pH-responsive antibacterial and antioxidant drug-loaded hydrogel and preparation method and application thereof

Abstract

The application relates to a pH-responsive antibacterial and antioxidant drug-loaded hydrogel as well as a preparation method and application thereof, and belongs to the field of biomedical materials. The pH-responsive antibacterial and antioxidant drug-loaded hydrogel is composed of gelatin, aldehyde-terminated Pluronic F127 drug-loaded micelles and magnesium micro-motors. An imine bond formed through Schiff base reaction between amino groups on the molecular chain of gelatin and aldehyde groups on the aldehyde-terminated Pluronic F127 micelles is used to construct a dynamic and reversible cross-linking network, and magnesium micro-motors for ROS removal are loaded in the network to obtain a pH-responsive antibacterial and antioxidant drug-loaded hydrogel dressing. The preparation process is simple, raw materials are easy to obtain, reaction conditions are mild, and no other initiators are needed. The prepared hydrogel has injectability and self-repairing capability, can be used as a drug release carrier, can accelerate drug release under an acidic microenvironment, can improve an inflammatory oxidative stress microenvironment, and has been proved to have positive therapeutic effects and broad application prospects in the treatment of diabetic chronic wound infection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and in particular relates to a pH-responsive antibacterial and antioxidant drug-loaded hydrogel, a preparation method thereof, and an application thereof. Background Art

[0002] Diabetes mellitus (DM) is a common chronic metabolic disease caused by hyperglycemia. With the increasing incidence of diabetes, difficult-to-heal chronic wounds, a common complication of diabetes, have placed a heavy economic burden on both patients and society. Promoting the rapid healing of chronic diabetic wounds and the orderly repair of skin tissue is a major clinical challenge. Diabetic wounds are moist and high in sugar, making them susceptible to bacterial infection, producing lactic acid and acetic acid, leading to local acidification (pH 4.5-6.5). Simultaneously, excessive reactive oxygen species (ROS) generated in the wound surface increase oxidative stress, exacerbate inflammatory responses, and hinder the re-epithelialization process. Therefore, we believe that scavenging ROS, reducing oxidative stress, and alleviating inflammation can be a therapeutic strategy to accelerate diabetic wound repair.

[0003] Hydrogels, consisting of a three-dimensional network structure formed by physical or chemical crosslinking of polymers, exhibit excellent biocompatibility and are widely used as functional drug carriers for wound infection treatment in biomedical fields such as tissue engineering and sustained drug release. Designing a pH-responsive, drug-loaded hydrogel based on the unique physiological microenvironment of diabetic wounds is of great research significance for promoting rapid wound healing.

[0004] Gelatin, a natural protein extracted from collagen, is a biomimetic peptide that promotes cell adhesion, proliferation, and differentiation. However, its poor mechanical properties and brittleness significantly limit its application. The numerous amino, carboxyl, and hydroxyl groups on its molecular chains are often modified or linked through chemical crosslinking to form various interpenetrating network and double-network composite hydrogels. Pluronic F127 is an amphiphilic triblock copolymer that self-assembles into micelles in water. Aldehyde groups on Pluronic F127 are modified in a one-step reaction, allowing them to react with amino groups on gelatin to form imine bonds under mild conditions via a Schiff base reaction. Imine bonds are a type of pH-sensitive, reversible, dynamic covalent bond that is relatively stable under neutral or alkaline conditions. Under acidic conditions (pH < 6.5), the protonation of the amino groups leads to hydrolysis and cleavage of the imine bond, enabling drug-responsive release. Imine bonds not only impart pH responsiveness to hydrogels but also impart injectability and self-healing abilities, improving the mechanical properties of pure gelatin.

[0005] A magnesium micromotor (Mg motor) is a novel in situ hydrogen storage system based on magnesium microparticles. It utilizes the reaction of magnesium with water in an aqueous medium to form magnesium hydroxide and release hydrogen to achieve sustained hydrogen delivery, scavenging ROS and pro-inflammatory factors at the site of inflammation. Recent studies have shown its application in treating various inflammatory diseases, including tumors and rheumatoid arthritis. Currently, micromotors are typically delivered orally or intravenously, but the complex fluid environment can damage their structure and lead to premature inactivation. Encapsulating micromotors in hydrogels can reduce environmental damage to the micromotors, prolong H2 release, effectively scavenge ROS, alleviate inflammation, and accelerate the healing process of diabetic wounds. Summary of the Invention

[0006] The present invention aims to provide a pH-responsive antimicrobial and antioxidant drug-loaded hydrogel, its preparation method, and application. The pH-responsive antimicrobial and antioxidant drug-loaded hydrogel is injectable and self-healing. In the slightly acidic environment of the wound surface, the imine bonds in the hydrogel break, accelerating drug release, ameliorating the inflammatory and oxidative stress microenvironment, and promoting the healing of chronic diabetic wounds.

[0007] The present invention is achieved through the following technical solutions:

[0008] A pH-responsive antibacterial and antioxidant drug-loaded hydrogel is composed of gelatin, terminal aldehyde-loaded Pluronic F127 drug-loaded micelles, and magnesium micromotors. A dynamic and reversible cross-linked network is constructed by using the imine bonds formed by the Schiff base reaction between the amino groups on the gelatin molecular chain and the aldehyde groups on the terminal aldehyde-loaded Pluronic F127 micelles, and the ROS scavenger magnesium micromotors are loaded into it.

[0009] The method for preparing the pH-responsive antibacterial and antioxidant drug-loaded hydrogel comprises the following steps:

[0010] Step 1: Synthesis of terminal aldehyde-terminated Pluronic F127 (FCHO)

[0011] Pluronic F127, p-formylbenzoic acid, 4-dimethylaminopyridine, and dicyclohexylcarbodiimide were ultrasonically mixed and dissolved in anhydrous tetrahydrofuran, and reacted at room temperature. The reaction solution was concentrated and precipitated with cold ether. The resulting white solid was filtered and dried, dissolved in pure water, and the supernatant was centrifuged and freeze-dried to obtain terminal aldehyde-terminated Pluronic F127 (FCHO).

[0012] Step 2: Preparation of FCHO drug-loaded micelles

[0013] The terminal aldehyde-modified Pluronic F127 and the hydrophobic anti-inflammatory drug were ultrasonically dissolved in an organic solvent, and a polymer film was obtained by rotary evaporation, which was then dissolved by hydration to obtain a drug-loaded FCHO micelle solution.

[0014] Step 3: Preparation of magnesium micromotor (Mg motor)

[0015] The magnesium microparticles were washed with acetone and dried under nitrogen; the magnesium microparticles were dispersed on a glass slide pre-coated with polyvinyl pyrrolidone; a layer of polylactic acid-co-glycolic acid copolymer was coated on the magnesium microparticle layer, dried, and then scraped off to obtain a magnesium micromotor.

[0016] Step 4: Preparation of pH-responsive hydrogel

[0017] After the gelatin solution and FCHO-loaded micelle solutions of different concentrations were evenly mixed, magnesium micromotors were added and allowed to stand at room temperature to obtain pH-responsive antibacterial and antioxidant drug-loaded hydrogels (Gel / FCHO / Mg motor).

[0018] In step 1, the reaction molar ratio of Pluronic F127, p-formylbenzoic acid, 4-dimethylaminopyridine, and dicyclohexylcarbodiimide is 1:(4-7.5):(0.4-0.6):(5-10); the amount ratio of Pluronic F127 and anhydrous tetrahydrofuran is 10 g:(50-100) mL; and the reaction time is 24-36 h.

[0019] In step 2, the hydrophobic anti-inflammatory drug is selected from any one of curcumin, berberine, quercetin, ibuprofen, indomethacin, celecoxib, and diclofenac sodium, preferably curcumin.

[0020] In the step 2, the mass ratio of the hydrophobic anti-inflammatory drug to the terminal aldehyde-terminated Pluronic F127 is 1:15-50.

[0021] In step 2, the organic solvent is any one of ethanol, methanol and acetone; the hydration solvent is PBS buffer (0.01M, pH=7.4); and the temperature of rotary evaporation and hydration is both 40°C.

[0022] In step 3, the polyvinyl pyrrolidone is dissolved in ethanol at a concentration of 1 to 4% (w / v) and a molecular weight of 24 to 30 kDa; the polylactic acid-glycolic acid copolymer is dissolved in any one of ethyl acetate, acetone, and dichloromethane at a concentration of 0.5 to 2% (w / v); the ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 75:25, and the molecular weight is 8 to 10 kDa.

[0023] In step 4, the concentration of the gelatin solution is 10-15% (w / v), the concentration of the FCHO drug-loaded micelle solution is 2.5-15% (w / v), and the concentration of Mg motor is 0.1-0.3% (w / v); the volume ratio of the gelatin solution to the FCHO drug-loaded micelle solution is 1:0.5-1.5.

[0024] The present invention also discloses the use of the pH-responsive antibacterial and antioxidant drug-loaded hydrogel prepared by the method as a wound dressing in the treatment of diabetic full-thickness skin wounds.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] (1) In the present invention, the terminal aldehyde-modified Pluronic F127 (FCHO) is used as an amphiphilic material to form micelles through the interaction between its internal non-polar hydrophobic groups and the hydrophobic side groups of hydrophobic drugs, thereby achieving drug encapsulation and effectively improving the drug loading capacity of the hydrogel for hydrophobic drugs.

[0027] (2) The preparation process provided by the present invention is simple, the raw materials are readily available, the reaction conditions are mild, and no other initiators are required. The dynamic imine bond formed by the Schiff base reaction between the amino groups on the gelatin molecular chain and the aldehyde groups on the surface of the FCHO micelles improves the mechanical properties of pure gelatin and gives the hydrogel a certain self-healing ability and injectability, allowing it to fill irregular wounds.

[0028] (3) The present invention uses anti-inflammatory drugs as a loading substance, which not only scavenges free radicals and reduces oxidative stress, but also destroys the integrity and permeability of bacterial cell membranes, leading to bacterial death. The addition of anti-inflammatory drugs gives the hydrogel excellent antioxidant and antibacterial properties, meeting practical application requirements.

[0029] (4) The amino groups on the gelatin molecular chain of the present invention will be protonated under the weakly acidic inflammatory microenvironment, thereby enhancing the intermolecular electrostatic interaction and breaking the imine bond, thereby achieving responsive and rapid release of the drug.

[0030] (5) The present invention utilizes an asymmetrically coated PLGA layer to fabricate a magnesium micromotor. A notch in the outer shell serves as a channel for the reaction of the core magnesium microparticles with water to generate hydrogen. Because the notch is limited in area, the reaction rate can be controlled, enabling sustained release of hydrogen.

[0031] (6) The present invention proposes the use of hydrogen therapy for the treatment of chronic diabetic wounds. Hydrogen (H2), as an endogenous small molecule gas with anti-inflammatory properties, can easily penetrate cell membranes, selectively scavenge highly toxic oxidative free radicals, downregulate the expression levels of pro-inflammatory factors, and does not affect the metabolism and signal transduction of normal cells. In addition, H2 can also exert antioxidant effects by activating endogenous antioxidant enzymes, making it a new type of ROS scavenger.

[0032] (7) The present invention utilizes hydrogel as a delivery carrier for magnesium micromotors, which provides a certain degree of protection for the micromotors and controls their release, greatly enhancing the application potential of micromotors in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the structure of the Gel / FCHO / Mg motor hydrogel prepared in the present invention;

[0034] Figure 2 The synthetic route of the terminal aldehyde-containing Pluronic F127 (FCHO) of the present invention is as follows;

[0035] Figure 3 The terminal aldehyde-terminated Pluronic F127 (FCHO) of the present invention 1 H-NMR spectrum;

[0036] Figure 4 The figure shows the preparation steps and structure of the Mg motor in the present invention;

[0037] Figure 5 This is the SEM image of the Mg motor in the present invention;

[0038] Figure 6 FT-IR images of Gel, FCHO and Gel / FCHO / Mg motor hydrogels in the present invention;

[0039] Figure 7 This is the SEM image of the Gel / FCHO / Mg motor hydrogel prepared in the present invention;

[0040] Figure 8 Demonstration diagrams and rheological characterizations of the self-healing process of the Gel / FCHO / Mg motor hydrogel prepared in the present invention; (a) and (b) illustrate the self-healing process of the pure gelatin hydrogel in Comparative Example 1 and the Gel / FCHO / Mg motor hydrogel in Example 1, respectively; (c) shows the continuous step strain (γ = 1% → 1000%) test results of the Gel / FCHO / Mg motor hydrogel for three consecutive cycles;

[0041] Figure 9The shear thinning properties and injectability test results of the Gel / FCHO / Mg motor hydrogel prepared in the present invention;

[0042] Figure 10 The in vitro pH-responsive drug release curve of the Gel / FCHO / Mg motor hydrogel prepared in the present invention;

[0043] Figure 11 The in vitro hydrogen release curves of the pure magnesium particles, magnesium micromotors, and Gel / FCHO / Mg motor hydrogel of the present invention are shown;

[0044] Figure 12 The scavenging efficiency of the Gel / FCHO / Mg motor hydrogel prepared by the present invention on DPPH free radicals;

[0045] Figure 13 The antibacterial effect of the Gel / FCHO / Mg motor hydrogel prepared by the present invention on Escherichia coli and Staphylococcus aureus;

[0046] Figure 14 This is a diagram showing the therapeutic effect of the Gel / FCHO / Mg motor hydrogel prepared in the present invention on diabetic full-thickness skin defect wounds. DETAILED DESCRIPTION

[0047] In order to better understand the present invention, the present invention is described in detail below in conjunction with specific embodiments, but the content of the present invention is not limited to the following embodiments.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the reagents, materials, etc. used in the following examples are all commercially available unless otherwise specified.

[0049] Example 1

[0050] A method for preparing a pH-responsive antibacterial and antioxidant drug-loaded hydrogel, the specific steps of which are as follows:

[0051] (1) Preparation of terminal aldehyde-terminated Pluronic F127 (FCHO):

[0052] Pluronic F127 (10.0 g), p-formylbenzoic acid (0.6 g), 4-dimethylaminopyridine (0.05 g) and dicyclohexylcarbodiimide (1.0 g) were ultrasonically mixed and dissolved in anhydrous tetrahydrofuran (50 mL), and reacted at room temperature for 36 hours. The reaction solution was concentrated by rotary evaporation at 40°C and filtered three times with cold ether precipitation. The dried white powder was dissolved in pure water, centrifuged at 12000 rpm for 15 minutes, and the supernatant was collected and freeze-dried to obtain terminal formyl Pluronic F127 (FCHO).

[0053] The synthetic route and chemical structure of terminal aldehyde Pluronic F127 are shown in Figure 2 , its H NMR spectrum ( 1 H-NMR) results are shown in Figure 3 The infrared spectrum (FT-IR) results are shown in Figure 6 .like Figure 2 As shown in the figure, the hydrogen atoms on the aromatic ring of the FCHO polymer showed signals at 8.14ppm and 7.88ppm, and the hydrogen atoms of the aldehyde group showed signals at 10.03ppm, indicating that p-formylbenzoic acid was successfully grafted onto Pluronic F127. Figure 6 As shown, compared with F127, FCHO has a -1 、1699cm -1 and 761cm -1 New absorption peaks appeared at , which were attributed to the stretching vibration of the ester bond C=O, the stretching vibration of the aldehyde group C=O and the out-of-plane bending of the aromatic ring CH, further proving that FCHO has been successfully synthesized.

[0054] (2) Preparation of FCHO drug-loaded micelles:

[0055] 100 mg FCHO and 4 mg curcumin were fully dissolved in 10 mL of anhydrous ethanol by ultrasonication, and the polymer film was obtained by rotary evaporation at 40°C. The film was then hydrated with 5 mL of PBS (0.01 M, pH = 7.4) at 40°C. The solution was centrifuged (8000 rpm, 10 min) and filtered through a 0.22 μm microporous filter membrane to obtain a curcumin-loaded FCHO micelle solution.

[0056] As shown in Table 1, when the Cur:FCHO mass ratio is between 1:15 and 1:50, the encapsulation efficiency can reach over 90%. Among them, when the Cur:FCHO mass ratio is 1:25, the encapsulation efficiency is the highest, reaching 97.17±1.81%.

[0057] Table 1 Encapsulation efficiency of curcumin in FCHO micelles at different dosing ratios

[0058]

[0059] (3) Preparation of magnesium micromotor (Mg motor):

[0060] The magnesium microparticles were washed with acetone and dried under N2. 1 mg of magnesium microparticles was dispersed on a glass slide pre-coated with a 100 μL thin film of PVP ethanol solution (2% w / v). 50 μL of PLGA ethyl acetate solution (1% w / v) was coated on the resulting magnesium microparticle layer and, after drying, carefully scraped from the glass slide to obtain a magnesium micromotor (Mg motor).

[0061] The preparation steps and structural diagram of the magnesium micromotor are shown in Figure 4 , the scanning electron microscope (SEM) is shown in Figure 5 It was demonstrated that the magnesium micromotor has a notch in its PLGA shell that can serve as a channel for H2 release.

[0062] (4) Preparation of hydrogel:

[0063] After evenly mixing 500 μL of gelatin solution (15% w / v) and 500 μL of FCHO-loaded micelle solution (10% w / v), 1 mg of Mg motor was added and allowed to stand to obtain the pH-responsive antibacterial and antioxidant drug-loaded hydrogel Gel / FCHO / Mg motor-2.

[0064] The schematic diagram of the hydrogel structure is shown in Figure 1 The infrared spectra (FT-IR) results of the hydrogel components are shown in Figure 6 .like Figure 6 As shown, GCM hydrogel has a peak at 1683 cm -1 The absorption peak that appeared was due to the newly formed C=N bond between the amino group on the gelatin molecular chain and the aldehyde group of FCHO, indicating that the hydrogel network had been successfully constructed through the Schiff base reaction.

[0065] Example 2

[0066] The same as Example 1, except that the concentration of the FCHO drug-loaded micelle solution was changed to 5% w / v in step (4), and other preparation process conditions and process parameters remained unchanged to obtain a pH-responsive antibacterial and antioxidant drug-loaded hydrogel Gel / FCHO / Mg motor-1.

[0067] Example 3

[0068] The same as Example 1, except that the concentration of the FCHO drug-loaded micelle solution was changed to 2.5% w / v in step (4), and the other preparation process conditions and process parameters remained unchanged to obtain a pH-responsive antibacterial and antioxidant drug-loaded hydrogel Gel / FCHO / Mg motor-0.5.

[0069] Comparative Example 1

[0070] Preparation of pure gelatin hydrogel

[0071] 500 μL of gelatin solution (15% w / v) and 500 μL of PBS solution were uniformly mixed and allowed to stand to obtain pure gelatin hydrogel Gel.

[0072] The following is a detailed analysis with reference to the accompanying drawings and experimental data. The hydrogel prepared in Example 1 was characterized and its performance tested as follows:

[0073] (1) Morphological characterization of hydrogels:

[0074] The prepared hydrogel was freeze-dried and then sprayed with a gold layer on its surface and cross-section, and its microstructure was characterized by scanning electron microscopy (SEM).

[0075] The experimental results are as follows Figure 7 As shown, the hydrogel presents an irregular porous network structure, indicating that this series of hydrogels has good water absorption and air permeability, which is conducive to drug release.

[0076] (2) Self-healing and injectability test of hydrogel:

[0077] The cut hydrogel was reassembled to observe its macroscopic self-healing properties. The experimental results are as follows Figure 8 As shown in (a) and (b), the pure gelatin hydrogel prepared in Comparative Example 1 cannot achieve self-repair, while the Gel / FCHO / Mg motor hydrogel can connect with each other to form a whole after a certain period of time, proving that the hydrogel has stable self-repairability.

[0078] The microscopic self-healing properties of the hydrogel were evaluated using a rheometer. The hydrogel was placed on the rheometer sample stage with a 20 mm diameter parallel plate. At a constant sweep frequency (1 Hz), the strain value was varied from 1% to 1000%, and the changes in the storage modulus (G') and loss modulus (G") of the hydrogel were observed over multiple cycles.

[0079] The experimental results are as follows Figure 8 As shown in (c), when a large strain (γ = 1000%) is applied, the G' of the hydrogel decreases significantly and is lower than G", indicating that the three-dimensional network structure inside the hydrogel is destroyed at this time. When switching to a small strain (γ = 1%), G' quickly rebounds, indicating that the internal structure of the hydrogel has been reconstructed, and after 3 cycles, G' and G" have no obvious changes, once again indicating that the hydrogel has good self-repair function and can quickly achieve its own structural recovery.

[0080] The shear thinning behavior of the hydrogel was evaluated using a rheometer. The hydrogel was placed on the sample stage of the rheometer, the steady-state viscosity scanning mode was selected, and the shear rate was set to 1-100 s. -1 .

[0081] The experimental results are as follows Figure 9 As shown in the figure, as the shear rate increases, the apparent viscosity of the hydrogel decreases rapidly, showing an obvious shear thinning phenomenon, indicating that the hydrogel has good injectability and can be easily squeezed out of the syringe.

[0082] (3) Investigation of pH-responsive drug release behavior of hydrogels:

[0083] The experimental results are as follows Figure 10As shown, the cumulative release rate of curcumin in a weakly acidic environment (pH = 5.0) was much higher than that in a normal physiological environment (pH = 7.4), indicating that the hydrogel has good pH responsiveness. In the weakly acidic physiological microenvironment of chronic diabetic wounds, the hydrogel accelerated drug release and demonstrated good therapeutic effects.

[0084] The experimental results are as follows Figure 11 As shown, compared with the hydrogen release behavior of pure magnesium particles and magnesium micromotors, hydrogels can prolong the action time of hydrogen and present a better sustained release effect.

[0085] (4) Antioxidant efficiency of hydrogel:

[0086] The antioxidant efficiency of the hydrogels was evaluated by measuring the DPPH free radical scavenging rate. The hydrogels were cut into homogenates using a tissue grinder. The required amount of hydrogel (9 mg, 18 mg, 27 mg, 36 mg, 45 mg) was dispersed in 3 mL of 100 μM DPPH ethanol solution and incubated in the dark for 30 minutes. The DPPH scavenging rate was calculated using the following formula (n = 3):

[0087]

[0088] Among them A Blank 、A Hydrogel These are the absorbances of blank solution (DPPH+ethanol) and hydrogel solution (DPPH+ethanol+hydrogel) at 517 nm, respectively.

[0089] The experimental results are as follows Figure 12 As shown in the results, the curcumin-loaded hydrogel exhibited significant antioxidant activity, and with the increase of the hydrogel content, its DPPH free radical scavenging rate also increased accordingly.

[0090] (5) In vitro antibacterial test of hydrogel:

[0091] Escherichia coli and Staphylococcus aureus were cultured in LB liquid medium at 37°C for 12 h, and the bacterial solution was gradiently diluted to 10 -5 times. 200 mg of hydrogel and 200 μL of PBS were added to a 48-well plate as a control. 100 μL of bacterial suspension was added to the hydrogel surface. The 48-well plate was placed in a 37°C incubator and cultured for 12 hours. Afterwards, 200 μL of sterilized PBS was added to each well and shaken vigorously for 30 minutes. 100 μL of the collected bacterial suspension was spread on an LB plate and cultured at 37°C for 24 hours. The bacterial growth on each LB plate was photographed and the number of colonies on the plate was counted. The sterilization rate was calculated by the following formula (n=3):

[0092]

[0093] The experimental results are as follows Figure 13 As shown, the killing rates of Gel / FCHO / Mg motor hydrogel against Escherichia coli and Staphylococcus aureus were 92.4% and 98.9%, respectively. Compared with the PBS control group and Comparative Example 1, it was demonstrated that Gel / FCHO / Mg motor hydrogel exhibited certain antibacterial activity against both Escherichia coli and Staphylococcus aureus.

[0094] (6) Investigation of the therapeutic effect of hydrogel on diabetic full-thickness wound defects:

[0095] A diabetic full-thickness wound defect model was established in mice. The mice were randomly divided into groups (n=8) and treated with different treatments: (a) no treatment group (Control) and (b) commercial dressing Tegaderm. TM (a) Film group; (b) Gel / FCHO / Mg motor hydrogel group. Photos were taken at different time points (days 0, 3, 7, and 14) to observe wound healing.

[0096] The experimental results are as follows Figure 14 As shown, the hydrogel's rapid promotion of wound healing can be seen throughout the treatment process. On day 14, the hydrogel group demonstrated superior therapeutic efficacy compared to the control group, with a smaller remaining wound area and a healing rate exceeding 95%, demonstrating a relatively intact skin structure. This demonstrates that the hydrogel can effectively promote the healing of full-thickness diabetic wounds and aid in effective skin repair.

[0097] In summary, the present invention successfully prepared a pH-responsive antibacterial and antioxidant hydrogel (Gel / FCHO / Mgmotor), which has stable performance and can be applied to the wound surface by injection to improve the coverage effect. The imine bond in the hydrogel structure gives it pH responsiveness, which can control drug release. At the same time, the hydrogel has excellent antibacterial, antioxidant and ROS scavenging capabilities, and has shown superiority to commercial dressings (Tegaderm) in the treatment of diabetic full-thickness skin defect wounds. TM film) has a broad application prospect.

Claims

1. A method for preparing a pH-responsive antibacterial and antioxidant drug-loaded hydrogel for treating diabetic full-thickness skin wounds, characterized in that: The following steps are involved: Step 1: Synthesis of FCHO Pluronic F127, p-formylbenzoic acid, 4-dimethylaminopyridine, and dicyclohexylcarbodiimide were ultrasonically mixed and dissolved in anhydrous tetrahydrofuran, and reacted at room temperature. The reaction solution was concentrated and precipitated with cold ether. The resulting white solid was filtered and dried, dissolved in pure water, centrifuged, and the supernatant was freeze-dried to obtain terminal aldehyde-terminated Pluronic F127, denoted as FCHO. Step 2: Preparation of FCHO drug-loaded micelles The hydrophobic anti-inflammatory drug and terminal aldehyde-modified Pluronic F127 are ultrasonically dissolved in an organic solvent at a mass ratio of 1:15-50, and a polymer film is obtained by rotary evaporation. The polymer film is then hydrated and dissolved to obtain a drug-loaded FCHO micelle solution; the hydrophobic anti-inflammatory drug is curcumin; Step 3: Preparation of magnesium micromotors The magnesium microparticles were washed with acetone and dried under nitrogen. The magnesium microparticles were dispersed on a glass slide pre-coated with polyvinyl pyrrolidone. A layer of polylactic acid-co-glycolic acid copolymer was then coated on the magnesium microparticle layer and scraped off after drying to obtain a magnesium micromotor. Step 4: Preparation of pH-responsive hydrogel After gelatin solution and FCHO drug-loaded micelle solution of different concentrations were evenly mixed, magnesium micromotors were added and allowed to stand at room temperature. The amino groups on the gelatin molecular chain reacted with the aldehyde groups on the FCHO micelles to form imine bonds, constructing a dynamic and reversible cross-linked network. ROS scavenger magnesium micromotors were then loaded into the network to obtain pH-responsive antibacterial and antioxidant drug-loaded hydrogels. The concentration of gelatin solution is 10~15% ( w / v ), the concentration of FCHO drug-loaded micelle solution is 2.5~15% ( w / v ), the concentration of magnesium micromotors is 0.1~0.3% ( w / v ); the volume ratio of gelatin solution to FCHO drug-loaded micelle solution is 1:0.5~1.

5.

2. The method for preparing the pH-responsive antibacterial and antioxidant drug-loaded hydrogel for treating diabetic full-thickness skin wounds according to claim 1, characterized in that: In step 1, the reaction molar ratio of Pluronic F127, p-formylbenzoic acid, 4-dimethylaminopyridine, and dicyclohexylcarbodiimide is 1:(4-7.5):(0.4-0.6):(5-10); the amount ratio of Pluronic F127 and anhydrous tetrahydrofuran is 10 g:50-100 mL; and the reaction time is 24-36 h.

3. The method for preparing the pH-responsive antibacterial and antioxidant drug-loaded hydrogel for treating diabetic full-thickness skin wounds according to claim 1, characterized in that: In step 2, the organic solvent is any one of ethanol, methanol and acetone; and the hydration solvent is 0.01 M PBS buffer solution with a pH of 7.

4.

4. The method for preparing the pH-responsive antibacterial and antioxidant drug-loaded hydrogel for treating diabetic full-thickness skin wounds according to claim 1, characterized in that: In step 3, the polyvinyl pyrrolidone is dissolved in ethanol at a concentration of 1-4% ( w / v ), with a molecular weight of 24~30 kDa; the polylactic acid-glycolic acid copolymer is dissolved in ethyl acetate, acetone or dichloromethane at a concentration of 0.5~2% ( w / v ); The ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 75:25, and the molecular weight is 8~10 kDa.

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