Enzyme-crosslinked polyvinyl protocatechuic aldehyde hydrogel as well as preparation method and application thereof

Abstract

The invention provides enzyme cross-linked polyvinyl protocatechuic aldehyde hydrogel as well as a preparation method and application thereof. The hydrogel is prepared from a cross-linked substance formed by a matrix and the balance of water or a phosphate buffer solution, the matrix is a polyvinyl protocatechuic aldehyde solution, and a cross-linking agent of the matrix is an enzyme catalysis system solution of horseradish peroxidase / hydrogen peroxide combination; the hydrogel has photo-thermal responsiveness to near infrared rays, and preferably, the hydrogel has photo-thermal responsiveness to near infrared rays with the wavelength of 808 nm. The hydrogel material is easy to prepare, green in synthesis and good in biological safety, the advantages of polyvinyl alcohol and protocatechualdehyde are combined, polyvinyl protocatechualdehyde is synthesized, and photo-thermal response type hydrogel is rapidly formed through catalysis of horseradish peroxidase / hydrogen peroxide. Extra photo-thermal corresponding materials do not need to be added, and the effects of rapidly killing bacteria at the wound part and promoting the wound to heal can be achieved by combining the characteristics of oxidation resistance, bacteria resistance and the like of the hydrogel and adopting a photo-thermal therapy method.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and specifically relates to an enzyme-crosslinked polyvinyl alcohol protocatechuic aldehyde hydrogel, a preparation method and application thereof, which is a hydrogel prepared by enzyme crosslinking based on polyvinyl alcohol protocatechuic aldehyde and its application in skin wounds. Background Art

[0002] The skin is the largest organ in the human body and has multiple important functions, including protection, temperature regulation, and sensation. Protection is particularly crucial. Acting as a barrier between the human body and the external environment, the skin effectively prevents the invasion of harmful external substances and microorganisms. When the skin is damaged, such as by cuts, abrasions, or burns, this barrier function is disrupted, and the local environment at the wound site changes, such as increased humidity and changes in pH, creating favorable conditions for bacterial growth. Once bacteria enter the wound, they can cause localized infection, manifesting as redness, swelling, heat, pain, pus, and even systemic infection in severe cases.

[0003] Antibiotics are a common treatment for wound infections. However, overuse and misuse of antibiotics can lead to bacterial resistance. Furthermore, antibiotics typically require oral administration or injection, potentially causing systemic side effects. Furthermore, topical antibiotics can sometimes be difficult to achieve effective concentrations, resulting in suboptimal therapeutic effects. Compared to antibiotic treatment, photothermal therapy (PTT) utilizes photothermally responsive materials to generate heat under specific wavelengths of light, thereby killing pathogens. This approach offers numerous advantages over antibiotics. For example, PTT does not rely on chemical agents and does not contribute to bacterial resistance. Furthermore, PTT's localized action allows for precise control of the treatment area, minimizing damage to surrounding healthy tissue. It can also rapidly and effectively kill bacteria, accelerating wound healing. However, current PTT materials, such as gold nanoparticles, carbon nanotubes, and graphene, have drawbacks in biosafety and biocompatibility. These materials can trigger immune responses, leading to inflammation or allergic reactions. Therefore, there is a need to develop biosafe and biodegradable PTT materials.

[0004] Hydrogel is a polymer material with a three-dimensional network structure that can absorb large amounts of water and has good biocompatibility and flexibility. Due to its high water content, it can maintain a moist environment in the wound, which is conducive to the healing process. It can also provide a physical barrier to prevent bacterial invasion. In addition, the hydrogel itself can load some materials that are beneficial to wound healing. In the prior art, in order to make hydrogel materials photothermal responsive, additional photothermal responsive materials are usually added to the hydrogel material and loaded with PPT to accelerate wound healing. However, this approach not only increases costs, but the photothermal responsive material itself also has certain biosafety risks. Polyvinyl alcohol (PVA), a synthetic polymer material commonly used to prepare hydrogels, has good biocompatibility, chemical stability, and mechanical properties. However, the lack of functional groups in the PVA molecular structure results in limited antibacterial properties and drug loading capacity. Protocatechualdehyde (Protocatechualdehyde or 3,4-Dihydroxybenzaldehyde) is a naturally occurring phenolic compound with significant antibacterial properties. When used alone, it has poor stability. High concentrations of PA can cause irritation, so its release needs to be controlled. Summary of the Invention

[0005] In response to the above-mentioned technical problems existing in the prior art, the present invention aims to provide an enzyme-crosslinked polyvinyl alcohol protocatechuic aldehyde hydrogel and its preparation method and application. The hydrogel is a hydrogel formed by enzyme crosslinking of polyvinyl alcohol protocatechuic aldehyde. The hydrogel itself is simple to prepare and low-cost. It has good biosafety, antibacterial, antioxidant, environmentally responsive sustained release, and photothermal responsiveness, thereby overcoming the technical problems existing in the prior art.

[0006] According to the first aspect of the technical solution of the present invention, the present invention provides a photothermal responsive hydrogel, which contains: a cross-linked product formed by a matrix, and the rest is water or phosphate buffer; the matrix is ​​a polyvinyl alcohol protocatechuic aldehyde solution, and the cross-linking agent of the matrix is ​​an enzyme catalytic system solution of a horseradish peroxidase / hydrogen peroxide combination; the hydrogel has photothermal responsiveness to near-infrared, preferably has photothermal responsiveness to near-infrared at 808nm.

[0007] Furthermore, in the mixed system of the matrix, cross-linker and solvent of the hydrogel, the proportions of each component are as follows: the final concentration of polyvinyl alcohol protocatechuic aldehyde is 5 wt% or more, preferably 5 wt%; the horseradish peroxidase is 4 U / mL or more, preferably 4 U / mL; the hydrogen peroxide is 0.02 wt% or more, preferably 0.02 wt%-0.08 wt%, and more preferably 0.04 wt%-0.08 wt%; the rest is water or phosphate buffer; the pH value of the polyvinyl alcohol protocatechuic aldehyde solution is initially prepared between 5 and 8.

[0008] The present invention provides a method for preparing a hydrogel, which is characterized by comprising the following steps: (1) dissolving polyvinyl alcohol protocatechualdehyde in water or phosphate buffer to prepare a 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, the pH value of the polyvinyl alcohol protocatechuic aldehyde solution initially prepared in step 1 is between 5 and 8; in the mixed system after the hydrogen peroxide solution is added in step 3, the final concentration of polyvinyl alcohol protocatechuic aldehyde is 5 wt% or more, preferably 5 wt%; the horseradish peroxidase concentration is 4 U / mL or more, preferably 4 U / mL; the hydrogen peroxide concentration is 0.02 wt% or more, preferably 0.02 wt%-0.08 wt%, and more preferably 0.04 wt%-0.08 wt%; and the remainder is water or phosphate buffer.

[0010] The present invention provides an in vitro antioxidant and / or antibacterial method, characterized in that the method is implemented using the hydrogel described herein, or a hydrogel obtained using the preparation method described herein. Furthermore, utilizing the photothermal responsiveness of the hydrogel, the hydrogel is irradiated with near-infrared light having a wavelength of 808 nm to enhance its antibacterial ability.

[0011] The present invention provides an application in the preparation of antioxidant and / or antibacterial drugs, wherein the application uses the hydrogel of the present invention, or the hydrogel obtained by the preparation method of the present invention to prepare the drug; utilizing the photothermal responsiveness of the hydrogel, the drug is irradiated with 808nm near-infrared rays to enhance the antibacterial ability of the drug containing the hydrogel.

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

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

[0014] The hydrogel material of the present invention is easy to prepare, has green synthesis and good biosafety, and combines the advantages of polyvinyl alcohol and protocatechuic aldehyde. The hydrogel quickly gels under the catalysis of horseradish peroxidase / hydrogen peroxide, and no additional photothermal response materials need to be added. The cross-linked structure generated under the catalysis of horseradish peroxidase itself has excellent photothermal response. Combined with the hydrogel's own antioxidant and antibacterial properties, photothermal therapy can achieve the effect of quickly killing bacteria in the wound and promoting healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The gelation time of the hydrogel of the present invention and the photothermal response change of the protocatechuic aldehyde solution before and after being treated with horseradish peroxidase and different contents of hydrogen peroxide, wherein Figure 1 a is the gelation time of the hydrogel of the present invention, Figure 1 b shows the photothermal response changes of protocatechuic aldehyde solution before and after treatment with horseradish peroxidase and different concentrations of hydrogen peroxide;

[0016] Figure 2 The degradation rates of the three hydrogels of the present invention in PBS (pH=6) and the degradation rates of the PPA4 hydrogel in PBS solutions at different pH values ​​are shown in Table 1. Figure 2 a is the degradation rate of the three hydrogels of the present invention in PBS (pH=6) environment, Figure 2 b is the degradation rate of the PPA4 hydrogel of the present invention in PBS solution under different pH conditions;

[0017] Figure 3 The three hydrogels of the present invention were subjected to 800 mW / cm 2 Temperature changes of near-infrared irradiation and temperature changes of PPA4 hydrogel using near-infrared irradiation of different powers, among which Figure 3 a is the three hydrogels of the present invention using 800 mW / cm 2 Temperature changes of near-infrared irradiation, Figure 3 b shows the temperature change of the PPA4 hydrogel of the present invention when irradiated with near-infrared radiation of different powers;

[0018] Figure 4 The cell activity of the three hydrogels of the present invention was evaluated by using the extract model combined with CCK8, as well as the hemolysis evaluation of the three hydrogels, wherein Figure 4 a is the cell activity of the three hydrogels of the present invention evaluated using the extract model combined with CCK8, Figure 4 b is the hemolysis evaluation of the three hydrogels of the present invention;

[0019] Figure 5 The tissue compatibility of the PPA4 hydrogel of the present invention was evaluated by H&M staining after 14 days of implantation in mice;

[0020] Figure 6 The scavenging rates of the three hydrogels of the present invention on DPPH free radicals and the scavenging rates of the three hydrogels on hydroxyl free radicals, wherein Figure 6 a is the scavenging rate of the three hydrogels of the present invention on DPPH free radicals, Figure 6 b is the scavenging rate of hydroxyl radicals by the three hydrogels of the present invention;

[0021] Figure 7 The intrinsic antibacterial rates of the three hydrogels of the present invention against Escherichia coli and Staphylococcus aureus and the photothermal antibacterial rates of the PPA4 hydrogel against Escherichia coli and Staphylococcus aureus are shown in FIG. Figure 7 a is the intrinsic antibacterial rate of the three hydrogels of the present invention against Escherichia coli and Staphylococcus aureus, Figure 7 b is the photothermal inhibition rate of the PPA4 hydrogel of the present invention against Escherichia coli and Staphylococcus aureus;

[0022] Figure 8 This is a diagram showing the healing of the PPA4 hydrogel of the present invention in a mouse infected wound model; DETAILED DESCRIPTION

[0023] 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.

[0024] The hydrogel disclosed in the present invention uses polyvinyl alcohol protocatechualdehyde (or polyvinyl-protocatechualdehyde-acetal, hereinafter referred to as PPA) as a base material. A horseradish peroxidase (HRP) solution is first added to a PPA solution and mixed uniformly. Finally, hydrogen peroxide (H2O2) is added to the solution to crosslink and form a gel to obtain a hydrogel material. The crosslinking principle of the present invention is as follows: the aldehyde groups of protocatechualdehyde (hereinafter referred to as PA) are grafted onto polyvinyl alcohol (hereinafter referred to as PVA) via acetal to form a PPA base material. When HRP solution and H2O2 solution are sequentially added to the PPA solution, the HRP enzyme catalyzes the catechol groups of PA, forming dimers or polymers of phenylene or oxyphenylene structures between the groups via C-C or C-O-C bonds, resulting in crosslinking of the PPA molecular chains to form a hydrogel. The catechol groups that are not catalyzed by HRP, as well as the dimers or polymers formed by the groups after being catalyzed by HRP, are rich in hydroxyl groups and can form multiple hydrogen bonds with themselves or with polyvinyl alcohol to further strengthen the cross-linking degree of the hydrogel.

[0025] In order to combine hydrogels with PTT therapy in the art, photothermal responsive materials are usually added to the hydrogel material and loaded. Unlike the prior art, the present invention finds that during the enzyme-catalyzed reaction of HRP and PPA, the dimer or polymer formed with CC or COC bonds between PA catechol groups can not only be used for hydrogel cross-linking, but also exhibits excellent photothermal responsiveness to near-infrared irradiation (hereinafter referred to as NIR). Thus, there is no need to add additional photothermal responsive materials to the enzyme-crosslinked hydrogel of the present invention. While utilizing the cross-linking of HRP, the photothermal response (near-infrared at a wavelength of 808nm) is imparted to the hydrogel of the present invention to further increase its functionality.

[0026] Experimental Example 1: Preparation of hydrogel substrate and preparation of hydrogel

[0027] The polyvinyl alcohol protocatechuic aldehyde disclosed in the present invention is prepared by forming an acetal bond from 1.12 g of PVA and 1.2 g of PA in an aqueous solution using acid catalysis. The specific synthesis process is as follows: (1) taking 16 ml of a 7 wt% aqueous solution of PVA (Shanghai Yuanye Biotechnology Co., Ltd., 1799S (L) type, CAS: 9002-89-5), weighing 1.2 g of protocatechuic aldehyde (Shanghai Yuanye Biotechnology Co., Ltd.; 3,4-Dihydroxybenzaldehyde; CAS: 139-85-5) and dissolving it in 30 ml of a 95% ethanol solution; (2) stirring the polyvinyl alcohol solution at 40° C., and gradually adding the protocatechuic aldehyde into the solution; The catechualdehyde solution forms a uniform mixed state; (3) HCl is added to the uniform mixed solution, the pH is adjusted to 1, and the activation reaction is stirred for 30-60 minutes; (4) After the activation reaction, the temperature is raised to 70°C and the stirring reaction is performed for 4 hours; (5) After the reaction is completed, the reaction solution is uniformly mixed into a mixture of anhydrous ethanol and sodium hydroxide aqueous solution (volume ratio, 99% anhydrous ethanol: sodium hydroxide aqueous solution = 10:3; the pH value of the sodium hydroxide aqueous solution is 12) to obtain a precipitate, and the collected precipitate is rinsed in the above-mentioned mixture of anhydrous ethanol and sodium hydroxide aqueous solution for multiple times to remove unreacted reactants. The final precipitate obtained is the purified reaction product of polyvinyl alcohol condensed protocatechualdehyde.

[0028] The preparation method of the hydrogel material disclosed in the present invention mainly comprises the following steps:

[0029] (1) Dissolve PPA in water or PBS to form a solution;

[0030] (2) Add HRP solution to the PPA solution and mix well;

[0031] (3) Adding H2O2 solution to the mixed solution in step (2) and stirring uniformly to form a hydrogel matrix.

[0032] The concentration of the appropriate PPA solution configured in step 1 should be appropriate. A concentration lower than 5wt% is not conducive to cross-linking, and a concentration that is too high will cause uneven dissolution of PPA. Since the cyclic acetal on PPA is unstable under strong acid conditions, and considering that the enzyme activity of HRP is better in an environment of pH = 5-9. The present invention has concluded through multiple experiments that if the pH value of the configured PPA solution is less than 5, the acetal bond is unstable in an acidic environment and is prone to protocatechualdehyde debranching, resulting in insufficient grafting and difficulty in gelation; if the pH value of the configured PPA solution is greater than 8, it may be due to the fact that PPA itself has a certain effect on the HRP enzyme activity in an alkaline environment, resulting in slower gelation and insufficient photothermal responsiveness. Therefore, the pH value of the PPA solution configured in step 1 is between 5-8, thereby ensuring that the hydrogel has a short cross-linking time, a suitable cross-linking degree and sufficient photothermal responsiveness. In addition, if the final concentration of HRP is lower than 4U / mL, it will result in slow gelation time, incomplete gelation, and uneven cross-linking.

[0033] After all solutions were added in step 3, the final mixed volume formed had a final concentration of 5 wt% PPA solution and a final concentration of 4 U / mL HRP. Three hydrogels were prepared according to the final H2O2 concentrations, namely PPA2, PPA4, and PPA8, representing final H2O2 concentrations of 0.02 wt%, 0.04 wt%, and 0.08 wt%, respectively, to explore the effects of different cross-linking degrees on the properties of PPA hydrogels.

[0034] Test Example 2: Characteristic test of hydrogel

[0035] The cross-linking process of horseradish peroxidase requires the participation of hydrogen peroxide. The amount of hydrogen peroxide added affects the speed of HRP enzyme reaction. Figure 1 As shown in Figure 1, the three hydrogels of the present invention gel very quickly, forming a photothermally responsive hydrogel within 20 seconds. Furthermore, the gelation time shortens with increasing amounts of hydrogen peroxide. Therefore, increasing the amount of hydrogen peroxide during the gelation process of the hydrogels of the present invention can increase the crosslinking speed and degree of the hydrogels.

[0036] The solution of protocatechuic aldehyde itself lacks photothermal response under near-infrared irradiation with a wavelength of 808 nm. Figure 1 As shown in (b), 100 μL of 0.5 wt% PA and 100 μL of HRP-treated PA were prepared in 1.5 mL EP tubes, with final concentrations of 0.5 wt% PA, 4 U / mL HRP, and 0.02 wt% (PA / HRP / 2), 0.4 wt% (PA / HRP / 4), and 0.8 wt% (PA / HRP / 8) H2O2. The cells were then heated at 800 mW / cm 2Under 808nm near-infrared irradiation, the results were recorded using a thermal imager (T120, Guide). By comparison, it can be seen that the PA treated with HRP exhibited good photothermal responsiveness, and the photothermal responsiveness of PA was significantly improved with the addition of hydrogen peroxide.

[0037] The present invention is based on the hydrogel formed by PVA, which has a certain stability. After PA is grafted onto PVA using acetal, the special properties of the acetal bond give the PPA hydrogel environmental response characteristics. In order to test its environmental degradation degree, as shown in the attached Figure 2 As shown in Figure a, 100 μL of PPA2, PPA4, and PPA8 hydrogels were prepared and immersed in 2 mL of PBS (pH = 6) at 37°C. The weight of the hydrogel was tested at regular intervals. It can be seen that as the amount of hydrogen peroxide decreased, the weight of the PPA hydrogel in the PBS environment increased. The increase in weight is due to the decrease in the degree of cross-linking of the hydrogel in the environment, which makes it easier to swell and thus absorb more water. This shows that the increase in hydrogen peroxide content will increase the cross-linking degree and stability of the hydrogel. As shown in the attached figure, Figure 2 As shown in Figure b, 100 μL of PPA4 was prepared and immersed in 2 mL of PBS with different pH values ​​at 37°C. It can be seen that as the pH value increases, the cross-linking degree of the hydrogel decreases and the weight increase caused by swelling becomes more obvious. Therefore, the PPA hydrogel of the present invention is responsive to the external acid-base environment.

[0038] The present invention also conducted a photothermal responsiveness test on three PPA hydrogels, as shown in the attached figure. Figure 3 As shown in a, 100 μL of PPA2, PPA4, and PPA8 were illuminated by near-infrared (800 mW / cm 2 ) After 5 minutes of irradiation, the maximum temperatures were approximately 25.3°C, 51.5°C, and 56.9°C. It can be seen that when insufficient hydrogen peroxide participates in the HRP enzyme reaction, although the PPA substrate can be cross-linked into a hydrogel, it cannot show good photothermal responsiveness due to incomplete cross-linking. When the final concentration of hydrogen peroxide reaches 0.04wt% and 0.08wt%, it can have good photothermal responsiveness to the near infrared. Figure 3 In b, at different power densities (600 mW / cm 2 , 800mW / cm 2 and 1000mW / cm 2 ) were used to study the temperature changes of the PPA4 hydrogel by irradiating a 100 μL volume of the hydrogel with 808 nm near-infrared radiation for 5 minutes. As the laser power density increased, the maximum temperature increased from 45.7°C to 61.8°C, demonstrating the efficient photothermal conversion capability of the PPA4 hydrogel.

[0039] Test Example 3: Biocompatibility test of hydrogel

[0040] In order to ensure that the hydrogel of the present invention has good biosafety when used as a dressing, the present invention uses the CCK8 method to evaluate the cell compatibility of the three hydrogels PPA2, PPA4, and PPA8. First, 100 μL of the three sterile hydrogels are soaked in 2 mL of 1640 complete culture medium for 24 hours to obtain a gel extract. At the same time, L929 cells are seeded in a 96-well plate at a density of 4000 cells per well. After culturing for 24 hours, the culture medium is replaced with the hydrogel extract. The control group is cultured with normal 1640 complete culture medium. After culturing for 24 and 48 hours, the extract and culture medium are removed, and the cell viability is measured using a CCK-8 kit. The results are shown in the attached figure. Figure 4 As shown in Figure a. Except for the PPA2 group, whose cytocompatibility did not exceed 90%, the cytocompatibility of the other two groups exceeded 90%. This may be due to insufficient hydrogen peroxide for HRP enzyme cross-linking, resulting in low PPA2 cross-linking and a large amount of free PPA substrate, leading to poor cytocompatibility. When the hydrogen peroxide content is sufficient, it not only imparts better photothermal responsiveness to the PPA hydrogel, but also increases the stability of the hydrogel material and improves cytocompatibility as the degree of cross-linking increases.

[0041] The hemolysis test tested the blood compatibility of the three hydrogels PPA2, PPA4, and PPA8 of the present invention. The specific experimental steps were as follows: incubate 50 μL of hydrogel with 1 mL of normal saline at 37°C for 30 minutes. The positive control and negative control used 1 mL of separate deionized water and normal saline, respectively. Then, 20 μL of fresh mouse heart blood was added to each group, and the incubation was continued at 37°C for 1 hour. After the incubation, the hydrogel was discarded and the sample was centrifuged at 2000 r / min for 5 minutes. After centrifugation, the absorbance of the supernatant at 545 nm was measured, and the morphology of red blood cells was observed under an optical microscope. The formula for calculating the hemolysis rate is: Hemolysis rate = (A hydrogel -A negative ) / (A positive -A negative )×100%, if attached Figure 4 As shown in Figure b, the hemolysis rates of the three hydrogels of the present invention are all as low as 5%, which is in line with the allowable limit of international standards, and thus have good blood compatibility.

[0042] The present invention also conducted a bio-tissue compatibility test by implanting 100 μL of PPA4 hydrogel into the back of mice. Fourteen days later, the main organs and muscles of the mice were taken and evaluated by H&E staining. No significant inflammation and physiological lesions were found. Figure 5 shown.

[0043] Test Example 3: Antioxidant Test of Hydrogel

[0044] Protocatechuic aldehyde is a naturally occurring phenolic compound that has certain antioxidant properties. It can give hydrogel dressings the ability to reduce the level of ROS at the wound, which is beneficial to wound healing. The present invention uses DPPH and safranin O to evaluate the free radical scavenging rates of the three hydrogels. The test method for the DPPH free radical scavenging rate is as follows: prepare 150 μL of PPA2, PPA4, and PPA8 hydrogels and soak them in 0.5 mL of anhydrous ethanol, then add 25 μL of DPPH (1 mM) solution, incubate in the dark for 1 hour, and use 150 μL of deionized water (DIW) instead of hydrogel in the control group. Finally, the absorbance of the sample at 517 nm is measured, and the DPPH free radical scavenging rate is calculated according to the following formula: DPPH scavenging rate (%) = (A control -A hydrogel ) / A control ×100%, as attached Figure 6 As shown in a, the scavenging rate of the hydrogel on DPPH free radicals is above 40%.

[0045] In addition, the scavenging ability of the hydrogel of the present invention for hydroxyl radicals was evaluated using safranin O. First, 150 μL of the three hydrogels of the present invention were prepared, mixed with 300 μL of FeSO4 (2 mM) and 250 μL of safranin O solution (360 μL / mL), and incubated for 10 minutes. Then 400 μL of H2O2 (6%) was added and incubated at 50°C for 1 hour. In the blank group, 150 μL of deionized water was used to replace the hydrogel; in the control group, 150 μL and 400 μL of deionized water were used to replace the hydrogel and H2O2 solution, 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 6 As shown in Figure b, the scavenging rate of hydroxyl radicals of all PPA hydrogels can reach more than 50%, and the scavenging rate increases slightly with the increase of cross-linking degree.

[0046] Test Example 4: Antibacterial test of hydrogel

[0047] The present invention used Escherichia coli and Staphylococcus aureus to test the intrinsic antibacterial properties of the three PPA hydrogels and the photothermal antibacterial properties of the PPA4 hydrogel. For the intrinsic antibacterial test, 100 μL of sterile PPA2, PPA4, and PPA8 hydrogels were prepared in a 96-well plate, and then 5 μL of Escherichia coli or Staphylococcus aureus bacterial suspension (1×10 7 CFU / mL, and the control group contained only 5 μL of bacterial suspension (1×10 7After incubation at 37°C for 6 hours, 200 μL of culture medium was added to suspend the bacterial solution. 100 μL of the suspension was added to fresh culture medium and diluted 10-fold. The cells were then incubated at 37°C for 12 hours. The absorbance at 600 nm was measured. The bacterial killing rate was calculated as follows: Bacterial killing rate = (A control -A hydrogel ) / (A control -A blank )×100%,(A blank is the absorbance of LB culture medium itself). Figure 7 As shown in a, PPA hydrogel has a certain inherent antibacterial rate against Escherichia coli and Staphylococcus aureus. When the amount of hydrogen peroxide reaches 0.08wt%, the antibacterial rate increases rapidly with the increase of cross-linking degree.

[0048] Therefore, PPA hydrogel itself has a certain inherent antibacterial rate. In actual treatment, its photothermal responsiveness can improve the antibacterial effect. In the present invention, 100 μL of sterile PPA4 hydrogel was prepared and placed in a 96-well plate, and 5 μL of diluted bacterial suspension (1×10 7 CFU / mL). The hydrogels were then exposed to near-infrared laser (808 nm, 0.8 W / cm 2 ) for 0, 1, 5, 10 and 15 minutes (the time starts from when the hydrogel temperature rises to 45°C). The control group is 5 μL bacterial suspension (1×10 7 CFU / mL). After treatment, the bacterial solution was first resuspended in 200 μL of culture medium, and 100 μL of the suspension was added to fresh culture medium for 10-fold dilution. After incubation of each diluted culture medium at 37°C for 24 hours, the absorbance at 600 nm was measured and the bacterial survival rate was calculated. Figure 7 As shown in Figure b, after 5 minutes of near-infrared irradiation, the killing rates of PPA hydrogel against Staphylococcus aureus and Escherichia coli can reach 61.1% and 49.43%. After 10 minutes of near-infrared irradiation, the killing rate of bacteria can reach more than 99%.

[0049] Test Example 5: Test on the effect of hydrogel on wound healing

[0050] Based on the above experimental tests, the PPA hydrogel of the present invention has good biosafety, antioxidant, antibacterial and photothermal responsiveness, and is suitable as a dressing for skin injuries. To verify this, the present invention established an infectious animal model, specifically creating a full-thickness skin wound with a diameter of 8 mm on the back of each Kunming mouse (30 g ± 3 g). Subsequently, 50 μL of Staphylococcus aureus solution (1×10 7CFU / mL) and left for 24 hours to induce infection. The mice were randomly divided into three groups (day 0): control group, PPA4 treatment group and PPA4+NIR treatment group. The wound of the control group was treated with 100μL PBS, the PPA4 group was treated with 100μL of PPA4 hydrogel, and the PPA4+NIR group was treated with NIR irradiation (800mW / cm^2, irradiation for 10 minutes) in addition to 100μL of PPA4 hydrogel. The wound area was photographed on day 0, day 3, day 7 and day 14 to evaluate the degree of healing. The results are shown in the attached figure. Figure 8 As shown, after 14 days, the wounds in the control group were still not fully healed and were accompanied by infection and inflammation. However, the wounds in the group treated with PPA4 alone were basically healed, but a large area of ​​newly healed skin tissue was still observed. Compared with the first two groups, the wounds treated with PPA4 and near-infrared irradiation significantly improved the degree of bacterial elimination. Therefore, the wound healing effect of the PPA4+NIR treatment group was more obvious, and the area of ​​newly healed skin tissue was smaller. Therefore, due to its inherent antioxidant, antibacterial, and photothermal properties, PPA hydrogel is suitable for combining with photothermal therapy to accelerate the healing process of skin injuries.

[0051] 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 responsive hydrogel, characterized in that: The hydrogel contains: a cross-linked substance formed by a matrix, and the rest is water or phosphate buffer; the matrix is ​​a polyvinyl alcohol protocatechualdehyde solution, and the cross-linking agent of the matrix is ​​an enzyme catalytic system solution of a horseradish peroxidase / hydrogen peroxide combination; the hydrogel has photothermal responsiveness to near-infrared, preferably has photothermal responsiveness to near-infrared at 808 nm.

2. The hydrogel according to claim 1, wherein 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 polyvinyl alcohol protocatechuic aldehyde is 5 wt% or more, preferably 5 wt%; the concentration of horseradish peroxidase is 4 U / mL or more, preferably 4 U / mL; the concentration of hydrogen peroxide is 0.02 wt% or more, preferably 0.02 wt%-0.08 wt%, and more preferably 0.04 wt%-0.08 wt%; The rest is water or phosphate buffer; the pH value of the polyvinyl alcohol protocatechuic aldehyde solution is initially prepared between 5 and 8.

3. The method for preparing the hydrogel according to any one of claims 1 to 2, characterized in that: The following steps are involved: (1) dissolving polyvinyl alcohol protocatechualdehyde in water or phosphate buffer to prepare a solution; (2) adding horseradish peroxidase solution to the mixed solution of step 1 and mixing uniformly; (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 The pH value of the polyvinyl alcohol protocatechuic aldehyde solution initially prepared in step 1 is between 5 and 8; in the mixed system after adding the hydrogen peroxide solution in step 3, the final concentration of polyvinyl alcohol protocatechuic aldehyde is 5 wt% or more, preferably 5 wt%; the horseradish peroxidase concentration is 4 U / mL or more, preferably 4 U / mL; the hydrogen peroxide concentration is 0.02 wt% or more, preferably 0.02 wt%-0.08 wt%, more preferably 0.04 wt%-0.08 wt%; and the remainder is water or phosphate buffer.

5. An in vitro antioxidant 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 The photothermal responsiveness of the hydrogel is utilized, and the hydrogel is irradiated with near infrared light having a wavelength of 808 nm to enhance the antibacterial ability of the hydrogel.

7. An application in the preparation of 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; utilizing the photothermal responsiveness of the hydrogel, the drug is irradiated with 808nm near-infrared rays to enhance the 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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