Photo-crosslinking hydrogel for promoting wound healing and preparation method thereof

Abstract

The invention relates to the technical field of medicines, in particular to photo-crosslinking hydrogel for promoting wound healing and a preparation method of the photo-crosslinking hydrogel. According to the preparation method, hyaluronic acid and methacrylic anhydride react by adopting a click chemical reaction, so that hyaluronic acid is grafted with double bonds, acrylamide and a photoinitiator are added, the double bonds of hyaluronic acid and the double bonds of acrylamide are irradiated by ultraviolet light under the action of a free radical photoinitiator, and carbon-carbon double bond addition polymerization between the double bonds can be completed at an extremely high speed; the hydrogel is formed through rapid curing. The hydrogel disclosed by the invention has good antibacterial performance, the addition of Fe < 3 + > has good antibacterial performance, so that bacterial infection can be reduced in a wound healing process, and the addition of glycyrrhizic acid can enable the hydrogel to relieve tissue pathological change and reduce inflammatory response. The photo-crosslinking hydrogel has the advantages that the photo-crosslinking hydrogel has a three-dimensional net-shaped structure, the water absorption performance is high, the moist environment of a wound surface is kept, the infection rate is reduced, and therefore wound healing is accelerated.

Description

Technical Field

[0001] The invention relates to the technical field of medicine, and in particular to a photo-crosslinked hydrogel that promotes wound healing and a preparation method thereof. Background Art

[0002] As an important organ of the human body, the skin has the functions of sensing external stimuli, regulating body temperature and protecting internal organs. However, due to the frequent contact between the skin and the external environment, it has also become one of the most vulnerable organs. Skin trauma poses a serious threat to human life, health and safety. The global annual cost of chronic wound care is approximately US$2.81-9.68 billion. Promoting wound healing has become a key scientific issue that needs to be urgently addressed in today's medical field. Wound dressings can provide a temporary barrier to wounds to avoid secondary injury and infection, and can also achieve effective drug delivery. In recent years, compared with synthetic polymer materials, biological wound dressings have good biocompatibility, degradability and anti-inflammatory and antibacterial properties, and therefore have attracted more and more attention. However, microbial infection during wound healing is still a very challenging problem in clinical treatment. Summary of the invention

[0003] The present invention provides a photo-crosslinked hydrogel for promoting wound healing and a preparation method thereof. The photo-crosslinked hydrogel has the advantages of a three-dimensional network structure, strong water absorption performance, maintaining a moist environment of the wound surface, reducing the infection rate, and thus accelerating wound healing.

[0004] In one aspect, the present invention provides a method for preparing a photo-crosslinked hydrogel for promoting wound healing, the preparation method comprising the following steps:

[0005] 1) Hyaluronic acid (HA) grafted double bonds

[0006] Add hyaluronic acid to deionized water and stir until dissolved; add methacrylic anhydride, adjust the pH value, react, dialyze with deionized water, freeze-dry the dialyzed sample, and obtain a sponge-like product recorded as HAMA.

[0007] 2) Photocrosslinked hydrogel HMG-Fe 3+ Preparation

[0008] Weigh HAMA and photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959) in deionized water, stir and dissolve to obtain solution ①.

[0009] Weigh acrylamide (AM) and photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959) in deionized water, stir and dissolve to obtain solution ②.

[0010] Weigh glycyrrhizic acid (GA) and photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959) into deionized water, stir and dissolve to obtain Solution ③.

[0011] Weigh ferric chloride hexahydrate (FeCl3·6H2O) and dissolve it in deionized water to obtain Solution ④;

[0012] Mix Solution ①, ②, and ③ in a volume ratio of 1:1:1. At this time, the solution is denoted as the HMG solution. Put the HMG solution in a mold and irradiate it with an ultraviolet lamp to form a gel to obtain the HMG hydrogel; Immerse the prepared HMG hydrogel in Solution ④ to obtain the photo-crosslinked hydrogel HMG-Fe 3+ 。

[0013] Preferably, the weight-to-volume ratio of hyaluronic acid to deionized water in step 1) is 1 g: 100 mL; the volume ratio of deionized water to methacrylic anhydride is 100:1.

[0014] Preferably, the pH value in step 1) is 8 - 8.5, the reaction temperature is 0 °C, and the reaction time is 24 h.

[0015] Preferably, the addition ratio of each substance in Solution ① is HAMA: photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959): deionized water = 0.05 g: 0.05 g: 5 mL.

[0016] Preferably, the addition ratio of each substance in Solution ② is acrylamide (AM): photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959): deionized water = 5 g: 0.05 g: 5 mL.

[0017] Preferably, the addition ratio of each substance in Solution ③ is glycyrrhizic acid (GA): photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959): deionized water = 0.05 g: 0.05 g: 5 mL.

[0018] Preferably, the concentration of FeCl3 in Solution ④ is 0.1 M.

[0019] Preferably, the irradiation power of the ultraviolet lamp is 10 W and the wavelength is 365 nm.

[0020] On the other hand, the present invention provides a photo-crosslinked hydrogel for promoting wound healing prepared by the above method.

[0021] On yet another aspect, the present invention provides the application of the photo-crosslinked hydrogel for promoting wound healing prepared by the above method in the preparation of drugs for treating wound healing.

[0022] The present invention uses click chemistry to react hyaluronic acid with methacrylic anhydride, grafting double bonds to hyaluronic acid. Acrylamide and a photoinitiator are added. Under the action of a free radical photoinitiator, the double bonds of hyaluronic acid and acrylamide are irradiated with ultraviolet light, and the addition polymerization of carbon-carbon double bonds can be completed at an extremely fast speed between the double bonds, achieving rapid curing to form a hydrogel. The hydrogel of the present invention has good antibacterial properties. The addition of Fe 3+ has good antibacterial properties, which can reduce bacterial infection during the wound healing process. The addition of glycyrrhizic acid can enable the hydrogel to relieve histopathological changes and reduce inflammatory reactions. This photo-crosslinked hydrogel has a three-dimensional network structure, strong water absorption performance, maintains a moist environment at the wound surface, reduces the infection rate, and thus accelerates wound healing and other advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the nuclear magnetic resonance spectrum (proton spectrum, 400 MHz) of hyaluronic acid (HA) and hyaluronic acid modified with methacrylic anhydride (HAMA).

[0024] Figure 2 is the SEM image of HMG and HMG-Fe 3+ .

[0025] Figure 3 is the swelling curve of a group of hydrogels.

[0026] Figure 4 is the rheological curve of the HM, HMG, and HMG-Fe 3+ groups.

[0027] Figure 5 is the antibacterial effect of the blank control, HM, HMG, and HMG-Fe 3+ groups of hydrogels.

[0028] Figure 6 is the relative cell viability of the HM, HMG, and HMG-Fe 3+ groups incubated with different concentrations of L929.

[0029] Figure 7 is the AO / EB staining morphology and survival status of the L929 cells incubated in the blank group, HM, HMG, and HMG-Fe 3+ groups at a concentration of 5000 μg / mL.

[0030] Figure 8 is the schematic diagram of wound healing in Example 7.

[0031] Figure 9 is the wound healing rate in Example 7.

[0032] Figure 10 is the embedding diagram in Example 7. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

[0034] Example 1 Preparation of Hydrogel

[0035] 1) Grafting double bonds onto hyaluronic acid (HA)

[0036] Add 1 g of hyaluronic acid to a three-necked flask containing 100 mL of deionized water, and stir until dissolved; then add 1 mL of methacrylic anhydride, and adjust the pH value to 8 - 8.5 with a 1 M NaOH solution, and react at 0 °C for 24 h. After the reaction, dialyze with deionized water using a dialysis bag with a molecular weight of 14000 for 72 h. Freeze-dry the dialyzed sample to obtain a sponge-like product denoted as HAMA.

[0037] 2) HAMA / AM / GA / Fe 3+ Preparation of hydrogel

[0038] Weigh 0.05 g of HAMA and 0.05 g of photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959) into a vial containing 5 mL of deionized water, stir and dissolve to prepare a 1% (w / v) solution, denoted as Solution ①.

[0039] Weigh 5 g of acrylamide (AM) and 0.05 g of I2959 into a vial with 5 mL of deionized water, stir and dissolve to prepare a 1% (w / v) solution, denoted as Solution ②.

[0040] Weigh 0.05 g of glycyrrhizic acid (GA) and 0.05 g of I2959 into a vial with 5 mL of deionized water, stir and dissolve to prepare a 1% (w / v) solution, denoted as Solution ③.

[0041] Weigh 2.703 g of ferric chloride hexahydrate (FeCl3·6H2O) and prepare a 100 mL 0.1 M solution, denoted as Solution ④.

[0042] Mix Solution ①, Solution ② and deionized water according to the volume ratio ①:②:H2O = 1:1:1, and denote the solution as HM at this time. Mix Solution ①, Solution ② and Solution ③ according to the volume ratio ①:②:③ = 1:1:1, and denote the solution as HMG solution at this time. Place 1 mL of Solution HM and HMG solution respectively in a mold with a length × width × height of 1 cm × 1 cm × 1 cm, and irradiate with ultraviolet light with a power of 10 W and a wavelength of 365 nm for 5 min to form a gel, and obtain HM and HMG hydrogels respectively. Place the prepared HMG hydrogel in Solution ④ and soak for 2 h to obtain the hydrogel denoted as HMG-Fe. 3+ .

[0043] Characterization of the hydrogel in Example 2

[0044] 1) Nuclear magnetic resonance spectroscopy

[0045] The degree of methacrylation of methacrylated hyaluronic acid was analyzed by nuclear magnetic resonance hydrogen spectroscopy. The prepared methacrylated hyaluronic acid (HAMA) was dissolved in deuterated water (D2O) at a ratio of 20 mg / mL at room temperature, and the solution was tested after it was completely dissolved and clarified. And unmodified hyaluronic acid (HA) was selected as the control group. During the test, the 1H-NMR spectra of HAMA and HA were detected using a frequency of 400 MHz. After the measured nuclear magnetic resonance hydrogen spectrum data was calibrated by MestReNova software, the degree of methacrylation of hyaluronic acid was calculated according to its integral area.

[0046] The grafting result of the double bond of hyaluronic acid is as Figure 1 shown. The results show that the methacrylation reaction of hyaluronic acid shows that the peak of methacrylate is located at 4.91 ppm, showing the proton nuclear magnetic resonance spectrum characteristics of this reaction. According to the relative integral intensity of methacrylic acid protons and methyl protons in hyaluronic acid (the peak value is 1.9 ppm), the degree of methacrylation of hyaluronic acid is calculated to be 63%, indicating that the double bond grafting is successful.

[0047] 2) Scanning electron microscopy (SEM)

[0048] The internal morphology of the gel was observed using scanning electron microscopy (Zeiss Sigma 500). Before the test, the hydrogels HMG and HMG-Fe 3+ were freeze-dried in a freeze dryer. After the hydrogels were freeze-dried, the cross-section was cut with a knife, and an appropriate amount of the sample was placed on the conductive adhesive, placed on the sample stage, sputter-coated with gold, and the cross-sectional morphology of the hydrogel was observed using a scanning electron microscope at an acceleration voltage of 3.0 kV.

[0049] The electron microscopy result images are as Figure 2 shown. The hydrogels in each group have a good pore structure. The results show that the hydrogel HMG before soaking has relatively large pore size and thin pore walls, resulting in strong ionic cross-linking between Fe 3+ and the carboxyl groups of glycyrrhizic acid and hyaluronic acid after the hydrogel was soaked in the Fe 3+ solution. The carboxylate precipitate generated by the coordination of Fe 3+ and the carboxyl group is mainly concentrated at the bottom, blocking the pores on one side of the bottom of the hydrogel. The pore size of the HMG-Fe 3+ hydrogel becomes significantly smaller, and the pore wall thickness is greatly increased. It shows that the introduction of Fe 3+ promotes the formation of a more compact network structure of the HMG hydrogel.

[0050] Example 3 Swelling performance experiment of the hydrogel

[0051] Hydrogel is a degradable three-dimensional network structure polymer with extremely hydrophilic functional characteristics and can quickly reach a swollen state in water. The swelling ability of the hydrogel was measured by the gravimetric method. Three groups of freeze-dried hydrogels HM, HMG, and HMG-Fe 3+ were weighed and placed in PBS solution with a pH of 7.4. Samples were taken at 1h, 2h, 4h, 6h, 8h, 24h, 48h, and 72h, the moisture was blotted with filter paper, and the weight was recorded. The swelling ratio was calculated according to the formula. The results are as Figure 3 shown. The results show that the hydrogels HM, HMG, and HMG-Fe 3+ all have good swelling properties. The hydrogels swell rapidly within 24h, and the swelling of the hydrogels reaches equilibrium after 72h. The swelling ratios of HM and HMG reach 2000%, and that of HMG-Fe 3+ reaches about 1800. HMG-Fe 3 + Because the hydrogel is immersed in 0.1M Fe 3+ solution, Fe 3+ will react with -COOH in hyaluronic acid and glycyrrhizic acid, making the internal cross-linking of the hydrogel tighter and the porosity lower. Therefore, its swelling ability is lower than the other two. This photo-crosslinked hydrogel can hold a large amount of water, which endows excellent swelling performance, similar to soft tissues, can absorb excess tissue exudate, maintain a moist wound environment, and isolate the invasion of external bacteria.

[0052] Example 4 Rheological Property Experiment of Hydrogel

[0053] Rheological experiments were carried out on a rheometer (TA instrument). A parallel plate with a diameter of 25mm was used, the sample gap was 1000μm, the test temperature was 25°C, and the hydrogel samples were tested multiple times to determine their mechanical properties and stability for response to treatment and biological applications. Under the conditions of a frequency of 10Hz and a strain of 1%, the single-frequency measurement was used to scan the material from time 0 - 300s, and the storage modulus (G’) and loss modulus (G”) of the material were obtained.

[0054] The results are shown in Figure 4 . The results show that G’ of the three hydrogel scaffolds HM, HMG, and HMG-Fe 3+ is greater than G”, indicating that both hydrogel scaffolds can maintain a gel state under relatively large pressures. And HM-G’ > HMG-G’ > HMG-Fe 3+ -G’, indicating that with the increase of components, the internal cross-linking of the hydrogel becomes tighter, making the hardness of the hydrogel gradually increase and the mechanical properties change.

[0055] Example 5 Antibacterial Experiment of Hydrogel

[0056] The hydrogel was co-incubated with Staphylococcus aureus. 3+ 0.3 g of each hydrogel was placed in 10 mL of Staphylococcus aureus suspension (sterile water, 10 6 CFU / mL) and incubate at 37°C for 6h. After the incubation, take 10μL of the cultured bacterial suspension and inoculate it on the surface of the solid nutrient medium. After culturing at 37°C for 24h, take out the culture dish and take pictures to count. The plate counting method is used to calculate and record the number of viable bacteria on the solid agar medium, and the antibacterial rate of the material (Ar, %) is calculated. Antibacterial rate. The negative control group is a bacterial suspension with the same activity without any treatment. For each group of samples, each bacterium is tested three times in parallel, and the antibacterial rate is calculated as follows:

[0057]

[0058] Where Nn is the number of colonies in the negative control group, and Ns is the number of colonies in the sample.

[0059] The results are as follows Figure 5 The results showed that the number of colonies in the blank control group was significantly higher than that in the other three groups. By calculation, the antibacterial rates of the HM group were 33.33%, the HMG group were 53.3%, and the HMG-Fe group were 53.3%. 3+ The group was 99.75%, indicating that the Fe 3+ After soaking, the hydrogel has obvious antibacterial effect.

[0060] Example 6 Cell experiment of hydrogel

[0061] 1) Cytotoxicity assay (MTT method)

[0062] The hydrogels HM, HMG, and HMG-Fe were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method using cultured mouse fibroblasts (L929). 3+ The L929 cell line was cultured in low-glucose medium (MEM) containing 10% fetal bovine serum, 500 μg / mL penicillin and 100 μg / mL streptomycin. The cells were cultured in an incubator (37°C, 5% CO2). L929 cells were cultured at 5×10 5Inoculate cells with a uniform density into a 96-well plate and culture them in an incubator for 24 h to evenly cover the bottom of the well plate with cells. Then add 180 μL of culture medium to each well. Immerse the hydrogel in PBS (pH = 7.4) and co-incubate at 37 °C for 24 h and 48 h. The concentration of the stock solution is 50000 μg / mL, and different concentrations of extraction solutions (500 μg / mL - 50000 μg / mL) are obtained by gradual dilution. Take 20 μL of the extraction solution and add it to each well to make the drug action concentration 50 μg / mL - 500 μg / mL. Add 20 μL of culture medium to the blank group. After culturing in an incubator for 24 h, add 20 μL of MTT (5 mg / mL) to each well, paying attention to operating in the dark. Culture for 4 h, aspirate the suspension, and then add 150 μL of dimethyl sulfoxide (DMSO). Oscillate in a 37 °C constant temperature shaker for 10 min, and measure the OD value at 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Among them, OD test is the absorbance value of the cell well after drug treatment, and OD control is the absorbance value of the blank control well. Calculate the relative cell viability:

[0063]

[0064] The results show that biocompatibility is one of the necessary conditions for a wound healing material to be qualified. The MTT colorimetric method is used to detect the compatibility of different materials with mouse fibroblasts (L929). Figure 6 As shown, for the hydrogels of the HMG and HMG-Fe 3+ groups with a hydrogel concentration in the range of 50 - 500 μg / mL, the survival rate of L929 cells is greater than 80% (>70%, no cytotoxicity). In addition, for the HMG-Fe 3+ group, as the concentration of the extraction solution increases, the cell viability increases significantly.

[0065] 2) Fluorescent staining of living cells

[0066] For fluorescent staining, according to the method reported in the literature, inoculate cells into 12 wells at a density of 1.5×10 5 per well, supplement the culture medium to 1 mL, and culture in a carbon dioxide incubator at 37 °C for 24 h. For the hydrogels HM, HMG, and HMG-Fe 3+The groups were co-incubated with the culture medium for 24 h at a concentration of 5000 μg / mL. After 24 h, the 12-well plates were taken out, and the culture medium was aspirated. In the control group, 1 mL of normal culture medium was added, and in the experimental group, the culture medium after co-incubation with the hydrogel was added, 1 mL per well. They were cultured in a carbon dioxide incubator at 37 °C for 24 h. Then the 12-well plates were taken out, the culture medium was aspirated, 1 mL of PBS (pH = 7.4) was added for washing 3 times, and 500 μL of cell suspension was retained in the last wash. 20 μL of the prepared fluorescent staining agent AO / EB working solution (AO:EB:AO / EB Dilution Buffer = 1:1:8) was added and incubated for 15 min. The liquid was aspirated, 1 mL of PBS was added for washing 3 times to wash away the excess staining agent, and the cell survival status was observed and photographed under a fluorescence microscope.

[0067] The results were as Figure 7 shown. The cell compatibility of different materials was qualitatively evaluated by AO / EB staining under a fluorescence microscope. Compared with the blank control group (Control), the hydrogels of the HM, HMG, and HMG-Fe 3+ groups had no obvious effect on cell morphology and survival status, indicating good in vitro cell compatibility.

[0068] Animal experiment of the hydrogel in Example 7

[0069] A mouse full-thickness skin infectious wound model was used to evaluate the wound healing ability of HMG-Fe 3+ hydrogel in ICR mice. After one week of adaptive feeding, ICR mice with an average body weight of 25 g were randomly divided into 4 groups (6 mice / group): blank control group, commercially available hydrogel (Coloplast, hydrocolloid dressing, model 33547) group, HMG group, and HMG-Fe 3+ group. The mice were anesthetized by intraperitoneal injection of 4% chloral hydrate (0.1 mL / 10 g). The hair on the back of the mice was removed with an animal electric hair clipper and 10% Na2S. A circular full-thickness skin wound (r = 8 mm) was formed on the back of each mouse using a sterile biopsy punch. 20 μL of Staphylococcus aureus (S. aureus) with a concentration of 1×10 6 CFU / mL was dropped on the wound. Then the same amount of (10 mm long, 10 mm wide, 2 mm high) dressings of each group were applied to the wound surface. The dressings were changed every other day, and the wounds of each mouse were photographed on days 0, 3, 7, and 14. The initial wound area (A0) and the wound area on days 3, 7, and 14 (A d ) were measured using ImageJ. The wound contraction (%) was calculated according to the following formula:

[0070]

[0071] A0 is the wound area on day 0, and A dare the wound areas on the 3rd, 7th, and 14th days.

[0072] The results are as Figure 8 and Figure 9 shown, the schematic diagrams of wound healing and the healing rates of the hydrogel on the 3rd day, 7th day, and 14th day respectively. We studied the blank group, the commercially available hydrogel group, the HMG group, and the HMG-Fe 3+ group. The results showed that: as the healing time extended, the wound healing area gradually decreased. The wound healing effect of the mice treated with the dressing HMG-Fe 3+ was the most obvious. The wound healing effect was the best, and nearly complete closure was achieved on the 14th day. At 3 days of healing, the wound areas of the blank control group, the commercially available hydrogel group, the HMG group, and the HMG-Fe 3+ group decreased in turn. Among them, the HMG-Fe 3+ group had the largest wound contraction rate, and the healing rate was 46.8%. At the 7th day, the wound areas of the HMG group and the HMG-Fe 3+ group were still smaller than those of the blank control group, while the commercially available hydrogel was smaller than the blank control group. At the 14th day, the wound contraction rate of the blank control group was 83.49%, that of the commercially available hydrogel group was 80.85%, that of the HMG group was 86.80%, and that of the HMG-Fe 3+ group was 90.06%.

[0073] Since the dressing is in direct contact with the skin, we embedded the material to further investigate the biocompatibility of the hydrogel in vivo. After one week of adaptive feeding of ICR mice, with an average body weight of 25 g, they were randomly divided into 4 groups (6 mice / group): blank control group, commercially available hydrogel group, HMG group, and HMG-Fe 3+ group. The mice were anesthetized by intraperitoneal injection of 4% chloral hydrate (0.1 mL / 10 g). The hair on the back of the mice was removed with an animal electric hair clipper and 10% Na2S. After disinfection with iodophor, a 1-cm-long wound was cut along the spine. Weigh 5 mg of hydrogel from the commercially available hydrogel group, the HMG group, and the HMG-Fe 3+ group respectively. After ultraviolet sterilization, they were implanted subcutaneously into the mice, and then the skin of the mice was sutured with surgical sutures. The control group was not implanted with hydrogel, and other operations were the same as those of the experimental group.

[0074] As Figure 10 shown, the materials were implanted into the dorsal skin of the mice and then sutured. The materials were taken out on the 10th day and 20th day respectively, and the skin at the implantation site, the kidneys, livers, and spleens of the mice were subjected to HE staining to evaluate the pathological structures of each part. According to the section results, the hydrogels of each group had no adverse effects on the reconstruction of the skin tissue structure of the mice and did not cause pathological abnormalities in the liver, spleen, and kidneys, indicating good in vivo biocompatibility.

[0075] Finally, it should also be noted that the above-listed is only a specific embodiment of the present invention. Obviously, the present invention is not limited to the above embodiments and there can be many variations. All variations that can be directly derived or associated by those of ordinary skill in the art from the disclosed content of the present invention should be considered within the protection scope of the present invention.

Claims

1. A preparation method of a photocrosslinkable hydrogel for promoting wound healing, characterized in that, The preparation method includes the following steps: 1) Grafting double bonds onto hyaluronic acid (HA) Add hyaluronic acid to deionized water and stir until dissolved; add methacrylic anhydride, adjust the pH value, react, dialyze the sample with deionized water, and freeze-dry the dialyzed sample to obtain a sponge-like product denoted as HAMA; 2) Preparation of photocrosslinked hydrogel HMG-Fe 3+ Preparation Weigh HAMA and photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959) in deionized water and stir to dissolve to obtain Solution ①; Weigh acrylamide (AM) and photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959) in deionized water and stir to dissolve to obtain Solution ②; Weigh glycyrrhizic acid (GA) and photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959) in deionized water and stir to dissolve to obtain Solution ③; Weigh ferric chloride hexahydrate (FeCl3·6H2O) and dissolve it in deionized water to obtain Solution ④; Mix solutions ①, ②, and ③ in a volume ratio of 1:1:

1. At this time, the solution is denoted as the HMG solution. Place the HMG solution in a mold and irradiate it with an ultraviolet lamp to form a gel, obtaining the HMG hydrogel. Immerse the prepared HMG hydrogel in solution ④ to obtain the photo-crosslinked hydrogel HMG-Fe 3+ .

2. The preparation method of a photo-crosslinkable hydrogel for promoting wound healing according to claim 1, wherein In step 1), the weight-to-volume ratio of hyaluronic acid to deionized water is 1 g: 100 mL; the volume ratio of deionized water to methacrylic anhydride is 100:

1.

3. The preparation method of a photo-crosslinkable hydrogel for promoting wound healing according to claim 1, characterized in that, In step 1), the pH value is 8 - 8.5, the reaction temperature is 0 °C, and the reaction time is 24 h.

4. The preparation method of a photo-crosslinkable hydrogel for promoting wound healing according to claim 1, characterized in that, In Solution ①, the addition ratio of each substance is HAMA: photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959): deionized water = 0.05 g: 0.05 g: 5 mL.

5. The preparation method of a photo-crosslinkable hydrogel for promoting wound healing according to claim 1, characterized in that, In Solution ②, the addition ratio of each substance is acrylamide (AM): photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959): deionized water = 5 g: 0.05 g: 5 mL.

6. The preparation method of a photo-crosslinkable hydrogel for promoting wound healing according to claim 1, characterized in that, In Solution ③, the addition ratio of each substance is glycyrrhizic acid (GA): photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959): deionized water = 0.05 g: 0.05 g: 5 mL.

7. The preparation method of a photocrosslinkable hydrogel for promoting wound healing according to claim 1, characterized in that, The concentration of FeCl3 in Solution ④ is 0.1 M.

8. The preparation method of a photo-crosslinkable hydrogel for promoting wound healing according to claim 1, characterized in that, The irradiation power of the ultraviolet lamp is 10 W and the wavelength is 365 nm.

9. A photo-crosslinked hydrogel for promoting wound healing prepared by the method according to any one of claims 1 - 8.

10. Use of the photo-crosslinked hydrogel for promoting wound healing according to claim 9 in the preparation of a drug for treating wound healing.

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