Preparation method and application of response type NO release anticoagulant hydrogel material

Abstract

The invention discloses a preparation method and application of a response type NO release anticoagulant hydrogel material. The preparation method comprises the following steps: providing methylacryloylated recombinant collagen; providing methacrylated hyaluronic acid; providing caffeic acid arginine amide modified hyaluronic acid; the response type NO release anticoagulant hydrogel material is obtained by taking methylacryloylated recombinant collagen, methylacryloylated hyaluronic acid and caffeic acid arginine amide modified hyaluronic acid as raw materials. The prepared hydrogel material has the advantages of resisting bacteria, promoting proliferation of endothelial cells, inhibiting proliferation of smooth muscle cells, resisting inflammation, promoting angiogenesis, inhibiting thrombosis and the like, arginine can serve as an endogenous NO donor in blood to release NO molecules, and therefore the effect of promoting vascular endothelial repair is achieved, and the hydrogel material has the advantages of being capable of resisting bacteria, promoting proliferation of endothelial cells, inhibiting proliferation of smooth muscle cells, resisting inflammation, promoting angiogenesis, inhibiting thrombosis and the like. The introduced NO molecules enable the hydrogel material to have good anticoagulant performance, and the hydrogel material is expected to be used for preparing an artificial blood vessel anticoagulant coating.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and in particular relates to a preparation method and application of a responsive NO-releasing anticoagulant hydrogel material. Background Art

[0002] In the process of treating cardiovascular diseases, due to the limitations of autologous blood vessels, artificial blood vessels are considered to be an important substitute for autologous grafts. Large-diameter vascular grafts (D>6mm) have been used clinically, however, there are great limitations in the application of small-diameter vascular grafts (D<6mm). It is reported that 75% of artificial vascular grafts fail within three years after implantation due to clinical complications such as infection, thrombosis, intimal hyperplasia, calcification and aneurysm formation.

[0003] The main causes of small-caliber graft failure are acute thrombosis, intimal hyperplasia, and infection. Therefore, improving endothelialization, scavenging reactive oxygen species to inhibit intimal hyperplasia, and preventing infection are promising strategies to reduce graft failure. Therefore, it is urgent to develop an anticoagulant hydrogel material that can responsively release NO. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing a responsive anticoagulant hydrogel material that releases NO in view of the deficiencies of the above-mentioned prior art. The hydrogel material prepared by the method has the advantages of antibacterial, promoting the proliferation of endothelial cells, inhibiting the proliferation of smooth muscle cells, anti-inflammatory, promoting angiogenesis and inhibiting thrombosis, among which arginine can release NO molecules as an endogenous NO donor in the blood, thereby promoting the repair of vascular endothelium. The introduced NO molecules make the hydrogel material have good anticoagulant properties, and it is expected to be used to prepare artificial vascular anticoagulant coatings.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for preparing a responsive anticoagulant hydrogel material that releases NO, characterized by comprising the following steps:

[0006] Step 1: dissolving methacrylylated recombinant collagen and a photoinitiator in deionized water under light-proof conditions to obtain liquid A; dissolving methacrylylated hyaluronic acid in deionized water to obtain liquid B; dissolving caffeic acid arginine amide-modified hyaluronic acid in deionized water to obtain liquid C;

[0007] Step 2: Mix the liquid A, liquid B and liquid C in step 1 to obtain a gel solution;

[0008] Step 3: Place the gel solution in step 2 in a refrigerator for 8 hours to 36 hours, irradiate with ultraviolet light for 3 minutes to 5 minutes for photo-crosslinking and curing, and sterilize to obtain a responsive anticoagulant hydrogel material that releases NO.

[0009] The above-mentioned method for preparing a responsive anticoagulant hydrogel material that releases NO is characterized in that the method for preparing the methacrylylated recombinant collagen described in step one specifically includes: adding glycidyl methacrylate to the recombinant collagen solution, stirring the reaction in a 37°C water bath for 6h to 8h, dialyzing, and freeze-drying to obtain the methacrylylated recombinant collagen.

[0010] The above-mentioned method for preparing a responsive anticoagulant hydrogel material that releases NO is characterized in that the recombinant collagen solution is a recombinant collagen solution obtained by dissolving recombinant collagen in deionized water, and the mass-volume concentration of the recombinant collagen solution is 1% to 2%; the relative molecular mass of the recombinant collagen is 97KDa; the amount of glycidyl methacrylate is 10mL to 30mL of glycidyl methacrylate per gram of recombinant collagen.

[0011] The above-mentioned method for preparing a responsive NO-releasing anticoagulant hydrogel material is characterized in that the preparation method of the methacryloyl hyaluronic acid described in step one specifically includes: adding glycidyl methacrylate to the hyaluronic acid solution, stirring in a 60°C water bath for 6h to 8h, dialyzing, and freeze-drying to obtain methacryloyl hyaluronic acid.

[0012] The above-mentioned method for preparing a responsive anticoagulant hydrogel material that releases NO is characterized in that the hyaluronic acid solution is a hyaluronic acid solution obtained by dissolving sodium hyaluronate in deionized water, and the mass-volume concentration of the hyaluronic acid solution is 1% to 2%; the relative molecular mass of the sodium hyaluronate is 10KDa to 500KDa; the amount of the glycidyl methacrylate is 10mL to 30mL of glycidyl methacrylate per gram of sodium hyaluronate.

[0013] The above-mentioned method for preparing a responsive NO-releasing anticoagulant hydrogel material is characterized in that the photoinitiator in step one is lithium phenyl-2,4,6-trimethylbenzoylphosphonate.

[0014] The above-mentioned method for preparing a responsive anticoagulant hydrogel material that releases NO is characterized in that the preparation method of the caffeic acid arginine amide modified hyaluronic acid in step 1 specifically includes:

[0015] Step 101, using 3-aminophenylboronic acid and sodium hyaluronate as raw materials to prepare 3-aminophenylboronic acid-modified hyaluronic acid; then dissolving the prepared 3-aminophenylboronic acid-modified hyaluronic acid in deionized water to obtain a 3-aminophenylboronic acid-modified hyaluronic acid solution;

[0016] Step 102, using caffeic acid and arginine as raw materials, and preparing caffeic acid arginine amide by thin layer chromatography purification; then dissolving the prepared caffeic acid arginine amide in deionized water to obtain a caffeic acid arginine amide solution;

[0017] Step 103, under stirring conditions, dropwise add the caffeic acid arginine amide solution described in step 102 to the 3-aminophenylboronic acid modified hyaluronic acid solution described in step 101, stir for 46 h to 48 h, dialyze, and freeze-dry to obtain caffeic acid arginine amide modified hyaluronic acid.

[0018] The above-mentioned method for preparing a responsive anticoagulant hydrogel material that releases NO is characterized in that the relative molecular mass of the sodium hyaluronate described in step 101 is 10KDa~500KDa, the molar ratio of 3-aminophenylboronic acid to sodium hyaluronate is 1:(1.5~2), and the mass-volume concentration of the 3-aminophenylboronic acid modified hyaluronic acid solution is 2%~5%.

[0019] The above-mentioned method for preparing a responsive anticoagulant hydrogel material that releases NO is characterized in that the mass of arginine in step 102 is 0.5 to 2 times the mass of caffeic acid, and the concentration of the caffeic acid arginine amide solution is 20 mg / mL to 50 mg / mL; in step 103, the volume ratio of the caffeic acid arginine amide solution to the 3-aminophenylboronic acid modified hyaluronic acid solution is 1: (1 to 2).

[0020] The above-mentioned method for preparing a responsive anticoagulant hydrogel material that releases NO is characterized in that the mass-volume concentration of methacryloyl recombinant collagen in the gel solution in step 2 is 5% to 10%, the mass-volume concentration of methacryloyl hyaluronic acid is 0.5% to 2%, the mass-volume concentration of the photoinitiator is 0.1% to 0.5%, and the concentration of caffeic acid arginine amide modified hyaluronic acid is 0.5 mg / mL to 1.5 mg / mL.

[0021] Furthermore, the present invention also provides an application of the anticoagulant hydrogel material prepared by the above method in preparing an artificial blood vessel anticoagulant coating.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] 1. The present invention utilizes the good biocompatibility, biodegradability and low risk of infection and immune response of recombinant collagen, which is beneficial to the growth of endothelial cells and realizes effective endothelialization; 3-aminophenylboronic acid and caffeic acid have anti-inflammatory properties due to their catechol structure, which prevent infection and realize the removal of ROS; the phenylboronic acid ester bond can respond to ROS breakage to release caffeic acid and arginine; arginine can release NO by acting on NO synthase produced by endothelial cells, thereby showing a good anticoagulant effect to maintain blood vessel patency, and can further stimulate the release of NO when the blood vessels are in a high active oxygen environment, reduce the level of ROS, inhibit the proliferation of smooth muscle cells, thereby inhibiting intimal hyperplasia, and hindering thrombosis. Recombinant collagen was creatively combined with caffeic acid arginine amide-modified hyaluronic acid to obtain a hydrogel material that induces NO release. The prepared hydrogel material has the advantages of antibacterial, promoting endothelial cell proliferation, inhibiting smooth muscle cell proliferation, anti-inflammatory, promoting angiogenesis and inhibiting thrombosis. Among them, arginine can serve as an endogenous NO donor in the blood to release NO molecules, thereby promoting vascular endothelial repair. The introduced NO molecules give the hydrogel material good anticoagulant properties, and it is expected to be used to prepare artificial vascular anticoagulant coatings.

[0024] 2. The raw materials of the present invention include recombinant collagen modified with glycidyl methacrylate, and its photosensitivity can effectively promote the UV curing process, which is beneficial to the growth of endothelial cells and realizes effective endothelialization.

[0025] 3. The present invention preferably first obtains caffeic acid arginine amide by thin layer chromatography, then modifies hyaluronic acid with the synthesized caffeic acid arginine amide by 3-aminophenylboronic acid, and then incorporates it into a hydrogel material, utilizing the characteristic of sustained release of NO in the hydrogel to exert the effect of inducing the migration and proliferation of endothelial cells and promoting angiogenesis by the hydrogel scaffold.

[0026] 4. The preparation method of the present invention has reliable principle and is conducive to popularization and application.

[0027] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the preparation process and physicochemical properties of each component of Example 1 of the present invention; wherein a is a schematic diagram of the process of preparing methacryloyl recombinant collagen; b is a Fourier transform infrared absorption spectrum analysis diagram of methacryloyl recombinant collagen; c is a Fourier transform infrared absorption spectrum analysis diagram of methacryloyl recombinant collagen 1H-NMR analysis results; d is a schematic diagram of the process for preparing methacryloyl hyaluronic acid; e is a Fourier transform infrared absorption spectrum analysis diagram of methacryloyl hyaluronic acid; f is a 1H-NMR analysis result of methacryloyl hyaluronic acid; g is a schematic diagram of the process for preparing caffeic acid arginine amide; h is a Fourier transform infrared absorption spectrum analysis diagram of caffeic acid arginine amide; i is a 1 H-NMR analysis results; j is a schematic diagram of the process for preparing 3-aminophenylboronic acid modified hyaluronic acid; k is a Fourier transform infrared absorption spectrum analysis diagram of 3-aminophenylboronic acid modified hyaluronic acid; l is a 3-aminophenylboronic acid modified hyaluronic acid 1 H-NMR analysis results; m is a schematic diagram of the process for preparing caffeic acid arginine amide modified hyaluronic acid; n is a Fourier transform infrared absorption spectrum analysis diagram of caffeic acid arginine amide modified hyaluronic acid; o is a diagram of caffeic acid arginine amide modified hyaluronic acid 1 H-NMR analysis results.

[0029] Figure 2 This is a scanning electron microscope image of the hydrogel prepared in Example 1 of the present invention at a scale of 100 μm.

[0030] Figure 3 These are the antibacterial performance test results of the hydrogel material prepared in Example 1 of the present invention; wherein a is a graph showing the number of bacteria after culturing in the antibacterial performance test of the hydrogel material; b is a graph showing the counting results after culturing Escherichia coli in the antibacterial performance test of the hydrogel material; and c is a graph showing the counting results after culturing Staphylococcus aureus in the antibacterial performance test of the hydrogel material.

[0031] Figure 4 This is the cell viability result calculated from the smooth muscle cell compatibility of the hydrogel material prepared in Example 1 of the present invention.

[0032] Figure 5 These are the cell proliferation ability test results of the hydrogel material prepared in Example 1 of the present invention; wherein a is the quantitative result in the test of the hydrogel material's ability to promote endothelial cell proliferation; b is the quantitative result in the test of the hydrogel material's ability to inhibit smooth muscle cell proliferation; a is the fluorescence staining result in the test of the hydrogel material's ability to promote endothelial cell proliferation; d is the fluorescence staining result in the test of the hydrogel material's ability to inhibit smooth muscle cell proliferation.

[0033] Figure 6 This is the fluorescence staining result of the anti-inflammatory ability test of the hydrogel material prepared in Example 1 of the present invention.

[0034] Figure 7 The photographic results of the angiogenesis ability test of the hydrogel material prepared in Example 1 of the present invention.

[0035] Figure 8 These are the anti-coagulation ability test results of the hydrogel material prepared in Example 1 of the present invention; wherein a is the anti-coagulation ability test result of the hydrogel material for platelet adhesion; b is the anti-coagulation ability test result of the hydrogel material for lactate dehydrogenase; c is the anti-coagulation ability test result of the hydrogel material for coagulation index; d is the anti-coagulation ability test result of the hydrogel material for activated partial thromboplastin time and prothrombin time; e is the anti-coagulation ability test result of the hydrogel material for fibrinogen adhesion; and f is the test result of the hydrogel material for fibrinogen adhesion fluorescence quantification. DETAILED DESCRIPTION

[0036] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the embodiments. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0037] The present invention provides a method for preparing a responsive anticoagulant hydrogel material for releasing NO, comprising the following steps:

[0038] Step 1: dissolving methacrylylated recombinant collagen and a photoinitiator in deionized water under light-proof conditions to obtain liquid A; dissolving methacrylylated hyaluronic acid in deionized water to obtain liquid B; dissolving caffeic acid arginine amide-modified hyaluronic acid in deionized water to obtain liquid C;

[0039] The preparation method of the methacryloyl recombinant collagen specifically comprises: adding glycidyl methacrylate to a recombinant collagen solution, stirring in a water bath at 37°C for 6h to 8h, dialyzing, and freeze-drying to obtain the methacryloyl recombinant collagen; the recombinant collagen solution is a recombinant collagen solution obtained by dissolving recombinant collagen in deionized water, and the mass-volume concentration of the recombinant collagen solution is 1% to 2%; the relative molecular mass of the recombinant collagen is 97KDa; the amount of glycidyl methacrylate is 10mL to 30mL of glycidyl methacrylate per gram of recombinant collagen;

[0040] The preparation method of the methacryloyl hyaluronic acid specifically comprises: adding glycidyl methacrylate to a hyaluronic acid solution, stirring in a water bath at 60° C. for a reaction for 6 h to 8 h, dialyzing, and freeze-drying to obtain the methacryloyl hyaluronic acid; the hyaluronic acid solution is a hyaluronic acid solution obtained by dissolving sodium hyaluronate in deionized water, and the mass-volume concentration of the hyaluronic acid solution is 1% to 2%; the relative molecular mass of the sodium hyaluronate is 10KDa to 500KDa; the amount of the glycidyl methacrylate is 10mL to 30mL of glycidyl methacrylate per gram of sodium hyaluronate;

[0041] The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphonate;

[0042] The preparation method of the caffeic acid arginine amide modified hyaluronic acid specifically comprises:

[0043] Step 101, using 3-aminophenylboronic acid and sodium hyaluronate as raw materials to prepare 3-aminophenylboronic acid-modified hyaluronic acid; then dissolving the prepared 3-aminophenylboronic acid-modified hyaluronic acid in deionized water to obtain a 3-aminophenylboronic acid-modified hyaluronic acid solution; the relative molecular mass of the sodium hyaluronate is 10KDa to 500KDa, the molar ratio of 3-aminophenylboronic acid to sodium hyaluronate is 1:(1.5 to 2), and the mass-volume concentration of the 3-aminophenylboronic acid-modified hyaluronic acid solution is 2% to 5%;

[0044] Step 102, using caffeic acid and arginine as raw materials, and preparing caffeic acid arginine amide by thin layer chromatography purification; then dissolving the prepared caffeic acid arginine amide in deionized water to obtain a caffeic acid arginine amide solution; the mass of the arginine is 0.5 to 2 times the mass of the caffeic acid, and the concentration of the caffeic acid arginine amide solution is 20 mg / mL to 50 mg / mL;

[0045] Step 103, under stirring conditions, dropwise adding the caffeic acid arginine amide solution described in step 102 to the 3-aminophenylboronic acid modified hyaluronic acid solution described in step 101, wherein the volume ratio of the caffeic acid arginine amide solution to the 3-aminophenylboronic acid modified hyaluronic acid solution is 1:(1-2), stirring for 46h-48h, dialyzing, and freeze-drying to obtain caffeic acid arginine amide modified hyaluronic acid;

[0046] Step 2, mixing the liquid A, liquid B and liquid C described in step 1 to obtain a gel solution; in the gel solution, the mass-volume concentration of methacryloyl recombinant collagen is 5% to 10%, the mass-volume concentration of methacryloyl hyaluronic acid is 0.5% to 2%, the mass-volume concentration of the photoinitiator is 0.1% to 0.5%, and the concentration of caffeic acid arginine amide modified hyaluronic acid is 0.5 mg / mL to 1.5 mg / mL;

[0047] Step 3: Place the gel solution in step 2 in a refrigerator at 4°C for 8h to 36h, irradiate with ultraviolet light for 3min to 5min for photo-crosslinking and curing, and sterilize to obtain a responsive anticoagulant hydrogel material that releases NO.

[0048] The present invention is specifically described below in conjunction with embodiments, but the following description is not intended to limit the present invention.

[0049] Example 1

[0050] This embodiment provides a method for preparing a responsive anticoagulant hydrogel material that releases NO, which specifically includes:

[0051] Step 1: providing methacrylylated recombinant collagen, specifically comprising:

[0052] Step 101, dissolving 1g of recombinant collagen in 100mL of deionized water to obtain a recombinant collagen solution; the recombinant collagen is obtained by reverse transcription of human collagen mRNA to generate cDNA, and then expressed in Escherichia coli BL21. Its structure and acquisition method can be referred to, for example, the patent application document with patent number ZL01106757.8 and patent name "A human-like collagen and its production method"; the recombinant collagen has a relative molecular mass of 97KDa;

[0053] Step 102, add 30 mL of glycidyl methacrylate dropwise to the recombinant collagen solution, stir in a water bath at 37° C. for 6 h, and collect the product;

[0054] Step 103, dialyzing the product in deionized water for 7 consecutive days, and freeze-drying to obtain methacrylylated collagen; the dialysis bag has a molecular weight cutoff of 3500 Da;

[0055] Step 2: providing methacrylylated hyaluronic acid, specifically comprising:

[0056] Step 201, dissolving 1 g of sodium hyaluronate in 100 mL of deionized water to obtain a hyaluronic acid solution; the relative molecular mass of the sodium hyaluronate is 10 KDa;

[0057] Step 202, adding 30 mL of glycidyl methacrylate dropwise to the hyaluronic acid solution, stirring in a water bath at 60° C. for 6 h, and collecting the product;

[0058] Step 203, dialyzing the product in deionized water for 7 consecutive days, and freeze-drying to obtain methacrylylated hyaluronic acid; the dialysis bag has a molecular weight cutoff of 3500 Da;

[0059] Step 3: providing a method for preparing caffeic acid arginine amide modified hyaluronic acid, which specifically comprises:

[0060] Step 301, adding 3-aminophenylboronic acid (93 mg, 0.50 mmol) to a mixture of water / dimethylformamide (water / DMF, 3:2 v / v, total solvent volume of 25 mL) containing 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride (212 mg, 0.77 mmol) and sodium hyaluronate (0.77 mmol, relative molecular mass of 10 KDa);

[0061] Step 302, adding 1M NaOH aqueous solution to the mixed solution of step 301 to adjust the pH value to 6.5; stirring the mixture at room temperature for 46 hours;

[0062] Step 303, dialyzing the mixed solution after adjusting the pH value in deionized water for 7 days, and freeze-drying to obtain 3-aminophenylboronic acid-modified hyaluronic acid; dissolving 3-aminophenylboronic acid-modified hyaluronic acid in deionized water to obtain a 3-aminophenylboronic acid-modified hyaluronic acid solution with a mass-volume concentration of 4%; the dialysis bag has a cut-off molecular weight of 3500Da;

[0063] Step 304, under nitrogen protection, 5 g of caffeic acid was dissolved in 100 mL of tetrahydrofuran, and 0.5 g of N-methylmorpholine and 0.5 mL of isobutyl chloroformate were added in sequence at 0° C. to react for 15 min to obtain a caffeic acid solution;

[0064] Step 305, adding 2.5 g of arginine L-Arg to the caffeic acid solution, reacting at 0°C, tracking the reaction by TLC, and detecting the reaction until there is no substrate by TLC, placing the reaction solution at room temperature for 10 h, and then concentrating the reaction solution under reduced pressure, extracting the solution with ethyl acetate three times (3x10 mL) after concentrating under reduced pressure, washing the organic phase with 5% citric acid solution and saturated saline solution in sequence, drying the organic layer with anhydrous Na2SO4, concentrating under reduced pressure to obtain a product, purifying it with thin layer chromatography to obtain caffeic acid arginine amide, and dissolving caffeic acid arginine amide in deionized water to obtain a caffeic acid arginine amide solution with a concentration of 40 mg / mL;

[0065] Step 306, under stirring conditions, dropwise adding the caffeic acid arginine amide solution described in step 305 to the 3-aminophenylboronic acid modified hyaluronic acid solution described in step 301, wherein the volume ratio of the caffeic acid arginine amide solution to the 3-aminophenylboronic acid modified hyaluronic acid solution is 1:1, stirring for 46 hours, dialyzing, and freeze-drying to obtain caffeic acid arginine amide modified hyaluronic acid;

[0066] Step 4: providing a method for preparing a responsive anticoagulant hydrogel material that releases NO, specifically comprising:

[0067] Step 401, placing 500 mg of methacrylated recombinant collagen and 12.5 mg of photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) in 2 mL of deionized water, sealing with tin foil to avoid light, and stirring at room temperature until completely dissolved to obtain solution A; the stirring time at room temperature may be 12 hours;

[0068] Step 402, placing 100 mg of methacryloyl hyaluronic acid in 2.5 mL of deionized water, stirring in a 37° C. water bath until completely dissolved, to obtain liquid B; the stirring time in the water bath may be 2 hours;

[0069] Step 403, dissolving 2.5 mg of caffeic acid arginine amide modified hyaluronic acid in 0.5 mL of deionized water, and sonicating until completely dissolved to obtain liquid C; the sonication time may be 1 hour;

[0070] Step 404, mixing the solution A in step 401, the solution B in step 402, and the solution C in step 403, and continuing stirring for 1 hour to obtain a gel solution; the gel solution has no obvious fluidity and is milky white;

[0071] Step 405, placing the gel solution in a refrigerator at 4°C for 12 hours to allow the gel solution to be fully cross-linked and defoamed;

[0072] Step 406: Expose the gel solution after standing in step 405 to ultraviolet light (365 nm, 10 mW / cm 2 ) and then irradiated for 3 minutes for photo-crosslinking and curing, and sterilized to obtain a responsive anticoagulant hydrogel material that releases NO.

[0073] Example 2

[0074] This embodiment provides a method for preparing a responsive anticoagulant hydrogel material that releases NO, which specifically includes:

[0075] Step 1: providing methacrylylated recombinant collagen, specifically comprising:

[0076] Step 101, dissolving 1.5 g of recombinant collagen in 100 mL of deionized water to obtain a recombinant collagen solution; the recombinant collagen is obtained by reverse transcription of human collagen mRNA to generate cDNA, and then expressed in Escherichia coli BL21. Its structure and acquisition method can be referred to, for example, the patent application document with patent number ZL01106757.8 and patent name "A human-like collagen and its production method"; the recombinant collagen has a relative molecular mass of 97 KDa;

[0077] Step 102, add 15 mL of glycidyl methacrylate dropwise to the recombinant collagen solution, stir in a water bath at 37° C. for 7 h, and collect the product;

[0078] Step 103, dialyzing the product in deionized water for 7 consecutive days, and freeze-drying to obtain methacrylylated recombinant collagen; the dialysis bag has a molecular weight cutoff of 3500Da;

[0079] Step 2: providing methacrylylated hyaluronic acid, specifically comprising:

[0080] Step 201, dissolving 2 g of sodium hyaluronate in 100 mL of deionized water to obtain a hyaluronic acid solution; the relative molecular mass of the sodium hyaluronate is 200 KDa;

[0081] Step 202, adding 20 mL of glycidyl methacrylate dropwise to the hyaluronic acid solution, stirring in a water bath at 60° C. for 7 h, and collecting the product;

[0082] Step 203, dialyzing the product in deionized water for 7 consecutive days, and freeze-drying to obtain methacrylylated hyaluronic acid; the dialysis bag has a molecular weight cutoff of 3500 Da;

[0083] Step 3: providing a method for preparing caffeic acid arginine amide modified hyaluronic acid, which specifically comprises:

[0084] Step 301, adding 3-aminophenylboronic acid (93 mg, 0.50 mmol) to a mixture of water / dimethylformamide (water / DMF, 3:2 v / v, total solvent volume of 25 mL) containing 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride (212 mg, 0.77 mmol) and sodium hyaluronate (1.0 mmol, relative molecular mass of 200 KDa);

[0085] Step 302, adding 1M NaOH aqueous solution to the mixed solution of step 301 to adjust the pH value to 6.5; stirring the mixture at room temperature for 47 hours;

[0086] Step 303, dialyzing the pH-adjusted mixed solution in deionized water for 7 consecutive days, and freeze-drying to obtain 3-aminophenylboronic acid-modified hyaluronic acid; dissolving 3-aminophenylboronic acid-modified hyaluronic acid in deionized water to obtain a 3-aminophenylboronic acid-modified hyaluronic acid solution with a mass-volume concentration of 2%; the dialysis bag has a molecular weight cutoff of 3500Da;

[0087] Step 304, under nitrogen protection, dissolving 10 g of caffeic acid in 100 mL of tetrahydrofuran, and then adding 1 g of N-methylmorpholine and 1 mL of isobutyl chloroformate in sequence at 0° C. to react for 15 min to obtain a caffeic acid solution;

[0088] Step 305, adding 10 g of arginine L-Arg to the caffeic acid solution, reacting at 0°C, tracking the reaction by TLC, and detecting the reaction until there is no substrate by TLC, placing the reaction solution at room temperature for 10 h, and then concentrating the reaction solution under reduced pressure, extracting the solution with ethyl acetate three times (3x10 mL) after concentrating under reduced pressure, washing the organic phase with 5% citric acid solution and saturated saline solution in sequence, drying the organic layer with anhydrous Na2SO4, concentrating under reduced pressure to obtain a product, purifying it with thin layer chromatography to obtain caffeic acid arginine amide, and dissolving caffeic acid arginine amide in deionized water to obtain a caffeic acid arginine amide solution with a concentration of 20 mg / mL;

[0089] Step 306, under stirring, adding the caffeic acid arginine amide solution described in step 305 dropwise to the 3-aminophenylboronic acid modified hyaluronic acid solution described in step 301, wherein the volume ratio of the caffeic acid arginine amide solution to the 3-aminophenylboronic acid modified hyaluronic acid solution is 1:2, stirring for 47 hours, dialyzing, and freeze-drying to obtain caffeic acid arginine amide modified hyaluronic acid;

[0090] Step 4: providing a method for preparing a responsive anticoagulant hydrogel material that releases NO, specifically comprising:

[0091] Step 401, placing 250 mg of methacrylated recombinant collagen and 25 mg of photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) in 2 mL of deionized water, sealing with tin foil to avoid light, and stirring at room temperature until completely dissolved to obtain solution A; the stirring time at room temperature may be 12 hours;

[0092] Step 402, placing 50 mg of methacryloyl hyaluronic acid in 2.5 mL of deionized water, stirring in a 37° C. water bath until completely dissolved, to obtain liquid B; the stirring time in the water bath may be 1 hour;

[0093] Step 403, dissolving 5 mg of caffeic acid arginine amide modified hyaluronic acid in 0.5 mL of deionized water, and sonicating until completely dissolved to obtain liquid C; the sonication time may be 1 hour;

[0094] Step 404, mixing the solution A in step 401, the solution B in step 402, and the solution C in step 403, and continuing stirring for 1 hour to obtain a gel solution; the gel solution has no obvious fluidity and is milky white;

[0095] Step 405, placing the gel solution in a refrigerator at 4°C for 8 hours to allow the gel solution to be fully cross-linked and defoamed;

[0096] Step 406: Expose the gel solution after standing in step 405 to ultraviolet light (365 nm, 10 mW / cm 2 ) and then irradiated for 5 minutes for photo-crosslinking and curing, and sterilized to obtain a responsive anticoagulant hydrogel material that releases NO.

[0097] The performance of the responsive NO-releasing anticoagulant hydrogel material of this embodiment is basically consistent with that of Example 1.

[0098] Example 3

[0099] This embodiment provides a method for preparing a responsive anticoagulant hydrogel material that releases NO, which specifically includes:

[0100] Step 1: providing methacrylylated recombinant collagen, specifically comprising:

[0101] Step 101, dissolving 2 g of recombinant collagen in 100 mL of deionized water to obtain a recombinant collagen solution; the recombinant collagen is obtained by reverse transcription of human collagen mRNA to generate cDNA, and then expressed in Escherichia coli BL21. Its structure and acquisition method can be referred to, for example, the patent application document with patent number ZL01106757.8 and patent name "A human-like collagen and its production method"; the recombinant collagen has a relative molecular mass of 97 KDa;

[0102] Step 102, add 40 mL of glycidyl methacrylate dropwise to the recombinant collagen solution, stir in a water bath at 37° C. for 8 h, and collect the product;

[0103] Step 103, dialyzing the product in deionized water for 7 consecutive days, and freeze-drying to obtain methacrylylated recombinant collagen; the dialysis bag has a molecular weight cutoff of 3500 Da;

[0104] Step 2: providing methacrylylated hyaluronic acid, specifically comprising:

[0105] Step 201, dissolving 1.5 g of sodium hyaluronate in 100 mL of deionized water to obtain a hyaluronic acid solution; the relative molecular mass of the sodium hyaluronate is 500 KDa;

[0106] Step 202, adding 30 mL of glycidyl methacrylate dropwise to the hyaluronic acid solution, stirring in a water bath at 60° C. for 8 h, and collecting the product;

[0107] Step 203, dialyzing the product in deionized water for 7 consecutive days, and freeze-drying to obtain methacrylylated hyaluronic acid; the dialysis bag has a molecular weight cutoff of 3500 Da;

[0108] Step 3: providing a method for preparing caffeic acid arginine amide modified hyaluronic acid, which specifically comprises:

[0109] Step 301, adding 3-aminophenylboronic acid (93 mg, 0.50 mmol) to a mixture of water / dimethylformamide (water / DMF, 3:2 v / v, total solvent volume of 25 mL) containing 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride (212 mg, 0.77 mmol) and sodium hyaluronate (0.9 mmol, relative molecular mass of 500 KDa);

[0110] Step 302, adding 1M NaOH aqueous solution to the mixed solution of step 301 to adjust the pH value to 6.5; stirring the mixture at room temperature for 48 hours;

[0111] Step 303, dialyzing the mixed solution after adjusting the pH value in deionized water for 7 days continuously, and freeze-drying to obtain 3-aminophenylboronic acid modified hyaluronic acid; dissolving 3-aminophenylboronic acid modified hyaluronic acid in deionized water to obtain a 3-aminophenylboronic acid modified hyaluronic acid solution with a mass-volume concentration of 5%; the dialysis bag has a cut-off molecular weight of 3500Da;

[0112] Step 304, under nitrogen protection, dissolving 7 g of caffeic acid in 100 mL of tetrahydrofuran, and then adding 0.5 g of N-methylmorpholine and 0.5 mL of isobutyl chloroformate in sequence at 0° C. to react for 15 min to obtain a caffeic acid solution;

[0113] Step 305, adding 14 g of arginine L-Arg to the caffeic acid solution, reacting at 0°C, tracking the reaction by TLC, and continuing until no substrate is detected by TLC, placing the reaction solution at room temperature for 10 h, and then concentrating the reaction solution under reduced pressure, extracting the solution with ethyl acetate three times (3x10 mL) after concentrating under reduced pressure, washing the organic phase with 5% citric acid solution and saturated saline solution in sequence, drying the organic layer with anhydrous Na2SO4, concentrating under reduced pressure to obtain a product, purifying it with thin layer chromatography to obtain caffeic acid arginine amide, and dissolving caffeic acid arginine amide in deionized water to obtain a caffeic acid arginine amide solution with a concentration of 50 mg / mL;

[0114] Step 306, under stirring, adding the caffeic acid arginine amide solution described in step 305 dropwise to the 3-aminophenylboronic acid modified hyaluronic acid solution described in step 301, wherein the volume ratio of the caffeic acid arginine amide solution to the 3-aminophenylboronic acid modified hyaluronic acid solution is 1:1.5, stirring for 48 hours, dialyzing, and freeze-drying to obtain caffeic acid arginine amide modified hyaluronic acid;

[0115] Step 4: providing a method for preparing a responsive anticoagulant hydrogel material that releases NO, specifically comprising:

[0116] Step 401, 350 mg of methacrylated recombinant collagen and 5 mg of photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) are placed in 2 mL of deionized water, sealed with tin foil to avoid light, and stirred at room temperature until completely dissolved to obtain solution A; the stirring time at room temperature may be 12 hours;

[0117] Step 402, placing 25 mg of methacryloyl hyaluronic acid in 2.5 mL of deionized water, stirring in a 37° C. water bath until completely dissolved, to obtain liquid B; the stirring time in the water bath may be 2 h;

[0118] Step 403:

[0119] .5 mg of caffeic acid arginine amide modified hyaluronic acid is dissolved in 0.5 mL of deionized water, and ultrasonicated until completely dissolved to obtain liquid C; the ultrasonication time can be 1 hour;

[0120] Step 404, mixing the solution A in step 401, the solution B in step 402, and the solution C in step 403, and continuing stirring for 1 hour to obtain a gel solution; the gel solution has no obvious fluidity and is milky white;

[0121] Step 405, placing the gel solution in a refrigerator at 4°C for 36 hours to allow the gel solution to be fully cross-linked and defoamed;

[0122] Step 406: Expose the gel solution after standing in step 405 to ultraviolet light (365 nm, 10 mW / cm 2 ) and then irradiated for 4 minutes for photo-crosslinking and curing, and sterilized to obtain a responsive anticoagulant hydrogel material that releases NO.

[0123] The performance of the responsive NO-releasing anticoagulant hydrogel material of this embodiment is basically consistent with that of Example 1.

[0124] Example 4

[0125] This embodiment is the same as embodiment 1, except that in step 3, the amount of methacrylylated recombinant collagen is 250 mg, the amount of photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) is 25 mg, and the cross-linking time is 5 min.

[0126] Example 5

[0127] This embodiment is the same as embodiment 1, except that in step 3, the amount of methacrylylated recombinant collagen is 250 mg, the amount of photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) is 5 mg, and the cross-linking time is 5 min.

[0128] Both Examples 4 and 5 can obtain responsive anticoagulant hydrogel materials that release NO.

[0129] Performance Evaluation

[0130] Physicochemical properties of each synthetic component

[0131] Figure 1 a is a schematic diagram of the process of preparing methacrylylated recombinant collagen in Example 1. Figure 1 b is the Fourier transform infrared absorption spectrum analysis diagram of methacrylylated recombinant collagen in Example 1, according to Figure 1 b is visible at 1653cm -1 The peak at the center is the stretching vibration peak of the carbon-carbon double bond, indicating that the recombinant collagen was successfully methacrylated. Figure 1 c is the methacrylylated recombinant collagen of Example 1 1 H-NMR analysis results. Figure 1 c As can be seen, there are olefin double peaks at 6.08 ppm and 5.70 ppm, confirming that the recombinant collagen was successfully grafted with double bond groups. Figure 1 d is a schematic diagram of the process of preparing methacryloyl hyaluronic acid in Example 1. Figure 1 e is the Fourier transform infrared absorption spectrum analysis diagram of methacryloyl hyaluronic acid in Example 1, according to Figure 1 e is visible at 1710cm -1 It is the stretching vibration peak of carbon-carbon double bond, indicating that hyaluronic acid is successfully methacrylated. Figure 1 f is the methacryloyl chitosan of Example 1 1 H-NMR analysis results. Figure 1 f shows that there are olefin double peaks at 6.17 ppm and 5.74 ppm, confirming that hyaluronic acid is successfully grafted with double bond groups. Figure 1 g is a schematic diagram of the process of preparing caffeic acid arginine amide in Example 1. Caffeic acid arginine amide is obtained. Figure 1 h is the Fourier transform infrared absorption spectrum analysis diagram of caffeic acid arginine amide in Example 1, according to Figure 1 h is visible at 3423cm -1 The broad peak at 2938 cm is the vibration peak of amino group (NH). -1 , 2850cm -1 Saturated (CH) stretching vibration peak, 1605 cm -1 The characteristic peak at 1465cm-1 is due to the vibration of the carbonyl group (C=O), and the characteristic peak at 1465cm-1 is due to the stretching vibration of the benzene ring, both indicating that the grafting of caffeic acid arginine amide was successful. Figure 1 i is the caffeic acid arginine amide of Example 1 1 H-NMR analysis results. Figure 1It can be seen that the characteristic peaks of the benzene ring protons of caffeic acid can be observed at 7.57ppm and 7.77ppm, and the characteristic peaks of arginine can be observed at 2.99ppm and 3.20ppm, indicating that the grafting of caffeic acid arginine amide was successful. Figure 1 j is a schematic diagram of the process for preparing 3-aminophenylboronic acid modified hyaluronic acid in Example 1. Figure 1 k is the Fourier transform infrared absorption spectrum analysis diagram of 3-aminophenylboronic acid modified hyaluronic acid prepared in Example 1, according to Figure 1 k is visible at 3606cm -1 , 3288cm -1 The broad peak at 1363 cm is the vibration peak of amino group (NH). -1 The characteristic peak at 1635 cm is due to the vibration of the boronic acid group (BO). -1 The characteristic peak at 1573 cm is due to the vibration of the carbonyl group (C=O). -1 , 1469cm -1 The characteristic peak at is due to the aromatic vibration of the benzene ring, indicating that the grafting of 3-aminophenylboronic acid modified hyaluronic acid was successful. Figure 1 l is the 1H-NMR analysis result of 3-aminophenylboronic acid modified hyaluronic acid in Example 1. Figure 1 It can be seen that the characteristic peaks of the benzene ring protons of the phenylboronic acid group can be observed at 7.45ppm, 7.6ppm, and 7.83ppm. These peaks correspond to the special absorption bands of the benzene ring, characteristically showing the presence of the phenylboronic acid group, confirming that the grafting of 3-aminophenylboronic acid modified hyaluronic acid was successful. Figure 1 m is a schematic diagram of the process of preparing caffeic acid arginine amide modified hyaluronic acid in Example 1. Figure 1 n is the Fourier transform infrared absorption spectrum analysis diagram of caffeic acid arginine amide modified hyaluronic acid in Example 1, according to Figure 1 n is visible at 13452cm -1 , 3046cm -1 The broad peak at 2560 cm is the vibration peak of amino group (NH). -1 , 2444cm -1 Saturated (CH) stretching vibration peak, 1755 cm -1 The characteristic peak at 1244 cm is due to the vibration of the carbonyl group (C=O). -1 The characteristic peak at is the stretching vibration of (BOH), indicating that caffeic acid arginine amide modified hyaluronic acid was successfully synthesized. Figure 1 o is the caffeic acid arginine amide modified hyaluronic acid of Example 1 1 H-NMR analysis results. Figure 1It can be seen that the characteristic peaks of caffeic acid can be observed at 7.45ppm, 7.7ppm, and 7.80ppm at the phenylboronic acid group, and the characteristic peaks of arginine can be observed at 2.90ppm and 3.19ppm, indicating that caffeic acid arginine amide modified hyaluronic acid was successfully synthesized.

[0132] Physicochemical properties of NO-releasing hydrogel materials

[0133] Figure 2 This is a scanning electron microscope image of a responsive NO-releasing anticoagulant hydrogel material in Example 1 at a scale of 100 μm. The testing method includes: freeze-drying the NO-releasing multifunctional hydrogel, cutting it into samples with a thickness of about 1 mm, adhering it to a sample stage with conductive glue, spraying gold, and observing its microscopic morphology through a scanning electron microscope (SEM). Figure 2 It can be seen that the hydrogel has a three-dimensional pore structure, which can provide a microenvironment for the growth of endothelial cells.

[0134] Antimicrobial properties

[0135] Figure 3 The antibacterial properties of a responsive NO-releasing anticoagulant hydrogel material in Example 1 are shown in FIG. 1 . The test method is to use Staphylococcus aureus and Escherichia coli as Gram-positive bacteria and Gram-negative bacteria to test the antibacterial activity of the hydrogel, specifically comprising: activating the bacteria with a liquid culture medium to obtain a bacterial suspension, diluting the suspension with physiological saline to 1×10 5 cfm, the hydrogel for preparing artificial blood vessels of Example 1 was added to the diluted bacterial suspension as the experimental group, and incubated on a shaker at 37°C and 120 rpm for 24 h. The bacterial suspension without hydrogel was used as the control group. The hydrogel concentration in the experimental group was 0.1 g / mL. After culturing for 24 h, the colony concentration of the control group was diluted to 1×10 3 cfm, and dilute the experimental group with the same dilution multiple, take 100 μL of each and apply it on the solid culture medium plate, place it in a 37℃ incubator for 24 hours, and count the colonies. Figure 3 As shown in a, the counting results are Figure 3 b and 3c, according to Figure 3b and 3c show that the survival rate of Escherichia coli in the hydrogel of the present invention is 36.10±2.005%, and the survival rate of Staphylococcus aureus is 13.01±0.1041%. The hydrogel of the present invention has a significantly improved antibacterial effect. Wherein, the preparation method of the solid culture medium is: add 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 15g of agar to a 1L volumetric flask, add deionized water to make up to 1L, ultrasonically dissolve and dispense into conical flasks, seal and sterilize at 121°C for 30min; the preparation method of the liquid culture medium includes: add 10g of tryptone, 5g of yeast extract and 10g of sodium chloride to a 1L volumetric flask, add deionized water to make up to 1L, ultrasonically dissolve and dispense into conical flasks, seal and sterilize at 121°C for 30min.

[0136] Cytocompatibility

[0137] Figure 4 This is a schematic diagram of the cytotoxicity test results of a responsive NO-releasing anticoagulant hydrogel material in Example 1, and the test method includes:

[0138] The scaffold extract is prepared, specifically comprising: placing the scaffold A of Example 1 in a 50 mL sterile centrifuge tube, adding 1 mL of smooth muscle cell complete culture medium per 0.1 g of hydrogel, and placing the centrifuge tube in an incubator at 37° C. and 5% CO 2 for 72 hours to obtain a hydrogel extract;

[0139] VSMCs were seeded in a 96-well plate at a density of 1×104 cells / well and cultured in a 37°C, 5% CO2 incubator for 24 hours. The old culture medium was removed and the hydrogel extract was added to continue the culture. The smooth muscle cell complete culture medium was used as the control group. After culturing for 24 hours and 48 hours, CCK-8 solution was added to the 96-well plate at 10 μL / well. After continuing to culture for 2 to 4 hours, the supernatant was aspirated and the OD value of each well was measured at 450 nm using an enzyme marker. The cell survival rate was calculated based on the OD value. The cell viability was calculated as follows:

[0140]

[0141] The cell viability calculation results are as follows Figure 4 As shown, according to Figure 4 It can be seen that the hydrogel material prepared by the present invention has no obvious cytotoxicity and can be used in organisms.

[0142] Figure 5The proliferation ability of a responsive NO-releasing anticoagulant hydrogel material of Example 1. The test method is based on the incorporation of thymidine analog EdU (5-ethynyl-2'-deoxyuridine) during DNA synthesis, and EdU is labeled with biotin through a subsequent click reaction, and then the added horseradish peroxidase-labeled streptavidin (HRP-Streptavidin) is combined with biotin, and finally TMB is used for color development. The OD value of each well is measured at 370nm using an enzyme reader, and the cell proliferation effect is quantified according to the OD value.

[0143] Figure 5 a is the test result of promoting endothelial cell proliferation ability of a responsive NO-releasing anticoagulant hydrogel material in Example 1, according to Figure 5 aIt can be seen that the hydrogel group of the present invention has the ability to significantly promote the proliferation of HUVECs.

[0144] Figure 5 b is the test result of the ability of a responsive NO-releasing anticoagulant hydrogel material to inhibit smooth muscle cell proliferation in Example 1, according to Figure 5 b It can be seen that the hydrogel group of the present invention has the ability to significantly inhibit the proliferation of VSMCs.

[0145] Figure 5 c is the immunofluorescence result of the ability of a responsive NO-releasing anticoagulant hydrogel material to promote endothelial cell proliferation in Example 1, according to Figure 5 c It can be seen that the hydrogel group of the present invention has the ability to significantly promote the proliferation of HUVECs.

[0146] Figure 5 d is the immunofluorescence result of the ability of a responsive NO-releasing anticoagulant hydrogel material to inhibit smooth muscle cell proliferation in Example 1. Figure 5 d It can be seen that the hydrogel group of the present invention has the ability to significantly inhibit the proliferation of VSMCs.

[0147] Anti-inflammatory ability

[0148] Figure 6 The anti-inflammatory ability of a responsive NO-releasing anticoagulant hydrogel material in Example 1 is tested by the following test method:

[0149] The DMEM complete culture medium is prepared, specifically comprising: adding fetal bovine serum and double antibodies to the DMEM basal culture medium to obtain the DMEM complete culture medium; in the DMEM complete culture medium, the mass percentage of the fetal bovine serum is 10%, the mass percentage of the double antibodies is 1%, and the double antibodies are streptomycin and penicillin;

[0150] The hydrogel extract is prepared, specifically comprising: placing the hydrogel of Example 1 in a 50 mL sterile centrifuge tube, adding 1 mL of DMEM complete culture medium per 0.1 g of hydrogel, and placing the centrifuge tube in an incubator at 37° C. and 5% CO2 for 72 hours to obtain a hydrogel extract;

[0151] RAW macrophages (264.7) were cultured at 5×10 5 The cells / well were seeded in a 6-well plate at a density of 1.54 μg / well and cultured in an incubator at 37°C and 5% CO2 for 24 h. The cells were treated with a culture medium containing LPS (1 μg / mL) for 12 h to induce M1 polarization. After 12 h, the old culture medium was aspirated and the hydrogel extract was added to continue the culture. DMEM complete culture medium was used as the control group. After 24 h of culture, the cells were washed three times with PBS in the 6-well plate, fixed with 4% paraformaldehyde at 4°C for 30 min, and permeabilized with 0.2% Triton X-100 for 5 min. Subsequently, CD86 and CD206 antibodies were added, and the cells were incubated at 4°C overnight for an additional period. The cell nuclei were then stained with DAPI for a total of 5 min. Finally, the stained cells were observed using a Leica TCSSP8 confocal laser microscope. According to Figure 6 It can be seen that the hydrogel material prepared by the present invention has good anti-inflammatory ability.

[0152] Angiogenesis

[0153] Figure 7 The angiogenesis ability of a responsive NO-releasing anticoagulant hydrogel material of Example 1 is tested by the following test method:

[0154] The hydrogel extract is prepared, specifically comprising: placing the hydrogel of Example 1 in a 50 mL sterile centrifuge tube, adding 1 mL of ECM complete culture medium per 0.1 g of hydrogel, and placing the centrifuge tube in an incubator at 37° C. and 5% CO 2 for 72 hours to obtain a hydrogel extract;

[0155] 4°C precooled matrix gel was spread on a 48-well plate, and then placed in a 37°C incubator for 30 minutes to allow the matrix gel to completely gel. HUVECs were seeded on the matrix gel at a number of 5×104 cells per well, and then the cells were cultured with ECM (2% serum) hydrogel extract medium, where ECM (2% serum) culture medium was used as the control group. After 6 hours of culture, the formation of cell tubular structures was observed with an inverted optical microscope and images were collected. Figure 7 It can be seen that the hydrogel material prepared by the present invention has good angiogenesis ability.

[0156] Anticoagulant ability

[0157] Figure 8a is a test of the anticoagulant ability of a responsive NO-releasing anticoagulant hydrogel material against platelet adhesion in Example 1. The test method includes:

[0158] The hydrogel was placed in a 24-well plate. 100 μL PRP was added to the sample. Platelet adhesion to the hydrogel was observed by incubating at 37°C for 2 hours. The sample surface was then rinsed with PBS solution to ensure that platelets not attached to the surface of the material were removed. The cleaned samples were fixed with 2.5% glutaraldehyde for 12 hours. The dried samples were then treated with alcohol of varying concentrations. The dried samples were treated with gold spraying and then observed under SEM. Figure 8 a It can be seen that the inner surface of the NO-releasing multifunctional hydrogel coating material prepared by the present invention shows slight platelet adhesion, but from the state of the platelets, it can be seen that the degree of platelet activation by the substance is not very obvious, thus indicating that it has the ability to inhibit thrombosis.

[0159] Figure 8 b is a test of the anticoagulant ability of a responsive NO-releasing anticoagulant hydrogel material against lactate dehydrogenase in Example 1. The test method includes:

[0160] Place the hydrogel in a 24-well plate. Add 100 μL of PRP to the sample. Incubate at 37°C for 1 hour. Then discard the supernatant. The sample is then repeatedly cleaned 3 times with PBS solution to ensure that platelets that are not attached to the surface of the material are removed. In addition, add 100 μL of 0.05% triton-100 lysate to the material and incubate at room temperature for 30 minutes. Then, add 10 μL of LDH releaser to the material and incubate at 37°C for 1 hour. Centrifuge the culture plate at 2000 rpm for 5 minutes. Measure the absorbance of 5 μL of supernatant at 440 nm to evaluate the number of platelets attached to the surface of the material. According to Figure 8 b As can be seen, the number of platelets adhered to by this substance is low, thus indicating that it has the ability to inhibit thrombosis.

[0161] Figure 8 c is a test of the anticoagulant ability of a responsive NO-releasing anticoagulant hydrogel material against the coagulation index in Example 1. The test method includes:

[0162] Place the material in a 50mL centrifuge tube and incubate at 37℃ for 5min. Take 100L of fresh anticoagulated blood and add it dropwise to the surface of the material. Immediately add 0.2M CaCl solution (20L) and start timing. After incubating at 37℃ for 5min, carefully add 25mL of deionized water along the edge of the centrifuge tube, followed by shaking at 30rpm for 5min at 37℃. Finally, take 200L of the upper clear liquid and test the absorbance (Abs) value using an enzyme reader (test wavelength set to 540nm). During the test, each sample was tested 3 times and the average value was taken. Use fresh anticoagulated blood (100μL) in 25mL of deionized water as a blank control. According to Figure 8 cIt can be seen that this substance can delay blood coagulation to a certain extent, thus indicating that it has the ability to inhibit thrombosis.

[0163] Figure 8 d is a test of the anticoagulant ability of a responsive NO-releasing anticoagulant hydrogel material in Example 1 for activated partial thromboplastin time and prothrombin time. Figure 8 d As can be seen, compared with the control group, the APTT values ​​of all groups were prolonged, indicating that the intrinsic coagulation pathway was not activated. In addition, compared with the control group, the PT values ​​of all groups were almost unchanged, indicating that the extrinsic coagulation pathway was not activated.

[0164] Figure 8 e is a test of the anticoagulant ability of a responsive NO-releasing anticoagulant hydrogel material against fibrinogen adhesion in Example 1. The test method includes:

[0165] The samples were incubated with FITC-hFg and whole protein plasma for 4 h, 1 day, or 3 days, and the protein adsorption behavior was evaluated based on the changes in surface fluorescence intensity and wettability.

[0166] Figure 8 f is a fluorescence quantitative test of a responsive NO-releasing anticoagulant hydrogel material for fibrinogen adhesion in Example 1. Figure 8 d and 8e show that the fluorescence of the hydrogel group samples is weak, indicating that the hydrophilicity of the hydrogel material effectively inhibits the adhesion of fibrinogen.

[0167] The present invention utilizes the good biocompatibility, biodegradability and low risk of infection and immune response of recombinant collagen, which is beneficial to the growth of endothelial cells and realizes effective endothelialization; 3-aminophenylboronic acid and caffeic acid have anti-inflammatory properties due to their catechol structures, which prevent infection and realize the removal of ROS at the same time; the phenylboronic acid ester bond can respond to ROS breakage to release caffeic acid and arginine; arginine can release NO by acting on NO synthase produced by endothelial cells, thereby showing a good anticoagulant effect to maintain blood vessel patency, and can further stimulate the release of NO when the blood vessels are in a high active oxygen environment, reduce the level of ROS, inhibit the proliferation of smooth muscle cells, thereby inhibiting intimal hyperplasia, and hindering thrombosis. Recombinant collagen was creatively combined with caffeic acid arginine amide-modified hyaluronic acid to obtain a hydrogel material that induces NO release. The prepared hydrogel material has the advantages of antibacterial, promoting endothelial cell proliferation, inhibiting smooth muscle cell proliferation, anti-inflammatory, promoting angiogenesis and inhibiting thrombosis. Among them, arginine can serve as an endogenous NO donor in the blood to release NO molecules, thereby promoting vascular endothelial repair. The introduced NO molecules give the hydrogel material good anticoagulant properties, and it is expected to be used to prepare artificial vascular anticoagulant coatings.

[0168] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a responsive anticoagulant hydrogel material that releases NO, characterized in that: The following steps are involved: Step 1: dissolving methacrylylated recombinant collagen and a photoinitiator in deionized water under light-proof conditions to obtain liquid A; dissolving methacrylylated hyaluronic acid in deionized water to obtain liquid B; dissolving caffeic acid arginine amide-modified hyaluronic acid in deionized water to obtain liquid C; Step 2: Mix the liquid A, liquid B and liquid C in step 1 to obtain a gel solution; Step 3: Place the gel solution in step 2 in a refrigerator for 8 hours to 36 hours, irradiate with ultraviolet light for 3 minutes to 5 minutes for photo-crosslinking and curing, and sterilize to obtain a responsive anticoagulant hydrogel material that releases NO.

2. The method for preparing a responsive anticoagulant hydrogel material for releasing NO according to claim 1, characterized in that: The method for preparing the methacryloyl recombinant collagen described in step 1 specifically comprises: adding glycidyl methacrylate to the recombinant collagen solution, reacting in a 37° C. water bath with stirring for 6 to 8 hours, dialyzing, and freeze-drying to obtain the methacryloyl recombinant collagen.

3. The method for preparing a responsive anticoagulant hydrogel material for releasing NO according to claim 2, characterized in that: The recombinant collagen solution is a recombinant collagen solution obtained by dissolving recombinant collagen in deionized water, and the mass-volume concentration of the recombinant collagen solution is 1% to 2%; the relative molecular mass of the recombinant collagen is 97KDa; the dosage of the glycidyl methacrylate is 10mL to 30mL of glycidyl methacrylate per gram of recombinant collagen.

4. The method for preparing a responsive anticoagulant hydrogel material for releasing NO according to claim 1, characterized in that: The preparation method of the methacryloyl hyaluronic acid in step 1 specifically comprises: adding glycidyl methacrylate dropwise to the hyaluronic acid solution, reacting in a 60° C. water bath with stirring for 6 to 8 hours, dialyzing, and freeze-drying to obtain the methacryloyl hyaluronic acid.

5. The method for preparing a responsive anticoagulant hydrogel material for releasing NO according to claim 4, characterized in that: The hyaluronic acid solution is a hyaluronic acid solution obtained by dissolving sodium hyaluronate in deionized water, and the mass-volume concentration of the hyaluronic acid solution is 1% to 2%; the relative molecular mass of the sodium hyaluronate is 10KDa to 500KDa; and the amount of the glycidyl methacrylate is 10mL to 30mL of glycidyl methacrylate per gram of sodium hyaluronate.

6. The method for preparing a responsive anticoagulant hydrogel material for releasing NO according to claim 1, characterized in that: The photoinitiator in step 1 is lithium phenyl-2,4,6-trimethylbenzoylphosphonate.

7. The method for preparing a responsive anticoagulant hydrogel material for releasing NO according to claim 1, characterized in that: The preparation method of caffeic acid arginine amide modified hyaluronic acid in step 1 specifically comprises: Step 101, using 3-aminophenylboronic acid and sodium hyaluronate as raw materials to prepare 3-aminophenylboronic acid-modified hyaluronic acid; then dissolving the prepared 3-aminophenylboronic acid-modified hyaluronic acid in deionized water to obtain a 3-aminophenylboronic acid-modified hyaluronic acid solution; Step 102, using caffeic acid and arginine as raw materials, and preparing caffeic acid arginine amide by thin layer chromatography purification; then dissolving the prepared caffeic acid arginine amide in deionized water to obtain a caffeic acid arginine amide solution; Step 103, under stirring conditions, dropwise add the caffeic acid arginine amide solution described in step 102 to the 3-aminophenylboronic acid modified hyaluronic acid solution described in step 101, stir for 46 h to 48 h, dialyze, and freeze-dry to obtain caffeic acid arginine amide modified hyaluronic acid.

8. The method for preparing a responsive anticoagulant hydrogel material for releasing NO according to claim 7, characterized in that: The relative molecular mass of sodium hyaluronate described in step 101 is 10KDa to 500KDa, the molar ratio of 3-aminophenylboronic acid to sodium hyaluronate is 1:(1.5 to 2), and the mass-volume concentration of the hyaluronic acid solution modified with 3-aminophenylboronic acid is 2% to 5%; the mass of arginine described in step 102 is 0.5 to 2 times the mass of caffeic acid, and the concentration of the caffeic acid arginine amide solution is 20 mg / mL to 50 mg / mL; and the volume ratio of the caffeic acid arginine amide solution to the hyaluronic acid solution modified with 3-aminophenylboronic acid is 1:(1 to 2) in step 103.

9. The method for preparing a responsive anticoagulant hydrogel material for releasing NO according to claim 1, characterized in that: In the gel solution of step 2, the mass-volume concentration of methacryloyl recombinant collagen is 5% to 10%, the mass-volume concentration of methacryloyl hyaluronic acid is 0.5% to 2%, the mass-volume concentration of the photoinitiator is 0.1% to 0.5%, and the concentration of caffeic acid arginine amide modified hyaluronic acid is 0.5 mg / mL to 1.5 mg / mL.

10. Use of the anticoagulant hydrogel material prepared by the method of claim 1 in preparing an anticoagulant coating for an artificial blood vessel.

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