阅读说明：本技术 一种抗凝血共聚物的制备方法及其应用 (Preparation method and application of anticoagulant copolymer ) 是由 欧阳晨曦 徐天成 焦培 龙行 吴瑶 刘思诗 于 2020-05-18 设计创作，主要内容包括：本发明属于医疗技术领域,公开了一种抗凝血共聚物的制备方法及其应用,该共聚物首先由单体A、B、C三种单体按比例加聚形成聚合物,其中,单体A含有双键和磺酸基团,单体B为含有双键和吡咯环的亲水性物质,单体C为含有双键的疏水性物质,然后通过将聚合物侧链的吡咯环打开,通过酯化反应接枝上维生素和薯蓣皂苷元即可制得。该共聚物能对聚氨酯材料表面进行改性,改性后的聚氨酯表面同时具有良好的抗凝血、抗感染、抗蛋白吸附、抗血栓性能,有良好的应用前景。(The invention belongs to the technical field of medical treatment, and discloses a preparation method and application of an anticoagulant copolymer, wherein the copolymer is formed by the polyaddition of A, B, C monomers according to a proportion, wherein the A monomer contains double bonds and sulfonic acid groups, the B monomer is a hydrophilic substance containing double bonds and a pyrrole ring, the C monomer is a hydrophobic substance containing double bonds, and then the anticoagulant copolymer is prepared by opening the pyrrole ring of the side chain of the polymer and grafting vitamins and diosgenin through esterification reaction. The copolymer can modify the surface of a polyurethane material, and the modified polyurethane surface has good anticoagulation, anti-infection, anti-protein adsorption and anti-thrombus performances, and has good application prospects.)

1. A method for preparing an anticoagulant copolymer, comprising the steps of:

s1, dissolving a monomer A, B, C in an organic solvent, stirring uniformly under the protection of nitrogen, adding an initiator, and reacting for 1-48 hours at 15-80 ℃ to obtain a polymer; wherein, the monomer A contains double bonds and sulfonic acid groups, the monomer B is a hydrophilic substance containing double bonds and pyrrole rings, and the monomer C is a hydrophobic substance containing double bonds;

s2, heating the polymer prepared in the step S1 in an aqueous solution of sodium hydroxide and N, N-dimethylacetamide for 1-10 h, adjusting the pH value to be acidic, adding vitamins and diosgenin, and carrying out esterification reaction;

and S3, removing residual sodium ions, chloride ions and N, N-dimethylacetamide from the solution obtained by the reaction of S2 by a dialysis method, and freeze-drying to obtain the copolymer.

2. The method according to claim 1, wherein the monomer A: the mass ratio of (B + C) is 99: 1-1: 99, wherein the mass ratio of the monomer B: the mass ratio of C is 99: 1-1: 99.

3. The method according to claim 1, wherein the organic solvent in step S1 is dimethyl sulfoxide, N-methylpyrrolidone, or N, N-dimethylacetamide.

4. The method according to claim 1 or 2, wherein the monomer A is sodium styrene sulfonate, the monomer B is vinylpyrrolidone, and the monomer C is vinyltriethoxysilane.

5. The method of claim 1, wherein the vitamin is one or more of vitamin A, vitamin C, and vitamin E.

6. The use of the copolymers prepared according to any of claims 1 to 5 in modified polyurethanes, characterized in that the modification is carried out by the following steps:

the method comprises the following steps: sequentially activating the surface of a polyurethane material by using a sodium hydroxide solution and a potassium permanganate solution to obtain a polyurethane material with a surface containing active carboxyl functional groups;

step two: dissolving the copolymer in an aqueous solution of N, N-dimethylacetamide, carrying out amidation reaction on the copolymer and the surface of activated polyurethane under the catalytic action of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, cleaning and drying.

7. The use according to claim 6, wherein the activation treatment of step one is in particular: the surface of the polyurethane is firstly placed in 0.1-5% of sodium hydroxide solution for 1-10 hours and then placed in 0.1-5% of potassium permanganate solution for 1-10 hours, and the reaction temperature is 15-25 ℃.

8. Use according to claim 6, wherein the molar ratio of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride to N-hydroxysuccinimide in step two is 6: 4.

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to an anticoagulant, anti-infection, anti-protein adsorption and antithrombotic copolymer capable of being used for surface modification of a polyurethane material, and a preparation method and application thereof.

Background

The polycarbonate polyurethane material is widely applied to the field of biomedicine due to good stability in organisms, good biocompatibility and excellent physical and mechanical properties. However, as the material of the medical device for implantation/intervention, during the process that polyurethane is in contact with tissues or blood, due to the inherent biological inertia of the surface and the lack of effective anticoagulation, anti-infection, anti-protein adsorption and anti-thrombus performance, the serious problems of coagulation, bacterial infection, inflammatory reaction and the like caused by the microbial contamination on the surface of the material are easily caused. Therefore, the establishment of efficient anticoagulant, anti-infection, anti-protein adsorption and anti-thrombus coatings on the surface of polyurethane materials is a key problem to be solved in the modification process of polyurethane medical materials.

The surface modification is a way to effectively improve or enhance the surface performance of the material, and can endow the material surface with new performance and function while keeping the original basic physicochemical performance of the material. In the past two decades, the research of improving the properties of materials such as anticoagulation, anti-infection, anti-protein adsorption, and antithrombotic property by activating the polyurethane surface and then introducing specific functional groups has become a hot spot. Previous studies have often modified polyurethanes for one of anticoagulant, antiinfective, antiproteogenic, and antithrombotic properties. For example, heparin is introduced into the surface of polyurethane to obtain polyurethane with heparin coating so as to improve the anticoagulation performance of the material. In practical applications, the polyurethane material often needs to face various problems such as blood coagulation, infection, protein adsorption, thrombosis and the like when contacting with blood or tissue of a human body, and a novel coating material needs to be developed to solve the problems at the same time.

Disclosure of Invention

In view of the above, the present invention provides an anticoagulant copolymer and a preparation method thereof, wherein the anticoagulant copolymer can simultaneously and effectively improve anticoagulant, anti-infection, anti-protein adsorption and antithrombotic properties of a polyurethane material after being grafted to a polyurethane surface.

In order to achieve the purpose, the invention specifically adopts the following technical scheme:

a preparation method of an anticoagulant copolymer comprises the following steps:

s1, dissolving a monomer A, B, C in an organic solvent, stirring uniformly under the protection of nitrogen, adding an initiator, and reacting for 1-48 hours at 15-80 ℃ to obtain a polymer; wherein, the monomer A contains double bonds and sulfonic acid groups, the monomer B is a hydrophilic substance containing double bonds and pyrrole rings, and the monomer C is a hydrophobic substance containing double bonds;

s2, heating the polymer prepared in the step S1 in an aqueous solution of sodium hydroxide and N, N-dimethylacetamide for 1-10 h, adjusting the pH value to be acidic, and sequentially adding vitamins and diosgenin to perform an esterification reaction, wherein the addition amount of the vitamins and the diosgenin is 1-20% of the amount of the monomer B substance.

And S3, removing residual sodium ions, chloride ions and N, N-dimethylacetamide from the solution obtained by the reaction of S2 by a dialysis method, and freeze-drying to obtain the copolymer.

In the above technical solution, the monomer a: the mass ratio of the (B + C) substance is 99:1 to 1: 99. In practical application, the proportion is adjusted according to different flow rates and pressures of the coating material contacting blood; specifically, the method comprises the following steps: the anticoagulant is applied to a blood flow environment with low aortic vascular pressure and high flow speed, preferably, the anticoagulant effect is dominant, the protein adsorption effect is auxiliary, and A: the mass ratio of (B + C) is preferably 99:1 to 50:50, more preferably 75: 25; the method is applied to a blood flow environment with high pressure and low flow speed of the peripheral small-caliber blood vessel, preferably, the anti-protein adsorption effect is mainly used, the anticoagulation effect is assisted, and A: the mass ratio of (B + C) is preferably 50:50 to 1:99, more preferably 25: 75.

Further, in the above technical solution, the monomer B: the mass ratio of C is 99: 1-1: 99. In practical applications, B: the mass ratio of C is preferably 99:1 to 50:50, and more preferably 75: 25.

Preferably, the organic solvent in step S1 is dimethyl sulfoxide, N-methylpyrrolidone, or N, N-dimethylacetamide.

As a preferred technical scheme, the monomer A is sodium styrene sulfonate, the monomer B is vinyl pyrrolidone, and the monomer C is vinyl triethoxysilane;

preferably, the vitamin is one or more of vitamin A, vitamin C and vitamin E.

Preferably, the pH value in step S2 is 3-6.

The invention also provides application of the copolymer prepared by the method in modified polyurethane, and the copolymer can be modified by the following steps:

the method comprises the following steps: sequentially activating the surface of a polyurethane material by using a sodium hydroxide solution and a potassium permanganate solution to obtain a polyurethane material with a surface containing active carboxyl functional groups;

step two: dissolving the copolymer in an aqueous solution of N, N-dimethylacetamide, carrying out amidation reaction on the copolymer and the surface of activated polyurethane under the catalytic action of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, cleaning and drying.

As a preferred technical scheme, the activation treatment in the first step is specifically as follows: the surface of the polyurethane is firstly placed in 0.1-5% of sodium hydroxide solution for 1-10 hours, and then placed in 0.1-5% of potassium permanganate solution for 1-10 hours, and the temperature is room temperature.

As a preferable technical scheme, the molar ratio of the catalyst 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride to the N-hydroxysuccinimide in the second step is 6: 4. Further, the molar ratio of the monomer B to the catalysts 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in the copolymer polymerization reaction is 10-1: 6:4, preferably 5-1: 6:4, and more preferably 1:6: 4.

The invention has the beneficial effects that: 1) the monomer containing anticoagulant and anti-protein adsorption functional groups forms macromolecules in an addition polymerization mode, then pyrrolidone five-membered rings are opened through base catalysis to form carboxyl and amino, and finally an esterification reaction is carried out under an acidic condition to graft anti-infection and anti-thrombosis substances on the end parts of side chains. 2) The amino of the molecular side chain of the copolymer coating and the carboxyl on the surface of the activated polyurethane can be firmly bonded on the surface of the polyurethane through simple amidation condensation polymerization reaction to form a stable polymer coating, and the anticoagulant, anti-infection, anti-protein adsorption and antithrombotic properties of the surface of the polyurethane material are obviously improved.

Drawings

FIG. 1 is a schematic diagram of the structure of a synthetic polymer intermediate prepared in example 1;

FIG. 2 is a schematic diagram showing the structure of a synthetic polymer intermediate prepared in example 1, after the five-membered ring of pyrrolidone is opened;

FIG. 3 is a schematic structural diagram of a synthesized copolymer prepared in example 1 (A is diosgenin, B is vitamin E);

FIG. 4 shows the surface of the polyurethane material without coating treatment (the left image is a low-magnification overall view, and the right image is a high-magnification local part);

FIG. 5 is a surface of a polyurethane material treated with a copolymer coating prepared in example 1 (the left image is a low-magnification overview, and the right image is a high-magnification part);

FIG. 6 shows the surface of a polyurethane material treated with a copolymer coating prepared in example 2 (the left is a low-magnification overview, and the right is a high-magnification detail).

Detailed Description

The present invention is further illustrated by the following specific examples, which should not be construed as limiting the scope of the invention.

Measuring the dissolved amount of diosgenin

Preparing a test solution: according to a surface area of 6cm²1mL of the sample was immersed in water at 37 ℃ for 72 hours, and all the liquid was collected as a test solution.

The detection method comprises the following steps: the amount of elution was calculated from the standard curve obtained by measuring the peak area at the 209nm wavelength position with an ultraviolet detector using a reversed-phase C18 column and methanol-water (95:5) as the mobile phase.

Measurement of amount of eluted vitamin E

Preparing a test solution: according to a surface area of 6cm²1mL of the sample was immersed in water at 37 ℃ for 72 hours, and all the liquid was collected as a test solution.

The detection method comprises the following steps: according to the high performance liquid chromatography method described in GB/T17812-1999.

Third, platelet adhesion experiment method for polyurethane material

After the surface of the polyurethane material is cleaned by normal saline, the residual normal saline on the product is removed. After blood collection from human veins, heparin was added to 50U/ml immediately. The blood was packed in 10mL of the polyurethane material within 10 minutes after the blood collection, and shaken at 37 ℃ for 1 hour. Thereafter, the polyurethane material was washed with 100ml of physiological saline, and the blood components were fixed with 2.5% glutaraldehyde physiological saline and washed with 200ml of distilled water. After cleaningCutting the dried polyurethane material to an appropriate size, attaching the cut polyurethane material to a sample stage of a scanning electron microscope using a double-sided tape, forming a Pt-Pd thin film on the surface of the sample by sputtering to obtain a sample, observing the surface of the sample with a field emission type scanning electron microscope at a magnification of 1500 times, and counting 1 field of view (4.3 × 10)³Micron meter²) The mean value of the number of adhered platelets in 10 fields different in the longitudinal direction near the center of the sample was taken as the number of adhered platelets (one/4.3 × 10)³Micron meter²)