High-adhesion enzymatic hydrogel capable of promoting endometrial repair and preparation method of high-adhesion enzymatic hydrogel

Abstract

The invention discloses a high-adhesion enzymatic hydrogel for promoting vascular intima repair and a preparation method of the high-adhesion enzymatic hydrogel, and belongs to the technical field of hydrogel preparation. The preparation method comprises the following steps: S1, carrying out double-bond modification on hyaluronic acid by utilizing methacrylic anhydride, and then cross-linking with dopamine to prepare dopamine modified double-bond hyaluronic acid; s2, deoxidizing the hyaluronic acid solution, adding a cross-linking agent and tyramine hydrochloride, and reacting in an acidic environment to prepare tyramine modified hyaluronic acid; s3, mixing the dopamine modified double-bond hyaluronic acid, the tyramine modified hyaluronic acid and deionized water to prepare a hydrogel precursor liquid; and S4, carrying out enzymatic reaction to obtain the hydrogel. The method designed by the invention is simple in process, no extra photocuring equipment needs to be added, and the constructed hydrogel is similar to an extracellular matrix. And the coating can be coated into different shapes to adapt to different environments. The gel is formed through enzymatic reaction, and the gel is rapid in gel forming, good in tissue adhesion, good in biocompatibility and suitable for being applied to vascular repair.

Description

Technical Field

[0001] The present invention belongs to the technical field of hydrogel preparation, and particularly relates to a highly adhesive enzyme-catalyzed hydrogel for promoting vascular intima repair and a preparation method thereof. Background Art

[0002] Atherosclerosis is the main cause of cardiovascular diseases. Vascular stent implantation is one of the important clinical treatment means for cardiovascular diseases, which can improve the vascular occlusion by dilating the lesion site. However, it is accompanied by the risks of restenosis, late thrombosis, and late neointimal atherosclerosis, etc. It may require reoperation, bringing more risks to patients, and the stent is non-degradable after implantation and needs to exist in the human body for a long time.

[0003] Hydrogel is a material similar to the extracellular matrix, often used for skin wound repair, etc. Changing the gel components can adjust its gelation time and adhesion ability, etc. Such repair-promoting hydrogels are often applied to the surface of static tissues such as the skin, and are prone to falling off when experiencing fluid scouring, and are usually made using molds, resulting in incomplete anastomosis with the vascular intima. At the same time, the current hydrogels usually require additional addition of photoinitiators and irradiation with ultraviolet light for curing, and the operation is more cumbersome. Moreover, in the vascular intima environment, there is a high-speed blood flow scouring, and a large number of platelets are present. The anti-platelet adhesion performance of the hydrogel is also a great challenge. Summary of the Invention

[0004] In view of the above deficiencies in the prior art, the present invention provides a highly adhesive enzyme-catalyzed hydrogel for promoting vascular intima repair and a preparation method thereof. The method designed by the present invention has a simple process, does not require additional addition of a photocuring device, and the constructed hydrogel is similar to the extracellular matrix. It can be coated into different shapes to adapt to different environments. The gelation is achieved by an enzyme-catalyzed reaction, with rapid gelation, good adhesion to tissues, and good biocompatibility, and is suitable for application in vascular repair after atherectomy of atherosclerotic plaques.

[0005] To achieve the above object, the technical solution adopted by the present invention to solve its technical problems is:

[0006] The object of the present invention is to provide a preparation method of a highly adhesive enzyme-catalyzed hydrogel for promoting vascular intima repair, comprising the following steps:

[0007] S1. Dopamine-modified double-bonded hyaluronic acid (HMD)

[0008] The hyaluronic acid is modified with double bonds using methacrylic anhydride, and then crosslinked with dopamine to obtain dopamine-modified double-bonded hyaluronic acid;

[0009] S2. Tyramine-modified hyaluronic acid (HT)

[0010] After deoxygenating the hyaluronic acid solution, a crosslinking agent and tyramine hydrochloride are added, and the reaction is carried out in an acidic environment for 4-8 h to obtain tyramine-modified hyaluronic acid;

[0011] S3. Mix the dopamine-modified double-bonded hyaluronic acid solution and the tyramine-modified hyaluronic acid solution to obtain a hydrogel precursor solution in which the mass concentrations of both dopamine-modified double-bonded hyaluronic acid and tyramine-modified hyaluronic acid are 2-3 wt%;

[0012] S4. Then, use an enzymatic reaction to prepare a highly adhesive enzymatic hydrogel for promoting vascular intima repair.

[0013] Furthermore, the process of modifying hyaluronic acid with methacrylic anhydride to introduce double bonds is as follows:

[0014] Add methacrylic anhydride to the hyaluronic acid solution, adjust the pH to 8-9, react for 2-3 h, add ethanol to precipitate, wash the precipitate with alcohol, dissolve it in deionized water, and dialyze and freeze-dry to obtain it;

[0015] Among them, the mass concentration of hyaluronic acid is 1-1.5 wt%, and the mass concentration of methacrylic anhydride is 4.5-5 wt%.

[0016] Furthermore, the process of modifying double-bonded hyaluronic acid with dopamine is as follows:

[0017] Dissolve the double-bonded modified hyaluronic acid in MES buffer to obtain a solution with a mass concentration of 1-1.5 wt%, then add a crosslinking agent and dopamine (DOPA), deoxygenate, adjust the pH to 5-6, react for 4-8 h, then adjust the pH to acidic, and dialyze and freeze-dry to obtain it;

[0018] Among them, the concentration of MES buffer is 100-120 mM, and the mass concentration of dopamine is 0.3-0.5 wt%.

[0019] Furthermore, the crosslinking agent used is NHS / EDC, among which the mass concentration of NHS is 0.2-0.3 wt%, and the mass concentration of EDC is 0.3-0.4 wt%.

[0020] Furthermore, in S2, the mass concentration of hyaluronic acid is 0.5-1.5 wt%, and the concentration of tyramine hydrochloride is 1.2-1.5 mM.

[0021] Furthermore, the crosslinking agent used in S2 is NHS / EDC, among which the mass concentration of NHS is 0.2-0.3 wt%, and the mass concentration of EDC is 0.3-0.4 wt%.

[0022] Furthermore, in S3, the volume ratio of the dopamine-modified double-bonded hyaluronic acid solution to the tyramine-modified hyaluronic acid solution is 1:0.5-0.5:1.

[0023] Further, the process of the enzymatic reaction is as follows:

[0024] The hydrogel precursor solution is evenly divided into Solution A and Solution B. Hydrogen peroxide is added to Solution A, and horseradish peroxidase is added to Solution B. Then, Solutions A and B are mixed together, and the hydrogel for promoting vascular intima repair can be prepared by using the enzymatic reaction.

[0025] Further, the concentration of hydrogen peroxide is 0.009%-0.019%, and the concentration of horseradish peroxidase is 10-30 U / mL.

[0026] Another object of the present invention is to provide a highly adhesive enzymatic hydrogel for promoting vascular intima repair, which is prepared by the above method.

[0027] Advantages of the present invention:

[0028] 1. The present invention provides a method for constructing a hydrogel. First, the hydrogel precursor solution containing hydrogen peroxide is coated on the surface, and then the hydrogel precursor solution containing an enzyme is coated on the surface and polymerized into a gel. This method has a simple process, does not require an external light-curing device, and the constructed hydrogel structure is similar to the extracellular matrix, having good biocompatibility.

[0029] 2. The hydrogel prepared by the method of the present invention can promote the adhesion, growth and migration of endothelial cells, and thus can promote the repair of the vascular intima. And it can inhibit the adhesion, aggregation and activation of platelets, having excellent anticoagulant properties.

[0030] 3. The method of the present invention has a simple process, does not require an additional light-curing device, and the constructed hydrogel is similar to the extracellular matrix. It can be coated into different shapes to adapt to different environments. Moreover, the present invention uses an enzymatic reaction to form a gel, which forms a gel quickly, adheres well to tissues, has good biocompatibility, and is suitable for application in vascular repair after atherectomy of atherosclerotic plaques. Description of the Drawings

[0031] Figure 1 It is the Fourier transform infrared spectroscopy analysis chart of dopamine-modified double-bonded hyaluronic acid and tyramine-modified hyaluronic acid prepared in Example 1;

[0032] Figure 2 It is the nuclear magnetic resonance hydrogen spectrum analysis chart of dopamine-modified double-bonded hyaluronic acid and tyramine-modified hyaluronic acid prepared in Example 1;

[0033] Figure 3 It is the gel formation and tissue adhesion test chart of the hydrogel precursor solution prepared by the present invention;

[0034] Figure 4 It is the detection of platelet adhesion and activation of the hydrogel prepared by the present invention;

[0035] Figure 5To detect the adhesion of endothelial cells to the hydrogel prepared by the present invention;

[0036] Figure 6 To detect the growth of endothelial cells on the hydrogel prepared by the present invention;

[0037] Figure 7 To detect the migration of endothelial cells on the hydrogel prepared by the present invention. Detailed implementation manners

[0038] The following describes the detailed implementation manners of the present invention to facilitate the understanding of the present invention by those skilled in the art. It should be clear, however, that the present invention is not limited to the scope of the detailed implementation manners. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions made using the concept of the present invention are within the scope of protection.

[0039] Example 1

[0040] A highly adhesive enzyme - promoted hydrogel for promoting vascular intima repair, and its preparation method includes the following steps:

[0041] S1. Synthesis of dopamine - modified double - bond - functionalized hyaluronic acid

[0042] Add methacrylic anhydride to the hyaluronic acid solution, adjust the pH to 8, react for 3 h, add ethanol to precipitate, wash the precipitate with alcohol, dissolve it in deionized water, dialyze and then freeze - dry. After freeze - drying, dissolve it in MES buffer to obtain a solution with a mass concentration of 1 wt%, then add NHS, EDC, DOPA, deoxygenate, adjust the pH to 5.5, react for 4 h, then adjust the pH of the system to acidic, dialyze and then freeze - dry;

[0043] The concentrations of each component in the above process are: hyaluronic acid 1 wt%, methacrylic anhydride 4.5 wt%, MES 100 mM, NHS 0.288 wt%, EDC 0.388 wt%, DOPA 0.471 wt%.

[0044] S2. Synthesis of tyramine - modified hyaluronic acid

[0045] After deoxygenating the hyaluronic acid solution, add NHS, EDC, and tyramine hydrochloride, adjust the pH to 4.5, react for 4 h, dialyze and then freeze - dry;

[0046] The concentrations of each component in the above process are: hyaluronic acid 1 wt%, NHS 0.288 wt%, EDC 0.388 wt%, tyramine hydrochloride 1.25 mM;

[0047] S3. Mix dopamine-modified double-bonded hyaluronic acid, tyramine-modified hyaluronic acid, and deionized water to prepare a hydrogel precursor solution with a mass concentration of 2 wt% for both dopamine-modified double-bonded hyaluronic acid and tyramine-modified hyaluronic acid. Moreover, the volume ratio of the HMD solution to the HT solution is 1:2.

[0048] S4. Divide the hydrogel precursor solution into two equal parts, namely A and B. Add hydrogen peroxide to solution A and horseradish peroxidase to solution B, where the concentration of hydrogen peroxide is 0.009% and the concentration of horseradish peroxidase is 20 U / mL.

[0049] S5. Blend solutions A and B and use an enzymatic reaction to prepare a highly adhesive enzyme-induced hydrogel for promoting vascular intima repair.

[0050] Example 2

[0051] A highly adhesive enzyme-induced hydrogel for promoting vascular intima repair, and its preparation method includes the following steps:

[0052] S1. Synthesis of dopamine-modified double-bonded hyaluronic acid

[0053] Add methacrylic anhydride to the hyaluronic acid solution, adjust the pH to 8, react for 3 h, add ethanol to precipitate, wash the precipitate with alcohol, dissolve it in deionized water, dialyze, and then lyophilize. After lyophilization, dissolve it in MES buffer to obtain a solution with a mass concentration of 1.5 wt%. Then add NHS, EDC, and DOPA, deoxygenate, adjust the pH to 5.5, react for 4 h, then adjust the pH of the system to acidic, dialyze, and lyophilize.

[0054] The concentrations of each component in the above process are as follows: hyaluronic acid 1.5 wt%, methacrylic anhydride 5 wt%, MES 120 mM, NHS 0.288 wt%, EDC 0.388 wt%, DOPA 0.5 wt%.

[0055] S2. Synthesis of tyramine-modified hyaluronic acid

[0056] After deoxygenating the hyaluronic acid solution, add NHS, EDC, and tyramine hydrochloride, adjust the pH to 4.5, react for 4 h, dialyze, and then lyophilize.

[0057] The concentrations of each component in the above process are as follows: hyaluronic acid 1 wt%, NHS 0.288 wt%, EDC 0.388 wt%, tyramine hydrochloride 1.5 mM.

[0058] S3. Mix dopamine-modified double-bonded hyaluronic acid, tyramine-modified hyaluronic acid, and deionized water to prepare a hydrogel precursor solution with a mass concentration of 2.5 wt% for both dopamine-modified double-bonded hyaluronic acid and tyramine-modified hyaluronic acid. Moreover, the volume ratio of the HMD solution to the HT solution is 1:1.

[0059] S4. Divide the hydrogel precursor solution into two equal parts, namely A and B. Add hydrogen peroxide to solution A and horseradish peroxidase to solution B. Among them, the concentration of hydrogen peroxide is 0.009%, and the concentration of horseradish peroxidase is 20 U / mL.

[0060] S5. Blend solutions A and B and use an enzymatic reaction to prepare a highly adhesive enzyme-catalyzed hydrogel for promoting vascular intima repair.

[0061] Example 3

[0062] A highly adhesive enzyme-catalyzed hydrogel for promoting vascular intima repair, and its preparation method includes the following steps:

[0063] S1. Synthesis of dopamine-modified double-bonded hyaluronic acid

[0064] Add methacrylic anhydride to the hyaluronic acid solution, adjust the pH to 8, react for 3 h, add ethanol to precipitate, wash the precipitate with alcohol, dissolve it in deionized water, dialyze and then freeze-dry; after freeze-drying, dissolve it in MES buffer solution to obtain a solution with a mass concentration of 1 wt%, then add NHS, EDC, DOPA, deoxygenate, adjust the pH to 5.5, react for 4 h, then adjust the pH of the system to acidic, dialyze and then freeze-dry;

[0065] The concentrations of each component in the above process are: hyaluronic acid 1 wt%, methacrylic anhydride 4.5 wt%, MES 110 mM, NHS 0.288 wt%, EDC 0.388 wt%, DOPA 0.3 wt%.

[0066] S2. Synthesis of tyramine-modified hyaluronic acid

[0067] After deoxygenating the hyaluronic acid solution, add NHS, EDC, and tyramine hydrochloride, adjust the pH to 4.5, react for 4 h, dialyze and then freeze-dry;

[0068] The concentrations of each component in the above process are: hyaluronic acid 1 wt%, NHS 0.288 wt%, EDC 0.388 wt%, tyramine hydrochloride 1.2 mM;

[0069] S3. Mix dopamine-modified double-bonded hyaluronic acid, tyramine-modified hyaluronic acid and deionized water to prepare a hydrogel precursor solution with the mass concentrations of dopamine-modified double-bonded hyaluronic acid and tyramine-modified hyaluronic acid both being 3 wt%; and the volume ratio of the HMD solution to the HT solution is 2:1;

[0070] S4. Divide the hydrogel precursor solution into two equal parts, namely A and B. Add hydrogen peroxide to solution A and horseradish peroxidase to solution B. Among them, the concentration of hydrogen peroxide is 0.009%, and the concentration of horseradish peroxidase is 20 U / mL.

[0071] S5. The S, A, and B solutions are blended and an enzyme-catalyzed reaction is used to prepare a highly adhesive enzyme-catalyzed hydrogel for promoting vascular intima repair.

[0072] Example 4

[0073] A highly adhesive enzyme-catalyzed hydrogel for promoting vascular intima repair, and its preparation method includes the following steps:

[0074] S1. Synthesis of dopamine-modified double-bonded hyaluronic acid

[0075] Add methacrylic anhydride to the hyaluronic acid solution, adjust the pH to 8, react for 2 h, add ethanol to precipitate, wash the precipitate with alcohol, dissolve it in deionized water, dialyze and then lyophilize; after lyophilization, dissolve it in MES buffer to obtain a solution with a mass concentration of 1 wt%, then add NHS, EDC, DOPA, deoxygenate, adjust the pH to 5.5, react for 4 h, then adjust the pH of the system to acidic, dialyze and then lyophilize;

[0076] The concentrations of each component in the above process are: hyaluronic acid 1 wt%, methacrylic anhydride 5 wt%, MES 100 mM, NHS 0.28 wt%, EDC 0.38 wt%, DOPA 0.46 wt%;

[0077] S2. Synthesis of tyramine-modified hyaluronic acid

[0078] After deoxygenating the hyaluronic acid solution, add NHS, EDC, and tyramine hydrochloride, adjust the pH value to 5, react for 8 h, dialyze and then lyophilize;

[0079] The concentrations of each component in the above process are: hyaluronic acid 1 wt%, NHS 0.28 wt%, EDC 0.38 wt%, tyramine hydrochloride 1.2 mM.

[0080] S3. Mix dopamine-modified double-bonded hyaluronic acid, tyramine-modified hyaluronic acid, and deionized water to prepare a precursor hydrogel solution with a mass concentration of 3 wt% for both dopamine-modified double-bonded hyaluronic acid and tyramine-modified hyaluronic acid; and, the volume ratio of the HMD solution to the HT solution is 1:1;

[0081] S4. Divide the precursor hydrogel solution into two equal parts, A and B. Add hydrogen peroxide to solution A and horseradish peroxidase to solution B. Among them, the concentration of hydrogen peroxide is 0.019%, and the concentration of horseradish peroxidase is 10 U / mL;

[0082] S5: Blend solutions A and B and use an enzyme-catalyzed reaction to prepare a highly adhesive enzyme-catalyzed hydrogel for promoting vascular intima repair.

[0083] Example 5

[0084] A highly adhesive enzyme-catalyzed hydrogel for promoting vascular intima repair, and its preparation method includes the following steps:

[0085] S1. Synthesis of dopamine-modified double-bonded hyaluronic acid

[0086] Add methacrylic anhydride to the hyaluronic acid solution, adjust the pH to 8, react for 3 h, add ethanol to precipitate, wash the precipitate with alcohol, dissolve it in deionized water, dialyze and then freeze-dry; after freeze-drying, dissolve it in MES buffer to obtain a solution with a mass concentration of 1 wt%, then add NHS, EDC, DOPA, deoxygenate, adjust the pH to 5.5, react for 4 h, then adjust the pH of the system to acidic, dialyze and then freeze-dry;

[0087] The concentrations of each component in the above process are as follows: hyaluronic acid 1 wt%, methacrylic anhydride 5 wt%, MES 100 mM, NHS 0.29 wt%, EDC 0.39 wt%, DOPA 0.48 wt%;

[0088] S2. Synthesis of tyramine-modified hyaluronic acid

[0089] After deoxygenating the hyaluronic acid solution, add NHS, EDC, and tyramine hydrochloride, adjust the pH value to 5, react for 4 h, dialyze and then freeze-dry;

[0090] The concentrations of each component in the above process are as follows: hyaluronic acid 1 wt%, NHS 0.29 wt%, EDC 0.39 wt%, tyramine hydrochloride 1.3 mM.

[0091] S3. Mix dopamine-modified double-bonded hyaluronic acid, tyramine-modified hyaluronic acid and deionized water to prepare a hydrogel precursor solution with the mass concentrations of dopamine-modified double-bonded hyaluronic acid and tyramine-modified hyaluronic acid both being 2 wt%; and, the volume ratio of the HMD solution to the HT solution is 1:1;

[0092] S4. Divide the hydrogel precursor solution into two equal parts, A and B. Add hydrogen peroxide to solution A and horseradish peroxidase to solution B, where the concentration of hydrogen peroxide is 0.019% and the concentration of horseradish peroxidase is 30 U / mL;

[0093] S5. Blend solutions A and B and use an enzymatic reaction to prepare a highly adhesive enzymatic hydrogel for promoting vascular intima repair.

[0094] Test Example

[0095] Taking the preparation method in Example 1 as an example, the prepared product was tested as follows:

[0096] 1. Fourier transform infrared spectroscopy analysis

[0097] Grind and tablet the raw material hyaluronic acid, dopamine-modified double-bonded hyaluronic acid and tyramine-modified hyaluronic acid prepared in Example 1 of the present invention with potassium bromide (KBr) respectively. Through FT-IR in the range of 4000 - 500 cm -1An infrared absorption peak is obtained at the wavenumber.

[0098] As Figure 1 shown, compared with hyaluronic acid, the C-O stretching vibration of phenol can be observed at 1262 cm -1 , indicating that dopamine and tyramine were successfully grafted onto the molecular chain of hyaluronic acid respectively.

[0099] 2. 1H NMR analysis

[0100] The raw material hyaluronic acid, dopamine-modified double-bonded hyaluronic acid and tyramine-modified hyaluronic acid prepared in Example 1 of the present invention were respectively dissolved in deuterated water for 1H NMR analysis to obtain 1H NMR spectra characterizing the structures.

[0101] As Figure 2 shown, the analysis results of MestReNova software indicate that the NMR peak at the chemical shift δ = 2.95 ppm is the characteristic peak of the two methylene groups connecting the benzene ring of dopamine and tyramine, indicating that dopamine and tyramine were successfully grafted onto the molecular chain of hyaluronic acid.

[0102] 3. Gelation and tissue adhesion test

[0103] The hydrogel prepared in Example 1 was coated on the inner wall of the rabbit thoracic aorta, and the gel formed and adhered firmly. Then, the gel coating was twisted, stretched and washed with water flow, and the gel coating could still adhere firmly to the tissue without breakage and shedding, as Figure 3 shown. It shows that the coating prepared from the gel precursor solution of the present invention has high adhesion to wet tissue.

[0104] 4. Platelet adhesion and activation

[0105] The platelet adhesion and activation tests were carried out on the hydrogel coatings prepared in Examples 1-3 of the present invention. The number and morphology of platelets were observed by scanning electron microscopy. The specific test method is as follows:

[0106] After the platelet-rich plasma was co-incubated with the hydrogels prepared in Examples 1-3, it was fixed with 2.5% glutaraldehyde solution for 24 h, dehydrated successively with ethanol aqueous solutions with concentrations of 30-100%, and observed by scanning electron microscopy after drying. The results are shown in Figure 4 , where HMDHT0.5 is the hydrogel prepared in Example 1, HMDHT1 is the hydrogel prepared in Example 2, and HMDHT2 is the hydrogel prepared in Example 3.

[0107] As Figure 4 shown, compared with the 316L stainless steel group, the platelet adhesion amount was significantly reduced and platelet activation was less, indicating that the anticoagulant effect of the hydrogel coating prepared by the present invention is very excellent.

[0108] 5. Endothelial cell adhesion, growth and migration

[0109] The gel coatings prepared in Examples 1-3 of the present invention were subjected to endothelial cell adhesion tests, CCK-8 tests and cell migration tests, and the results are shown in Figures 5 to 7 , and the specific process is as follows:

[0110] (1) Endothelial cell adhesion test:

[0111] The prepared hydrogel product was sterilized and placed in a 24-well plate. 500 μl of tracer-stained endothelial cell suspension (cell density about 25,000 cells per well) was added. After incubating in an incubator for 4 h, the surface was gently rinsed and then photographed with a confocal microscope.

[0112] (2) CCK-8 test:

[0113] Endothelial cells were seeded at 5,000 cells per well in a 48-well plate. After adhesion, the culture medium was replaced with the hydrogel product culture medium extract. After 1 day and 3 days, the culture medium was aspirated, 200 μl of CCK-8 solution was added per well and incubated for 2 h, and then 100 μl of the culture medium was aspirated into a 96-well plate and the absorbance value of the solution was measured with an enzyme-linked immunosorbent assay (ELISA) reader.

[0114] (3) Cell migration test:

[0115] One side of a stainless steel sheet folded into an L shape was coated with the hydrogel product and sterilized. The uncoated side of the stainless steel sheet was placed at the bottom of the well plate, and tracer-stained endothelial cells (25,000 / well) were seeded. After the cells adhered, the stainless steel sheet was transferred to another 24-well plate so that the side with the hydrogel was at the bottom of the well plate. At this time, the endothelial cells on the vertical plane would start to migrate. After 24 hours, the cell migration images on the hydrogel side were photographed with a confocal microscope.

[0116] As Figure 5 shown, it can be seen that compared with the control group, the amount of endothelial cell adhesion in the hydrogel coating group prepared by the present invention is more. The CCK-8 results are as Figure 6 shown, indicating that the hydrogel coating group prepared by the present invention can promote the growth of endothelial cells. The results of the cell migration experiment are as Figure 7 shown. It can be seen from the figure that the hydrogel coating group prepared by the present invention can better promote the migration of endothelial cells.

[0117] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a high-adhesion enzymatic hydrogel for promoting vascular endothelial repair, characterized in that: The following steps are involved: S1, dopamine modified double bond hyaluronic acid Hyaluronic acid is double-bonded by using methacrylic anhydride, and then cross-linked with dopamine to obtain dopamine-modified double-bonded hyaluronic acid. S2, tyrosine modified hyaluronic acid After deoxygenation of the hyaluronic acid solution, a crosslinking agent and tyramine hydrochloride are added, and the mixture is reacted in an acidic environment for 4 to 8 hours to obtain tyramine-modified hyaluronic acid; S3, mixing the dopamine-modified double-bonded hyaluronic acid solution and the tyramine-modified hyaluronic acid solution to obtain a hydrogel precursor solution in which the mass concentrations of the dopamine-modified double-bonded hyaluronic acid and the tyramine-modified hyaluronic acid are both 2-3 wt %; S4. Then, the enzymatic reaction is used to prepare a high-adhesion enzymatic hydrogel that promotes vascular endothelial repair.

2. The preparation method according to claim 1, characterized in that: The process of double-bonding hyaluronic acid using methacrylic anhydride is as follows: Add methacrylic anhydride to the hyaluronic acid solution, adjust the pH to 8-9, react for 2-3 hours, add ethanol to precipitate, wash the precipitate with alcohol, dissolve it in deionized water, dialyze and freeze-dry it to obtain the product; The mass concentration of hyaluronic acid is 1-1.5wt%, and the mass concentration of methacrylic anhydride is 4.5-5wt%.

3. The preparation method according to claim 1 or 2, characterized in that: The process of dopamine-modified double-bonded hyaluronic acid is as follows: The double-bond modified hyaluronic acid is dissolved in MES buffer to obtain a solution with a mass concentration of 1 to 1.5 wt%, and then a cross-linking agent and dopamine are added, and the pH is adjusted to 5 to 6 after deoxygenation, and after reacting for 4 to 8 hours, the pH is adjusted to acidic, and dialyzed and freeze-dried to obtain the solution; The concentration of the MES buffer is 100-120 mM, and the mass concentration of dopamine is 0.3-0.5 wt %.

4. The preparation method according to claim 3, characterized in that: The cross-linking agent used is NHS / EDC, wherein the mass concentration of NHS is 0.2-0.3wt%, and the mass concentration of EDC is 0.3-0.4wt%.

5. The preparation method according to claim 1, characterized in that: The mass concentration of hyaluronic acid in S2 is 0.5-1.5wt%, and the concentration of tyramine hydrochloride is 1.2-1.5mM.

6. The preparation method according to claim 1, characterized in that: The cross-linking agent used in S2 is NHS / EDC, wherein the mass concentration of NHS is 0.2-0.3 wt %, and the mass concentration of EDC is 0.3-0.4 wt %.

7. The preparation method according to claim 1, characterized in that: The volume ratio of the dopamine-modified double-bonded hyaluronic acid solution to the tyramine-modified hyaluronic acid solution in S3 is 1:0.5 to 0.5:

1.

8. The preparation method according to claim 1, characterized in that: The process of the enzymatic reaction is: The hydrogel precursor liquid is evenly divided into liquid A and liquid B, hydrogen peroxide is added to liquid A, horseradish peroxidase is added to liquid B, and then liquids A and B are mixed to obtain a hydrogel that promotes vascular endothelial repair by an enzymatic reaction.

9. The preparation method according to claim 8, characterized in that: The concentration of hydrogen peroxide is 0.009%-0.019%, and the concentration of horseradish peroxidase is 10-30 U / mL.

10. A high-adhesion enzymatic hydrogel for promoting vascular endothelial repair, characterized in that: The method is prepared by any one of claims 1 to 9.

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