.0% w / v of oxidized hyaluronic acid (OHA) solution to the coordination mixture, mix evenly, and react at room temperature to obtain a metal coordination-driven dynamic composite hydrogel.

2. The method as described in claim 1, characterized in that, In step (1), the relative molecular mass of the sodium hyaluronate is 800,000 to 1,500,000 Da; In step (1), the volume ratio of the sodium hyaluronate aqueous solution to the sodium periodate solution is 100 mL: 0.5 mL to 1 mL.

3. The method as described in claim 1, characterized in that, In step (1), 0.5~1.0 mL of ethylene glycol is added after the reaction is completed to quench the reaction.

4. The method as described in claim 1, characterized in that, In step (1), the dialysis specifically involves transferring the reaction solution to a dialysis bag for dialysis treatment. The dialysis time is 3 days, and the external dialysis solution is replaced every 8 hours.

5. The method as described in claim 1, characterized in that, In step (2), the molar ratio of dopamine hydrochloride to ferric chloride in the coordination mixture is 3:

1.

6. The method as described in claim 1, characterized in that, In step (3), the volume ratio of the coordination mixture and the oxidized hyaluronic acid (OHA) solution is 1:8~17.

7. The metal coordination-driven dynamic composite hydrogel prepared by the method described in claims 1 to 6.

8. The use of the metal coordination-driven dynamic composite hydrogel of claim 7 in the preparation of a medicament for treating diabetic wounds infected with multidrug-resistant bacteria.

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