Black rice extract loaded photo-thermal response enhanced hydrogel as well as preparation method and application thereof

Abstract

The invention provides photo-thermal response enhanced hydrogel loaded with black rice extract as well as a preparation method and application of the photo-thermal response enhanced hydrogel loaded with the black rice extract, and the photo-thermal response enhanced hydrogel is formed by taking a mixture of hyaluronic acid / tyrosine conjugate and the black rice extract as a base material under the cross-linking action of horse radish peroxidase / catalase, horseradish peroxidase acts on the black rice extract while serving as a cross-linking agent, the near-infrared photo-thermal responsiveness of the black rice extract after reaction of the horseradish peroxidase is greatly enhanced, and good oxidation resistance, inflammation resistance and photo-thermal responsiveness can be achieved only through a small amount of black rice extract in a hydrogel system.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and specifically relates to a photothermal response enhanced hydrogel loaded with black rice extract, a preparation method thereof, and an application thereof. The hydrogel is formed by cross-linking a hyaluronic acid / tyrosine conjugate and a black rice extract through horseradish peroxidase, and its application in treating skin damage. Background Art

[0002] Skin infection occurs when a wound becomes infected by pathogenic microorganisms after skin injury, triggering a local or systemic inflammatory response. This type of infection poses a serious threat to patients' health and quality of life. Although antibiotics play an important role in treating bacterial infections, they have some significant shortcomings in the treatment of skin infection, such as drug resistance, side effects, and difficulty in controlling the uniform release of local drug concentrations. Photothermal therapy (PTT) uses the heat generated by photothermal-responsive materials under irradiation with light of specific wavelengths to kill pathogens and promote wound healing. It physically kills bacteria without developing drug resistance, can achieve local treatment, and can be combined with antibacterial and anti-inflammatory materials to enhance the therapeutic effect of wounds. However, PTT therapy requires the addition of photothermal materials, which poses a potential risk to biosafety.

[0003] Compared to traditional wound dressings, hydrogels are three-dimensional mesh structures composed of hydrophilic polymers that can absorb large amounts of water while maintaining good flexibility and biocompatibility. They can also be loaded with drugs, active ingredients, or photothermal responsive materials in combination with other means to enhance wound healing. Hyaluronic acid (HA) is a naturally occurring polysaccharide with excellent biocompatibility and moisturizing properties. After HA is grafted with tyrosine via amide, it can be cross-linked using horseradish peroxidase to form a hyaluronic acid / tyramine conjugate hydrogel with high biocompatibility and certain antibacterial, antioxidant, and anti-inflammatory effects. However, in this hydrogel system, the antibacterial, antioxidant, and anti-inflammatory functions produced by tyrosine alone are relatively limited, and the hydrogel system lacks photothermal responsive materials, making it impossible to combine with PTT therapy to improve wound healing.

[0004] Black rice is a cereal grain rich in various bioactive ingredients. Black rice extract is rich in anthocyanidins and other polyphenols, with cyanidin-3-glucoside being the primary anthocyanidin. While the anthocyanidins and polyphenols in black rice extract exhibit a certain degree of photothermal responsiveness at 808nm near-infrared radiation, their photothermal conversion efficiency is insufficient, requiring very high concentrations and high near-infrared irradiation intensities to reach the appropriate antibacterial temperature (45-50°C), posing potential biosafety risks and increasing costs.

[0005] In response to the above-mentioned technical problems existing in the prior art, the present invention aims to provide a hyaluronic acid / tyrosine conjugate mixed with black rice extract, which is then cross-linked using HRP enzyme to form a hydrogel. At the same time, HRP also acts on the black rice extract. The black rice extract after enzyme catalysis exhibits enhanced photothermal response properties. Only a small amount of black rice extract is required in the hydrogel system to achieve good antioxidant, anti-inflammatory and photothermal responsiveness. Therefore, the hydrogel itself is simple to prepare, reduces cost, and improves biosafety, thereby overcoming the technical problems existing in the prior art. Summary of the Invention

[0006] The present invention provides a black rice extract-loaded photothermal response enhanced hydrogel, which comprises a cross-linked product formed by a matrix and a solution of water or phosphate buffer, wherein the matrix is ​​a mixture of hyaluronic acid / tyrosine conjugate and black rice extract, and the cross-linking agent of the matrix is ​​an enzyme catalytic system composed of horseradish peroxidase / hydrogen peroxide; the photothermal response of the hydrogel is enhanced in the near-infrared band, preferably at 808 nm and an irradiation power of 400 mW / cm 2 The above near infrared has enhanced photothermal responsiveness. The mixture of hyaluronic acid / tyrosine conjugate and black rice extract, and the enzyme catalytic system composed of horseradish peroxidase / hydrogen peroxide are all reacted in the form of solution.

[0007] In the mixed system of the matrix, cross-linking agent and solvent of the hydrogel of the present invention, the proportions of the components are as follows: the final concentration of the hyaluronic acid / tyrosine conjugate is 1 wt% or more, preferably 1 wt%; the final concentration of the black rice extract is 0.1 wt% or more, preferably 0.1-0.5 wt%, and more preferably 0.3 wt%; the horseradish peroxidase is 5 U / mL or more, preferably 5 U / mL; the hydrogen peroxide is 0.05 wt% or more, preferably 0.05 wt%; and the rest is water or phosphate buffer.

[0008] The present invention provides a method for preparing a hydrogel, comprising the following steps: (1) preparing a solution of a hyaluronic acid / tyrosine conjugate and a black rice extract, and uniformly mixing the solution; (2) adding a horseradish peroxidase solution to the mixed solution of step 1, and uniformly mixing the solution; and (3) adding a hydrogen peroxide solution to the mixed solution of step 2, and uniformly stirring the solution to form a hydrogel.

[0009] Furthermore, in the mixed system after adding the hydrogen peroxide solution in step 3, the final concentration of the hyaluronic acid / tyrosine conjugate is 1 wt% or more, preferably 1 wt%; the final concentration of the black rice extract is 0.1 wt% or more, preferably 0.1-0.5 wt%, and more preferably 0.3 wt%; the horseradish peroxidase concentration is 5 U / mL or more, preferably 5 U / mL; the hydrogen peroxide concentration is 0.05 wt% or more, preferably 0.05 wt%; and the remainder is water or phosphate buffer.

[0010] The present invention provides an in vitro anti-inflammatory and / or anti-oxidative and / or antibacterial method, which utilizes the hydrogel described herein, or a hydrogel obtained by the preparation method of the present invention. Leveraging the hydrogel's enhanced photothermal responsiveness, the hydrogel is irradiated with near-infrared light at a wavelength of 808 nm to enhance its anti-inflammatory and / or antibacterial capabilities.

[0011] The present invention provides an application in the preparation of anti-inflammatory and / or antioxidant and / or antibacterial drugs, wherein the application uses the hydrogel described in the present invention, or the hydrogel obtained by any preparation method described in the present invention to prepare the drug; preferably, the photothermal responsiveness of the hydrogel is utilized, and the drug is irradiated with near-infrared rays to enhance the anti-inflammatory and / or antibacterial ability of the drug containing the hydrogel.

[0012] The present invention also provides an application in preparing a drug for repairing skin damage, wherein the application uses the hydrogel according to the present invention to prepare a drug for repairing skin damage, or uses the hydrogel obtained by the preparation method of the present invention to prepare a drug for repairing skin damage.

[0013] The present invention further provides a wound dressing for repairing skin wounds, wherein the wound dressing contains the hydrogel of the present invention, or the wound dressing contains the hydrogel obtained by the preparation method of the present invention.

[0014] The hydrogel uses hyaluronic acid / tyrosine conjugate as a matrix, and a mixed solution including the hyaluronic acid / tyrosine conjugate and black rice extract is catalyzed and cross-linked by a horseradish peroxidase / catalase catalytic system to form the hydrogel.

[0015] The hydrogel material of the present invention has low production cost, contains natural black rice extract, has good biosafety, adhesion, anti-inflammatory, antioxidant and antibacterial properties, and has enhanced photothermal response to 808nm near-infrared. Only a relatively low concentration of black rice extract is used to achieve an ideal photothermal conversion temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The left figure is a schematic diagram of the gelation time of the hydrogel of the present invention and a comparison diagram before and after gelation in a vial; the right figure is a comparison diagram before and after gelation in a vial;

[0017] Figure 2 is the in vitro degradation rate of the hydrogel of the present invention;

[0018] Figure 3 The photothermal response changes of black rice extract solutions with different concentrations before and after treatment with horseradish peroxidase;

[0019] Figure 4 The temperature change of the hydrogel of the present invention under near-infrared irradiation intensity includes: Figure 4 a is the temperature change of the hydrogel of the present invention under the same near-infrared irradiation intensity, Figure 4 b is HTB of the present invention 0.3 Temperature changes of hydrogels under different near-infrared irradiation intensities;

[0020] Figure 5 The left figure is a pigskin stretching test diagram and adhesion test results of the hydrogel of the present invention; the right figure is a schematic diagram of the adhesion test results;

[0021] Figure 6 Schematic diagram of hydrogel antibacterial rate, which includes: Figure 6 a is the inherent antibacterial rate of the hydrogel of the present invention, Figure 6 b is HTB of the present invention 0.3 Photothermal antibacterial rate of hydrogel;

[0022] Figure 7 Schematic diagram of the free radical scavenging rate of the hydrogel of the present invention, which includes: Figure 7 a is the DPPH free radical scavenging rate of the hydrogel of the present invention, Figure 7 b is the hydroxyl radical scavenging rate of the hydrogel of the present invention;

[0023] Figure 8 Schematic diagram of the compatibility of the hydrogel of the present invention, which includes: Figure 8 a is the cell compatibility of the hydrogel of the present invention evaluated using the extract model combined with CCK8, Figure 8 b is the blood compatibility of the hydrogel tested in the present invention;

[0024] Figure 9 The histocompatibility of the hydrogel of the present invention was evaluated by H&M staining and GOT, GPT and AKP blood biochemical tests after implantation into mice for 14 days;

[0025] Figure 10 This is a diagram of the wound healing area of ​​each group of hydrogels of the present invention;

[0026] Figure 11 These are the immunofluorescence staining images of CD86, CD206, CD31, and α-SMA in the wound skin of the infection model treated with the hydrogel groups of the present invention on the 14th day. DETAILED DESCRIPTION

[0027] The following will describe the embodiments of the present invention in detail with reference to examples, so as to fully understand and implement the process of how the present invention applies technical means to solve technical problems and achieve technical effects.

[0028] The hydrogel disclosed in the present invention uses a hyaluronic acid-tyramine conjugate (HT) as a base material. A black rice extract (BRE) solution (main components: 5-25% anthocyanidins and anthocyanins, purchased from Tianjin Jianfeng Natural Products Research and Development Co., Ltd.) is uniformly added to the HT solution. A horseradish peroxidase (HRP; HRP, ≥150 units / mg, solid) solution and a hydrogen peroxide (H2O2) solution are then added sequentially. The hydrogel material is then enzymatically cross-linked to form a hydrogel. The cross-linking principle of the hydrogel of the present invention is that under the catalytic action of HRP, the p-hydroxyphenyl groups of tyramine on the HT form phenylene or oxyphenylene structures via C-C or C-O-C bonds, resulting in cross-linking of the molecular chains between the HT to form a hydrogel system. At the same time, HRP will also act on the black rice extract, causing the substances in the black rice extract to undergo enzyme-catalyzed reactions. The substances in the black rice extract are rich in hydroxyl groups. The black rice extract with or without HRP catalysis can form multiple hydrogen bonds with HT macromolecules to further strengthen the cross-linking inside the hydrogel.

[0029] In the prior art, in order to combine hydrogel with PTT therapy, photothermal responsive materials are usually added to the hydrogel material and loaded. Studies have reported that black rice extract alone has certain photothermal responsiveness, but it requires a higher concentration (10 mg / mL) and a higher near-infrared irradiation (hereinafter referred to as NIR) intensity (808 nm, 1 W / cm 2) to reach around 50-55°C. In the present invention, HRP enzyme-catalyzed crosslinking of the HT and BRE mixture not only catalyzes the crosslinking, but also reacts with BRE to significantly enhance the photothermal responsiveness of the BRE-loaded hydrogel, allowing the ideal photothermal conversion temperature to be achieved using only a relatively low concentration of BRE.

[0030] Experimental Example 1: Preparation of hydrogel substrate and preparation of hydrogel.

[0031] The synthesis of hyaluronic acid / tyramine conjugate (HT) was based on existing studies. Specifically, 0.5 g of sodium hyaluronate (Aladdin, CAS: 9067-32-7) was dissolved in 100 mL of 50 mM MES [(2-(N-morpholino)ethanesulfonicacid] solution. The carboxyl groups of the hyaluronic acid were then activated by adding 25 mM EDC [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide] and 47 mM NHS (N-Hydroxysuccinimide) for 30 minutes. The activated hyaluronic acid was reacted with 73 mM tyramine (Aladdin, CAS: 51-67-2) at room temperature for 24 hours. The resulting HT solution was transferred to a dialysis bag, dialyzed against distilled water for 3 days, and freeze-dried to obtain the synthesized HT.

[0032] The preparation method of the hydrogel disclosed in the present invention mainly comprises the following steps:

[0033] (1) Completely dissolve HT or BRE in water or PBS to form a solution;

[0034] (2) Add BRE solution to HT solution and mix well;

[0035] (3) Add HRP solution to the HT-BRE mixed solution in step 2 and mix well;

[0036] (4) Adding H2O2 solution to the mixed solution in step 3 and stirring uniformly, the mixture is cross-linked by enzyme catalysis to form a hyaluronic acid / tyrosine conjugate loaded black rice extract hydrogel (HT-BRE Hydrogel, hereinafter referred to as HTB).

[0037] In the final mixed system formed after all the solutions were added in step 4, the final concentration of HT was 1 wt%, the final concentration of HRP was 5 U / mL, and the final concentration of H2O2 was 0.05 wt%. In order to explore the effect of BRE content on the hydrogel system, the present invention set HT1, HTB 0.1 、HTB 0.3 and HTB 0.5The four hydrogels all used the above final concentrations of HT, HRP and H2O2, except that HT1 hydrogel did not contain BRE, HTB 0.1 、HTB 0.3 and HTB 0.5 Respectively, they represent BRE with a final concentration of 0.1wt%, 0.3wt%, and 0.5wt% in the hydrogel system. After multiple attempts by the present invention, it was found that the final concentrations of HRP and H2O2 should not be too low. If they are lower than 5U / mL or 0.05wt%, the gelation time of the HTB hydrogel will be significantly slowed, cross-linking will be uneven, and the photothermal responsiveness enhancement will be low. If the final concentration of HT is lower than 1wt%, the overall integrity of the HTB hydrogel will be poor, the gelation time will be slow, and the degree of cross-linking will be low. If the final concentration of black rice extract is lower than 0.1wt%, the photothermal responsiveness enhancement effect will be lacking.

[0038] Test Example 2: Characteristic test of hydrogel

[0039] Attachment Figure 1 The gelation time of the three hydrogels of the present invention and the pictures before and after gelation in the vials. In the pictures before and after gelation in the vials, the upper left is the state of HT1 before gelation, the upper right is the state of HT1 after gelation; the lower left is the state of HTB 0.3 Before gelation, the lower right is HTB 0.3 After gelation. With increasing amounts of BRE, gelation time increases significantly. This is likely because HRP acts on the p-hydroxyphenyl groups of tyramine on HT, forming phenylene or oxyphenylene crosslinks via C-C or C-O-C bonds. Furthermore, HRP also acts on components within BRE, so the introduction of BRE competes with the crosslinking process of HT. Furthermore, HRP enzymatic crosslinking requires hydrogen peroxide. The anthocyanidins and plant polyphenols contained in BRE are natural hydroxyl radical scavengers that consume hydrogen peroxide, slowing the HRP enzymatic crosslinking process.

[0040] In order to evaluate the stability of the four hydrogels of the present invention, 100 μL of HT1, HTB and 0.1 、HTB 0.3 and HTB 0.5 The hydrogels were immersed in 2 mL of PBS at 37°C and the weight of the hydrogels was measured regularly. Figure 2As shown, the HT1 hydrogel without BRE exhibited good stability, lasting approximately 21 days. Adding 0.1 wt% BRE to the HT hydrogel system reduced its stability, leading to complete degradation on the 14th day. Adding 0.3 wt% BRE achieved optimal stability, preventing the hydrogel system from completely degrading within 21 days. However, increasing the BRE concentration to 0.5 wt% likely resulted in insufficient cross-linking due to excessive H2O2 consumption, causing the hydrogel to swell rapidly in the PBS environment and completely degrade after 14 days.

[0041] HA, tyrosine, and HT themselves lack photothermal responsiveness. Although BRE solutions have certain photothermal responsiveness, they require high concentrations and strong irradiation. In order to evaluate the enhancing effect of HRP on the photothermal responsiveness of BRE, the present invention prepared 100 μL of separate BRE solutions containing 0.1wt%, 0.3wt%, and 0.5wt% concentrations in 1.5 mL EP tubes, referring to the final concentration of the hydrogel HTB cross-linked mixed system, and three 100 μL HRP-treated BRE solutions (final concentrations of 0.1wt%, 0.3wt%, and 0.5wt% BRE, 5U / mL HRP, and 0.05wt% H2O2, respectively) and respectively at 800 mW / cm 2 The images were recorded using a thermal imager (T120, Guide) under 808nm near-infrared irradiation. Figure 3 Compared with the maximum temperature of BRE alone after five minutes of near-infrared irradiation, the temperature of the three concentrations of BRE after HRP treatment increased by 7.8℃, 14.8℃, and 16.8℃ respectively. 0.3 +HRP、BRE 0.5 The solution with HRP can reach a maximum of 45.1℃ and 50.8℃, which shows that the photothermal responsiveness of BRE itself will be greatly improved after HRP treatment.

[0042] In the hydrogel system, the HT1 hydrogel alone was detected at (808 nm, 800 mW / cm 2 ) There was almost no photothermal response under near-infrared radiation for 5 minutes. Under the same conditions, HTB was evaluated 0.1 、HTB 0.3 and HTB 0.5 The performance (attached Figure 4(a). When the BRE content was 0.1%, the temperature of the hydrogel only increased by about 12°C from room temperature to a maximum of 38.8°C. As the BRE content increased to 0.3%, the temperature increased by about 30.5°C from room temperature to a maximum of 53.9°C. When the BRE content was further increased to 0.5%, the maximum temperature of the hydrogel under near-infrared radiation under the same conditions reached 96.1°C. In order to measure HTB 0.3 The response of hydrogel to different near-infrared radiation intensities, such as Figure 4 As shown in b, at 400mW / cm 2 , 600mW / cm 2 , 800mW / cm 2 and 1000mW / cm 2 The results showed that when the power did not exceed 800mW / cm 2 When HTB 0.3 The temperature of the hydrogel gradually increases with the increase of radiation intensity. However, when the radiation intensity reaches 1000mW / cm 2 When irradiated for approximately 3.5 minutes, the temperature exceeded 150°C, exceeding the maximum measurable temperature of the infrared thermal imager. This indicates that after HRP treatment, BRE can exhibit excellent photothermal responsiveness at low concentrations and low near-infrared irradiation intensities, significantly enhancing the photothermal responsiveness of BRE.

[0043] The present invention tests the effect of BRE content on the adhesion of hydrogels through a pigskin tensile test. First, fresh pigskin is cut into 20mm×70mm rectangles, and 200μL of hydrogel sample is gelled between two prepared 20mm×20mm pigskins. During the gelling process, ensure that the assembly is aligned and completely bonded. Then, the pigskin is allowed to fully contact with the hydrogel for 5 minutes, and an electronic tensile device (CMT2102, MTS) is used to perform a pigskin lap shear test at a tensile speed of 20mm / min, and the maximum tensile strength at the breaking point is recorded. The results are shown in the attached figure. Figure 5 As shown, HT1, HTB 0.1 、HTB 0.3 and HTB 0.5The adhesion elastic moduli of the hydrogel groups were approximately 17.5 kPa, 46.6 kPa, 65.1 kPa, and 55 kPa, respectively. The HT1 hydrogel system inherently exhibits adhesion due to its rich phenolic hydroxyl functional groups, as these groups can interact with peptides on the surface of biological tissues. Compared with the HT1 hydrogel, the introduction of more BRE (which is rich in anthocyanidins and plant polyphenols) enhances the adhesion and tensile properties of the hydrogel by participating in the gelation and HRP reaction processes. These anthocyanidins and plant polyphenols strengthen the internal connection of the hydrogel through hydrogen bonds. On the other hand, when the concentration of BRE is too high, it may lead to incomplete cross-linking of the HT hydrogel system, thereby reducing the adhesion and tensile strength of the hydrogel.

[0044] Test Example 3: Antibacterial test of hydrogel

[0045] Hydrogel dressings with antibacterial properties can alleviate inflammation, poor wound healing, and other problems caused by bacterial infection. To evaluate the inherent and photothermal antibacterial effects of the HTB hydrogel of the present invention, two common bacteria associated with wound infection were selected: Staphylococcus aureus and Escherichia coli. For the inherent antibacterial test: In a pollution-free environment, 100 μL of hydrogel samples (HT1, HTB) were prepared and distributed. 0.1 、HTB 0.3 、HTB 0.5 ) into a 96-well plate. Then, 5 μL of bacterial suspension (containing Staphylococcus aureus or Escherichia coli, with a concentration of 1×10 7 CFU / mL), and the control group had no contact with the hydrogel. After incubation for 6 hours, 200 μL of LB liquid medium was added to each well to resuspend the bacteria. Then 100 μL of the suspension was extracted from each sample and added to fresh LB liquid medium for dilution. After an additional 12 hours of incubation, the optical density of the bacterial suspension at 600 nm was measured to calculate the number of viable bacteria. The bacterial survival rate was calculated as follows: Bacterial survival rate = (A hydroge 1A blank ) / (A contro 1A blank )×100%(A blank is the absorbance of LB itself). Figure 6 As shown in Figure a, since BRE is a natural antibacterial agent, the overall antibacterial effect of HTB hydrogel is higher than that of HT1 hydrogel. With the increase of BRE content, the antibacterial effect of HTB hydrogel also increases slightly.

[0046] Based on the inherent antibacterial properties, the present invention also tested HTB 0.3 The specific steps are to prepare and distribute 100μL of HTB0.3 The hydrogels were placed in a 96-well plate, and then 5 μL of bacterial suspension (containing Staphylococcus aureus or Escherichia coli, with a concentration of 1×10 7 CFU / mL). The hydrogel was exposed to 808 nm, 0.8 W / cm 2 The near-infrared radiation was applied for 0, 1, 3, 5, 10 and 15 minutes (timed after the surface temperature of the hydrogel reached 45°C). After near-infrared irradiation, 200 μL of LB liquid culture medium was added to each well to resuspend the bacteria. 100 μL of the suspension was then extracted from each sample, and each sample was diluted by adding fresh LB liquid culture medium twice. The samples were incubated in a constant temperature incubator at 37°C for 18-24 hours. The optical density (OD) of the bacterial suspension at 600 nm was measured to estimate the number of viable bacteria. The bacterial survival rate was calculated by the following formula: Bacterial survival rate = (A hydrogel -A blank ) / (A control -A blank )×100%(A blank is the absorbance of LB itself). Figure 6 As shown in b, after 5 minutes of near-infrared irradiation, HTB 0.3 The antibacterial efficiency of the hydrogel against Escherichia coli and Staphylococcus aureus was 66.5% and 79.1%, respectively. After 10 minutes of irradiation, both bacteria were almost completely eliminated. These results indicate that HTB 0.3 The hydrogel exhibited effective photothermal antibacterial capabilities, highlighting its potential in preventing and effectively treating bacterial infections at wound sites.

[0047] Test Example 4: Antioxidant Test of Hydrogel

[0048] Hydrogel wound dressings with antioxidant properties are helpful in alleviating excessive reactive oxygen species (ROS) in skin wounds. The present invention uses DPPH and hydroxyl radical scavenging efficiency tests to evaluate the antioxidant capacity of HTB hydrogel in vitro. First, the DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging capacity is specifically: prepare 150 μL of HT1, HTB in a 48-well plate. 0.1 、HTB 0.3 and HTB 0.5 The hydrogel sample was used as the experimental group; 150 μL of deionized water was added to the well plate instead of the hydrogel as the control group; 500 μL of ethanol and 25 μL of 1 mM DPPH solution were added to all groups in sequence; the plate was incubated in the dark for 1 hour, and then the absorbance of the solution in each well at a wavelength of 517 nm was recorded. DPPH scavenging rate (%) = (A control -A hydrogel ) / Acontrol ×100%. As attached Figure 7 As shown in a, HT1, HTB 0.1 、HTB 0.3 and HTB 0.5 The scavenging efficiencies of the hydrogels were 15.9%, 14.7%, 39.8%, and 48.9%, respectively. These results indicate that HTB hydrogels are very effective in scavenging nitrogen radicals with increasing BRE content.

[0049] The hydroxyl radical scavenging ability test is as follows: prepare 150 μL of HT1, HTB 0.1 、HTB 0.3 and HTB 0.5 Hydrogel sample; 300 μL of FeSO4 solution (2 mM) and 250 μL of Safranin O (360 μg / mL) were added to the hydrogel sample and incubated for 10 minutes; then 400 μL of 6% H2O2 was added and incubated at 50°C for 1 hour; the blank group replaced the hydrogel with 150 μL of deionized water; in the control group, the hydrogel and H2O2 solution were replaced with 150 μL and 400 μL of deionized water, respectively. The absorbance of the sample was measured at 492 nm. The hydroxyl radical scavenging rate was calculated according to the formula: Hydroxyl radical scavenging rate (%) = (A hydrogel -A blank ) / (A control -A blank )×100%. Figure 7 As shown in b, HT1, HTB 0.1 、HTB 0.3 and HTB 0.5 The scavenging efficiencies of the hydrogels were 84.4%, 83.19%, 87.2%, and 89.1%, respectively. These results indicate that both HT and HTB hydrogels can effectively neutralize hydroxyl radicals, and their hydroxyl radical scavenging efficiencies slightly increase with increasing BRE content.

[0050] Test Example 5: Biocompatibility test of hydrogel

[0051] The present invention evaluates the biosafety of four hydrogels by cell compatibility, blood compatibility and tissue compatibility to ensure that they meet the requirements for wound dressing. First, for cell compatibility, the present invention adopts CCK-8 detection method: initially prepare 100 μL of sterile hydrogel (HT1, HTB 0.1 ,HTB 0.3 ,HTB 0.5 ), and soaked in 2 mL of 1640 complete medium for 24 hours to obtain the hydrogel extract. At the same time, L929 (mouse fibroblasts) were seeded into 96-well plates at a density of 4×10 3After culturing for 24 hours, the control group was replaced with normal 1640 complete medium, while the experimental group was added with hydrogel extract. After culturing for another 24 and 48 hours, the culture medium of both the control and experimental groups was replaced with a medium containing 10% CCK-8 and incubated for another 2 hours. The absorbance was then measured at 450 nm. Figure 8 As shown in a, the viability of L929 cells in all HTB groups exceeded 90%, which met the requirement of greater than 80% in the international standard (ISO 10993-5).

[0052] The blood compatibility test steps are as follows: prepare 50 μL of hydrogel (HT1, HTB 0.1 ,HTB 0.3 ,HTB 0.5 ) and placed in 1 mL of normal saline, incubated at 37 ° C for 30 minutes, and then added with 20 μL of anticoagulated blood as the hydrogel group (denoted as A hydrogel At the same time, 20 μL of anticoagulated blood was added to 1 mL of deionized water as a positive control group (denoted as A positive ), and then add 20 μL of anticoagulated blood to 1 mL of normal saline as the negative control group (denoted as A negative All groups were incubated at 37°C for 1 hour. After incubation, each group was centrifuged at 2000 rpm for 5 minutes, and the absorbance of the supernatant of each group was measured at 545 nm. The hemolysis rate was calculated as follows: Hemolysis rate = (A hydrogel -A negative ) / (A positive -A negative )×100%. Figure 8 As shown in b, HT1, HTB 0.1 、HTB 0.3 and HTB 0.5 The hemolysis rates of the hydrogels were 1.78%, 1.46%, 1.29% and 1.62%, respectively, which met the standard for non-hemolytic materials (ISO 10993-4), ie, a hemolysis rate of less than 5%.

[0053] The present invention also conducted a tissue compatibility test, specifically, 100 μL of HTB 0.3 The hydrogel was implanted subcutaneously on the back of mice, and histocompatibility was assessed by H&E staining on day 14. Figure 9 As shown in the figure, no obvious inflammatory reaction was observed in the skin tissue around the hydrogel implantation. In addition, compared with the control group, no obvious pathological changes were observed in the main organs of the hydrogel implantation group. The results of blood biochemical analysis showed that compared with the normal group, HTB 0.3 The hydrogel had no significant effect on the activities of GOT, GPT, and AKP, indicating that the bioactive hydrogel did not cause hepatotoxicity during implantation. 0.3The hydrogel was found to be safe for in vivo implantation. These results comprehensively demonstrate the cytocompatibility, hemocompatibility, and tissue compatibility of the HTB hydrogel, indicating its potential safety and effectiveness in medical applications.

[0054] Test Example 6: Test on the ability of hydrogel materials to promote wound healing.

[0055] Taking into account the above-mentioned properties of the hydrogel of the present invention, the healing effect of the hydrogel as a wound dressing on infected wounds was tested. The specific steps were as follows: a full-thickness skin wound with a diameter of 8 mm was applied to the back of each mouse (Kunming mouse, 30 g ± 3 g), and then 50 μL of Staphylococcus aureus (1 x 10 7 CFU / mL), and the infection was induced for 24 hours. Then the mice were randomly divided into four groups: control group, HT1 group, HTB group 0.3 Groups and HTB 0.3 +NIR group, control group received 100 μL PBS treatment; HT1 group received 100 μL HT hydrogel treatment; HTB 0.3 Group received 100 μL of HTB 0.3 Hydrogel treatment; HTB 0.3 +NIR group used 100 μL HTB 0.3 Hydrogel treatment and additional near-infrared irradiation (800 mW / cm 2 , 10 minutes). Pictures of the wound area were taken on day 0, day 3, day 7, and day 14, the wound area was measured using ImageJ software, and the wound healing rate was calculated using the following formula: Wound healing rate = (A day0 -A dayN ) / A day0 ×100%.A day0 and A dayN The wound areas are on day 0 and day N respectively. Figure 10 As shown, HTB 0.3 The +NIR group showed the fastest wound healing speed, followed by HTB 0.3 group, HT1 group and control group. These results confirmed that HTB 0.3 The hydrogel has a significant effect in promoting the healing of infected wounds after combining with photothermal therapy.

[0056] To further elucidate the mechanism of hydrogel in promoting wound healing, the present invention selected CD86 (M1 macrophage marker) and CD206 (M2 macrophage marker) to evaluate the inflammatory level of injured skin tissue on the 14th day (see Appendix Figure 11 The control group had a higher expression level of CD86, indicating an obvious inflammatory state; while in HT1 and HTB 0.3In the hydrogel-treated group, the expression of CD86 decreased significantly and gradually; 0.3 The +NIR group showed the lowest expression level of CD86 and the highest expression level of CD206, while the HTB 0.3 These data indicate that HTB 0.3 The hydrogel reduced wound inflammation and promoted the polarization of M2 macrophages, an effect further enhanced by combining it with NIR irradiation.

[0057] In order to study the effect of hydrogel on angiogenesis in vivo, the expression of CD31 and α-SMA was determined by immunofluorescence staining. α-SMA is often used as a specific marker of vascular smooth muscle cells to locate new blood vessels in regenerated tissues, while CD31 is used to detect the presence of vascular endothelial cells and evaluate angiogenesis. 0.3 Groups and HTB 0.3 The expression levels of CD31 and α-SMA in the +NIR group were higher than those in the other groups on day 14. 0.3 The +NIR group had the highest expression levels of CD31 and α-SMA. 0.3 The hydrogel showed enhanced angiogenesis effect at 14 days, and its ability to promote angiogenesis was even stronger after NIR treatment, indicating that HTB 0.3 The +NIR group had significant advantages in inhibiting inflammation and promoting angiogenesis.

[0058] In the present invention, all experiments were performed in at least three replicates, and statistical analyses were performed using t-test, one-way ANOVA, and two-way ANOVA in GraphPad Prism. A p-value less than 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0059] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A photothermal response enhanced hydrogel, characterized in that: The hydrogel contains: a cross-linked product formed by a matrix and a solution of water or phosphate buffer as the remainder, wherein the matrix is ​​a mixed solution of hyaluronic acid / tyrosine conjugate and black rice extract, and the cross-linking agent of the matrix is ​​an enzyme catalytic system solution of a horseradish peroxidase / hydrogen peroxide combination; the photothermal responsiveness of the hydrogel to near-infrared radiation is enhanced, preferably to near-infrared radiation of 808 nm.

2. The hydrogel according to claim 2, characterized in that The proportions of the components in the mixed system of the matrix, cross-linking agent and solvent of the hydrogel are as follows: the final concentration of the hyaluronic acid / tyrosine conjugate is 1 wt% or more, preferably 1 wt%; the final concentration of the black rice extract is 0.1 wt% or more, preferably 0.1-0.5 wt%, and more preferably 0.3 wt%; the horseradish peroxidase is 5 U / mL or more, preferably 5 U / mL; the hydrogen peroxide is 0.05 wt% or more, preferably 0.05 wt%; and the remaining components are water or phosphate buffer.

3. The method for preparing the hydrogel according to any one of claims 1 to 2, characterized in that: The method comprises the following steps: (1) preparing a solution of hyaluronic acid / tyrosine conjugate and black rice extract, and uniformly mixing the solution; (2) adding a horseradish peroxidase solution to the mixed solution of step 1, and uniformly mixing the solution; (3) Add hydrogen peroxide solution to the mixed solution in step 2 and stir evenly to form a hydrogel.

4. The method according to claim 3, characterized in that In the mixed system after adding the hydrogen peroxide solution in step 3, the final concentration of the hyaluronic acid / tyrosine conjugate is 1 wt% or more, preferably 1 wt%; the final concentration of the black rice extract is 0.1 wt% or more, preferably 0.1-0.5 wt%, more preferably 0.3 wt%; the horseradish peroxidase is 5 U / mL or more, preferably 5 U / mL; the hydrogen peroxide is 0.05 wt% or more, preferably 0.05 wt%; and the remainder is water or phosphate buffer.

5. An in vitro anti-inflammatory and / or anti-oxidative and / or antibacterial method, characterized in that: The method is achieved by using the hydrogel according to any one of claims 1-2, or the hydrogel obtained by the preparation method according to any one of claims 3-4.

6. The method according to claim 5, characterized in that By utilizing the enhanced photothermal responsiveness of the hydrogel, the hydrogel is irradiated with near infrared light having a wavelength of 808 nm to enhance the anti-inflammatory and / or antibacterial capabilities of the hydrogel.

7. A use in the preparation of anti-inflammatory and / or antioxidant and / or antibacterial drugs, characterized in that: The application uses the hydrogel according to any one of claims 1-2, or the hydrogel obtained by the preparation method according to any one of claims 3-4 to prepare the drug; preferably, the photothermal responsiveness of the hydrogel is utilized, and the drug is irradiated with near-infrared rays to enhance the anti-inflammatory and / or antibacterial ability of the drug containing the hydrogel.

8. An application in the preparation of a drug for repairing skin damage, characterized in that: The application is to use the hydrogel according to any one of claims 1-2 to prepare a drug for repairing skin damage, or to use the hydrogel obtained by the preparation method according to any one of claims 3-4 to prepare a drug for repairing skin damage.

9. A wound dressing for repairing skin wounds, characterized in that: The wound dressing contains the hydrogel according to any one of claims 1-2, or the wound dressing contains the hydrogel obtained by the preparation method according to any one of claims 3-4.

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