Preparation method and application of a responsive NO-releasing anticoagulant hydrogel material

Abstract

The present invention discloses a preparation method and application of a responsive anticoagulant hydrogel material that releases NO, comprising: providing methacryloyl-recombinant collagen; providing methacryloyl-recombinant hyaluronic acid; providing caffeic acid arginine amide-modified hyaluronic acid; and obtaining a responsive anticoagulant hydrogel material that releases NO using methacryloyl-recombinant collagen, methacryloyl-recombinant hyaluronic acid, and caffeic acid arginine amide-modified hyaluronic acid as raw materials. 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. Arginine can act 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 is expected to be used in the preparation of artificial vascular anticoagulant coatings.

Description

Technical Field

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

[0002] Due to the limitations of autologous blood vessels in the treatment of cardiovascular diseases, artificial blood vessels are considered an important alternative to autologous grafts. Large-diameter vascular grafts (D>6mm) have been used clinically. However, there are significant 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, the development of an anticoagulant hydrogel material capable of responsively releasing NO is urgently needed. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the shortcomings of the above-mentioned existing technologies and provide a method for preparing an anticoagulant hydrogel material that releases NO in a responsive manner. The hydrogel material prepared by this method has the advantages of antibacterial properties, promoting endothelial cell proliferation, inhibiting smooth muscle cell proliferation, anti-inflammatory, promoting angiogenesis, and inhibiting thrombosis. Arginine can act 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 excellent anticoagulant properties, and it is expected to be used in the preparation of anticoagulant coatings for artificial blood vessels.

[0005] 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 methacryloyl recombinant collagen and a photoinitiator in deionized water in the dark to obtain solution A; dissolving methacryloyl hyaluronic acid in deionized water to obtain solution B; and dissolving caffeic acid arginine amide-modified hyaluronic acid in deionized water to obtain solution C;

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

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

[0009] The above-mentioned method for preparing a responsive NO-releasing anticoagulant hydrogel material is characterized in that the preparation method of the methacrylated recombinant collagen described in step 1 specifically includes: dropwise adding glycidyl methacrylate to the recombinant collagen solution, stirring in a 37°C water bath for 6h to 8h, dialyzing, and freeze-drying to obtain the methacrylated 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; and 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 methacrylated hyaluronic acid described in step 1 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 methacrylated 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; and 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 1 is lithium phenyl-2,4,6-trimethylbenzoylphosphonate.

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

[0015] Step 101: preparing 3-aminophenylboronic acid-modified hyaluronic acid using 3-aminophenylboronic acid and sodium hyaluronate as raw materials; 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, purifying by thin layer chromatography to prepare caffeic acid arginine amide; 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, the caffeic acid arginine amide solution described in step 102 is added dropwise to the 3-aminophenylboronic acid modified hyaluronic acid solution described in step 101, stirred for 46 h to 48 h, dialyzed, and freeze-dried 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 to 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% to 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 NO-releasing anticoagulant hydrogel material 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 a use 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 to promote the growth of endothelial cells and achieve effective endothelialization; 3-aminophenylboronic acid and caffeic acid have anti-inflammatory properties due to their catechol structure, preventing infection while achieving ROS clearance; the phenylboronic acid ester bond can respond to ROS cleavage to release caffeic acid and arginine; arginine can release NO by reacting with NO synthase produced by endothelial cells, thereby exhibiting a good anticoagulant effect to maintain blood vessel patency. When the blood vessels are in a highly reactive oxygen environment, it can further stimulate the release of NO, reduce the level of ROS, inhibit the proliferation of smooth muscle cells, and thus inhibit intimal hyperplasia, thereby hindering thrombosis. By creatively combining recombinant collagen with caffeic acid arginine amide-modified hyaluronic acid, a hydrogel material that induces NO release was obtained. 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 act 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, which has photosensitivity that can effectively promote the UV curing process, is beneficial to the growth of endothelial cells, and achieves 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. By utilizing the property of sustained release of NO in the hydrogel, the hydrogel scaffold induces the migration and proliferation of endothelial cells and promotes angiogenesis.

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

[0027] The technical solution of the present invention is further described in detail below with reference to 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 for preparing methacryloyl recombinant collagen; b is a Fourier transform infrared absorption spectrum analysis diagram of methacryloyl recombinant collagen; c is a diagram of the 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 diagram of 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 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 cultivation in the antibacterial performance test of the hydrogel material; b is a graph showing the counting results after cultivation of Escherichia coli in the antibacterial performance test of the hydrogel material; and c is a graph showing the counting results after cultivation of 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 of the hydrogel material's ability to promote endothelial cell proliferation test; b is the quantitative result of the hydrogel material's ability to inhibit smooth muscle cell proliferation test; a is the fluorescence staining result of the hydrogel material's ability to promote endothelial cell proliferation test; d is the fluorescence staining result of the hydrogel material's ability to inhibit smooth muscle cell proliferation test.

[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 These are 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 against platelet adhesion; b is the anti-coagulation ability test result of the hydrogel material against lactate dehydrogenase; c is the anti-coagulation ability test result of the hydrogel material against coagulation index; d is the anti-coagulation ability test result of the hydrogel material against activated partial thromboplastin time and prothrombin time; e is the anti-coagulation ability test result of the hydrogel material against fibrinogen adhesion; and f is the test result of the hydrogel material against fibrinogen adhesion fluorescence quantification. DETAILED DESCRIPTION

[0036] To make the above-mentioned objects, features and advantages of the present invention more clearly understood, the following detailed description of the specific embodiments of the present invention is given in conjunction with the following examples. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

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

[0038] Step 1: dissolving methacryloyl recombinant collagen and a photoinitiator in deionized water in the dark to obtain solution A; dissolving methacryloyl hyaluronic acid in deionized water to obtain solution B; and dissolving caffeic acid arginine amide-modified hyaluronic acid in deionized water to obtain solution C;

[0039] The method for preparing methacrylated recombinant collagen specifically comprises: dropwise adding glycidyl methacrylate to a recombinant collagen solution, stirring in a water bath at 37° C. for 6 to 8 hours, dialyzing, and freeze-drying to obtain the methacrylated 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 recombinant collagen has a relative molecular mass of 97 kDa; and the amount of glycidyl methacrylate used is 10 mL to 30 mL per gram of recombinant collagen;

[0040] The preparation method of the methacrylated hyaluronic acid specifically comprises: adding glycidyl methacrylate dropwise to a hyaluronic acid solution, stirring in a water bath at 60° C. for 6 to 8 hours, dialyzing, and freeze-drying to obtain the methacrylated 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; and the amount of glycidyl methacrylate used is 10mL to 30mL 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: preparing 3-aminophenylboronic acid-modified hyaluronic acid using 3-aminophenylboronic acid and sodium hyaluronate as raw materials; 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 10 KDa to 500 KDa, 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% to 5%;

[0044] Step 102: Using caffeic acid and arginine as raw materials, purifying and preparing caffeic acid arginine amide by thin layer chromatography; 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, the caffeic acid arginine amide solution described in step 102 is added dropwise 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 46-48 hours, dialyzing, and freeze-drying to obtain caffeic acid arginine amide-modified hyaluronic acid;

[0046] Step 2: Mixing Solution A, Solution B, and Solution C described in Step 1 to obtain a gel solution; the mass-volume concentration of methacryloyl recombinant collagen in the gel solution 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 8 to 36 hours, irradiate with ultraviolet light for 3 to 5 minutes for photocrosslinking and curing, and sterilize to obtain a responsive anticoagulant hydrogel material that releases NO.

[0048] The present invention is described in detail below with reference to the 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, specifically comprising:

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

[0052] Step 101: Dissolve 1 g of recombinant collagen in 100 mL of deionized water to obtain a recombinant collagen solution. The recombinant collagen solution is obtained by reverse-transcribing human collagen mRNA to generate cDNA, which is then expressed in Escherichia coli BL21. For example, the structure and acquisition method of the recombinant collagen solution can be found in patent application number ZL01106757.8, entitled "A Human-like Collagen and Its Production Method." The recombinant collagen solution has a relative molecular mass of 97 kDa.

[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 methacryloyl collagen; the dialysis bag has a molecular weight cutoff of 3500 Da;

[0055] Step 2: providing methacryloyl 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: add 30 mL of glycidyl methacrylate dropwise to the hyaluronic acid solution, stir in a water bath at 60° C. for 6 h, and collect the product;

[0058] Step 203: dialyzing the product in deionized water for 7 consecutive days and freeze-drying to obtain methacryloyl 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, specifically comprising:

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

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

[0062] Step 303: dialyzing the pH-adjusted mixed solution in deionized water for 7 days and freeze-drying to obtain 3-aminophenylboronic acid-modified hyaluronic acid; dissolving the 3-aminophenylboronic acid-modified hyaluronic acid in deionized water to obtain a 3-aminophenylboronic acid-modified hyaluronic acid solution having a mass-volume concentration of 4%; the dialysis bag having a molecular weight cutoff of 3500 Da;

[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. and reacted for 15 minutes to obtain a caffeic acid solution;

[0064] Step 305: Add 2.5 g of arginine L-Arg to the caffeic acid solution, react at 0° C., track the reaction by TLC, and continue reaction until no substrate is detected by TLC. The reaction solution is allowed to react at room temperature for 10 h, and then concentrated under reduced pressure. After concentration under reduced pressure, the reaction solution is extracted three times with ethyl acetate (3×10 mL). The organic phase is washed sequentially with 5% citric acid solution and saturated saline solution. The organic layer is dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain a product, which is purified by thin layer chromatography to obtain caffeic acid arginine amide. Caffeic acid arginine amide is dissolved in deionized water to obtain a 40 mg / mL caffeic acid arginine amide solution.

[0065] Step 306: Under stirring, the caffeic acid arginine amide solution described in step 305 is added 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. The mixture is stirred for 46 hours, dialyzed, and freeze-dried 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: Place 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, cover with tin foil to protect from light, and stir at room temperature until completely dissolved to obtain solution A; the stirring time at room temperature may be 12 hours;

[0068] Step 402: Place 100 mg of methacryloyl hyaluronic acid in 2.5 mL of deionized water and stir in a 37° C. water bath until completely dissolved to obtain Solution 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 Solution C; the sonication time may be 1 hour;

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

[0071] Step 405: Place the gel solution in a refrigerator at 4°C for 12 hours to allow the gel solution to fully crosslink and defoam;

[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 photocrosslinking 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: Dissolve 1.5 g of recombinant collagen in 100 mL of deionized water to obtain a recombinant collagen solution. The recombinant collagen solution is obtained by reverse-transcribing human collagen mRNA to generate cDNA, which is then expressed in Escherichia coli BL21. For example, the structure and acquisition method of the recombinant collagen solution can be found in patent application number ZL01106757.8, entitled "A Human-like Collagen and Its Production Method." The recombinant collagen solution 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 methacryloyl recombinant collagen; the dialysis bag has a molecular weight cutoff of 3500 Da;

[0079] Step 2: providing methacryloyl 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: add 20 mL of glycidyl methacrylate dropwise to the hyaluronic acid solution, stir in a water bath at 60° C. for 7 h, and collect the product;

[0082] Step 203: dialyzing the product in deionized water for 7 consecutive days and freeze-drying to obtain methacryloyl 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, specifically comprising:

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

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

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

[0087] Step 304: Under nitrogen protection, 10 g of caffeic acid was dissolved in 100 mL of tetrahydrofuran, and 1 g of N-methylmorpholine and 1 mL of isobutyl chloroformate were added in sequence at 0° C. and reacted for 15 minutes 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. and tracking the reaction by TLC until no substrate is detected by TLC. The reaction solution is allowed to react at room temperature for 10 h, and then concentrated under reduced pressure. After concentration under reduced pressure, the reaction solution is extracted three times with ethyl acetate (3×10 mL). The organic phase is washed sequentially with 5% citric acid solution and saturated saline solution. The organic layer is dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain a product, which is purified by thin layer chromatography to obtain caffeic acid arginine amide. Caffeic acid arginine amide is dissolved in deionized water to obtain a 20 mg / mL caffeic acid arginine amide solution.

[0089] Step 306: Under stirring, the caffeic acid arginine amide solution described in step 305 is added 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. The mixture is stirred for 47 hours, dialyzed, and freeze-dried 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: Place 250 mg of methacrylated recombinant collagen and 25 mg of photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) in 2 mL of deionized water, cover with tin foil to avoid light, and stir at room temperature until completely dissolved to obtain solution A; the stirring time at room temperature may be 12 hours;

[0092] Step 402: Place 50 mg of methacryloyl hyaluronic acid in 2.5 mL of deionized water and stir in a 37° C. water bath until completely dissolved to obtain Solution 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 Solution C; the sonication time may be 1 hour;

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

[0095] Step 405: Place the gel solution in a refrigerator at 4°C for 8 hours to allow the gel solution to fully crosslink and defoam;

[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 photocrosslinking 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 the same as 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 solution is obtained by reverse-transcribing human collagen mRNA to generate cDNA, which is then expressed in Escherichia coli BL21. For example, the structure and acquisition method of the recombinant collagen solution can be found in patent application number ZL01106757.8, entitled "A Human-like Collagen and Its Production Method." The recombinant collagen solution 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 methacryloyl recombinant collagen; the dialysis bag has a molecular weight cutoff of 3500 Da;

[0104] Step 2: providing methacryloyl 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: add 30 mL of glycidyl methacrylate dropwise to the hyaluronic acid solution, stir in a water bath at 60° C. for 8 h, and collect the product;

[0107] Step 203: dialyzing the product in deionized water for 7 consecutive days and freeze-drying to obtain methacryloyl 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, specifically comprising:

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

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

[0111] Step 303: dialyzing the pH-adjusted mixed solution in deionized water for 7 days and freeze-drying to obtain 3-aminophenylboronic acid-modified hyaluronic acid; dissolving the 3-aminophenylboronic acid-modified hyaluronic acid in deionized water to obtain a 3-aminophenylboronic acid-modified hyaluronic acid solution having a mass-volume concentration of 5%; the dialysis bag having a molecular weight cutoff of 3500 Da;

[0112] Step 304: Under nitrogen protection, 7 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. and reacted for 15 minutes to obtain a caffeic acid solution;

[0113] Step 305: Add 14 g of arginine L-Arg to the caffeic acid solution, react at 0° C., track the reaction by TLC, and continue reaction until no substrate is detected by TLC. The reaction solution is allowed to react at room temperature for 10 h, and then concentrated under reduced pressure. After concentration under reduced pressure, the reaction solution is extracted three times with ethyl acetate (3×10 mL). The organic phase is washed sequentially with 5% citric acid solution and saturated saline solution. The organic layer is dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain a product, which is purified by thin layer chromatography to obtain caffeic acid arginine amide. Caffeic acid arginine amide is dissolved in deionized water to obtain a 50 mg / mL caffeic acid arginine amide solution.

[0114] Step 306: Add the caffeic acid arginine amide solution described in step 305 dropwise to the 3-aminophenylboronic acid-modified hyaluronic acid solution described in step 301 under stirring, wherein the volume ratio of the caffeic acid arginine amide solution to the 3-aminophenylboronic acid-modified hyaluronic acid solution is 1:1.5. Stir for 48 hours, dialyze, and freeze-dry 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, covered with tin foil to avoid light, and stirred at room temperature until completely dissolved to obtain solution A; the stirring time at room temperature can be 12 hours;

[0117] Step 402: Place 25 mg of methacryloyl hyaluronic acid in 2.5 mL of deionized water and stir in a 37° C. water bath until completely dissolved to obtain Solution B. The stirring time in the water bath may be 2 hours.

[0118] Step 403:

[0119] 0.5 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 solution C; the ultrasonication time can be 1 hour;

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

[0121] Step 405: Place the gel solution in a refrigerator at 4°C for 36 hours to allow the gel solution to fully crosslink and defoam;

[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 photocrosslinking 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 the same as that of Example 1.

[0124] Example 4

[0125] This embodiment is the same as embodiment 1, except that in step 3, the amount of methacrylated 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 Example 1, except that in step 3, the amount of methacrylated 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 synthetic components

[0131] Figure 1 a is a schematic diagram of the process for preparing methacrylylated recombinant collagen in Example 1. Figure 1 b is the Fourier transform infrared absorption spectrum analysis diagram of methacryloyl recombinant collagen in Example 1, according to Figure 1 b is visible at 1653 cm -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 methacryloyl 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 for 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 1710 cm -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, it can be seen that there are olefin double peaks at 6.17 ppm and 5.74 ppm, which confirms that the double bond groups are successfully grafted onto hyaluronic acid. Figure 1 g is a schematic diagram of the process for preparing caffeic acid arginine amide in Example 1. Caffeic acid arginine amide was 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 1465 cm-1 is due to the vibration of the carbonyl group (C=O), and the characteristic peak at 1465 cm-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 7.57 ppm and 7.77 ppm, and the characteristic peaks of arginine can be observed at 2.99 ppm and 3.20 ppm, 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 carbonyl (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 is successful. Figure 1 1 is the 1H-NMR analysis result of 3-aminophenylboronic acid modified hyaluronic acid in Example 1. Figure 1 As can be seen, characteristic peaks of the phenyl ring protons of the phenylboronic acid group can be observed at 7.45ppm, 7.6ppm, and 7.83ppm. These peaks correspond to the specific absorption bands of the benzene ring and characteristically indicate the presence of phenylboronic acid groups, confirming the successful grafting of 3-aminophenylboronic acid-modified hyaluronic acid. 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 carbonyl (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 characteristic peaks of caffeic acid can be observed at 7.45ppm, 7.7ppm, and 7.80ppm at the phenylboronic acid group, and 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 it with gold, and observing its microscopic morphology using 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] Antibacterial properties

[0135] Figure 3 The antibacterial properties of a responsive NO-releasing anticoagulant hydrogel material according to Example 1 were tested using Staphylococcus aureus and Escherichia coli as Gram-positive and Gram-negative bacteria, respectively. Specifically, the antibacterial activity of the hydrogel was tested by activating the bacteria with a liquid culture medium to obtain a bacterial suspension, which was then diluted with physiological saline to a concentration of 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 spread 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 as follows Figure 3 b and 3c, according to Figure 3As shown in Figures b and 3c, 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. The solid culture medium is prepared by adding 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 15g of agar to a 1L volumetric flask, adding deionized water to make the volume to 1L, ultrasonically dissolving, and then dispensing into conical flasks, sealing, and sterilizing at 121°C for 30min. The liquid culture medium is prepared by adding 10g of tryptone, 5g of yeast extract and 10g of sodium chloride to a 1L volumetric flask, adding deionized water to make the volume to 1L, ultrasonically dissolving, and then dispensing into conical flasks, sealing, and sterilizing 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 according to Example 1. The test method includes:

[0138] The scaffold extract was prepared by 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 culturing 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 at a density of 1×104 cells / well in a 96-well plate 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 further culture for 2 to 4 hours, the supernatant was aspirated and the OD value of each well was measured at 450 nm using a microplate reader. 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 5This is the proliferation capacity of a responsive NO-releasing anticoagulant hydrogel material from Example 1. The testing method is based on the incorporation of the thymidine analog EdU (5-ethynyl-2'-deoxyuridine) during DNA synthesis. A click reaction then labels EdU with biotin. Horseradish peroxidase-labeled streptavidin (HRP) is then added to bind to the biotin, and finally, TMB is used for color development. The OD value of each well is measured at 370 nm using a microplate reader, and the cell proliferation effect is quantified based on 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 a It 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 the responsive NO-releasing anticoagulant hydrogel material to inhibit smooth muscle cell proliferation in Example 1. 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. 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 the responsive anticoagulant hydrogel material releasing NO to inhibit the proliferation of smooth muscle cells 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 according to Example 1 is tested by the following method:

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

[0150] The hydrogel extract was prepared by 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 culturing the centrifuge tube in an incubator at 37° C. and 5% CO 2 for 72 hours to obtain a hydrogel extract;

[0151] RAW macrophages (264.7) were cultured at a rate of 5×10 5 The cells / well were seeded in a 6-well plate at a density of 100 μg / well and cultured in an incubator at 37°C and 5% CO2 for 24 hours. The cells were treated with a culture medium containing LPS (1 μg / mL) for 12 hours to induce M1 polarization. After 12 hours, the old culture medium was removed and the hydrogel extract was added to continue the culture. DMEM complete culture medium was used as the control group. After 24 hours of culture, the cells were washed three times with PBS in a 6-well plate, fixed with 4% paraformaldehyde at 4°C for 30 minutes, and permeabilized with 0.2% Triton X-100 for 5 minutes. Subsequently, CD86 and CD206 antibodies were added, and the cells were incubated at 4°C for an additional overnight. The cell nuclei were then stained with DAPI for a total of 5 minutes. 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 ability

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

[0154] The hydrogel extract was prepared by 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 culturing the centrifuge tube in an incubator at 37° C. and 5% CO 2 for 72 h to obtain a hydrogel extract;

[0155] 4℃ pre-cooled matrix gel was spread on a 48-well plate, and then placed in a 37℃ 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 washed sample was fixed with 2.5% glutaraldehyde for 12 hours. The dried sample was then treated with alcohol of different concentrations. The dried sample was treated with gold spraying and then observed under SEM. Figure 8 It can be seen from a that the inner surface of the NO-releasing multifunctional hydrogel coating material prepared by the present invention exhibits slight platelet adhesion, but from the state of the platelets it can be seen that the degree of platelet activation by the material 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 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 amount of platelet adhesion of this substance is low, 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 according to Example 1 against the coagulation index. 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, and then shake at 30rpm for 5min at 37℃. Finally, take 200L of the upper clear liquid and test the absorbance (Abs) value with an enzyme marker (test wavelength set to 540nm). During the test, each sample was tested 3 times and the average value was taken. Fresh anticoagulated blood (100μL) in 25mL of deionized water was used as a blank control. According to Figure 8 c It 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 against activated partial thromboplastin time and prothrombin time. Figure 8 d As shown in Figure d, 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 As shown in Figures d and 8e, the fluorescence of the hydrogel group samples was weak, indicating that the hydrophilicity of the hydrogel material effectively inhibited the adhesion of fibrinogen.

[0167] The present invention utilizes the good biocompatibility, biodegradability, and low risk of infection and immune response of recombinant collagen to promote the growth of endothelial cells and achieve effective endothelialization; 3-aminophenylboronic acid and caffeic acid have anti-inflammatory properties due to their catechol structures, preventing infection while achieving ROS clearance; the phenylboronic acid ester bond can respond to ROS cleavage to release caffeic acid and arginine; arginine can release NO by reacting with NO synthase produced by endothelial cells, thereby exhibiting a good anticoagulant effect to maintain blood vessel patency. When the blood vessels are in a highly reactive oxygen environment, the release of NO can be further stimulated, the level of ROS can be reduced, the proliferation of smooth muscle cells can be inhibited, and thus the intimal hyperplasia can be inhibited, thereby hindering thrombosis. By creatively combining recombinant collagen with caffeic acid arginine amide-modified hyaluronic acid, a hydrogel material that induces NO release was obtained. 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 act 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 based on the technical essence of the invention shall still fall within the scope of protection 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 methacryloyl recombinant collagen and a photoinitiator in deionized water in the dark to obtain solution A; dissolving methacryloyl hyaluronic acid in deionized water to obtain solution B; and dissolving caffeic acid arginine amide-modified hyaluronic acid in deionized water to obtain solution C; The preparation method of the caffeic acid arginine amide modified hyaluronic acid specifically comprises: Step 101: preparing 3-aminophenylboronic acid-modified hyaluronic acid using 3-aminophenylboronic acid and sodium hyaluronate as raw materials; 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, purifying by thin layer chromatography to prepare caffeic acid arginine amide; then dissolving the prepared caffeic acid arginine amide in deionized water to obtain a caffeic acid arginine amide solution; Step 103: adding the caffeic acid arginine amide solution in step 102 dropwise to the 3-aminophenylboronic acid-modified hyaluronic acid solution in step 101 under stirring, stirring for 46 to 48 hours, dialyzing, and freeze-drying to obtain caffeic acid arginine amide-modified hyaluronic acid; Step 2: Mix the solution A, solution B and solution C in step 1 to obtain a gel solution; Step 3: The gel solution in step 2 is placed in a refrigerator for 8 hours to 36 hours, irradiated with ultraviolet light for 3 minutes to 5 minutes for photo-crosslinking and curing, and sterilized to obtain a responsive anticoagulant hydrogel material that releases NO.

2. The method for preparing a responsive anticoagulant hydrogel material that releases NO according to claim 1, characterized in that: The preparation method of the methacryloyl recombinant collagen in step 1 specifically includes: dropwise adding glycidyl methacrylate to the recombinant collagen solution, stirring in a 37° C. water bath 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 that releases 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; and the amount of glycidyl methacrylate used is 10mL to 30mL of glycidyl methacrylate per gram of recombinant collagen.

4. The method for preparing a responsive anticoagulant hydrogel material that releases 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, stirring in a 60° C. water bath 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 that releases 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 glycidyl methacrylate used is 10mL to 30mL of glycidyl methacrylate per gram of sodium hyaluronate.

6. The method for preparing a responsive anticoagulant hydrogel material that releases 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 that releases NO according to claim 1, characterized in that: In step 101, the relative molecular mass of sodium hyaluronate is 10 KDa to 500 KDa, 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% to 5%; in step 102, the mass of arginine 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 in step 103, the volume ratio of the caffeic acid arginine amide solution to the 3-aminophenylboronic acid-modified hyaluronic acid solution is 1:(1-2).

8. The method for preparing a responsive anticoagulant hydrogel material that releases 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.

9. 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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