Diabetic foot wound anti-inflammatory gel and preparation method thereof

Abstract

The present application belongs to the field of biological medical dressings, and particularly relates to a diabetic foot wound anti-inflammatory gel and a preparation method thereof. The composition comprises the following raw materials: sodium hyaluronate, NaIO4, carboxymethyl chitosan, tannic acid, a boronic acid compound, dexamethasone, dopamine hydrochloride, sodium citrate, a PBS buffer and a Tris buffer. The diabetic foot wound anti-inflammatory gel is based on in-situ gelation dynamic cross-linking network, maintains a moist healing microenvironment, and reduces dressing change pulling damage; the introduction of polydopamine realizes stable wet adhesion, and balances the balance between fixity and dressing friendliness. Mild ion competition is used to control bacteria and break membranes, drug-loaded particles target anti-inflammatory and cooperate with tannic acid, and through multifunctional coupling design, the internal conflicts such as traditional dressing irritation and dressing damage are resolved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical dressings, specifically relating to an anti-inflammatory gel for diabetic foot wounds and its preparation method. Background Technology

[0002] Diabetic foot ulcers are a common and serious chronic complication of diabetes. Their development is usually related to the combined effects of multiple factors, including peripheral neuropathy, peripheral vascular disease, and immune and metabolic abnormalities. Patients experience decreased sensation, abnormal pressure distribution, and difficulty detecting minor injuries in the foot. Local tissues are also chronically hypoxic and nutrient-deficient due to insufficient blood supply and microcirculatory disturbances. Simultaneously, the high-glucose environment leads to decreased chemotaxis and phagocytic function of immune cells, weakening their ability to fight infection. This makes wounds prone to recurrent infections and hinders normal repair processes. Clinically, diabetic foot wounds often present with excessive exudation, maceration of the wound edges and surrounding skin, slow or even stagnant granulation tissue growth, and are often accompanied by odor, pain or numbness, atypical local redness, swelling, heat, and pain, and recurrent signs of infection. In prolonged cases, deep tissue involvement and an increased risk of osteomyelitis may occur, and in severe cases, amputation may be necessary, significantly impacting patients' quality of life and medical burden.

[0003] Compared to typical acute wounds, the more prominent pathological feature of diabetic foot wounds lies in the chronic, inflammatory state of the wound microenvironment. Infection and residual necrotic tissue continuously activate inflammatory pathways, leading to elevated levels of pro-inflammatory factors and impaired macrophage phenotypic conversion, making the transition from the inflammatory phase to the proliferative phase difficult. Simultaneously, proteases (such as matrix metalloproteinases) are often overexpressed, easily causing degradation of the extracellular matrix and growth factors, and unstable granulation tissue structure, further weakening re-epithelialization and angiogenesis. Adding to the challenge is the frequent presence of bacterial biofilms in diabetic foot wounds. These biofilms form a protective barrier through the extracellular polymer matrix, enabling bacteria to become resistant to antibiotics and immune clearance, resulting in prolonged infection, recurrent infections, and poor efficacy of conventional debridement and dressing treatments. Furthermore, diabetic foot wounds often involve exudation and fragile skin; frequent dressing changes can easily cause adhesions to newly formed tissue, leading to secondary damage and pain, and even bleeding and inflammatory rebound, further prolonging the healing period.

[0004] For diabetic foot wounds, existing dressings and local treatment materials mainly include traditional covering materials such as gauze and cotton pads, foam dressings and hydrocolloid dressings, alginate dressings, hydrogel dressings, as well as antibacterial dressings containing silver, iodine, etc., and composite functional dressings. Traditional covering materials are low in cost but have limited moisturizing and barrier properties, are prone to adhesion to the wound surface, and require frequent changes; foam and alginate dressings have a certain ability to absorb exudate, but are insufficient in regulating biofilms and the chronic inflammatory microenvironment; hydrocolloid and some hydrogel dressings can provide a moist environment, reduce pain, and promote autolytic debridement, but are prone to maceration, insufficient fixation, or fluctuations in efficacy when there is high exudation or infection; antibacterial dressings can reduce the surface bacterial load to a certain extent, but have limited ability to penetrate and destroy mature biofilms, and some strong antibacterial systems may introduce irritation or affect cell repair; in addition, many dressings are prone to displacement in a moist environment or require secondary fixation, and may still cause traction on fragile granulation tissue when removed, affecting the dressing change experience and wound stability. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide an anti-inflammatory gel for diabetic foot wounds and its preparation method.

[0006] The technical effects described in this invention are achieved through the following technical solution: an anti-inflammatory gel for diabetic foot wounds, comprising the following raw materials: sodium hyaluronate, carboxymethyl chitosan, tannic acid, boric acid compounds, dexamethasone, dopamine hydrochloride, sodium citrate, sodium gluconate, PBS buffer and Tris buffer. Furthermore, the molecular weight of the sodium hyaluronate ranges from 200 to 500 kDa; Further, the boric acid compound is any one of borax, phenylboronic acid, and 4-carboxyphenylboronic acid; more preferably, it is 4-carboxyphenylboronic acid. Furthermore, the pH of the PBS buffer is 7.2–7.4; Furthermore, the pH of the Tris buffer solution is 8.2–8.8; Furthermore, another aspect of the present invention provides a method for preparing an anti-inflammatory gel for diabetic foot wounds, specifically comprising the following steps: S1: Dissolve sodium hyaluronate in deionized water and stir at 4°C until completely dissolved to prepare a 10-15 mg / mL aqueous solution. Then, add NaIO4 in three batches at 10-20 min intervals under light-protected conditions. React at 4-8°C for 3-5 h. Add ethylene glycol to terminate the reaction. After 30 min, dialyze the solution using a 5-12 kDa MWCO dialysis bag. Pre-freeze at -80°C for 6-12 h and freeze-dry under vacuum at -40°C for 24-48 h to obtain oxidized modified sodium hyaluronate. S2: Dissolve carboxymethyl chitosan in PBS buffer and stir until clear and homogeneous to prepare a 20-30 mg / mL chitosan solution; S3: Dissolve tannic acid in deionized water and stir until completely dissolved to prepare a 5-10 mg / mL TA solution; add dexamethasone to the TA solution and stir for 10-30 min to disperse evenly, then add 10-15 mM boric acid compound solution dropwise, and adjust the pH of the system to 8.2-8.8 with Tris buffer, continue stirring for 60-90 min, let stand for aging for 6-12 h, centrifuge at 3000g for 5 min and collect the supernatant to obtain the TA-boric acid dynamic network drug-loaded particle dispersion; S4: Add dopamine hydrochloride to the chitosan solution from step S2, controlling the final concentration of dopamine hydrochloride to 0.5–1.5 mg / mL. Adjust the pH of the system to 8.2–8.8 using Tris buffer. Stir the reaction at room temperature for 20–30 min to trigger the in-situ formation of polydopamine adhesion precursor microdomains. Then add the TA-boric acid dynamic network drug-loaded particle dispersion from step S3 and ultrasonically disperse it evenly. After dispersion, adjust the pH of the system to 7.2–7.4 to obtain solution B. S5: Dissolve the oxidized modified sodium hyaluronate from step S1 in PBS buffer, stir until dissolved, and prepare a 15-30 mg / mL solution. Then add sodium citrate and sodium gluconate, stir until completely dissolved, so that the total final concentration of the two is 10-20 mM, and obtain solution A. S6: Mix the B solution from step S4 and the A solution from step S5 at a volume ratio of 1:1 through a static mixing head with a mixing core structure of 10 to 20 sections, coat it on a PTFE mold, place it in a relatively humid environment at 37°C to form a gel in situ, with a coating thickness of 1 to 2 mm and a complete gelation time of 5 to 10 minutes to obtain an anti-inflammatory gel. Further, in step S1, the mass ratio of NaIO4 to sodium hyaluronate is 0.15 to 0.25:1; the mass ratio of ethylene glycol to NaIO4 is 0.4 to 0.8 mL:1 g. Furthermore, in step S1, the water is changed every 2-4 hours for the first 3 purification dialysis cycles, and then every 6-8 hours thereafter, for a total of 48-72 hours of dialysis; Further, in step S2, the pH of the chitosan solution is 7.2–7.4; the pH is adjusted using PBS buffer. Further, in step S3, the mass ratio of dexamethasone to tannic acid is 0.1 to 0.15:1; Further, in step S3, the boric acid compound solution is prepared by dissolving boric acid compounds in deionized water and adding Tris buffer to assist in dissolution; the volume ratio of the boric acid compound solution to the TA solution is 0.1 to 0.3:1. It should be noted that in step S3, the average particle size of the TA-boric acid dynamic network drug-loaded particle dispersion was measured to be 0.5-3 μm by DLS. Further, in step S4, the amount of the TA-boric acid dynamic network drug-loaded particle dispersion added is 3-5% of the chitosan solution volume; It should be noted that in step S4, after adding the TA-boric acid dynamic network drug-loaded particle dispersion and ultrasonically dispersing it evenly, it should be immediately mixed with solution A to form a gel. Further, in step S5, the molar ratio of sodium citrate and sodium gluconate is 1 to 2:1.

[0007] The beneficial effects of this invention are as follows: Compared to existing technologies, the anti-inflammatory gel for diabetic foot wounds of this invention is based on an in-situ gelling network. Through the formation of a dynamic cross-linked structure between oxidized modified hyaluronic acid and carboxymethyl chitosan under aqueous conditions, the dressing can quickly adhere to and form a continuous covering layer in a moist, exudative environment. This stabilizes the microenvironment required for moist healing and reduces re-irritation to the wound from external contamination and mechanical friction. This dynamic network possesses a certain degree of structural rearrangement, ensuring both integrity and coverage during its service life while reducing the risk of traction on newly formed tissue during dressing changes, providing a gentler user experience for fragile wounds such as diabetic foot wounds.

[0008] Regarding adhesion and fixation, this invention introduces dopamine hydrochloride and triggers the in-situ formation of polydopamine adhesion microdomains through a short-term weak alkali treatment. This allows the gel to establish a stable wet adhesion interface even in the presence of exudate, significantly reducing secondary contamination caused by dressing displacement, edge lifting, and leakage. Simultaneously, the polydopamine interface exhibits reversible hydration, facilitating smooth detachment under wet conditions during removal. This achieves a balance between fixation and dressing change friendliness, reducing secondary damage and inflammatory rebound during dressing changes. This characteristic is particularly relevant to the clinical challenges of repeated dressing changes and easy tissue bleeding and breakage in diabetic foot wounds. Regarding biofilm and bacterial load control, this invention employs a mild ion competition strategy rather than relying on a highly irritating and strong bactericidal system. By using a compounded mild competitive ion component, the ion bridge stabilization structure in the biofilm matrix is ​​weakened, promoting biofilm loosening and peeling, thereby reducing the barrier effect of the biofilm on exogenous antibacterial agents and the body's immune clearance. This strategy works synergistically with the physical barrier formed by in-situ gelation to reduce the chances of new bacterial invasion and mitigate the chronic inflammatory maintenance effect caused by continuous stimulation of existing biofilms, thus reducing the driving factors that keep the wound in a long-term inflammatory phase at the source. The introduction of mild ion competition also avoids the cytotoxicity and delayed healing problems that may arise from traditional strong antibacterial systems, ensuring that bacterial control and repair promotion no longer hinder each other. Regarding the regulation of inflammation and protease imbalance, this invention loads dexamethasone into drug-loaded particles constructed from tannic acid and boric acid crosslinking units, resulting in a more uniform distribution of anti-inflammatory components within the gel network and more controllable release behavior. These drug-loaded particles are more prone to structural relaxation and enhanced drug release in wound environments with greater oxidative stress, thus making anti-inflammatory intervention more likely to work in stages of more severe inflammation and stronger tissue stress, promoting the transformation of the wound from a long-term stagnant inflammatory state to a proliferative repair stage, and reducing the risk of damage to the extracellular matrix and granulation tissue stability caused by inflammation-mediated protease overexpression. The antioxidant and microenvironment buffering effects of tannic acid itself further complement anti-inflammatory drugs, making anti-inflammatory regulation more gradual and more in line with the treatment needs of diabetic foot wounds that require "continuous but not excessive" treatment.

[0009] In summary, this invention achieves functional synergy and resolves the inherent conflicts of traditional dressings through a coupled design of "membrane breaking and barrier reduction, anti-inflammatory and de-stimulation, adhesion and stable coverage, and reversible damage reduction": it weakens the biomembrane structure through gentle ion competition, reducing the irritation caused by relying on high-intensity sterilization; it achieves reliable fixation through polydopamine wet adhesion microdomains, while relying on the reversible hydration properties to alleviate the damage caused by strong adhesion during dressing changes; and it uses drug-loaded particles to achieve an anti-inflammatory release trend that better fits the wound condition, avoiding the contradiction between stimulation and repair inhibition caused by excessive release at one time. Attached Figure Description

[0010] Figure 1 The graph shows the test results of the wet shear adhesion strength of the gel in Examples 1-3 and Comparative Examples 1-4 of this invention. Figure 2 The graph shows the biofilm biomass results of the gel biofilm disruption test in Example 1 and Comparative Examples 1-4 of this invention. Figure 3 The graph shows the relative biofilm residue results of the gel biofilm destruction test in Example 1 and Comparative Examples 1-4 of this invention; Figure 4 The graph shows the TNF-α concentration results of the gel anti-inflammatory test in Example 1 and Comparative Examples 1-4 of this invention. Figure 5 The graph shows the anti-inflammatory rate results of the gel anti-inflammatory test in Example 1 and Comparative Examples 1-4 of this invention; Figure 6 The graph shows the gel protease inhibition test results of Example 1 and Comparative Examples 1-4 of this invention; Figure 7 The FTIR infrared spectra of sodium hyaluronate and oxidized modified sodium hyaluronate in Example 1 of this invention are shown. Detailed Implementation

[0011] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the raw materials involved in the present invention are all purchased through conventional commercial channels. Experimental methods without specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0012] Example 1: An anti-inflammatory gel for diabetic foot wounds, comprising the following ingredients: sodium hyaluronate, carboxymethyl chitosan, tannic acid, boric acid compounds, dexamethasone, dopamine hydrochloride, sodium citrate, sodium gluconate, PBS buffer, and Tris buffer. The molecular weight of the sodium hyaluronate is 300 kDa; The pH of the PBS buffer is 7.3; The pH of the Tris buffer solution is 8.5; The preparation method of the anti-inflammatory gel for diabetic foot wounds specifically includes the following steps: S1: Dissolve 1.2g of sodium hyaluronate in 100mL of deionized water and stir at 4℃ until completely dissolved to prepare a 12mg / mL aqueous solution. Then, under light-protected conditions, add 0.24g of NaIO4 in three batches, with each batch 15min apart. React at 4℃ for 4h. Add 0.144mL of ethylene glycol to terminate the reaction. After 30min, dialyze using a dialysis bag with MWCO 8kDa for purification. Change the water every 3h for the first three dialysis cycles, and then every 7h thereafter, for a total of 60h of dialysis. Pre-freeze at -80℃ for 10h and freeze-dry under vacuum at -40℃ for 36h to obtain oxidized modified sodium hyaluronate. S2: Dissolve 2.5g of carboxymethyl chitosan in 100mL of PBS buffer and stir until clear and homogeneous to prepare a 25mg / mL chitosan solution with a pH of 7.3; S3: Dissolve 0.8g of tannic acid in 100mL of deionized water and stir until completely dissolved to prepare an 8mg / mL TA solution; add 0.08g of dexamethasone to the TA solution, stir for 20min to disperse evenly, then add 20mL of 12mM 4-carboxyphenylboronic acid solution, and adjust the pH of the system to 8.5 with Tris buffer, continue stirring for 80min, let stand for aging for 10h, centrifuge at 3000g for 5min and take the supernatant to obtain the TA-boronic acid dynamic network drug-loaded particle dispersion; S4: Add 0.05 g of dopamine hydrochloride to 50 mL of chitosan solution from step S2, controlling the final concentration of dopamine hydrochloride to 1 mg / mL. Adjust the pH of the system to 8.5 with Tris buffer. Stir the reaction at room temperature for 25 min to trigger the in-situ formation of polydopamine adhesion precursor microdomains. Then add 2 mL of TA-boric acid dynamic network drug-loaded particle dispersion from step S3. Disperse evenly by ultrasonication. After dispersion, adjust the pH of the system to 7.3 to obtain solution B. S5: Dissolve 1g of oxidized sodium hyaluronate from step S1 in 50mL of PBS buffer, stir until dissolved, prepare a 20mg / mL solution, then add 0.129g of sodium citrate and 0.059g of sodium gluconate, stir until completely dissolved, so that the total final concentration of the two is 15mM, and obtain solution A. S6: Mix liquid B from step S4 and liquid A from step S5 at a volume ratio of 1:1 through a static mixing head with a 15-section mixing core structure, coat the mixture onto a PTFE mold, and place it in a relatively humid environment at 37°C to form a gel in situ. The coating thickness is 1.5 mm, and the complete gelation time is 8 min to obtain an anti-inflammatory gel.

[0013] Example 2: An anti-inflammatory gel for diabetic foot wounds, comprising the following ingredients: sodium hyaluronate, carboxymethyl chitosan, tannic acid, boric acid compounds, dexamethasone, dopamine hydrochloride, sodium citrate, sodium gluconate, PBS buffer, and Tris buffer. The molecular weight of the sodium hyaluronate is 200 kDa; The pH of the PBS buffer is 7.4; The pH of the Tris buffer solution is 8.8; The preparation method of the anti-inflammatory gel for diabetic foot wounds specifically includes the following steps: S1: Dissolve 1.5g of sodium hyaluronate in 100mL of deionized water and stir at 4℃ until completely dissolved to prepare a 15mg / mL aqueous solution. Then, under light-protected conditions, add 0.225g of NaIO4 in three batches, with an interval of 20min between each batch. React at 6℃ for 5h. Add 0.18mL of ethylene glycol to terminate the reaction. After 30min, dialyze using a dialysis bag with MWCO 12kDa for purification. Change the water every 2h for the first three dialysis cycles, and then every 6h thereafter, for a total of 48h of dialysis. Pre-freeze at -80℃ for 12h and freeze-dry under vacuum at -40℃ for 48h to obtain oxidized modified sodium hyaluronate. S2: Dissolve 3g of carboxymethyl chitosan in 100mL of PBS buffer and stir until clear and homogeneous to prepare a 30mg / mL chitosan solution with a pH of 7.4; S3: Dissolve 1g of tannic acid in 100mL of deionized water and stir until completely dissolved to prepare a 10mg / mL TA solution; add 0.1g of dexamethasone to the TA solution, stir for 30min to disperse evenly, then add 10mL of 15mM 4-carboxyphenylboronic acid solution, and adjust the pH of the system to 8.8 with Tris buffer, continue stirring for 90min, let stand for aging for 12h, centrifuge at 3000g for 5min and take the supernatant to obtain the TA-boronic acid dynamic network drug-loaded particle dispersion; S4: Add 0.075 g of dopamine hydrochloride to 50 mL of chitosan solution from step S2, controlling the final concentration of dopamine hydrochloride to 1.5 mg / mL. Adjust the pH of the system to 8.8 with Tris buffer. Stir the reaction at room temperature for 30 min to trigger the in-situ formation of polydopamine adhesion precursor microdomains. Then add 2.5 mL of TA-boric acid dynamic network drug-loaded particle dispersion from step S3. Disperse evenly by ultrasonication. After dispersion, adjust the pH of the system to 7.4 to obtain solution B. S5: Dissolve 1.5g of oxidized sodium hyaluronate from step S1 in 50mL of PBS buffer, stir until dissolved, and prepare a 30mg / mL solution. Then add 0.129g of sodium citrate and 0.118g of sodium gluconate, stir until completely dissolved, and make the total final concentration of the two 20mM to obtain solution A. S6: Mix liquid B from step S4 and liquid A from step S5 at a volume ratio of 1:1 through a static mixing head with a 20-section mixing core structure, coat the mixture onto a PTFE mold, and place it in situ in a relatively humid environment at 37°C to form a gel. The coating thickness is 2 mm, and the complete gelation time is 10 min to obtain an anti-inflammatory gel.

[0014] Example 3: An anti-inflammatory gel for diabetic foot wounds, comprising the following ingredients: sodium hyaluronate, carboxymethyl chitosan, tannic acid, boric acid compounds, dexamethasone, dopamine hydrochloride, sodium citrate, sodium gluconate, PBS buffer, and Tris buffer. The molecular weight of the sodium hyaluronate is 500 kDa; The pH of the PBS buffer is 7.2; The pH of the Tris buffer solution is 8.2; The preparation method of the anti-inflammatory gel for diabetic foot wounds specifically includes the following steps: S1: Dissolve 1g of sodium hyaluronate in 100mL of deionized water and stir at 4℃ until completely dissolved to prepare a 10mg / mL aqueous solution. Then, under light-protected conditions, add 0.25g of NaIO4 in three batches, with each batch 10min apart. React at 8℃ for 3h. Add 0.1mL of ethylene glycol to terminate the reaction. After 30min, dialyze using a dialysis bag with MWCO 5kDa for purification. Change the water every 4h for the first three dialysis cycles, and then every 8h thereafter, for a total of 72h of dialysis. Pre-freeze at -80℃ for 6h and freeze-dry under vacuum at -40℃ for 24h to obtain oxidized modified sodium hyaluronate. S2: Dissolve 2g of carboxymethyl chitosan in 100mL of PBS buffer and stir until clear and homogeneous to prepare a 20mg / mL chitosan solution with a pH of 7.2; S3: Dissolve 0.5g of tannic acid in 100mL of deionized water and stir until completely dissolved to prepare a 5mg / mL TA solution; add 0.075g of dexamethasone to the TA solution, stir for 10min to disperse evenly, then add 30mL of 10mM phenylboronic acid solution dropwise, and adjust the pH of the system to 8.2 with Tris buffer, continue stirring for 60min, let stand for aging for 6h, centrifuge at 3000g for 5min and take the supernatant to obtain the TA-boronic acid dynamic network drug-loaded particle dispersion; S4: Add dopamine hydrochloride to the chitosan solution from step S2, controlling the final concentration of dopamine hydrochloride to 0.5 mg / mL. Adjust the pH of the system to 8.2 with Tris buffer. Stir the reaction at room temperature for 20 min to trigger the in-situ formation of polydopamine adhesion precursor microdomains. Then add 1.5 mL of the TA-boric acid dynamic network drug-loaded particle dispersion from step S3. Disperse evenly by ultrasonication. After dispersion, adjust the pH of the system to 7.2 to obtain solution B. S5: Dissolve 0.75g of oxidized sodium hyaluronate from step S1 in 50mL of PBS buffer, stir until dissolved, and prepare a 15mg / mL solution. Then add 0.051g of sodium citrate and 0.071g of sodium gluconate, stir until completely dissolved, and make the total final concentration of the two 10mM to obtain solution A. S6: Mix liquid B from step S4 and liquid A from step S5 at a volume ratio of 1:1 through a static mixing head with a 10-section mixing core structure, coat the mixture onto a PTFE mold, and place it in situ in a relatively humid environment at 37°C to form a gel. The coating thickness is 1 mm, and the complete gelation time is 5 min to obtain an anti-inflammatory gel.

[0015] Comparative Example 1: Comparative Example 1 is basically the same as Example 1. The main difference is that in Comparative Example 1, the process of weak base triggering dopamine to form polydopamine adhesion precursor microdomains in situ is not carried out in step S4. That is, after adding dopamine hydrochloride to chitosan solution, the pH of the system is not adjusted to 8.5 and a 25-minute pre-reaction is not carried out. Instead, the pH of the system is directly controlled at 7.3 before adding the drug-loaded particles in step S3 and dispersing them. Then, they are immediately mixed with solution A to form a gel. The remaining steps and parameters are consistent with those of Example 1.

[0016] Comparative Example 2: Comparative Example 2 is basically the same as Example 1, except that sodium citrate and sodium gluconate are not added as ion-competing components in step S5 of Comparative Example 2; in order to maintain the ionic strength and osmotic pressure conditions of solution A basically the same, an equimolar amount of sodium chloride is used to replace the total amount of the two salts added above in Comparative Example 2; the remaining steps and parameters are the same as those in Example 1.

[0017] Comparative Example 3: Comparative Example 3 is basically the same as Example 1, except that the tannic acid-boric acid dynamic network drug-loaded particle dispersion of step S3 is not prepared in Comparative Example 3. Instead, dexamethasone is directly dissolved or dispersed in solution B in an equal amount. In order to maintain the polyphenol background and solid content in solution B, an equal mass of tannic acid solution is added to solution B in Comparative Example 3, but 4-carboxyphenylboronic acid is not added to form a dynamic network. The remaining steps and parameters are consistent with those in Example 1.

[0018] Comparative Example 4: Comparative Example 4 is basically the same as Example 1, except that tannic acid is not added in step S3 of Comparative Example 4, and PEG-6000 with equal solid content and equal particle size is used as a substitute component for particle formation and dispersion stabilization; the remaining steps and parameters are consistent with those of Example 1.

[0019] Performance testing: Wet adhesion strength test: Fresh pigskin was used as the wet tissue simulation substrate. Subcutaneous fat was removed and the samples were cut into uniform sizes (20mm × 20mm). The samples were moistened with PBS and equilibrated in a 37℃ incubator for 30 minutes. Anti-inflammatory gel samples (before gel formation) prepared in Examples 1-3 and Comparative Examples 1-4 were mixed in equal volumes using a static mixing head and immediately applied to the center of the contact surface of two pigskin samples to form an adhesive layer (1cm × 1cm), approximately 1mm thick. The mixture was then placed in a relatively humid environment at 37℃ until fully gelled and set. The two pigskin samples were then joined together with an overlap area of ​​1cm × 1cm and placed under the same conditions for another 10 minutes to ensure sufficient interface contact. The joined samples were then mounted on a material tensile / shear testing device (universal testing machine) and subjected to a shear peel test at a constant tensile rate of 5mm / min. The maximum shear load at which interface slippage or separation occurred was recorded and converted to wet shear adhesion strength (kPa) based on the overlap area. The results are as follows: Figure 1 As shown.

[0020] based on Figure 1 Results analysis showed that the anti-inflammatory gels of Examples 1-3 all exhibited high wet shear adhesion strength on the moist pigskin substrate, indicating that the present invention, through the synergistic effect of "polydopamine adhesion precursor microdomain construction" and "sodium oxidized hyaluronic acid-carboxymethyl chitosan gelling network," can form stable interface anchoring and provide sufficient cohesive support in a moist environment. Although the adhesion levels of Examples 1-3 fluctuated to some extent due to slight differences in process parameters, they were all at a high level overall, indicating that the system has good process adaptability and stability. In Comparative Example 1, after the weak base-triggered pre-reaction was removed, dopamine was unable to fully form polydopamine adhesion precursor microdomains, and the wet interface lacked effective anchoring points, resulting in a significant decrease in wet shear adhesion strength. In Comparative Example 2, after removing the ion-competing components in solution A, since its main effect was on weakening the biofilm rather than interface adhesion construction, the impact on the adhesion module and the main gelling network was relatively limited, resulting in the wet adhesion strength remaining at a high level and close to that of the examples. In Comparative Example 3, after removing the tannic acid-boric acid dynamic network drug-loaded particle structure and replacing it with direct addition of dexamethasone, although the polydopamine adhesion microdomains still existed, the energy dissipation and microscopic contact stability of the gel interface and internal structure decreased, resulting in a moderate decrease in wet shear adhesion strength. In Comparative Example 4, after removing tannic acid and using PEG as a site filler, the interfacial synergistic effect brought about by the polyphenol system was lost, further reducing interfacial stability and adhesion retention capacity, manifested in a more significant decrease in wet adhesion strength.

[0021] Biofilm disruption test: *Pseudomonas aeruginosa* was selected as a representative biofilm-forming strain. The bacterial culture was inoculated into LB medium and cultured at 37°C with shaking until the logarithmic growth phase, then diluted to a uniform initial concentration of 1×10⁻⁶. 6 CFU / mL; Add 200 μL of the diluted bacterial suspension to each well of a 96-well plate and incubate at 37°C for 48 h to form a stable biofilm; Discard the supernatant and gently wash with PBS to remove airborne bacteria; Take 100 mg of the anti-inflammatory gel samples prepared in Example 1 and Comparative Examples 1-4 respectively and add 10 mL of each gel to 10 mL of the plate. Gel extracts were prepared by incubation in PBS at 37°C and 120 rpm for 4 h on a shaker. After incubation, insoluble matter was removed by centrifugation and the extract was filtered through a 0.22 μm filter to obtain a clear extract. Each extract was added to the wells of the biofilm formed (200 μL per well), incubated at 37°C for 24 h, the solution was discarded, and the extract was washed with PBS. The extracts were then stained with 0.1% crystal violet solution for 15 min to remove free dye and allowed to air dry. Subsequently, the bound dye was dissolved in an ethanol / acetone decolorizing solution, and the absorbance at 570 nm was measured. The absorbance value was used as an indicator of biofilm biomass, and the relative biofilm residue (%) was calculated as OD group / OD model × 100%. Results are as follows: Figure 2 and Figure 3 As shown.

[0022] based on Figure 2 and Figure 3 Results analysis showed that the gel extract of the examples had a more significant weakening effect on Pseudomonas aeruginosa biofilm, with a significant reduction in biofilm residue. The introduction of sodium citrate and sodium gluconate as ion-competing components in solution A of the examples effectively weakened the divalent ion-bridge-dependent structure in the biofilm matrix, making the biofilm structure looser and easier to detach during PBS washing. Although Comparative Example 1 weakened the construction of polydopamine adhesion precursor microdomains, the biofilm rupture effect of the PBS extract used in this experiment still mainly came from the ion-competing components in solution A, resulting in a relatively strong biofilm weakening ability in Comparative Example 1, with a residue level only slightly higher than the examples. In Comparative Example 2, the removal of sodium citrate and sodium gluconate and their replacement with equimolar NaCl resulted in the loss of the key mechanism for ion-competitive weakening of the biofilm, leading to a significant reduction in the destructive effect of the extract on the stable structure of the biofilm and a significant increase in biofilm residue. Both Comparative Examples 3 and 4 retained ion-competing salts, resulting in a less significant decrease in their membrane-breaking ability compared to Comparative Example 2. However, due to the absence of drug-loaded particle structures or tannic acid-polyphenol synergy, the polyphenol-related auxiliary perturbation and structural weakening effects in the extract were reduced, and the biofilm residue was between that of the Examples and Comparative Example 2.

[0023] Anti-inflammatory test: Mouse macrophage line RAW264.7 was seeded in DMEM complete medium containing 10% fetal bovine serum and cultured at 37°C with 5% CO2 until the logarithmic growth phase; cells were collected and the cell density was adjusted to approximately 1×10⁻⁶. 5Cells were seeded at a rate of 200 μL per well in a 96-well plate and cultured overnight until the cells adhered stably. Anti-inflammatory gel dressing samples prepared in Example 1 and Comparative Examples 1-4 were taken and prepared aseptically to obtain sample extracts: 100 mg of each gel sample was cut and weighed, added to 10 mL of DMEM medium containing 1% fetal bovine serum (solid-liquid ratio 10 mg / mL), and incubated at 37°C on a shaker at 120 rpm for 4 h to release the leaching components from the gel into the medium. After incubation, the extract was centrifuged to remove insoluble particles and filtered through a 0.22 μm sterile filter membrane to obtain a sterile, clear extract for use in inflammatory cell experiments. Preliminary experiments were conducted using the CCK-8 method to determine the safe dilution factor for each extract that would not affect cell survival, and working solutions for each sample were prepared accordingly. The blank control group received no LPS and no sample; the model control group received LPS to a final concentration of 1 μg / mL but no sample; each treatment group received LPS (final concentration 1 μg / mL) and corresponding working solution (final concentration 100 μg / mL based on gel equivalent mass), with 3 parallel wells per group. After incubation at 37℃ and 5% CO2 for 24 hours, the supernatant from each well was collected. The supernatant from each group was added to an ELISA plate pre-coated with TNF-α capture antibody. Incubation, washing, and color development were performed according to the kit instructions. The absorbance at 450 nm was measured, and the concentration of TNF-α in the supernatant of each group was calculated using a standard curve. The anti-inflammatory rate (%) was further calculated as follows: (average TNF-α concentration in the model control group - average TNF-α concentration in each treatment group) / average TNF-α concentration in the model control group × 100%. The results are shown below. Figure 4 and Figure 5 As shown.

[0024] based on Figure 4 and Figure 5Results analysis showed that Example 1 exhibited superior TNF-α inhibition and anti-inflammatory activity in the RAW264.7 cell LPS-induced inflammation model. Dexamethasone, through a dynamic network of tannic acid-boric acid drug-loaded particles, achieved more uniform dispersion and more controllable release background. Combined with the microenvironmental regulation effect of tannic acid polyphenols, this allowed the anti-inflammatory active ingredients to exert their effects more fully and stably under extraction conditions, thereby significantly reducing TNF-α secretion. Simultaneously, the compatibility between the polydopamine adhesion microdomain and the polyphenol system helped maintain stable component distribution within the system, reducing the loss of active ingredients due to phase separation or aggregation. Comparative Example 1, without weak base triggering to form polydopamine adhesion precursor microdomains, mainly affected wet adhesion fixation and interface construction. Since this experiment used the extract to treat cells, interface adhesion construction was not the dominant factor determining the anti-inflammatory effect of the extract, resulting in only a certain degree of reduction in its anti-inflammatory activity. In Comparative Example 2, after removing the ion-competing salt and replacing it with an equimolar amount of NaCl, the anti-inflammatory effect remained close to that of the Example 2, as the ion-competing salt was not the main determinant of TNF-α inhibition in this in vitro inflammation model. In Comparative Example 3, after removing the drug-loaded particle structure and replacing it with direct dissolution or dispersion of dexamethasone, the lack of a carrier structure to regulate drug dispersion and release reduced the stability of the active ingredient, decreased the TNF-α inhibitory effect, and resulted in significantly weaker anti-inflammatory activity compared to the Example 2. In Comparative Example 4, after removing tannic acid and replacing it with PEG, the synergistic effect of polyphenol microenvironment buffering and auxiliary anti-inflammatory function was lost. Simultaneously, the stability of carrier structure formation and dispersion decreased, leading to higher TNF-α levels and further reduced anti-inflammatory activity.

[0025] Protease test: Sampling was the same as the anti-inflammatory test described above. The supernatant from each group was added to an MMP-9 activity assay kit to determine the MMP-9 activity in the culture medium. The supernatant from each group was reacted with the MMP-9 specific substrate according to the kit instructions. After incubation at the specified temperature and time, the absorbance was measured. The MMP-9 activity level of each group was calculated based on the standard curve / relative activity. Using the MMP-9 activity of the model control group as 100% baseline, the relative inhibition rate of MMP-9 activity in each treatment group was calculated. Inhibition rate (%) = (Average MMP-9 activity of the model control group - Average MMP-9 activity of each treatment group) / Average MMP-9 activity of the model control group × 100%. The test results are as follows: Figure 6 As shown.

[0026] based on Figure 6Results analysis showed that Example 1 exhibited a more significant inhibitory effect on MMP-9 activity. This is because Example 1 reduced the inflammatory cascade response through more effective anti-inflammatory intervention, thereby alleviating the inflammation-related upregulation of MMP-9; simultaneously, the buffering effect of the tannic acid polyphenol microenvironment on oxidative stress and protein interactions helped improve the imbalance of excessive protease, resulting in a higher MMP-9 inhibition rate. Comparative Example 1 mainly weakened the construction of the polydopamine adhesion precursor microdomain, while the anti-inflammatory active components in the extract remained, resulting in only a moderate decrease in the MMP-9 inhibition rate. After removing the ion-competitive membrane-breaking salt, Comparative Example 2 had limited direct impact on the cellular inflammation-protease pathway, and with isomolar NaCl maintaining the ionic strength background, its MMP-9 inhibition rate remained close to that of Example 1. The lack of drug-loaded particle structure in Comparative Example 3 led to reduced stability of the anti-inflammatory components and insufficient inflammation suppression, resulting in a decrease in the MMP-9 inhibition rate; Comparative Example 4 further lacked the synergistic effect of tannic acid polyphenols and microenvironment regulation, making the anti-inflammatory and protease imbalance-relieving chain weaker, resulting in the lowest MMP-9 inhibition rate.

[0027] Spectral analysis: The spectra of the sodium hyaluronate sample prepared in Example 1 before and after oxidation modification were measured using an FTIR spectrometer. The results are as follows: Figure 7 As shown.

[0028] based on Figure 7 As a result, compared with sodium hyaluronate (HA), oxidized modified sodium hyaluronate (OHA) showed a lower concentration at approximately 1720 cm⁻¹. -1 The presence / enhancing carbonyl (C=O) absorption band, while retaining the broad hydroxyl peak and fingerprint region characteristic peak of the polysaccharide backbone, indicates that sodium hyaluronate was successfully introduced into the aldehyde structure after periodate oxidation and the main chain backbone was not completely destroyed.

[0029] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An anti-inflammatory gel for diabetic foot wounds, characterized in that, Its composition includes the following ingredients: sodium hyaluronate, carboxymethyl chitosan, tannic acid, borate compounds, dexamethasone, dopamine hydrochloride, sodium citrate, sodium gluconate, PBS buffer and Tris buffer; The molecular weight range of the sodium hyaluronate is 200–500 kDa; The boric acid compound is any one of borax, phenylboronic acid, and 4-carboxyphenylboronic acid; The preparation of the anti-inflammatory gel for diabetic foot wounds includes the following steps: S1: Dissolve sodium hyaluronate in deionized water and stir until completely dissolved to prepare an aqueous solution. Then, add NaIO4 in batches under light-protected conditions. After the reaction is complete, add ethylene glycol to terminate the reaction. Purify and dialyze, pre-freeze, and freeze-dry under vacuum to obtain oxidized modified sodium hyaluronate. S2: Dissolve carboxymethyl chitosan in PBS buffer and stir until clear and homogeneous to prepare a chitosan solution; S3: Dissolve tannic acid in deionized water and stir until completely dissolved to prepare a TA solution; add dexamethasone to the TA solution, stir to disperse evenly, then add boric acid compound solution dropwise, adjust the pH of the system with Tris buffer, continue stirring, let stand for aging, centrifuge to collect the supernatant, and obtain TA-boric acid dynamic network drug-loaded particle dispersion. S4: Add dopamine hydrochloride to the chitosan solution in step S2, adjust the pH of the system with Tris buffer, stir the reaction at room temperature, then add the TA-boric acid dynamic network drug-loaded particle dispersion from step S3, and ultrasonically disperse it evenly. After dispersion, adjust the pH of the system to obtain solution B. S5: Dissolve the oxidized modified sodium hyaluronate from step S1 in PBS buffer, stir until dissolved and prepared into a solution, then add sodium citrate and sodium gluconate, stir until completely dissolved, and obtain solution A; S6: Mix liquid B from step S4 and liquid A from step S5 through a static mixing head, coat the mixture onto a PTFE mold, and place it in situ in a relatively humid environment to form an anti-inflammatory gel. In step S1, the mass ratio of NaIO4 to sodium hyaluronate is 0.15–0.25:1; the mass ratio of ethylene glycol to NaIO4 is 0.4–0.8 mL:1 g. In step S2, the concentration of the chitosan solution is 20–30 mg / mL; In step S3, the concentration of the TA solution is 5–10 mg / mL; the mass ratio of dexamethasone to tannic acid is 0.1–0.15:1; the concentration of the boric acid compound solution is 10–15 mM; the boric acid compound solution is prepared by dissolving boric acid compounds in deionized water and adding Tris buffer to assist in dissolution; the volume ratio of the boric acid compound solution to the TA solution is 0.1–0.3:1; and the Tris buffer is used to adjust the pH of the system to 8.2–8.

8. In step S4, the final concentration of dopamine hydrochloride in the chitosan solution is 0.5–1.5 mg / mL; the Tris buffer solution is used to adjust the pH of the system to 8.2–8.8; the stirring reaction time at room temperature is 20–30 min; the amount of TA-boric acid dynamic network drug-loaded particle dispersion added is 3–5% of the volume of the chitosan solution; after dispersion, the pH of the system is adjusted to 7.2–7.

4. In step S5, the concentration of the oxidized modified sodium hyaluronate solution is 15-30 mg / mL; the molar ratio of sodium citrate to sodium gluconate is 1-2:1, and the total final concentration of both in solution A is 10-20 mM. In step S6, the volume ratio of liquid B to liquid A is 1:

1.

2. The anti-inflammatory gel for diabetic foot wounds according to claim 1, characterized in that, In step S1, the water is changed every 2-4 hours for the first 3 purification dialysis cycles, and then every 6-8 hours thereafter, for a total of 48-72 hours of dialysis.

3. The anti-inflammatory gel for diabetic foot wounds according to claim 1, characterized in that, In step S2, the pH of the chitosan solution is 7.2 to 7.4; the pH is adjusted using PBS buffer.

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