Hyaluronic acid-based hydrogel as well as preparation method and application thereof

Abstract

The invention provides hyaluronic acid-based hydrogel as well as a preparation method and application thereof, and belongs to the technical field of chemical synthesis and biomedicine. The hyaluronic acid-based hydrogel is obtained by crosslinking a hydrogel main body structure as shown in a formula 1 and genipin. The invention also provides an application of the hyaluronic acid-based hydrogel in preparation of a medicine for treating wound healing caused by bacterial infection. The hyaluronic acid-based hydrogel provided by the invention has excellent bioluminescence performance, and can detect the intensity of inflammation at a wound part. The prepared hydrogel material has photo-thermal, photodynamic, Fenton and carbon monoxide gas release combined anti-inflammatory and antibacterial and wound healing effects.

Description

Technical Field

[0001] The present invention belongs to the field of chemical synthesis and biomedical technology, and particularly relates to a hyaluronic acid-based hydrogel and a preparation method and application thereof. Background Art

[0002] In recent years, wound healing has become a complex biological process by which the body responds to tissue injury, involving the coordinated actions of multiple stages, including inflammation regulation, cell migration, angiogenesis, and matrix remodeling. However, chronic wounds (such as diabetic ulcers and pressure sores) and infected wounds often experience delayed healing and even systemic complications due to microenvironmental imbalances (such as persistent inflammation, hypoxia, and bacterial colonization). While traditional dressings (such as gauze and hydrocolloids) provide physical protection, they struggle to dynamically respond to wound changes or actively regulate the repair process. There is an urgent need to develop novel materials that combine bioactivity with intelligent adaptability. Hyaluronic acid, a glycosaminoglycan composed of disaccharide units of D-glucuronic acid and N-acetylglucosamine, is widely present in various tissues of the human body. Influenced by various factors and hormones, it can lead to a wide range of diseases in the human body. As a core component of the extracellular matrix (ECM), hyaluronic acid has become a star molecule in the field of wound repair due to its excellent antimicrobial and anti-inflammatory properties, biocompatibility, and specific binding to the CD44 receptor on the cell surface. Furthermore, the porphyrin metal ring formed by the addition of transition metals not only enhances photothermal performance but also utilizes the transition metal's valence-shifting mechanism to further deplete hydrogen peroxide and reactive oxygen species. Hydrogels, a type of gel with a network-like cross-linked structure, exhibit excellent biocompatibility and biodegradability, making them commonly used in the treatment of various diseases. Therefore, this study designed a hyaluronic acid-based hydrogel material for the treatment of bacterially infected wounds.

[0003] At the same time, photodynamic therapy (PDT), photothermal therapy (PTT) and chemodynamic therapy (CDT) are emerging external stimulation methods with broad application prospects in the field of wound healing. Compared with traditional methods, these methods can effectively remove harmful reactive species (ROS) through light initiation and chemical reactions, thereby achieving antibacterial and anti-inflammatory effects at the wound site. Previous studies have confirmed that carbonyl manganese (Mn2[CO] 10) can react with hydrogen peroxide to generate manganese ions and carbon monoxide (CO), and the concentration of CO acts as a gaseous signal messenger to regulate various physiological and pathological processes of the body, such as the anti-inflammatory response mediated by the HO-1 pathway, thereby having a therapeutic effect on a variety of diseases. In 2020, Professor Zhao Chunshun's team at Sun Yat-sen University developed an injectable and near-infrared (NIR) light-triggered ROS-degradable hyaluronic acid hydrogel platform as a local delivery vehicle for the photosensitizer prophorphyrin IX (PpIX) and the anticancer drug doxorubicin (DOX) to achieve excellent chemotherapy-photodynamic combination therapy and light-adjustable on-demand drug release. Therefore, hyaluronic acid-based hydrogels are expected to become the next generation of "all-in-one" wound dressings, pushing regenerative medicine towards precision treatment. Summary of the Invention

[0004] The present invention provides a hyaluronic acid-based hydrogel and a preparation method and application thereof. The hyaluronic acid-based hydrogel of the present invention first introduces a porphyrin derivative into Fe metal, then connects amino-modified hyaluronic acid to the porphyrin derivative, and finally forms a dynamic Schiff base bond cross-link with genipin, so that the hydrogel can simultaneously achieve the synergistic treatment effects of photothermal therapy, photodynamic therapy and chemodynamic therapy, thereby achieving the effects of antibacterial and anti-inflammatory and promoting wound healing.

[0005] The present invention first provides a hyaluronic acid-based hydrogel, which is obtained by cross-linking the hydrogel main structure shown in Formula 1 and genipin.

[0006]

[0007] The present invention also provides a method for preparing a hyaluronic acid-based hydrogel, comprising the following steps:

[0008] Step 1: Dissolve FeCl3·6H2O and tetracarboxyphenylporphyrin in a solvent, and heat under reflux to obtain a porphyrin derivative TCPP-Fe;

[0009] Step 2: Dissolve HA in water, add adipic acid dihydrazide (ADH) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), stir, dialyze, and freeze-dry to obtain amino-modified hyaluronic acid HA-ADH;

[0010] Step 3: The TCPP-Fe obtained in step 1 is mixed with EDC and NHS to obtain a TCPP-Fe solution, and the HA-ADH obtained in step 2 is mixed with the TCPP-Fe solution, stirred, dialyzed, and freeze-dried to obtain the hydrogel main body HA-ADH-TCPP (Fe) shown in Formula 1;

[0011] Step 4: Mix the hydrogel body shown in Formula 1 and genipin, add them into water, add carbonyl manganese powder, stir, and place in a water bath to obtain a hyaluronic acid-based hydrogel.

[0012] Preferably, the reaction temperature of step 1 is 120-130° C., and the reaction time is 6-8 h.

[0013] Preferably, the molar ratio of the tetracarboxyphenylporphyrin in step 1 to FeCl3·6H2O is 1:(1-1.5).

[0014] Preferably, the mass ratio of HA to adipic acid dihydrazide in step 2 is 1:3, and the mass ratio of HA to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:1.

[0015] Preferably, the stirring time in step 3 is 40-48 hours, and the dialysis time is 40-48 hours.

[0016] Preferably, the mass ratio of HA-ADH to TCPP-Fe in step three is 20:1.

[0017] Preferably, in the step 4, the mass ratio of the hydrogel body represented by formula 1, the carbonyl manganese powder and genipin is 30:1:1.

[0018] Preferably, the water bath temperature in step 4 is 60° C. and the placement time is 40-60 min.

[0019] The present invention also provides the use of the hyaluronic acid-based hydrogel in preparing a drug for treating bacterial infection wound healing.

[0020] Beneficial effects of the present invention

[0021] The present invention provides a hyaluronic acid-based hydrogel and its preparation method and application. The present invention designs and synthesizes a self-healing injectable biosafety hydrogel in a simple and convenient way. First, hyaluronic acid can use its anti-inflammatory properties to activate M1 macrophages to transform into an anti-inflammatory phenotype (M2 type) through a CD44 receptor-mediated signaling pathway, inhibit chronic inflammation, and thus achieve anti-inflammatory and antibacterial effects. In addition, a porphyrin-based photosensitizer is grafted thereon to enable it to have excellent reactive oxygen species production capacity under 635nm laser irradiation, so that the material has the dual therapeutic effects of dual-mode PTT / PDT, and due to the presence of variable valence metal Fe ions, it can react with H2O2 and produce reactive oxygen species by chemical kinetics, thereby effectively improving its antioxidant capacity. At the same time, a CO release precursor is also introduced to achieve multimodal combined antibacterial and promote wound healing. The hydrogel not only has good antibacterial and antioxidant capabilities in vitro, but also achieves the treatment and wound healing effects of bacterial infection on the skin surface of rats. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1Schematic diagram of the preparation of HA-ADH-TCPP(Fe) prepared in Example 1 of the present invention;

[0023] Figure 2 The ultraviolet absorption spectrum and fluorescence emission spectrum of HA-ADH-TCPP(Fe) prepared in Example 1 of the present invention in aqueous solution;

[0024] Figure 3 The HA-ADH-TCPP (Fe) prepared in Example 1 of the present invention is 1 O2 production diagram;

[0025] Figure 4 This is a diagram showing the generation of ·OH in aqueous solution of HA-ADH-TCPP(Fe) prepared in Example 1 of the present invention;

[0026] Figure 5 This is a photothermal data diagram of the HAT-Fe / GPCO hydrogel prepared in Example 2 of the present invention;

[0027] Figure 6 This is a thermal imaging data diagram of the HAT-Fe / GPCO hydrogel prepared in Example 2 of the present invention;

[0028] Figure 7 This is a graph showing the antioxidant capacity of the HAT-Fe / GPCO hydrogel prepared in Example 2 of the present invention;

[0029] Figure 8 This is a carbon monoxide release test graph of the HAT-Fe / GPCO hydrogel prepared in Example 2 of the present invention;

[0030] Figure 9 This is a hemolysis test diagram of the HAT-Fe / GPCO hydrogel prepared in Example 2 of the present invention;

[0031] Figure 10 This is a graph showing the antibacterial test of the HAT-Fe / GPCO hydrogel prepared in Example 2 of the present invention against different bacterial species;

[0032] Figure 11 This is a graph showing the cell viability of the HAT-Fe / GPCO hydrogel prepared in Example 2 of the present invention after being cultured in HUVEC cells for 24 hours;

[0033] Figure 12 This is a diagram showing the generation of intracellular ROS after HUVEC cells were cultured with the HAT-Fe / GPCO hydrogel prepared in Example 2 of the present invention at different concentrations;

[0034] Figure 13 This is a photothermal imaging test image of the HAT-Fe / GPCO hydrogel prepared in Example 2 of the present invention;

[0035] Figure 14 This is a diagram showing the effect of HAT-Fe / GPCO hydrogel prepared in Example 2 of the present invention on the wound surface healing of rats;

[0036] Figure 15 The rheological stress and frequency spectrum of the HAT-Fe / GPCO hydrogel prepared in Example 2 of the present invention.

[0037] Figure 16 This is a diagram showing the injectability and self-healing properties of the HAT-Fe / GPCO hydrogel prepared in Example 2 of the present invention.

[0038] Figure 17 This is the infrared spectrum of HA-ADH-TCPP(Fe) prepared in Example 1 of the present invention.

[0039] Figure 18 HA-ADH-TCPP prepared in Comparative Example 1 of the present invention 1 H NMR spectrum. DETAILED DESCRIPTION

[0040] The present invention first provides a hyaluronic acid-based hydrogel, which is obtained by cross-linking the hydrogel main structure shown in Formula 1 and genipin.

[0041]

[0042] In formula 1, n represents the degree of polymerization.

[0043] The present invention also provides a method for preparing a hyaluronic acid-based hydrogel, such as Figure 1 As shown, the following steps are included:

[0044] Step 1: Dissolve FeCl3·6H2O and tetracarboxyphenylporphyrin in a solvent, and heat under reflux to obtain a porphyrin derivative TCPP-Fe;

[0045] Step 2: Dissolve HA in water, add adipic acid dihydrazide (ADH) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), stir, dialyze, and freeze-dry to obtain amino-modified hyaluronic acid HA-ADH;

[0046] Step 3: The TCPP-Fe obtained in step 1 is mixed with EDC and NHS to obtain a TCPP-Fe solution, and the HA-ADH obtained in step 2 is mixed with the TCPP-Fe solution, stirred, dialyzed, and freeze-dried to obtain the hydrogel main body HA-ADH-TCPP (Fe) shown in Formula 1;

[0047] Step 4: Mix the hydrogel body shown in Formula 1 and genipin, add them into water, add carbonyl manganese powder, stir, and place in a water bath to obtain a hyaluronic acid-based hydrogel.

[0048] According to the present invention, a tetracarboxyphenylporphyrin ligand TCPP and ferric chloride hexahydrate are added to a reaction vessel, followed by a solvent, preferably anhydrous N,N-dimethylformamide. The reaction is refluxed at a temperature of preferably 120-130°C, more preferably 130°C, for a reaction time of preferably 6-8 hours, preferably 6 hours. After the reaction is completed and cooled to room temperature, deionized water is added to the solution, and a purple precipitate precipitates from the reaction solution. The suspension is filtered, the product is washed with deionized water until the filtrate is colorless, and the resulting solid product is dissolved in a NaOH solution to obtain a dark green solution. Concentrated HCl is slowly added dropwise to the solution until no more precipitate forms, the resulting suspension is filtered again, and the product is washed with deionized water until the filtrate is colorless and the pH is neutral. The solid product is dried to obtain a purple solid powder, which is TCPP-Fe. The molar ratio of the tetracarboxyphenylporphyrin ligand TCPP to ferric chloride hexahydrate is preferably 1:(1-1.5).

[0049] According to the present invention, hyaluronic acid HA is added to a reaction vessel, followed by deionized water, stirred and dissolved, adipic acid dihydrazide (ADH) is added and stirred and dissolved, the solution pH is adjusted to 4.75, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) is added. After dissolution, the pH is maintained at 4.75 and stirred. The stirring time is preferably 40-48 hours. The solution is dialyzed, and the dialysis time is preferably 40-48 hours. After lyophilization, the obtained white flocculent solid is HA-ADH. The mass ratio of HA to adipic acid dihydrazide is preferably 1:3, and the mass ratio of HA to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is preferably 1:1.

[0050] According to the present invention, TCPP-Fe is added to a reaction vessel, followed by a solvent, preferably dimethyl sulfoxide, and then EDC and NHS. The reaction is stirred in the dark for a period of preferably 2-3 hours to obtain a TCPP-Fe solution. HA-ADH is then dissolved in deionized water, and the TCPP-Fe solution is slowly added dropwise to the HA-ADH aqueous solution with stirring for a period of preferably 40-48 hours. The solution is then dialyzed for a period of preferably 40-48 hours and freeze-dried to obtain a green flocculent solid, which is HA-ADH-TCPP(Fe) as shown in Formula 1. The mass ratio of HA-ADH to TCPP-Fe is preferably 20:1, and the mass ratio of TCPP-Fe, EDC, and NHS is preferably 2:4:3.

[0051] According to the present invention, HA-ADH-TCPP(Fe) represented by Formula 1 is dissolved in deionized water, a crosslinking agent, genipin, is added, and the mixture is stirred. Manganese carbonyl powder is then added, stirred again, and placed in a water bath for heating and solidification. The water bath temperature is preferably 50-60°C, and the placement time is preferably 40-60 minutes. The mass ratio of HA-ADH-TCPP(Fe) represented by Formula 1, manganese carbonyl powder, and genipin is preferably 30:1:1; and the concentration of the genipin solution is preferably 1 mg / mL.

[0052] The present invention also provides the use of the hyaluronic acid-based hydrogel in preparing a drug for treating bacterial infection wound healing.

[0053] The present invention is further described by way of examples below, which do not limit the present invention in any way. Without departing from the technical solution of the present invention, any modification or alteration of the present invention that can be easily implemented by a person skilled in the art will fall within the scope of the claims of the present invention.

[0054] Example 1 Preparation of HA-ADH-TCPP(Fe)

[0055] Step 1: To a 100 mL single-necked flask, add the tetracarboxyphenylporphyrin ligand TCPP (0.1 mmol, 0.079 g) and ferric chloride hexahydrate (0.1 mmol, 0.27 g). Reflux the mixture for 6 h using 20 mL of DMF as the solvent. After the reaction is complete and cooled to room temperature, 30 mL of deionized water is added, causing a purple precipitate to form in the reaction solution. The solution is then filtered using a suction filtration device and washed with deionized water until the filtrate is colorless. The resulting solid product is then dried and dissolved in NaOH solution to yield a dark green solution. Concentrated HCl is slowly added dropwise to the solution until no further precipitation occurs. The resulting suspension is then filtered again and washed with deionized water until the filtrate is colorless and the pH is neutral. The solid product is then dried to yield a purple solid powder, TCPP-Fe.

[0056] Step 2: Add 1 g of HA to a 500 mL beaker, use 100 mL of deionized water as the solvent for the reaction, stir to dissolve, then add 3 g of ADH, add 0.1 mol / L NaOH solution to adjust the pH to 4.75, then add 1 g of EDC, maintain the pH at 4.75 with the above concentration of NaOH solution, stir overnight, then dialyze for 2 days and freeze-dry to obtain a white flocculent solid, which is HA-ADH.

[0057] Step 3: Add 600 mg of HA-ADH to a 250 mL conical flask, use deionized water as the reaction solvent, stir to dissolve, then add 30 mg of TCPP-Fe to a 50 mL conical flask, use DMSO as the reaction solvent, stir to dissolve, then add 60 mg of EDC and 45 mg of NHS, stir in the dark for 2 hours, then slowly add the solution dropwise to the HA-ADH aqueous solution, stir in the dark for 48 hours, then dialyze for 2 days, and lyophilize to obtain a green flocculent solid which is HA-ADH-TCPP (Fe).

[0058] Example 2 Preparation of HAT-Fe / GPCO Hydrogel

[0059] 30 mg of HA-ADH-TCPP(Fe) prepared in Example 1 was dissolved in deionized water, and 1 mg of genipin GP and 1 mg of Mn2[CO] were added. 10 , heating at 60℃ for 60min, the HAT-Fe / GPCO hydrogel was obtained.

[0060] Comparative Example 1

[0061] The preparation process and conditions are the same as those in Example 1, except that TCPP-Fe in step 3 is replaced with TCPP to obtain HA-ADH-TCPP; the hyaluronic acid-based hydrogel precursor HA-ADH-TCPP and amino-modified hyaluronic acid HA-ADH prepared in Comparative Example 1 have the following hydrogen nuclear magnetic spectra: Figure 18 shown.

[0062] The performance of the hyaluronic acid-based hydrogels prepared in Comparative Example 1, Example 1, and Example 2 was characterized as follows:

[0063] 1. Photophysical properties of hyaluronic acid-based hydrogel precursors

[0064] The photophysical properties of HA-ADH-TCPP and HA-ADH-TCPP(Fe) in the present invention were measured in aqueous solution. Figure 2 is the ultraviolet absorption of HA-ADH-TCPP(Fe) in aqueous solution ( Figure 2 A) and fluorescence emission ( Figure 2 B) As can be seen from the figure, HA-ADH-TCPP and HA-ADH-TCPP(Fe) exhibit bright red emission in aqueous solution, with an emission peak at 660nm. The absorption is due to the incorporation of Fe metal into the porphyrin core, which eliminates the two original Q bands of the porphyrin. The emission spectrum undergoes fluorescence quenching due to the introduction of the metal heteronuclear element.

[0065] 2. Singlet oxygen generation ability of hyaluronic acid-based hydrogel precursors

[0066] Figure 3 HA-ADH-TCPP prepared by the present invention ( Figure 3 B), HA-ADH-TCPP(Fe)( Figure 3 A), In vitro singlet oxygen production experiment under light and DPBF conditions and the related kinetic curves ( Figure 3 C), from Figure 3 It can be seen that HA-ADH-TCPP(Fe) has excellent singlet oxygen generation ability.

[0067] 3. Hydroxyl radical generating ability of hyaluronic acid-based hydrogel precursor:

[0068] Figure 4 The HA-ADH-TCPP (Fe) prepared by the present invention was subjected to an in vitro hydroxyl radical generation experiment in the presence of H2O2 with the addition of methylene blue (MB) and carbonyl manganese. Figure 4 A is a comparison chart of hydroxyl radicals produced by different samples to degrade methylene blue. Figure 4 B is the degradation diagram of methylene blue solution after adding carbonyl manganese to HA-ADH-TCPP(Fe). Figure 4 It can be seen that HA-ADH-TCPP(Fe) has excellent hydroxyl radical generation ability.

[0069] 4. Photothermal properties of hyaluronic acid-based hydrogels

[0070] Figure 5 The photothermal properties of HAT-Fe / GPCO hydrogel were measured, and the results showed that HAT-Fe / GPCO had better photothermal generation ability than the control group HA-ADH, HA-ADH-TCPP, and drug group ( Figure 5 A), and the higher the power, the higher the heat generation and temperature rise ( Figure 5 B), and after five cycles of heating and cooling, it can be seen that the drug has good photothermal cycling ability ( Figure 5 C).

[0071] Figure 6 This is the thermal imaging image of the HAT-Fe / GPCO hydrogel corresponding to the photothermal data. Under light conditions, the solution temperature increases with the extension of the illumination time.

[0072] 5. Antioxidant performance experiment of hyaluronic acid-based hydrogel:

[0073] Figure 7 The test kit was used to test the antioxidant capacity of the HAT-Fe / GPCO hydrogel of the present invention. When the drug concentration reached 5 mg / mL, the consumption rate of DPPH could reach 81.5% ( Figure 7 A). The consumption rate of hydrogen peroxide was detected to be nearly 85.2% ( Figure 7 B) The content of reactive oxygen species, especially hydrogen peroxide, at the site of infected wounds far exceeds that of normal tissue. The consumption of hydrogen peroxide will effectively accelerate wound healing. This also proves that HAT-Fe / GPCO hydrogel has excellent antioxidant and anti-inflammatory capabilities, and can have a good therapeutic effect on inflammation in the body.

[0074] 6. CO release experiment of hyaluronic acid-based hydrogel:

[0075] Figure 8 Graph showing the CO release experiment of the HAT-Fe / GPCO hydrogel of the present invention. Figure 8 A uses the hemoglobin assay to measure the concentration of released CO. In the presence of H2O2, Figure 8 A hemoglobin assay was used to detect the decrease in absorbance at 430 nm and the increase in absorbance at 410 nm, thereby detecting that the HAT-Fe / GPCO hydrogel can effectively release CO. Figure 8 B is the concentration of released CO actually calculated by the formula.

[0076] 7. Hemolysis experiment of hyaluronic acid-based hydrogel:

[0077] Figure 9 For the hemolysis test of the HAT-Fe / GPCO hydrogel of the present invention, HAT-Fe / GPCO hydrogels of different concentrations were co-incubated with red blood cells in a 37°C oven for 2 hours. The PBS group served as the negative control group, and the water group served as the positive control group. Panel a) shows a visual image of the corresponding concentration of HAT-Fe / GPCO hydrogel, and panel b) shows a visual image of the corresponding concentration of HAT-Fe / GPCO hydrogels co-incubated with red blood cells in hot water at 37°C for 2 hours followed by centrifugation. The measured hemolysis rate of the drug at 5 mg / mL was still less than 5%, indicating good biosafety.

[0078] 8. Antibacterial experiment of HAT-Fe / GPCO hydrogel:

[0079] Figure 10 The antibacterial properties of the HAT-Fe / GPCO hydrogel of the present invention were measured. Compared with the control groups HA-ADH and HA-ADH-TCPP, the drug group HAT-Fe / GPCO had a better ability to inhibit the growth of Escherichia coli ( Figure 10 A), and better ability to inhibit the growth of Staphylococcus aureus ( Figure 10 B).

[0080] 9. Cell therapy experiments of HAT-Fe / GPCO hydrogel:

[0081] Figure 11Figure 2 shows the cell viability of the HAT-Fe / GPCO hydrogel after 24 hours of incubation in human umbilical vein endothelial cells (HUVEC). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay of the HAT-Fe / GPCO hydrogel was used to investigate the potential cytotoxicity of the HAT-Fe / GPCO hydrogel on HUVEC cells. The MTT assay for drug cytotoxicity was performed as follows: HUVEC cells were seeded at a density of 10,000 cells per well in a 96-well plate. The cells were incubated in a 37°C, 5% CO2 incubator for 24 hours. After aspirating the old culture medium, drug intervention was added by adding 100 μL of DMEM medium containing a gradient of drug concentrations (0-6 mg / mL) to each well. Four hours later, the original culture medium was replaced with 100 μL of fresh DMEM medium. The cells were placed in an incubator for 24 hours. MTT (20 μL) at a concentration of 5 mg / mL was added to each well. The cells were placed in an incubator for 4 hours. After 4 hours, DMSO (150 μL) was added to each well to replace the original culture medium. The absorbance of the sample at 570 nm was detected by a microplate reader. Figure 11 It can be seen that even when the drug HAT-Fe / GPCO concentration reaches a higher concentration of 6 mM, the survival rate of cells incubated with HAT-Fe / GPCO hydrogel reaches 96%, indicating that it has low cytotoxicity.

[0082] Figure 12 The total ROS production in cells under incubation of different control groups of the HAT-Fe / GPCO hydrogel of the present invention ( Figure 12 A) and superoxide anion O2 · -( Figure 12 B) The production of ROS. It can be seen that the green fluorescence of DCFH probe and the red fluorescence of DHE probe in the control group HAT-Fe / GPCO hydrogel are the weakest or even absent, showing a high ability to remove ROS and O2 · -ability.

[0083] 10. In vivo therapeutic experiments of HAT-Fe / GPCO hydrogel:

[0084] Figure 13 These are the in vivo photothermal images corresponding to different control groups of the HAT-Fe / GPCO hydrogel of the present invention. Under light conditions, the skin surface temperature increases with the extension of the light exposure time.

[0085] Figure 14The wound healing effect diagram of different control groups of HAT-Fe / GPCO hydrogel of the present invention is shown. After the model is successfully established, it is applied to the wound surface and laser (635nm 0.8W / cm 2 ) irradiated the wound site, and the results showed that it had a good wound healing effect. Figure 14 B is a schematic diagram of the wound healing ratio of rats after treatment with different samples on days 3, 6, 9, and 12. 123 in the figure represent the three samples HA-ADH, HA-ADH-TCPP, and HAT-Fe / GPCO, respectively.

[0086] 11. Rheological properties test of HAT-Fe / GPCO hydrogel:

[0087] Figure 15 is the rheological stress-strain spectrum of the HAT-Fe / GPCO hydrogel of the present invention ( Figure 15 A) and rheological frequency spectrum ( Figure 15 B). It can be seen from the figure that the material has good mechanical properties.

[0088] Figure 16 The self-healing ability of the HAT-Fe / GPCO hydrogel of the present invention is shown in FIG Figure 16 A) and injectability diagram ( Figure 16 B), it can be seen from the figure that the material has self-healing properties and injectable properties.

Claims

1. A hyaluronic acid-based hydrogel, characterized in that The hyaluronic acid-based hydrogel is obtained by cross-linking the hydrogel main structure shown in Formula 1 with genipin.

2. The method for preparing a hyaluronic acid-based hydrogel according to claim 1, wherein: The following steps are involved: Step 1: Dissolve FeCl3·6H2O and tetracarboxyphenylporphyrin in a solvent, and heat under reflux to obtain a porphyrin derivative TCPP-Fe; Step 2: Dissolve HA in water, add adipic acid dihydrazide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, stir, dialyze and freeze-dry to obtain amino-modified hyaluronic acid HA-ADH; Step 3: The TCPP-Fe obtained in step 1 is mixed with EDC and NHS to obtain a TCPP-Fe solution, and the HA-ADH obtained in step 2 is mixed with the TCPP-Fe solution, stirred, dialyzed, and freeze-dried to obtain the hydrogel main body HA-ADH-TCPP (Fe) shown in Formula 1; Step 4: Mix the hydrogel body shown in Formula 1 and genipin, add them into water, add carbonyl manganese powder, stir, and place in a water bath to obtain a hyaluronic acid-based hydrogel.

3. The method for preparing a hyaluronic acid-based hydrogel according to claim 2, wherein: The reaction temperature of step 1 is 120-130° C., and the reaction time is 6-8 hours.

4. The method for preparing a hyaluronic acid-based hydrogel according to claim 2, wherein: The molar ratio of the tetracarboxyphenylporphyrin and FeCl3·6H2O in step 1 is 1:(1-1.5).

5. The method for preparing a hyaluronic acid-based hydrogel according to claim 2, wherein: The mass ratio of HA to adipic acid dihydrazide described in step 2 is 1:3, and the mass ratio of HA to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:

1.

6. The method for preparing a hyaluronic acid-based hydrogel according to claim 2, wherein: The stirring time in step 3 is 40-48 hours, and the dialysis time is 40-48 hours.

7. The method for preparing a hyaluronic acid-based hydrogel according to claim 2, wherein: The mass ratio of HA-ADH and TCPP-Fe in step three is 20:

1.

8. The method for preparing a hyaluronic acid-based hydrogel according to claim 2, wherein: In the step 4, the mass ratio of the hydrogel body, carbonyl manganese powder and genipin shown in formula 1 is 30:1:

1.

9. The method for preparing a hyaluronic acid-based hydrogel according to claim 2, wherein: The water bath temperature in step 4 is 60° C. and the placement time is 40-60 min.

10. Use of the hyaluronic acid-based hydrogel according to claim 1 in preparing a drug for treating bacterial infection and wound healing.

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