A photo-enzyme coupled polymeric hydrogel, its preparation method and application

Abstract

The present invention relates to a photo-enzyme coupled polymerization hydrogel, a preparation method thereof and an application thereof. The photo-enzyme coupled polymerization hydrogel is composed of the following raw materials in parts by weight: 50 parts - 300 parts of acrylated hydrogel matrix; 10 parts - 100 parts of acrylated amino acid; 0.05 parts - 0.3 parts of flavoenzyme; 1000 parts of water. The preparation method is to mix the above raw materials and irradiate them under visible light, and then the photo-enzyme coupled polymerization hydrogel can be obtained. The photo-enzyme coupled polymerization hydrogel can be applied to the preparation of wound dressings. Compared with the prior art, the preparation process of the present invention is simple and efficient, has good repeatability, and the raw materials are cheap and easily available, which conforms to the development concept of green chemistry and can be widely promoted and applied on a large scale, and has good application prospects and broad development space in the field of preparing wound dressings.

Description

Technical Field

[0001] The present invention relates to the technical field of gel materials, and particularly relates to a photo-enzyme coupled polymerization hydrogel and a preparation method and application thereof. Background Art

[0002] A wound is caused by a deep incision or tear in the skin. Wound healing is a very complex biological process, which is divided into two types: acute and chronic. The main purpose of treatment is to quickly heal the wound without leaving a scar. Acute wound healing usually takes 8 - 12 weeks, while chronic wounds heal more slowly due to problems such as increased inflammation and bacterial infection. Moreover, the infection of unhealed wounds and healing scars can cause serious physical and mental health problems, bringing expensive costs to patients and the medical system. In recent years, the number of people suffering from unhealed wounds and related health risks has increased significantly. Therefore, it is of great practical significance to study wound healing materials.

[0003] At present, many different types of temporary skin substitutes have been developed and used as wound dressings to promote wound closure and induce tissue generation. For example, many dry wound dressings, such as bandages, cotton, gauze, etc. These traditional dressings need to be replaced regularly, and their usage efficiency is not very high. In addition, these dressings are too dry to provide a moist healing environment for wound healing, and are very likely to stick to the wound, causing secondary physical injury when the dressing is removed. Moreover, traditional dressings do not have a regulatory effect on the microenvironment of the wound, and only external drugs such as antibiotics can be used to regulate the microenvironment of the wound, but problems such as drug resistance will occur with the externally added antibiotics, etc. Therefore, it is necessary to develop new wound dressings to overcome the disadvantages of traditional dressings.

[0004] Hydrogel materials are a kind of biomaterials with a three-dimensional network structure that can absorb and retain a large amount of water. They have a structure similar to the extracellular matrix (ECM), can maintain the activity of bioactive agents and various cells, enabling cells to obtain good adhesion, spreading and proliferation. At the same time, the three-dimensional through-network structure is conducive to the transport of nutrients and gas exchange. Compared with other dressings, their higher water content can keep the wound surface moist, and will not stick to the wound to cause further damage. In addition, the gel has adjustable mechanical properties, can meet the mechanical requirements of the tissues at the wound, has no irritating effect on the tissues, and will not generate great mechanical friction with the tissues during the implantation process, thereby promoting wound healing and reducing the pain of patients.

[0005] Conventional photoinitiators used for preparing hydrogels are common diazonium salts, onium salts, ethers, etc. Their stability is poor, and the initiators and their cleavage products usually have certain toxic and side effects, which are not suitable for materials. Patent CN109316621A discloses a preparation method of a hydrogel dressing. The hydrogel dressing is prepared by the following method: after mixing a monomer, a crosslinking agent, a photoinitiator and a thickening agent, it is coated on a substrate and irradiated with ultraviolet light to obtain a support layer gel, and then an antibacterial and wound-healing monomer is added on the basis of it and irradiated with light to obtain the hydrogel dressing. This preparation process is complex and not suitable for large-scale production. The biocompatibility of the support layer monomer and the photoinitiator is poor, and it is easy to bring toxic and side effects to tissues. Summary of the Invention

[0006] The purpose of the present invention is to provide a photo-enzyme coupled polymerization hydrogel, its preparation method and application, in order to overcome the defects of poor stability and toxic and side effects existing in the above-mentioned prior art.

[0007] As a protein with high catalytic performance, enzymes can regulate the metabolic process of cells through complex and orderly biochemical reactions in living organisms, and can quickly, efficiently and gently initiate free radical polymerization in material synthesis to achieve in-situ forming of gels. By adopting a photo-enzyme coupling system, not only can the toxic and side effects brought by traditional photoinitiators be avoided, but also rapid and efficient gelation can be achieved. At the same time, the in-situ immobilization of enzymes can regulate the microenvironment of the wound site, thereby promoting the healing of the wound site.

[0008] The purpose of the present invention can be achieved by the following technical solutions:

[0009] One of the technical solutions of the present invention is to provide a photo-enzyme coupled polymerization hydrogel, which is composed of the following raw materials in parts by weight:

[0010]

[0011] Further, the acrylated hydrogel matrix is selected from any one or more of acrylated gelatin, acrylated chondroitin sulfate, acrylated chitosan or acrylated hyaluronic acid.

[0012] Further, the acrylated amino acid is selected from any one or more of acrylated lysine, acrylated serine, acrylated cysteine or acrylated glycine.

[0013] Further, the flavoenzyme is selected from any one or more of glucose oxidase, diaphorase or xanthine oxidase.

[0014] Another technical solution of the present invention is to provide a preparation method of a photo-enzyme coupled polymerization hydrogel, which includes the following steps:

[0015] (1) After dissolving the hydrogel matrix into a transparent solution, it is graft-modified with glycidyl methacrylate to prepare an acrylated hydrogel matrix;

[0016] (2) The amino acid is graft-modified with acryloyl chloride to prepare acryloylated amino acid;

[0017] (3) The acrylated hydrogel matrix in step (1) and the acryloylated amino acid in step (2) are mixed, and an enzyme is added and mixed evenly to obtain a precursor solution;

[0018] (4) The precursor solution in step (3) is irradiated under visible light to prepare a photo-enzyme coupled polymerization hydrogel.

[0019] Further, in step (1), the hydrogel matrix is selected from any one or more of gelatin, chondroitin sulfate, chitosan or hyaluronic acid, and the ratio of the hydrogel matrix to the glycidyl methacrylate is 1 g: 1 mL.

[0020] Further, in step (2), the amino acid is selected from any one or more of lysine, serine, cysteine or glycine, and the ratio of the amino acid to the acryloyl chloride is 5 mmol: 1 mL.

[0021] Further, in step (4), the visible light is selected from any one of blue light, red light, ultraviolet light or sunlight.

[0022] Further, in step (4), the time of visible light irradiation is 3 - 30 min.

[0023] The acrylated hydrogel matrix serves as a macromonomer, and the acryloylated amino acid serves as a small molecule monomer. Both have good biocompatibility and will not bring toxic and side effects to cells and tissues. There is a certain steric hindrance effect in the macromonomer, which will lead to a slowdown in the polymerization process, while the small molecule monomer will reduce the steric hindrance effect and accelerate the polymerization rate. Therefore, the addition amounts of the macromonomer and the small molecule monomer will affect the final mechanical properties of the hydrogel. If the addition amount of the small molecule monomer is too high, the degree of polymerization will be too high, the pore size of the hydrogel will be too small, which is not conducive to cell spreading, adhesion, growth, and the transport of nutrients and the excretion of metabolic wastes; if the addition amount of the macromonomer is too low, the mechanical properties of the hydrogel will be poor and it cannot maintain stability.

[0024] The isoalloxazine structure of the flavoenzyme will undergo a redox reaction under light irradiation, thereby initiating polymerization. And as a protein, the flavoenzyme has no toxic and side reactions and can regulate the microenvironment of the wound site, and thus is used to prepare wound dressings.

[0025] The third technical solution of the present invention is to provide an application of a photo-enzyme coupled polymeric hydrogel, and the photo-enzyme coupled polymeric hydrogel is applied to the field of preparing wound dressings.

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

[0027] (1) The photo-enzyme coupled polymeric hydrogel of the present invention has a structure similar to the extracellular matrix, has a high water content, can keep the wound surface moist, and avoids secondary physical damage to the wound.

[0028] (2) The photo-enzyme coupled polymeric hydrogel of the present invention has a three-dimensional through structure, good biocompatibility and mechanical properties, which are beneficial to cell adhesion, spreading and proliferation.

[0029] (3) The photo-enzyme coupled polymeric hydrogel of the present invention has good free radical scavenging ability, can effectively scavenge hydroxyl radicals, DPPH radicals and hydrogen peroxide, can significantly improve the oxidative stress response at the wound, and has long-term and efficient antioxidant ability.

[0030] (4) On the one hand, the flavoenzyme in the present invention is used as a component for initiating gelation to avoid the toxic and side effects brought by traditional initiators. On the other hand, the flavoenzyme is in-situ loaded into the hydrogel matrix, which is beneficial to the subsequent regulation of the microenvironment at the wound site. The present invention realizes the synthesis of the hydrogel, the loading of the enzyme and the subsequent utilization of the enzyme through a one-step method, which is simple and efficient.

[0031] (5) The present invention can adjust the mechanical properties of the photo-enzyme coupled polymeric hydrogel only by adjusting the amounts of the macromonomer and the small molecule monomer added.

[0032] (6) The preparation process of the present invention is simple and efficient, has good repeatability, the raw materials are cheap and easy to obtain, conforms to the development concept of green chemistry, can be widely promoted and applied, and has good application prospects and broad development space in the field of preparing wound dressings. Description of the Drawings

[0033] Figure 1 It is a physical picture of the photo-enzyme coupled hydrogel of Example 1;

[0034] Figure 2 It is a scanning electron microscope picture of the photo-enzyme coupled hydrogel of Example 1;

[0035] Figure 3 It is an electron paramagnetic resonance spectrum of the photo-enzyme coupled hydrogel of Example 1;

[0036] Figure 4 It is a compression curve graph and a compression modulus graph of the photo-enzyme coupled hydrogel of Example 1;

[0037] Figure 5CCK-8 graph of cells after the treatment of the precursor solution in Example 1;

[0038] Figure 6 CCK-8 graph of cells after the treatment of the leaching solution of the photo-enzyme conjugate hydrogel in Example 1;

[0039] Figure 7 Live / dead cell staining graph of cells after the treatment of the photo-enzyme conjugate hydrogel in Example 1;

[0040] Figure 8 Results of the effect of the leaching solution of the photo-enzyme conjugate hydrogel in Example 1 on cell migration and statistical graph of migration rate;

[0041] Figure 9 Statistical graph of the efficiency of the photo-enzyme conjugate hydrogel in Example 1 for scavenging free radicals in vitro;

[0042] Figure 10 Fluorescence detection graph of the photo-enzyme conjugate hydrogel in Example 1 for scavenging free radicals in vivo;

[0043] Figure 11 Physical graph of the degree of wound healing at the wound site of mice by the photo-enzyme conjugate hydrogel in Example 1;

[0044] Figure 12 Statistical result graph of the efficiency of the photo-enzyme conjugate hydrogel in Example 1 for wound healing in mice. Detailed implementation manners

[0045] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0046] In the following examples and comparative examples, unless otherwise specified, the raw materials or processing techniques are all conventional commercially available raw material products or conventional processing techniques in the art.

[0047] Preparation method of the leaching solution of the photo-enzyme conjugate polymer hydrogel: Add 1 mL of the prepared photo-enzyme conjugate polymer hydrogel to the DMEM high-glucose medium containing 10% FBS and 1% double antibody (penicillin-streptomycin) at a ratio of 0.1 g / mL, and place it in a sealed condition at 37°C for extraction for 1 d, 3 d, 5 d and 7 d respectively. After sucking the supernatant and filtering, 100% leaching solutions of different days are obtained and used for subsequent cell experiments (the reference is DOI: 10.1002 / adhm.202101722).

[0048] The double antibody (penicillin-streptomycin) is BaselMedia, 100X, sourced from Shanghai Yuanpei Biotechnology Co., Ltd.

[0049] Example 1:

[0050] A photo-enzyme conjugate hydrogel, the preparation method comprising the following steps:

[0051] (1) After dissolving gelatin into a transparent solution, it was graft-modified with glycidyl methacrylate at a ratio of 1 g:1 mL to prepare acrylated gelatin.

[0052] (2) Lysine was graft-modified with acryloyl chloride at a ratio of 5 mmol:1 mL to prepare acryloylated lysine.

[0053] (3) 50 mg of the prepared acrylated gelatin and 30 mg of acryloylated lysine were mixed, 100 μL of myocardial xanthine oxidase with a concentration of 1 mg / mL and 900 μL of deionized water were added, and vortexed and sonicated to form a homogeneous and transparent precursor solution.

[0054] (4) Under room temperature conditions, the precursor solution was irradiated with 455 nm blue light for 10 min to prepare a photo-enzyme coupled polymeric hydrogel.

[0055] The prepared photo-enzyme coupled polymeric hydrogel was tested as follows:

[0056] 1. Morphology characterization of the photo-enzyme coupled polymeric hydrogel:

[0057] (1) The prepared photo-enzyme coupled polymeric hydrogel is as Figure 1 shown, and the precursor solution ( Figure 1 a) is in a flowing state and turns into a gel state ( Figure 1 b) after being irradiated with visible light.

[0058] (2) The prepared photo-enzyme coupled polymeric hydrogel was observed by scanning electron microscopy. As Figure 2 shown, the photo-enzyme coupled polymeric hydrogel has a uniform and stable three-dimensional porous structure with a pore size of about 30 μm.

[0059] 2. Gelation mechanism characterization of the photo-enzyme coupled polymeric hydrogel:

[0060] The precursor solution without acrylated gelatin and acryloylated lysine was irradiated with visible light and then tested by electron paramagnetic resonance spectroscopy. The results are as Figure 3 shown. The irradiated group is labeled as Light, and the control group without visible light irradiation is labeled as Dark. It can be seen from the figure that there are a large number of hydroxyl radicals in the precursor solution after irradiation, so it can initiate the polymerization of acrylated gelatin and acryloylated lysine to form a gel.

[0061] 3. Mechanical property characterization of the photo-enzyme coupled polymeric hydrogel:

[0062] The prepared photo-enzyme coupled polymeric hydrogel was tested for compressive properties on a universal testing machine. The results are as Figure 4 shown. Figure 4a and Figure 4 b show that when the content of LysMA (acrylated lysine) is fixed at 3%, with the increase of the content of GelMA (acrylated gelatin), the compressive stress and compressive modulus of the photo - enzyme coupled polymerization hydrogel gradually increase; Figure 4 c and 4d show that when the content of GelMA is fixed at 10%, with the increase of the content of LysMA, the compressive properties of the photo - enzyme coupled polymerization hydrogel gradually increase. This result indicates that the mechanical properties of the photo - enzyme coupled polymerization hydrogel can be regulated by changing the addition ratio of the monomers, and then different ratios of monomers can be screened for the preparation of corresponding photo - enzyme coupled polymerization hydrogels according to different specific application purposes.

[0063] 4. Evaluation of biocompatibility and cytotoxicity of photo - enzyme coupled polymerization hydrogel:

[0064] After treating 3T3 cells with the extracts of acrylated gelatin, acrylated lysine and photo - enzyme coupled polymerization hydrogel at different concentrations respectively, the toxicity of 3T3 cells was evaluated by CCK - 8 assay. The photo - enzyme coupled polymerization hydrogel was co - cultured with 373 cells, and the cell viability was evaluated by AM / PI live - dead staining, in which live cells would be stained green and dead cells would be stained red.

[0065] The results are as Figures 5 - 7 shown, Figure 5 a is the result of the cytotoxicity test of acrylated gelatin at different concentrations on cells, Figure 5 b is the result of the cytotoxicity test of acrylated lysine at different concentrations on 3T3 cells. The figure shows that compared with the blank control (labeled as Ctr), neither acrylated gelatin nor acrylated lysine has obvious toxic side effects on 3T3 cells. Figure 6 is the result of the cytotoxicity test of the extracts of photo - enzyme coupled polymerization hydrogel soaked for different days on 3T3 cells. The figure shows that the extracts of the photo - enzyme coupled polymerization hydrogel do not cause the death of 373 cells, but have a promoting effect on the growth of 3T3 cells, indicating good biocompatibility. Figure 7 is the AM / PI live - dead staining map of 373 cells cultured with photo - enzyme coupled polymerization hydrogel for different days. In the figure, all 3T3 cells are green and in good growth condition, indicating that the photo - enzyme coupled polymerization hydrogel is beneficial to the adhesion, growth and proliferation of 3T3 cells.

[0066] 5. Influence of photo - enzyme coupled polymerization hydrogel on cell migration:

[0067] Inoculate fibroblasts in a 24 - well plate. When the fibroblasts cover the whole well plate, use the tip of a pipette gun to scratch a cell - free area. Add the extract of the photo - enzyme coupled polymerization hydrogel to the cell - free area and co - culture with fibroblasts, and observe at different times.

[0068] The results are as Figure 8 shown, Figure 8 a shows the migration of fibroblasts co-cultured with the leaching solution of the photo-enzyme coupled polymerization hydrogel on the 7th day, Figure 8 b shows the migration rate of fibroblasts co-cultured with the leaching solution of the photo-enzyme coupled polymerization hydrogel on the 7th day. The experimental group is labeled as Gel, and the blank control group is labeled as Ctr. The figures all show that the fibroblasts in the experimental group proliferate significantly and have covered the cell-free area after 48 h, indicating that the photo-enzyme coupled polymerization hydrogel can promote the proliferation and migration of fibroblasts.

[0069] 6. Test on the free radical scavenging ability of the photo-enzyme coupled polymerization hydrogel in vitro and in vivo:

[0070] (1) Scavenging of H2O2: Mash 1 mL of the photo-enzyme coupled polymerization hydrogel and incubate it with 3 mL of H2O2 solution at 37 °C for 1 h. Set the corresponding background group (1 mL of the photo-enzyme coupled polymerization hydrogel co-incubated with 3 mL of H2O) and the blank control group (3 mL of H2O2). Take the supernatant, mix it with 0.5 mL of Ti(SO4)2 solution for 5 min, measure the absorbance at 410 nm, and calculate the scavenging rate of H2O2.

[0071] According to the titanium sulfate method for determining hydrogen peroxide content, that is, H2O2 reacts with titanium sulfate to form a yellow titanium peroxide complex precipitate, which has a characteristic absorption peak at 410 nm. Thus, it is calculated according to the scavenging rate formula of H2O2:

[0072] Scavenging rate (%) = [1 - (A s - A j ) / A0] * 100, where A s is the absorbance of the sample group, A j is the absorbance of the background group, and A0 is the absorbance of the blank control group.

[0073] (2) Scavenging of DPPH: Incubate 3 mL of DPPH free radical solution (0.1 mM, absolute ethanol) with 1 mL of the photo-enzyme coupled polymerization hydrogel at 37 °C for 60 min. Set the corresponding background group (1 mL of the photo-enzyme coupled polymerization hydrogel mashed and co-incubated with 3 mL of absolute ethanol) and the blank control group (0.1 mM DPPH), measure the absorbance at 517 nm, and calculate the scavenging rate of DPPH. The scavenging rate formula is calculated as follows:

[0074] Scavenging rate (%) = [1 - (A s - A j ) / A0] * 100, where A s is the absorbance of the sample group, A j is the absorbance of the background group, and A0 is the absorbance of the blank control group.

[0075] (3) Scavenging of hydroxyl radicals: The amount of hydroxyl radicals was determined by the salicylic acid colorimetric method. After mashing 1 mL of the photo-enzyme coupled polymeric hydrogel, it was mixed with 0.15 mL of salicylic acid (10 mM, absolute ethanol), 0.15 mL of FeSO4 (10 mM, H2O), 1.5 mL of H2O2 (100 mM), and 1.2 mL of H2O. The final volume was 3.0 mL. Incubation was carried out in the dark for 60 min. A corresponding background group (only replacing 0.15 mL of the FeSO4 solution with 0.15 mL of H2O, and the rest remained unchanged) and a blank control group (without adding the photo-enzyme coupled polymeric hydrogel) were set. The absorbance at 510 nm was measured, and the hydroxyl radical scavenging rate was calculated. The calculation formula for the scavenging rate is as follows:

[0076] Scavenging rate (%) = [1 - (A s - A j ) / A0] * 100, where A s is the absorbance of the sample group, A j is the absorbance of the background group, and A0 is the absorbance of the blank control group.

[0077] (4) Scavenging of free radicals in vivo: NIH 3T3 mouse embryonic fibroblasts were seeded in 48-well plates and incubated with 10 μL of PBS, 10 μL of H2O2 (final concentration 100 μM), and 10 μL of H2O2 + 100 μL of the photo-enzyme coupled polymeric hydrogel for 2 h. Then, the cells were incubated with DCFH-DA (5 μm) for 20 min, and then gently washed 3 times with PBS. The intracellular reactive oxygen species (ROS) level was evaluated by detecting the fluorescence intensity through Confocal. Among them, co-incubation with PBS and co-incubation with H2O2 were used as control groups respectively.

[0078] As shown in Figure 9 a - c, the experimental group was labeled as Gel, the blank control group was labeled as CTR. The scavenging efficiencies of the photo-enzyme coupled polymeric hydrogel for H2O2, DPPH, and hydroxyl radicals were 96%, 51%, and 47% respectively, indicating that the photo-enzyme coupled polymeric hydrogel can efficiently scavenge free radicals.

[0079] As shown in Figure 10 a - c, compared with the H2O2 control group( Figure 10 a) and the PBS control group( Figure 10 b), the ROS level of the experimental group co-incubated with the photo-enzyme coupled polymeric hydrogel( Figure 10 c) decreased, further indicating that the photo-enzyme coupled polymeric hydrogel can effectively reduce ROS and has effective antioxidant properties.

[0080] 7. Animal experiments of the photo-enzyme coupled polymeric hydrogel:

[0081] Male Balb / c mice (SPF grade, Shanghai Slack Experimental Animal Co., Ltd.) weighing about 18 - 22 g at 6 - 8 weeks were used for wound modeling. After cleaning the wound surface with normal saline, the photo - enzyme conjugate polymer hydrogel was placed on the wound as the experimental group (labeled as Gel). Meanwhile, a blank control group (labeled as Ctr) was set up to observe the change in the area of the wound surface after culturing for different times.

[0082] The wound surface condition of the mice is as Figures 11 - 12 shown. It can be seen from Figure 11 that compared with the blank control group, after 7 days, the wound surface area of the mice in the experimental group decreased significantly, and after 14 days, the wounds of the mice in the experimental group were basically completely healed.

[0083] Example 2:

[0084] A photo - enzyme conjugate hydrogel, the preparation method comprises the following steps:

[0085] (1) After dissolving gelatin into a transparent solution, it was graft - modified with glycidyl methacrylate at a ratio of 1 g:1 mL to prepare acrylated gelatin;

[0086] (2) Lysine was graft - modified with acryloyl chloride at a ratio of 5 mmol:1 mL to prepare acryloylated lysine;

[0087] (3) 200 mg of the prepared acrylated gelatin and 30 mg of acryloylated lysine were mixed, 50 μL of flavin - containing enzyme with a concentration of 1 mg / mL and 950 μL of deionized water were added, and vortexed and ultrasonicated to form a homogeneous and transparent precursor solution;

[0088] (4) At room temperature, the precursor solution was irradiated with ultraviolet light for 8 min to prepare the photo - enzyme conjugate polymer hydrogel.

[0089] Example 3:

[0090] A photo - enzyme conjugate hydrogel, the preparation method comprises the following steps:

[0091] (1) After dissolving gelatin into a transparent solution, it was graft - modified with glycidyl methacrylate at a ratio of 1 g:1 mL to prepare acrylated gelatin;

[0092] (2) Lysine was graft - modified with acryloyl chloride at a ratio of 5 mmol:1 mL to prepare acryloylated lysine;

[0093] (3) 150 mg of the prepared acrylated gelatin and 30 mg of acryloylated lysine were mixed, 200 μL of glucose oxidase with a concentration of 1 mg / mL and 800 μL of deionized water were added, and vortexed and ultrasonicated to form a homogeneous and transparent precursor solution;

[0094] (4) At room temperature, irradiate the precursor solution with 455 nm blue light for 5 min to prepare the photo-enzyme coupled polymeric hydrogel.

[0095] Example 4:

[0096] A photo-enzyme coupled hydrogel, the preparation method comprising the following steps:

[0097] (1) After dissolving gelatin into a transparent solution, carry out graft modification with glycidyl methacrylate according to the ratio of 1 g: 1 mL to prepare acrylated gelatin.

[0098] (2) Carry out graft modification with lysine and acryloyl chloride according to the ratio of 5 mmol: 1 mL to prepare acryloylated lysine.

[0099] (3) Mix 200 mg of the prepared acrylated gelatin and 30 mg of acryloylated lysine, add 100 μL of myocardial xanthine oxidase with a concentration of 1 mg / mL and 900 μL of deionized water, and vortex and ultrasonicate to form a homogeneous and transparent precursor solution.

[0100] (4) At room temperature, irradiate the precursor solution with ultraviolet light for 3 min to prepare the photo-enzyme coupled polymeric hydrogel.

[0101] Example 5:

[0102] A photo-enzyme coupled hydrogel, the preparation method comprising the following steps:

[0103] (1) After dissolving gelatin into a transparent solution, carry out graft modification with glycidyl methacrylate according to the ratio of 1 g: 1 mL to prepare acrylated gelatin.

[0104] (2) Carry out graft modification with lysine and acryloyl chloride according to the ratio of 5 mmol: 1 mL to prepare acryloylated lysine.

[0105] (3) Mix 150 mg of the prepared acrylated gelatin and 50 mg of acryloylated lysine, add 100 μL of myocardial xanthine oxidase with a concentration of 1 mg / mL and 900 μL of deionized water, and vortex and ultrasonicate to form a homogeneous and transparent precursor solution.

[0106] (4) At room temperature, irradiate the precursor solution with 455 nm blue light for 10 min to prepare the photo-enzyme coupled polymeric hydrogel.

[0107] Example 6:

[0108] A photo-enzyme coupled hydrogel, the preparation method comprising the following steps:

[0109] (1) After dissolving gelatin into a transparent solution, graft modification is carried out with glycidyl methacrylate at a ratio of 1 g: 1 mL to prepare acrylated gelatin;

[0110] (2) Lysine is subjected to graft modification with acryloyl chloride at a ratio of 5 mmol: 1 mL to prepare acryloylated lysine;

[0111] (3) 150 mg of the prepared acrylated gelatin and 100 mg of acryloylated lysine are mixed, 150 μL of xanthine oxidase with a concentration of 1 mg / mL and 850 μL of deionized water are added, and vortexed and ultrasonicated to form a homogeneous and transparent precursor solution;

[0112] (4) Under room temperature conditions, the precursor solution is irradiated with ultraviolet light for 5 min to prepare a photo-enzyme coupled polymer hydrogel.

[0113] Example 7:

[0114] A photo-enzyme coupled hydrogel, and the preparation method comprises the following steps:

[0115] (1) After dissolving hyaluronic acid into a transparent solution, graft modification is carried out with glycidyl methacrylate at a ratio of 1 g: 1 mL to prepare acrylated hyaluronic acid;

[0116] (2) Serine is subjected to graft modification with acryloyl chloride at a ratio of 5 mmol: 1 mL to prepare acryloylated serine;

[0117] (3) 100 mg of the prepared acrylated hyaluronic acid and 50 mg of acryloylated serine are mixed, 100 μL of cardiomyozyme with a concentration of 1 mg / mL and 900 μL of deionized water are added, and vortexed and ultrasonicated to form a homogeneous and transparent precursor solution;

[0118] (4) Under room temperature conditions, the precursor solution is irradiated with 455 nm blue light for 15 min to prepare a photo-enzyme coupled polymer hydrogel.

[0119] Example 8:

[0120] A photo-enzyme coupled hydrogel, and the preparation method comprises the following steps:

[0121] (1) After dissolving chitosan into a transparent solution, graft modification is carried out with glycidyl methacrylate at a ratio of 1 g: 1 mL to prepare acrylated chitosan;

[0122] (2) Serine is subjected to graft modification with acryloyl chloride at a ratio of 5 mmol: 1 mL to prepare acryloylated serine;

[0123] (3) Mix the prepared 150 mg of acrylated chitosan and 70 mg of acrylated serine, add 200 μL of glucose oxidase with a concentration of 1 mg / mL and 800 μL of deionized water, and vortex and ultrasonicate to form a homogeneous and transparent precursor solution;

[0124] (4) Under room temperature conditions, irradiate the precursor solution with ultraviolet light for 10 min to obtain the photo-enzyme conjugate polymer hydrogel.

[0125] Example 9:

[0126] A photo-enzyme conjugate hydrogel, the preparation method comprising the following steps:

[0127] (1) After dissolving hyaluronic acid into a transparent solution, carry out graft modification with glycidyl methacrylate according to a ratio of 1 g:1 mL to obtain acrylated hyaluronic acid;

[0128] (2) Carry out graft modification of lysine and acryloyl chloride according to a ratio of 5 mmol:1 mL to obtain acrylated lysine;

[0129] (3) Mix the prepared 200 mg of acrylated hyaluronic acid and 50 mg of acrylated lysine, add 200 μL of xanthine oxidase with a concentration of 1 mg / mL and 800 μL of deionized water, and vortex and ultrasonicate to form a homogeneous and transparent precursor solution;

[0130] (4) Under room temperature conditions, irradiate the precursor solution with ultraviolet light for 10 min to obtain the photo-enzyme conjugate polymer hydrogel.

[0131] Example 10:

[0132] A photo-enzyme conjugate hydrogel, the preparation method comprising the following steps:

[0133] (1) After dissolving chondroitin sulfate into a transparent solution, carry out graft modification with glycidyl methacrylate according to a ratio of 1 g:1 mL to obtain acrylated chondroitin sulfate;

[0134] (2) Carry out graft modification of cysteine and acryloyl chloride according to a ratio of 5 mmol:1 mL to obtain acrylated cysteine;

[0135] (3) Mix the prepared 300 mg of acrylated chondroitin sulfate and 100 mg of acrylated cysteine, add 300 μL of xanthine oxidase with a concentration of 1 mg / mL and 700 μL of deionized water, and vortex and ultrasonicate to form a homogeneous and transparent precursor solution;

[0136] (4) Under room temperature conditions, irradiate the precursor solution with red light for 30 min to obtain the photo-enzyme conjugate polymer hydrogel.

[0137] Example 11:

[0138] A photo-enzyme coupled hydrogel, the preparation method comprising the following steps:

[0139] (1) After dissolving hyaluronic acid into a transparent solution, graft modification is carried out with glycidyl methacrylate according to a ratio of 1 g: 1 mL to prepare acrylated hyaluronic acid;

[0140] (2) Graft modification is carried out with glycine and acryloyl chloride according to a ratio of 5 mmol: 1 mL to prepare acryloylated glycine;

[0141] (3) Mix 200 mg of the prepared acrylated hyaluronic acid and 10 mg of acryloylated glycine, add 200 μL of xanthine oxidase with a concentration of 1 mg / mL and 800 μL of deionized water, and vortex and ultrasonicate to form a homogeneous and transparent precursor solution;

[0142] (4) Under room temperature conditions, irradiate the precursor solution with sunlight for 10 min to prepare the photo-enzyme coupled polymer hydrogel.

[0143] The above description of the embodiments is for the convenience of those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A photo-enzyme coupled polymeric hydrogel, characterized in that, It consists of the following raw materials in parts by weight:

2. The photo-enzyme coupled polymeric hydrogel according to claim 1, wherein The acrylated hydrogel matrix is selected from any one or more of acrylated gelatin, acrylated chondroitin sulfate, acrylated chitosan or acrylated hyaluronic acid.

3. The photo-enzyme coupled polymeric hydrogel according to claim 1, wherein The acryloylated amino acid is selected from any one or more of acryloylated lysine, acryloylated serine, acryloylated cysteine or acryloylated glycine.

4. The photo-enzyme coupled polymer hydrogel according to claim 1, wherein The flavoenzyme is selected from any one or more of glucose oxidase, diaphorase or xanthine oxidase.

5. A method for preparing a photo-enzyme coupled polymeric hydrogel as described in any one of claims 1-4, characterized in that, It includes the following steps: (1) After dissolving the hydrogel matrix into a transparent solution, graft modification is carried out with glycidyl methacrylate to prepare an acrylated hydrogel matrix; (2) Graft modification is carried out between the amino acid and acryloyl chloride to prepare an acryloylated amino acid; (3) Mix the acrylated hydrogel matrix in step (1) and the acryloylated amino acid in step (2), add flavoenzyme and deionized water, and mix evenly to obtain a precursor solution; (4) Irradiate the precursor solution in step (3) under visible light to prepare a photo-enzyme coupled polymerized hydrogel.

6. The preparation method of a photo-enzyme coupled polymeric hydrogel according to claim 5, characterized in that, In step (1), the hydrogel matrix is selected from any one or more of gelatin, chondroitin sulfate, chitosan or hyaluronic acid, and the ratio of the hydrogel matrix to glycidyl methacrylate is 1 g: 1 mL.

7. The preparation method of a photo-enzyme coupled polymer hydrogel according to claim 5, characterized in that, In step (2), the amino acid is selected from any one or more of lysine, serine, cysteine or glycine, and the ratio of the amino acid to acryloyl chloride is 5 mmol: 1 mL.

8. A method for preparing a photo-enzyme coupled polymeric hydrogel according to claim 5, characterized in that, In step (4), the visible light is selected from any one of blue light, red light or sunlight.

9. The preparation method of a photo-enzyme coupled polymer hydrogel according to claim 5, characterized in that, In step (4), the time of visible light irradiation is 3 - 30 min.

10. Use of a photo-enzyme coupled polymeric hydrogel according to any one of claims 1-4, characterized in that, The photo-enzyme coupled polymerized hydrogel is applied to the field of preparing wound dressings.

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