A photothermal and photodynamic antibacterial hydrogel with pH / glucose dual-responsive drug release, its preparation method and application

Abstract

The present invention discloses a photothermal and photodynamic antibacterial hydrogel with pH / glucose dual-responsive drug release, its preparation method and application. Grafting PBA onto the HA backbone can enhance the self-healing performance, swelling performance and mechanical strength of the system; after oxidizing CS, the self-healing performance and adhesion performance of the system can be increased; when the two macromolecular chains HAP and OCS are crosslinked, a dual dynamic network of Schiff base and phenylborate ester can be formed, endowing the system with pH and glucose dual-responsive properties; introducing GPC into the hydrogel system endows the hydrogel with good antioxidant performance, photothermal and photodynamic antibacterial performance, and electrical conductivity, which can prevent severe wound infections, accelerate wound healing, and the released PFD has good anti-inflammatory and angiogenesis-promoting effects to promote the healing of diabetic foot ulcer wounds.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and relates to a photothermal and photodynamic antibacterial hydrogel with pH / glucose dual-responsive drug release, a preparation method thereof, and an application thereof. Background Art

[0002] Diabetes is a serious chronic disease in the world, which can cause chronic complications affecting the life and health of patients, including: stroke, renal failure, diabetic foot ulcer (DFU), etc. Among them, factors such as hyperglycemia, peripheral neuropathy, peripheral arterial disease, blood transport disorders, and bacterial load leading to slow wound healing are the reasons for the high incidence rate of DFU up to 15% and the amputation rate up to 30%. DFU is difficult to heal due to problems such as oxidative stress, excessive infection and inflammation, and difficulty in angiogenesis, and has become a research hotspot in the field of wound healing. The treatment methods of DFU include blood glucose regulation, non-surgical debridement, wound dressings, hyperbaric oxygen therapy, and negative pressure wound therapy. Due to the wide application range, easy portability, and long service life of wound dressings, they play a crucial role in the treatment process. However, traditional wound dressings are prone to falling off and breaking, and lack the biochemical functions to promote wound healing. Therefore, it is of great significance to design a wound dressing that not only has various biochemical functions such as antioxidant, tissue adhesion, infection prevention, anti-inflammatory, and blood vessel promotion, but also can meet the frequent movement of DFU.

[0003] Hydrogel wound dressings are easy to obtain good tissue adhesion, stretchability and self-healing properties, and can absorb wound exudate to maintain the microenvironment of the wound area, and have great potential in the treatment of DFU wounds.

[0004] Hyaluronic acid has good biocompatibility, viscoelasticity, moisture retention, and biodegradability, and can regulate soft tissue wound repair by promoting cell migration, promoting angiogenesis, and immunomodulation. Although hyaluronic acid hydrogel can fill the wound and absorb wound exudate, it is easy to rupture. At the same time, how to achieve the high-efficiency antioxidant, self-healing and adhesion of hyaluronic acid hydrogel, and solve the serious infection and vascular lesions in diabetic foot ulcers makes it of great significance in the design of diabetic foot ulcer wound dressings.

[0005] Overuse of antibiotics will ultimately lead to the emergence of bacterial drug resistance and make antibiotics ineffective. Photothermal antibacterial (PTA) hydrogels have a broad antibacterial spectrum and good tissue permeability, and are a new sterilization strategy. Single PTA only produces antibacterial effects after maintaining the temperature at 52°C for a period of time, but it also has adverse effects on surrounding tissues over time. Summary of the Invention

[0006] The object of the present invention is to overcome the above-mentioned disadvantages of the prior art, and provide a photothermal and photodynamic antibacterial hydrogel with pH / glucose dual-responsive drug release, its preparation method and application, so as to solve the problems in the prior art such as insufficient adhesion performance of diabetic foot ulcer wound dressings, low antioxidant property, poor self-healing performance, difficult blood vessel regeneration at the wound site, and inhibition of bacterial infection.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A preparation method of a photothermal and photodynamic antibacterial hydrogel with pH / glucose dual-responsive drug release, comprising the following steps:

[0009] Step 1, under the action of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole, adipic dihydrazide is used to carry out amino modification on hyaluronic acid to obtain amino-modified hyaluronic acid AHA;

[0010] Step 2, graft PBA onto amino-modified hyaluronic acid AHA to obtain HAP,

[0011] Step 3, after oxidizing chondroitin sulfate, chondroitin sulfate containing aldehyde groups is obtained, which is OCS;

[0012] Step 4, under alkaline conditions, a polydopamine coating is polymerized on the surface of graphene oxide to generate reduced graphene oxide rGO@PDA;

[0013] Step 5, mix glycine solution and fullerene solution, and obtain glycine-modified fullerene after reaction treatment;

[0014] Step 6, using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and NHS as catalysts, graft glycine-modified fullerene onto reduced graphene oxide rGO@PDA to generate GPC;

[0015] Step 7, dissolve HAP, OCS, GPC and pirfenidone in water respectively to form HAP solution, OCS solution, GPC solution and pirfenidone solution, and mix the four solutions evenly according to the volume ratio of 375:150:100:375 to obtain a photothermal and photodynamic antibacterial hydrogel with pH / glucose dual-responsive drug release.

[0016] A further improvement of the present invention lies in:

[0017] Preferably, the specific process of step 1 is as follows: adipic dihydrazide is added to the hyaluronic acid solution, and after adjusting the pH value with hydrochloric acid and NaOH, a mixed solution A is formed. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole are dissolved in solvent 1 to form a mixed solution B. The mixed solution B is added to the mixed solution A, and after adjusting the pH value, a reaction solution C is obtained. After terminating the reaction, the solution obtained by the reaction is dialyzed, precipitated, and freeze-dried to obtain amino-modified hyaluronic acid AHA.

[0018] Preferably, the specific process of step 2 is as follows: the amino-modified hyaluronic acid AHA is dissolved in 4-morpholineethanesulfonic acid buffer to obtain a mixed solution E. 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride is added to the mixed solution E, and then PBA is added. After the reaction, it is dialyzed and freeze-dried to obtain HAP.

[0019] Preferably, the specific process of step 3 is as follows: sodium periodate solution is dropped into the chondroitin sulfate solution, and after stirring, a mixed solution F is obtained. After dialyzing the mixed solution F, oxidized chondroitin sulfate is obtained.

[0020] Preferably, the specific process of step 4 is as follows: graphene oxide and dopamine hydrochloride are dissolved in Tris-HCl buffer, and after stirring, they are filtered, purified, and separated. The separated product is washed with water and ethanol to obtain an rGO@PDA solution. After drying the rGO@PDA solution, an rGO@PDA copolymer is obtained.

[0021] Preferably, the specific process of step 5 is as follows: fullerene is added to toluene to obtain a fullerene solution. Glycine and NaOH are added to solvent 2 to obtain a glycine solution. The fullerene solution is added to the glycine solution, and after stirring until the mixed solution becomes brownish-black, a mixed solution G is obtained. After removing the upper organic layer from the mixed solution G, the lower layer substance is washed and rotary-evaporated to obtain a process substance H. The process substance H is dissolved in water, precipitated after being washed with absolute ethanol, and after centrifugal separation, the centrifugal separation product is dissolved and then suction-filtered, and rotary-evaporated to obtain a process substance I. After acidifying the process substance I, black precipitate is obtained by centrifugal separation, and after drying the black precipitate, glycine-modified fullerene is obtained.

[0022] Preferably, the specific process of step 6 is as follows: the glycine-modified fullerene is dissolved in water to obtain a solution J. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and NHS are added to the solution J to obtain a solution K. The reduced graphene oxide rGO@PDA dispersion is dispersed in the solution K, and after stirring, it is washed and vacuum-dried to obtain GPC.

[0023] Preferably, in step 7, the concentration of the HAP solution is 2 wt%, the concentration of the OCS solution is 2.5 wt%, the concentration of the GPC solution is 1 wt% to 4 wt%, and the concentration of the pirfenidone solution is 0.08 wt%.

[0024] A photothermal and photodynamic antibacterial hydrogel with pH / glucose dual-responsive drug release prepared by any one of the above preparation methods, comprising HAP macromolecular chains and OCS macromolecular chains, and the two macromolecular chains are crosslinked and combined with each other; GPC and pirfenidone are embedded in the hydrogel.

[0025] An application of the above-mentioned photothermal and photodynamic antibacterial hydrogel with pH / glucose dual-responsive drug release as a wound dressing for diabetic wound healing.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention relates to a preparation method of a photothermal and photodynamic antibacterial hydrogel with pH / glucose dual-responsive drug release. In this preparation method, a solution containing HAP and a solution containing OCS are mixed, and crosslinked through Schiff base and phenylborate ester to form a pH / glucose dual-responsive HAP / OCS hydrogel. Meanwhile, GPC and pirfenidone are loaded into the HAP / OCS hydrogel to form a hydrogel with pH / glucose dual-responsive photothermal and photodynamic antibacterial properties. The hydrogel is crosslinked by two macromolecular chains. One macromolecular chain is HA, which has good biocompatibility, viscoelasticity, moisture retention, and biodegradability. First, it is aminated to endow it with reaction sites for Schiff base; then PBA is grafted onto the modified HA to obtain the final product HAP. The other macromolecular chain is CS, which is oxidized to obtain OCS. The aldehyde groups on OCS can form Schiff base crosslinking with the amino groups on HAP, endowing the hydrogel with good adhesion properties; the vicinal dihydroxy groups on OCS can form dynamic borate ester bonds with PBA on HAP. These dual dynamic networks endow the hydrogel with good self-healing properties and mechanical properties, and at the same time enable the system to have pH and glucose dual-responsive capabilities. In the hydrogel system, GPC with photothermal and photodynamic antibacterial properties is loaded. GO is coated with polydopamine to form reduced graphene oxide coated with polydopamine with good photothermal antibacterial properties (rGO@PDA), and C60 with photodynamic antibacterial properties is modified with glycine; according to the amide reaction, glycine-modified C60 is loaded onto rGO@PDA, which can combine rGO@PDA with photothermal effect and C60 with photodynamic antibacterial properties to obtain the final nanocomposite GPC, which endows the hydrogel with good antioxidant properties, electrical conductivity, photothermal and photodynamic antibacterial properties. The present invention introduces PFD into the entire hydrogel. The pH and glucose dual-responsive hydrogel constructed based on Schiff base and borate ester bonds has the ability to regulate the release of PFD on demand. Low concentration of pirfenidone (PFD) can promote the formation and migration of blood vessels, and accelerating angiogenesis is crucial for the healing of DFU wounds because it provides oxygen and nutrients to damaged tissues. In addition, PFD can reduce uncontrolled inflammation and oxidative stress, and promote the healing of DFU wounds.

[0028] The present invention also discloses a photothermal and photodynamic antibacterial hydrogel with pH / glucose dual-responsive drug release. Grafting PBA onto the HA backbone can enhance the self-healing property, swelling property, and mechanical strength of the system. After oxidizing CS, the self-healing property and adhesion property of the system can be increased. When the two macromolecular chains HAP and OCS crosslink, a dual dynamic network of Schiff base and phenylborate ester can be formed, endowing the system with pH and glucose dual-responsive properties. Introducing GPC into the hydrogel system endows the hydrogel with good antioxidant property, photothermal and photodynamic antibacterial property, and electrical conductivity, which can prevent severe infection of wounds, accelerate wound healing, and the released PFD has good anti-inflammatory and angiogenesis-promoting effects to promote the healing of diabetic foot ulcer wounds. This hydrogel dressing has dual responsiveness and can release drugs controllably; it has photothermal and photodynamic antibacterial properties, can inhibit and kill Escherichia coli and Staphylococcus aureus, prevent severe infection of wounds, promote angiogenesis, and promote the healing of diabetic foot ulcer wounds.

[0029] The present invention also discloses the application of a photothermal and photodynamic antibacterial hydrogel with pH / glucose dual-responsive drug release. GPC in this hydrogel has good photothermal and photodynamic antibacterial properties. Whether it is Escherichia coli or Staphylococcus aureus, bacteria can be killed within a short time to prevent severe infection of diabetic foot ulcer wounds. At the same time, the hydrogel is endowed with good antioxidant property and electrical conductivity, which will promote the healing of diabetic foot ulcer wounds. Brief Description of the Drawings

[0030] Figure 1(a) is the swelling curve of HAP / OCS hydrogel; Figure 1(b) is the degradation curve of HAP / OCS hydrogel; Figure 1(c) is the rheological property diagram of HAP / OCS hydrogel; Figure 1(d) is the self-healing curve of HAP / OCS2 (hereinafter referred to as HPC) hydrogel.

[0031] Figure 2(a) is the release rate of metformin from HPC hydrogel under different pH conditions; Figure 2(b) is the conductivity of HPC-based hydrogels with different contents of GPC; Figure 2(c) is the adhesion ability of HPC, HPCG1, HPCG2, and HPCG3 hydrogels; Figure 2(d) is the DPPH scavenging rate of HPC, HPCG1, HPCG2, and HPCG3 hydrogels; Figure 2(e) is the ability of HPC, HPCG1, HPCG2, and HPCG3 hydrogels to scavenge ROS generated by macrophages.

[0032] Figure 3(a) shows the in vitro antibacterial activities of HPC, HPCG1, HPCG2, and HPCG3 hydrogels against Staphylococcus aureus under near-infrared irradiation at different times; Figure 3(b) shows the in vitro antibacterial activities of HPC, HPCG1, HPCG2, and HPCG3 hydrogels against Escherichia coli under near-infrared irradiation at different times.

[0033] Figure 4(a) shows the quantitative data of the hemolysis rates of HPC, HPCG1, HPCG2, and HPCG3 hydrogels; Figure 4(b) shows the quantitative data of the coagulation indices of HPC, HPCG1, HPCG2, and HPCG3 hydrogels; Figure 4(c) shows the evaluation of cell compatibility, *P < 0.05, **P < 0.01.

[0034] Figure 5(a) shows Tegaderm TM Statistical graphs of the wound healing conditions of the film and the HPC, HPCG2, NIR / HPCG2, and NIR / HPCG2 / PFD hydrogel groups at 3 days, 7 days, 14 days, and 21 days respectively, *P < 0.05, **P < 0.01; Figure 5(b) shows the relative data statistics of inflammatory cells on the 7th day; Figure 5(c) shows the epidermal regeneration rate on the 7th day; Figure 5(d) shows the statistical thickness of granulation tissue on the 7th day; Figure 5(e) shows the angiogenesis statistics on the 14th day; Figure 5(f) shows the statistical content of collagen deposition in the new tissue on the 14th day, *P < 0.05, **P < 0.01.

[0035] Figure 6 is a schematic diagram of the structure of the hydrogel. Detailed implementation mode

[0036] The present invention will be further described in detail below with reference to the accompanying drawings:

[0037] See Figure 6 , the present invention discloses a preparation method of a pH / glucose dual-responsive photothermal and photodynamic antibacterial hydrogel, which specifically includes the following steps:

[0038] (1) Under the action of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole (HOBt), adipic dihydrazide (ADH) is used to modify the amino group of hyaluronic acid (HA);

[0039] The specific preparation steps for the amino group modification of HA by ADH include:

[0040] (1A) Dissolve 0.5 g of HA in 100 mL of deionized water, and stir it at room temperature to fully dissolve it to form a hyaluronic acid solution;

[0041] (1B) 6.53 g of ADH was added to the solution obtained in (1A), and the pH of the solution was adjusted to about 6.8 with hydrochloric acid and NaOH to form a mixed solution A;

[0042] (1C) 0.78 g of EDC and 0.77 g of HOBt were dissolved in a 1:1 (5 mL each) mixed solution of DMSO and H2O (solvent 1) to form a mixed solution B;

[0043] (1D) The solution obtained in (1C) was added to the solution in (1B), and the pH of the solution was adjusted to about 6.8 with 1 M hydrochloric acid and maintained for at least 4 hours to obtain a reaction solution C. After reacting overnight, the pH was adjusted to 7 to terminate the reaction;

[0044] (1E) The solution obtained in (1D) was dialyzed in deionized water for 24 hours, and NaCl was added to the dialyzed solution to form a solution containing 5% NaCl to obtain a mixed solution D;

[0045] (1F) The solution obtained in (1E) was precipitated in absolute ethanol, then centrifuged to remove absolute ethanol, and then dissolved in deionized water;

[0046] (1G) The solution obtained in (1F) was dialyzed in deionized water for 48 hours and freeze-dried to obtain the final product, amino-modified hyaluronic acid AHA.

[0047] (2) Graft 3-aminophenylboronic acid (PBA) onto the AHA obtained in step (1) to produce the product HAP with PBA grafted onto AHA;

[0048] The specific preparation steps for synthesizing HAP include:

[0049] (2A) 0.5 g of amino-modified hyaluronic acid AHA was dissolved in 150 mL of 4-morpholineethanesulfonic acid (MES) buffer (100 mM, pH 5.5) to obtain a mixed solution E;

[0050] (2B) 70 mg of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM) was added to the solution in (2A);

[0051] (2C) After 30 min, finally 57 mg of PBA was added to the solution obtained in (2B), and the reaction was carried out for 24 hours under dark conditions;

[0052] (2D) The solution obtained in (2C) was dialyzed in deionized water for 72 hours and then freeze-dried to obtain HAP.

[0053] (3) CS was oxidized to obtain OCS containing aldehyde groups;

[0054] The specific preparation steps for synthesizing OCS are as follows:

[0055] (3A) Dissolve 5 g of chondroitin sulfate (CS) in 100 mL of deionized water to obtain a chondroitin sulfate solution;

[0056] (3B) Dissolve 1.288 g of sodium periodate in deionized water, and gradually add all the obtained sodium periodate solution dropwise to the solution obtained in (3A). Stir for 12 hours under dark conditions to obtain a mixed solution F;

[0057] (3C) Dialyze the solution obtained in (3B) in deionized water for 72 hours, and then freeze-dry to obtain oxidized chondroitin sulfate (OCS).

[0058] (4) Prepare a polydopamine coating on the surface of GO by the self-polymerization of dopamine (DA) under alkaline conditions to generate reduced graphene oxide (rGO@PDA);

[0059] The specific preparation steps for synthesizing rGO@PDA are as follows:

[0060] (4A) Dissolve 20 mg of graphene oxide (GO) and 20 mg of dopamine hydrochloride (DA) in 40 mL of Tris-HCl buffer solution (pH = 8.5) and sonicate for 30 minutes;

[0061] (4B) Vigorously stir the mixture obtained in (4A) at room temperature for 24 hours for an oxidation-reduction reaction, and coat GO with polydopamine;

[0062] (4C) Purify and separate the mixture obtained in (4B) by filtration, and further wash the separated product several times with water and ethanol to obtain an rGO@PDA solution;

[0063] (4D) Dry the rGO@PDA solution obtained in (4C) in a vacuum oven at 60 °C for 48 hours to obtain an rGO@PDA copolymer.

[0064] (5) Modify fullerene (C60) with glycine to improve the water solubility of C60 and generate GC60;

[0065] The specific preparation steps for synthesizing GC60 are as follows:

[0066] (5A) Add 40 mg of fullerene (C60) to 35 mL of toluene solution, and sonicate to completely disperse it to obtain a C60 solution;

[0067] (5B) Take 1.5 g of glycine and 0.85 g of NaOH, and dissolve them in a mixture of 4 ml of deionized water and 20 ml of absolute ethanol (solvent II) to obtain a glycine solution;

[0068] (5C) The solution obtained in (5A) was added dropwise to the solution obtained in (5B) with continuous stirring for 10 days until the purple color of the organic layer completely disappeared and the aqueous solution changed from colorless to brownish-black, obtaining a mixed solution G;

[0069] (5D) The upper organic layer was separated by liquid separation in (5C), and the lower layer was washed twice in toluene solution, and then the solvent was removed by rotary evaporation to obtain a viscous brownish-black substance H;

[0070] (5E) A small amount of distilled water was added to dissolve the viscous substance obtained in (5D), and then it was precipitated with absolute ethanol, and then centrifuged;

[0071] (5F) The precipitate obtained in (5E) was dissolved in 40 mL of water, filtered by suction, and the filtrate was rotary evaporated to obtain a viscous black process substance I;

[0072] (5G) The product obtained in (5F) was acidified with dilute HCl (1 mol / L), and the black precipitate was obtained by centrifugation. After drying, glycine-modified fullerene was obtained.

[0073] (6) According to the amide reaction, using EDC and N-hydroxysuccinimide sodium salt (NHS) as catalysts, glycine-modified fullerene was grafted onto rGO@PDA. The carboxyl group after glycine modification can react with the amino group on rGO@PDA to obtain the product GPC with photothermal and photodynamic antibacterial properties;

[0074] The specific preparation steps for synthesizing GPC are as follows:

[0075] (6A) The product obtained in (5G) was dissolved in water and dispersed evenly to obtain a solution J;

[0076] (6B) 5 mg of EDC and 4 mg of NHS were added to the solution in (6A) to obtain a solution K;

[0077] (6C) 30 μL of rGO@PDA was dispersed in deionized water to prepare a 10 mg / mL rGO@PDA dispersion, which was added to the solution obtained in (6B), and stirred at room temperature for 24 hours;

[0078] (6D) The solution obtained in (6C) was washed three times with deionized water and dried in vacuo to obtain GPC.

[0079] (7) HAP was dissolved in deionized water to prepare a HAP solution with a mass concentration of 2 wt%;

[0080] (8) OCS was dissolved in deionized water to prepare an OCS solution with a mass concentration of 2.5 wt% - 10 wt%;

[0081] (9) Dissolve GPC in deionized water to prepare a GPC solution with a mass concentration of 1 wt% - 4 wt%, and dissolve pirfenidone in deionized water to prepare a pirfenidone solution with a mass concentration of 0.08 wt%.

[0082] (10) Mix 375 mL of HAP solution, 150 mL of OCS solution, 100 mL of GPC solution, and 375 mL of pirfenidone solution, and crosslink them through Schiff base and phenylborate ester to obtain a photothermal and photodynamic hydrogel that helps diabetic foot ulcer wounds heal and has pH / glucose dual responsiveness.

[0083] The finally formed hydrogel is crosslinked by two macromolecular chains, one of which is OCS and the other is HAP, which is loaded with GPC with photothermal and photodynamic antibacterial properties and PFD that can promote wound healing after release.

[0084] The photothermal and photodynamic hydrogel with pH / glucose dual-responsive drug release prepared by the present invention can be applied in diabetic wound healing, such as in wound dressings.

[0085] Example 1

[0086] (1) Under the action of EDC and HOBt, modify the amino group of hyaluronic acid with adipic dihydrazide (ADH).

[0087] The specific preparation steps for the amino group modification of HA with ADH include:

[0088] (1A) Dissolve 0.5 g of HA in 100 mL of deionized water and stir it at room temperature to dissolve it completely.

[0089] (1B) Add 6.53 g of ADH to the solution obtained in (1A), and adjust the pH of the solution to about 6.8 with hydrochloric acid and NaOH.

[0090] (1C) Dissolve 0.78 g of EDC and 0.77 g of HOBt in a 1:1 (5 mL each) mixed solution of DMSO and H2O.

[0091] (1D) Add the solution obtained in (1C) to the solution in (1B), and use 1 M hydrochloric acid to adjust the pH value of the solution to maintain it at about 6.8 for at least 4 hours. After reacting overnight, adjust the pH value to 7 to terminate the reaction.

[0092] (1E) Dialyze the solution obtained in (1D) in deionized water for 24 hours, and add NaCl to the dialyzed solution to form a hyaluronic acid solution containing 5% NaCl.

[0093] (1F) The solution obtained in (1E) was precipitated in absolute ethanol, and then centrifuged to remove the absolute ethanol, and then dissolved in deionized water;

[0094] (1G) The solution obtained in (1F) was dialyzed in deionized water for 48 hours and freeze-dried to obtain the final product, amino-modified hyaluronic acid AHA.

[0095] (2) Graft PBA onto the AHA obtained in step (1) to generate the product HAP with PBA grafted onto AHA;

[0096] The specific preparation steps for synthesizing HAP include:

[0097] (2A) Dissolve 0.5 g of HAP in 150 mL of MES buffer (100 mM, pH 5.5);

[0098] (2B) Add 70 mg of DMTMM to the solution in (2A);

[0099] (2C) After 30 min, finally add 57 mg of PBA to the solution obtained in (2B) and react for 24 hours under dark conditions;

[0100] (2D) The solution obtained in (2C) was dialyzed in deionized water for 72 hours and then freeze-dried to obtain HAP.

[0101] (3) Oxidize CS to obtain OCS containing aldehyde groups;

[0102] The specific preparation steps for synthesizing OCS include:

[0103] (3A) Dissolve 5 g of CS in 100 mL of deionized water;

[0104] (3B) Dissolve 1.288 g of sodium periodate in deionized water and add it dropwise to the solution obtained in (3A), and stir for 12 hours under dark conditions;

[0105] (3C) The solution obtained in (3B) was dialyzed in deionized water for 72 hours and then freeze-dried to obtain OCS;

[0106] (4) Dissolve HAP in deionized water to prepare a HAP solution with a mass concentration of 2 wt%;

[0107] (5) Dissolve OCS in deionized water to prepare an OCS solution with a mass concentration of 2.5 wt%;

[0108] (6) Mix 375 mL of the HAP solution, 150 mL of the OCS solution and 475 mL of deionized water and mix well to obtain a hydrogel with pH / glucose dual responsiveness, which is named HAP / OCS1.

[0109] Example 2

[0110] Different from Example 1, the 2.5 wt% OCS solution in step (5) was replaced with 5 wt%, and the prepared hydrogel was named HAP / OCS2.

[0111] Example 3

[0112] Different from Example 1, the 2.5 wt% OCS solution in step (5) was replaced with 7.5 wt%, and the prepared hydrogel was named HAP / OCS3.

[0113] Example 4

[0114] Different from Example 1, the 2.5 wt% OCS solution in step (5) was replaced with 10 wt%, and the prepared hydrogel was named HAP / OCS4.

[0115] Example 5

[0116] (1) Under the action of EDC and HOBt, hyaluronic acid was amino-modified with adipic dihydrazide (ADH).

[0117] The specific preparation steps for the amino-modification of hyaluronic acid with ADH include:

[0118] (1A) Dissolve 0.5 g of HA in 100 mL of deionized water and stir it well at room temperature for complete dissolution.

[0119] (1B) Add 6.53 g of ADH to the solution obtained in (1A), and adjust the pH of the solution to about 6.8 with hydrochloric acid and NaOH.

[0120] (1C) Dissolve 0.78 g of EDC and 0.77 g of HOBt in a 1:1 (5 mL each) mixed solution of DMSO and H2O.

[0121] (1D) Add the solution obtained in (1C) to the solution in (1B), and adjust the pH value of the solution to be maintained at about 6.8 with 1 M hydrochloric acid for at least 4 hours. After reacting overnight, adjust the pH value to 7 to terminate the reaction.

[0122] (1E) Dialyze the solution obtained in (1D) in deionized water for 24 hours, and add NaCl to the dialyzed solution to form an HA solution containing 5% NaCl.

[0123] (1F) Precipitate the solution obtained in (1E) in absolute ethanol, then centrifuge to remove the absolute ethanol, and then dissolve it in deionized water.

[0124] (1G) Dialyze the solution obtained in (1F) in deionized water for 48 hours and then perform lyophilization to obtain the final product, amino-modified hyaluronic acid AHA.

[0125] (2) Graft PBA onto the AHA obtained in step (1) to generate the product HAP with PBA grafted onto AHA;

[0126] The specific preparation steps for synthesizing HAP include:

[0127] (2A) Dissolve 0.5 g of HAP in 150 mL of MES buffer (100 mM, pH 5.5);

[0128] (2B) Add 70 mg of DMTMM to the solution in (2A);

[0129] (2C) After 30 min, finally add 57 mg of PBA to the solution obtained in (2B) and react for 24 hours under dark conditions;

[0130] (2D) Dialyze the solution obtained in (2C) in deionized water for 72 hours and then perform lyophilization to obtain HAP.

[0131] (3) Oxidize CS to obtain OCS containing aldehyde groups;

[0132] The specific preparation steps for synthesizing OCS include:

[0133] (3A) Dissolve 5 g of CS in 100 mL of deionized water;

[0134] (3B) Dissolve 1.288 g of sodium periodate in deionized water and gradually add it dropwise to the solution obtained in (3A), and stir for 12 hours under dark conditions;

[0135] (3C) Dialyze the solution obtained in (3B) in deionized water for 72 hours and then perform lyophilization to obtain OCS.

[0136] (4) Prepare a polydopamine coating on the surface of graphene oxide (GO) by the self-polymerization of DA under alkaline conditions to generate reduced graphene oxide (rGO@PDA);

[0137] The specific preparation steps for synthesizing rGO@PDA are as follows:

[0138] (4A) Dissolve GO (20 mg) and dopamine hydrochloride (20 mg) in 40 mL of Tris-HCl buffer (pH = 8.5) and sonicate for 30 minutes;

[0139] (4B) Vigorously stir the mixture obtained in (4A) at room temperature for 24 hours;

[0140] (4C) The mixture obtained in (4B) was purified and separated by filtration, and further washed several times with water and ethanol to obtain an rGO@PDA solution;

[0141] (4D) The rGO@PDA solution obtained in (4C) was dried in a vacuum oven at 60 °C for 48 hours to obtain an rGO@PDA copolymer.

[0142] (5) Modify fullerene (C60) with glycine to improve the water solubility of C60 and generate GC60;

[0143] The specific preparation steps for synthesizing GC60 are as follows:

[0144] (5A) Add 40 mg of C60 to 35 mL of toluene solution and sonicate to completely disperse it;

[0145] (5B) Take 1.5 g of glycine and 0.85 g of NaOH and dissolve them in a mixture of 4 mL of deionized water and 20 mL of absolute ethanol;

[0146] (5C) Dropwise add the solution obtained in (5A) to the solution obtained in (5B) and continuously stir for 10 days until the purple color of the organic layer completely disappears and the aqueous solution changes from colorless to brownish-black;

[0147] (5D) Separate the upper organic layer by liquid separation, wash it twice with toluene, and then remove the solvent by rotary evaporation to obtain a viscous brownish-black substance;

[0148] (5E) Add a small amount of distilled water to dissolve the viscous substance obtained in (5D), then precipitate it with absolute ethanol, and then centrifuge and separate;

[0149] (5F) Dissolve the precipitate obtained in (5E) in 40 mL of water, filter by suction, and rotary evaporate the filtrate to obtain a viscous black substance;

[0150] (5G) Acidify the product obtained in (5F) with dilute HCl (1 mol / L), centrifuge and separate to obtain a black precipitate, and dry it to obtain glycine-modified fullerene.

[0151] (6) According to the amide reaction, use EDC and NHS as catalysts to graft glycine-modified fullerene onto rGO@PDA to obtain a product GPC with photothermal and photodynamic antibacterial properties;

[0152] The specific preparation steps for synthesizing GPC are as follows:

[0153] (6A) Dissolve the product obtained in (5G) in water;

[0154] (6B) Add 5 mg of EDC and 4 mg of NHS to the solution in (6A);

[0155] (6C) Add 30 μL of rGO@PDA dispersion (10 mg / mL) to the solution obtained in (6B), and stir at room temperature for 24 hours;

[0156] (6D) Wash the solution obtained in (6C) three times with deionized water, and dry it under vacuum to obtain GPC.

[0157] (7) Dissolve HAP in deionized water to prepare a HAP solution with a mass concentration of 2 wt%;

[0158] (8) Dissolve OCS in deionized water to prepare an OCS solution with a mass concentration of 5 wt%;

[0159] (9) Dissolve GPC in deionized water to prepare a GPC solution with a mass concentration of 1 wt%;

[0160] (10) Mix 375 mL of HAP solution, 150 mL of OCS solution, 100 mL of GPC solution, and 375 mL of deionized water to obtain a pH / glucose dual-responsive photothermal and photodynamic antibacterial hydrogel, named HPCG1.

[0161] Example 6

[0162] Different from Example 5, replace the 1 wt% GPC solution in step (9) with 2 wt%, and the prepared hydrogel is named HPCG2.

[0163] Example 7

[0164] Different from Example 5, replace the 1 wt% GPC solution in step (9) with 4 wt%, and the prepared hydrogel is named HPCG3.

[0165] Example 8

[0166] Different from Example 6, dissolve PFD in deionized water to prepare a PFD solution with a mass concentration of 0.08 wt%, and replace 375 mL of deionized water in step (10) with 375 mL of pirfenidone solution, and the prepared hydrogel is named HPCG2 / PFD.

[0167] Example 9

[0168] Different from Example 8, in this example, the concentration of the OCS solution is 2.5 wt%.

[0169] Example 10

[0170] Different from Example 8, in this example, the concentration of the OCS solution is 7 wt%.

[0171] Example 11

[0172] Example 8 is different in that the concentration of the OCS solution in this example is 10 wt%.

[0173] Example 12

[0174] Different from Example 8, in this example, the concentration of the GPC solution is 1 wt%.

[0175] Example 13

[0176] Different from Example 8, in this example, the concentration of the GPC solution is 4 wt%.

[0177] The hydrogel adjuvant with pH and glucose dual-responsive photothermal and photodynamic antibacterial properties, having good self-healing performance, strong adhesion performance, electrical conductivity, and antioxidant performance in the present invention, is helpful for the repair of diabetic foot ulcer wounds. The hydrogel prepared by this method has stable performance, can absorb wound exudate, protect the wound microenvironment, has good biocompatibility with mouse fibroblasts (L929), its photothermal and photodynamic antibacterial performance is good in in vitro antibacterial tests, controls and prevents severe wound infections, and both the blank gel and the hydrogel loaded with GPC and pirfenidone have a better wound healing effect on the foot ulcers of diabetic rats than the commercial dressing Tegaderm^TM. The following is a detailed analysis in combination with the attached drawings and experimental data:

[0178] In the experimental part of the attached drawings, the HAP concentration is 2 wt%, the OCS concentrations are 2.5 wt%, 5 wt%, 7.5 wt%, 10 wt%, the GPC concentrations are 1 wt%, 2 wt%, 4 wt%, and the PFD concentration is 0.08 wt%. Without adding GPC and PFD, according to the OCS concentrations of 2.5 wt%, 5 wt%, 7.5 wt%, 10 wt% in the hydrogel, the hydrogels are named HAP / OCS1, HAP / OCS2, HAP / OCS3, HAP / OCS4 in sequence; using an OCS concentration of 5 wt% and a constant HAP concentration, only adding GPC, according to the GPC concentrations of 1 wt%, 2 wt%, 4 wt% in the hydrogel, the hydrogels are named HPCG1, HPCG2, HPCG3 in sequence; using an OCS concentration of 5 wt%, a constant HAP concentration, a GPC concentration of 2 wt%, and a PFD concentration of 0.08 wt%, the hydrogel HPCG2 / PFD is generated.

[0179] Comparing the hydrogel swelling behavior test results of Example 1, Example 2, Example 3, and Example 4 in Figure 1(a), it shows that in the HAP / OCS hydrogel, as the OCS content in the hydrogel increases, the swelling rate of the hydrogel gradually decreases. However, the swelling rate of the HAP / OCS1 hydrogel is the lowest, which may be due to the low cross-linking density and the inability to support the full swelling of the hydrogel.

[0180] The test results of the hydrogel degradation behavior in Figure 1(b) show that all the hydrogels prepared by this method have good degradation performance. After the hydrogels reach swelling equilibrium, as the concentration of OCS increases, the degradation rate of the hydrogels decreases. The HAP / OCS1 hydrogel degrades the fastest, which may be due to its low cross-linking density and the easy destruction of the cross-linking network. After 48 h, all the hydrogels are almost completely degraded, and the residual mass is less than 8%.

[0181] Figure 1(c) shows the storage modulus (G’) and loss modulus (G”) obtained from the rheological test of the hydrogels. As the concentration of OCS increases, the G′ of the HAP / OCS1, HAP / OCS2, HAP / OCS3, and HAP / OCS4 hydrogels increases from 21.3, 52.4, 83.3 Pa to 128.8 Pa, which is due to the increase in the cross-linking density of the hydrogel Schiff base and phenylborate ester.

[0182] Figure 1(d) further tests the self-healing behavior of the HAP / OCS2 hydrogel through continuous alternating strain. After high strain (600%), the G' of the hydrogel significantly decreases from 52.6 Pa to 11.0 Pa, and G">G', indicating that the hydrogel network is damaged. Under low strain (1%) conditions, the hydrogel returns to the state of G'>G", indicating that the dynamic Schiff base and phenylborate ester cross-linking network is restored. After 3 cycles of high strain and low strain, the G' and G" values of the hydrogel are almost the same. Through the above experiments, the hydrogel has good self-healing ability.

[0183] Figure 2(a) shows the release curve of PFD by the HAP / OCS2 hydrogel in the hydrogel dressing prepared by the present invention under different pH conditions. As the pH decreases, the release amount of PFD by the hydrogel increases significantly. At pH 5.5, the release amount of PFD is 92±3.3%, higher than 90±1.2% at pH 6.8 and higher than 85.6±2.9% at pH 7.4 (physiological environment). This is mainly because the Schiff base structure is more easily dissociated under acidic conditions, making the release of PFD faster, which is applicable to the acidic environment of chronic inflammation of DFU.

[0184] Figure 2(b) shows the test results of the conductivity of the hydrogel prepared by the present invention. The conductivity increases with the increase in the content of GPC.

[0185] Figure 2(c) shows the test results of the adhesion test of the hydrogel dressing prepared by the present invention. The adhesion performance of these hydrogels to the skin was estimated through experiments. The adhesion strengths of the HPC, HPCG1, HPCG2, and HPCG3 hydrogels are 7.3, 7.4, 5.6, and 4.0 kPa respectively. However, as the content of GPC increases, the adhesion force of the hydrogel decreases, which may be due to excessive bonding. Good adhesion performance ensures that the hydrogel is not easily detached from the moving wound surface and protects the wound surface environment from being damaged by the external environment.

[0186] Figure 2(d) shows the results of the DPPH free radical scavenging experiment of the hydrogel dressing prepared by the present invention. In HPC, when GPC was not added, the free radical scavenging rate of DPPH was very low. After adding GPC with antioxidant effect, with the increase of GPC concentration, the free radical scavenging rate of DPPH also increased. The free radical scavenging rates of HPCG1, HPCG2 and HPCG3 hydrogels on DPPH were 72.6%, 76.9% and 82.5% respectively.

[0187] Figure 2(e) shows the ability of the hydrogel dressing prepared by the present invention to scavenge ROS produced by macrophages. The LPS group was used as the negative control, and the group without LPS was used as the positive control. The fluorescence intensity of the HPCG hydrogel group was quantified by setting the fluorescence intensity of the LPS group to 100%. The fluorescence intensity of the LPS group (Figures J, K) was 24.4 times that of the positive control group, and the fluorescence intensity of the HPC group was 19.2 times that of the positive control group, which was due to the antioxidant effect of the catechol groups in the hydrogel. After adding GPC with better antioxidant performance, the fluorescence intensity decreased significantly (**P < 0.01), about 2.5 times that of the positive control group, which was consistent with the DPPH free radical scavenging results. It demonstrated the excellent antioxidant activity of the HPCG hydrogel.

[0188] Figure 3(a) shows the antibacterial effect test of the hydrogel dressing prepared by the present invention against Staphylococcus aureus and Escherichia coli in vitro under near-infrared irradiation. The results showed that with the increase of GPC concentration in the hydrogel and the near-infrared irradiation time of the hydrogel, the hydrogel group showed strong antibacterial ability against both Escherichia coli and Staphylococcus aureus. Within 5 minutes, the HPCG hydrogel group could kill 90% of Escherichia coli and Staphylococcus aureus. Within 10 minutes, all Escherichia coli and Staphylococcus aureus could be killed. Among them, the HPCG3 hydrogel could kill more than 95% of Escherichia coli and Staphylococcus aureus within 3 minutes and almost all bacteria within 5 minutes. These experimental results indicate that the hydrogel prepared by the high method has good photothermal and photodynamic antibacterial ability, can prevent and control the infection of diabetic foot ulcer wounds, and promote wound healing.

[0189] Figure 4(a) shows the results of the in vitro hemolysis experiment of the hydrogel dressing prepared by the present invention. The hemolysis rate of the positive group (0.1% triton) was set to 100%, and the hemolysis rates of all hydrogel groups were less than 5%, showing good blood compatibility.

[0190] Figure 4(b) shows the results of the in vitro coagulation experiment of the hydrogel dressing prepared by the present invention. The coagulation performance of the HPCG hydrogel was determined by the coagulation index. The larger the BCI value, the poorer the coagulation performance of the hydrogel. Using the gelatin sponge group and the PBS group as the control groups, within 3 minutes, it was observed that the amount of uncoagulated blood and the macroscopic blood cell suspension in the hydrogel group were lower than those in the blank control group, and the effect was better than that of the commercial gelatin sponge group. The HPCG hydrogel has certain coagulation potential.

[0191] Figure 4(c) shows the results of the cell compatibility of the hydrogel dressing prepared by the present invention in vitro. L929 (mouse) fibroblasts were used to evaluate the cell compatibility of the hydrogel. After the hydrogel was in direct contact with the cells, as the GPC content increased, the cell viability gradually decreased. The cell survival rates of the HPCG1, HPCG2, and HPCG3 hydrogels were 98.9±2.1%, 88.3±1.0%, and 82.9±3.0% respectively. It proved the good cell compatibility of the hydrogel.

[0192] Figure 5(a) shows the statistical results of the wound closure rate of the hydrogel dressing prepared by the present invention in the diabetic wound repair experiment. After 3 days of treatment, compared with the Tegaderm TM film group, the wound closure degrees of the HPC, HPCG2, NIR / HPCG2, and NIR / HPCG2 / PFD hydrogels were significantly higher (*P < 0.05). And on the 21st day of wound repair, the NIR / HPCG2 / PFD group had the best healing effect, almost no wound marks, and even hair grew on the skin. Compared with the Tegaderm TM film, it showed that the NIR / HPCG2 / PFD hydrogel had a better wound healing effect; compared among the hydrogel groups, it showed that the photothermal and photodynamic antibacterial ability of GPC and PFD in the NIR / HPCG2 / PFD group both had a positive promoting effect on the healing of diabetic foot ulcer wounds.

[0193] Figure 5(b) shows the statistical results of the relative number of inflammatory cells of the hydrogel dressing prepared by the present invention in the diabetic foot ulcer repair experiment. On the 7th day of wound repair, the number of inflammatory cells in the Tegaderm TM film, HPC, HPCG2, NIR / HPCG2, and NIR / HPCG2 / PFDt groups decreased in turn. The results showed that the hydrogel prepared by the present invention had a good effect of reducing the inflammatory response in the diabetic foot ulcer repair experiment (*P < 0.5).

[0194] Figure 5(c) shows the statistics of the epidermal regeneration rate of the hydrogel dressing prepared by the present invention in the diabetic foot ulcer wound repair experiment. On the 7th day of wound repair, compared with the Tegaderm TMCompared with the film group, the epidermal regeneration rates of HPC, HPCG2, NIR / HPCG2, and NIR / HPCG2 / PFD hydrogels gradually increased. The epidermal regeneration rate of the NIR / HPCG2 / PFD hydrogel reached 64%, which was better than that of the NIR / HPCG2 hydrogel group (61%). There were extremely significant differences in the epidermal regeneration rates between the NIR / HPCG2 / PFD hydrogel group and the HPC and HPCG2 hydrogel groups (49% and 50.9%, respectively) (*P<0.05), and an extremely significant difference compared with the control group (38.1%) (**P<0.01). This showed that the hydrogel prepared by the present invention had a good promoting effect on epidermal regeneration in the wound healing of diabetic foot ulcers.

[0195] Figure 5(d) shows the statistical results of the granulation tissue thickness in the diabetic foot ulcer repair experiment of the hydrogel dressing prepared by the present invention. The thickness of the granulation tissue can reflect the quality of wound repair. Compared with the Tegaderm TM film group, the granulation tissue thickness of the four hydrogel groups gradually increased (*P<0.5); compared among the hydrogel groups, the NIR / HPCG2 / PFD group had the thickest granulation tissue. This showed that the hydrogel prepared by the present invention had a good promoting effect on the formation of granulation tissue in the diabetic foot ulcer repair experiment.

[0196] Figure 5(e) shows the statistics of angiogenesis in the diabetic foot ulcer repair experiment of the hydrogel dressing prepared by the present invention. On the 14th day of wound repair, compared with the Tegaderm^TM film group, the angiogenesis rates of HPC, HPCG2, NIR / HPCG2, and NIR / HPCG2 / PFD hydrogels increased in sequence. This showed that the hydrogel prepared by the present invention had an excellent effect on improving the angiogenesis rate in the diabetic foot ulcer wound repair experiment (*P<0.5).

[0197] Figure 5(f) shows the statistics of collagen deposition in the diabetic foot ulcer repair experiment of the hydrogel dressing prepared by the present invention. On the 14th day of wound repair, the collagen deposition levels of the Tegaderm^TM film, HPC, HPCG2, NIR / HPCG2, and NIR / HPCG2 / PFD groups increased in sequence. The results showed that the hydrogel prepared by the present invention had a significant effect on promoting collagen metabolism in the diabetic foot ulcer repair experiment (*P<0.5).

[0198] The present invention discloses a preparation method of a pH / glucose dual-responsive photothermal and photodynamic hydrogel and its application in diabetic foot ulcer wound dressings.

[0199] The experimental results show that the rheology, swelling ratio, adhesiveness, etc. of the hydrogel prepared by the present invention can be adjusted by changing the content of OCS in the hydrogel. The experimental results verify that the double-dynamic bond crosslinking endows the hydrogel with pH and glucose dual-responsive release and good self-healing performance, oxidized chondroitin sulfate endows the hydrogel with tissue adhesiveness, and GPC endows the hydrogel with good electrical conductivity, photothermal-photodynamic antibacterial performance and antioxidant performance. In addition, the hemolysis experiment and the co-culture experiment of L929 cells verify its good in vitro biocompatibility. Histological results: Collagen metabolism, granulation tissue thickness, epidermal regeneration, inflammatory reaction, angiogenesis, and the results of immunofluorescence staining of IL-6, CD31, and α-SMA confirm the good effect of the hydrogel in promoting the healing of diabetic foot ulcer wounds. Therefore, this multifunctional hydrogel system has good application prospects in the field of promoting the healing of diabetic foot ulcer wounds.

[0200] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of a photothermal and photodynamic antibacterial hydrogel with pH / glucose dual-responsive drug release, characterized in that, It includes the following steps: Step 1: Under the action of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole, adipic dihydrazide is used to carry out amino modification on hyaluronic acid to obtain amino-modified hyaluronic acid AHA; Step 2: Graft PBA onto amino-modified hyaluronic acid AHA to obtain HAP; Step 3: After oxidizing chondroitin sulfate, chondroitin sulfate containing aldehyde groups is obtained, which is OCS; Step 4: Under alkaline conditions, a polydopamine coating is polymerized on the surface of graphene oxide to generate reduced graphene oxide rGO@PDA; Step 5: Mix the glycine solution and the fullerene solution, and after reaction treatment, glycine-modified fullerene is obtained; Step 6: Using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and NHS as catalysts, graft glycine-modified fullerene onto reduced graphene oxide rGO@PDA to generate GPC; Step 7: Dissolve HAP, OCS, GPC, and pirfenidone in water respectively to form HAP solution, OCS solution, GPC solution, and pirfenidone solution. After mixing the four solutions evenly according to the volume ratio of 375:150:100:375, a photothermal and photodynamic antibacterial hydrogel with pH / glucose dual-responsive drug release is obtained.

2. The preparation method of a photothermal and photodynamic antibacterial hydrogel with pH / glucose dual-responsive drug release according to claim 1, characterized in that, The specific process of Step 1 is as follows: Add adipic dihydrazide to the hyaluronic acid solution, adjust the pH value with hydrochloric acid and NaOH to form a mixed solution A. Dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole in Solvent 1 to form a mixed solution B. Add the mixed solution B to the mixed solution A, adjust the pH value, and obtain a reaction solution C. After terminating the reaction, dialyze, precipitate, and freeze-dry the resulting solution to obtain amino-modified hyaluronic acid AHA.

3. The preparation method of a photothermal and photodynamic antibacterial hydrogel with pH / glucose dual-responsive drug release according to claim 1, characterized in that, The specific process of Step 2 is as follows: Dissolve amino-modified hyaluronic acid AHA in 4-morpholineethanesulfonic acid buffer solution to obtain a mixed solution E. Add 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride to the mixed solution E, then add PBA, and after reaction, dialyze and freeze-dry to obtain HAP.

4. The preparation method of a photothermal and photodynamic antibacterial hydrogel with pH / glucose dual-responsive drug release according to claim 1, characterized in that, The specific process of Step 3 is as follows: Drop sodium periodate solution into the chondroitin sulfate solution, stir to obtain a mixed solution F, and after dialyzing the mixed solution F, obtain oxidized chondroitin sulfate.

5. The preparation method of a photothermal and photodynamic antibacterial hydrogel with pH / glucose dual-responsive drug release according to claim 1, characterized in that, The specific process of Step 4 is as follows: Dissolve graphene oxide and dopamine hydrochloride in Tris-HCl buffer solution, stir, filter, purify, and separate. After washing the separated product with water and ethanol, obtain an rGO@PDA solution. After drying the rGO@PDA solution, obtain an rGO@PDA copolymer.

6. The preparation method of a photothermal and photodynamic antibacterial hydrogel with pH / glucose dual-responsive drug release according to claim 1, characterized in that, The specific process of Step 5 is as follows: Add fullerene to toluene to obtain a fullerene solution. Add glycine and NaOH to Solvent 2 to obtain a glycine solution; Add the fullerene solution to the glycine solution and stir until the mixed solution turns brownish black to obtain the mixed solution G. After removing the upper organic layer from the mixed solution G, wash and rotary evaporate the lower layer substance to obtain the process substance H. Dissolve the process substance H in water, precipitate after washing with absolute ethanol, and after centrifugal separation, dissolve the centrifuged product and perform suction filtration, and then rotary evaporate to obtain the process substance I. After acidifying the process substance I, perform centrifugal separation to obtain a black precipitate, and after drying the black precipitate, obtain glycine-modified fullerene.

7. The preparation method of a photothermal and photodynamic antibacterial hydrogel with pH / glucose dual-responsive drug release according to claim 1, characterized in that, The specific process of step 6 is as follows: dissolve the glycine-modified fullerene in water to obtain solution J, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and NHS to solution J to obtain solution K, disperse the reduced graphene oxide rGO@PDA dispersion in solution K, stir and wash, and after vacuum drying, obtain GPC.

8. The preparation method of a photothermal and photodynamic antibacterial hydrogel with pH / glucose dual-responsive drug release according to claim 1, characterized in that, In step 7, the concentration of the HAP solution is 2 wt%, the concentration of the OCS solution is 2.5 wt%, the concentration of the GPC solution is 1 wt% - 4 wt%, and the concentration of the pirfenidone solution is 0.08 wt%.

9. A photothermal and photodynamic antibacterial hydrogel with pH / glucose dual-responsive drug release prepared by the preparation method according to any one of claims 1-8, characterized in that, It includes HAP macromolecular chains and OCS macromolecular chains, and the two macromolecular chains are crosslinked and combined with each other; GPC and pirfenidone are embedded in the hydrogel.

10. An application of the photothermal and photodynamic antibacterial hydrogel with pH / glucose dual-responsive drug release according to claim 9, characterized in that, As a wound dressing for diabetic wound healing.

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