CN111748053A

CN111748053A - Preparation method and application of anticoagulant copolymer

Info

Publication number: CN111748053A
Application number: CN202010419077.8A
Authority: CN
Inventors: 欧阳晨曦; 徐天成; 焦培; 龙行; 吴瑶; 刘思诗
Original assignee: Wuhan Yangsen Biotech Co ltd
Current assignee: Wuhan Yangsen Biotech Co ltd
Priority date: 2020-05-18
Filing date: 2020-05-18
Publication date: 2020-10-09
Also published as: CN113045695A; CN113045695B

Abstract

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.

Description

Preparation method and application of anticoagulant copolymer

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

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)

Example 1

The preparation method of the copolymer suitable for modifying the large-caliber blood vessels comprises the following specific steps:

(1) preparation of anticoagulant copolymers

The proportions of the monomers in this example are as follows:

the molar ratio of sodium p-styrenesulfonate (A), N-vinylpyrrolidone (B), vinyltriethoxysilane (C) and azobisisobutyronitrile (D) is A (B + C), D is 75:25:1.5, wherein B: and C is 75: 25.

Weighing an appropriate amount of A, B, C monomers according to the proportion, dissolving the A, B, C monomers in 200mL of DMAc solution, stirring the solution for 30 minutes under the protection of nitrogen, adding an initiator of azobisisobutyronitrile, and reacting the mixture for 20 hours at 80 ℃ to ensure that 3 monomers containing double bonds are polymerized into macromolecules in an addition mode.

Heating the polymerized high molecular polymer in an aqueous solution of 5 wt% of sodium hydroxide and N, N-dimethylacetamide (the mass ratio of the N, N-dimethylacetamide to the water is 1:1) for 10 hours to open a pyrrolidone five-membered ring on a side chain of the polymer to generate an amino group and a carboxyl group.

Titrating with 5% hydrochloric acid solution to change the solution from alkalinity to acidity, controlling the pH value to be 4, removing water and soluble inorganic ions in the solution by dialysis, adding diosgenin under the catalysis and dehydration of concentrated sulfuric acid for reacting for 2 hours at 70 ℃, then adding vitamin E for reacting, and reacting for 4 hours at 70 ℃ to fully complete the esterification reaction of carboxyl and alcohol, specifically, the esterification reaction of carboxyl on a macromolecule side chain and hydroxyl in diosgenin and vitamin E; wherein the addition amount of diosgenin and vitamin E is 10% of monomer B.

In vitamin E, alcoholic hydroxyl is connected with a benzene ring structure with a strong electron-withdrawing effect, so that hydrogen ions are easy to lose, and therefore, the reaction activity is strong, while alcoholic hydroxyl in diosgenin is connected with a cyclohexane structure with a weak electron-withdrawing effect, so that hydrogen ions are difficult to lose, and therefore, the reaction activity is weak. Therefore, the diosgenin and the vitamin E are added separately in sequence, so that the diosgenin can be grafted on the polymer fully.

Removing residual sodium ions, chloride ions and N, N-dimethylacetamide from the reacted solution by dialysis, and freeze-drying to remove residual water.

(2) Surface modification of polyurethanes

Polyurethane surface activation: the polyurethane surface was soaked in 5% sodium hydroxide solution for 10 hours to obtain hydrolysis of the polycarbonate linkages of the surface thermoplastic polymer to form hydroxyl and carboxyl groups. And then soaking the surface of the product in 5 percent potassium permanganate solution for 10 hours to oxidize functional groups such as hydroxyl, aldehyde and the like in the polyurethane on the surface into carboxyl, thereby completing the activation treatment of the surface of the polyurethane material.

Dissolving the polymer obtained in the step (1) in an aqueous solution of N, N-dimethylacetamide, and carrying out amidation reaction on amino groups in the polymer and carboxyl groups on the surface of activated polyurethane under the catalysis of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) (EDC: NHS ═ 6:4), wherein the molar ratio of the polymer to the catalyst is that of the polymer: EDC: NHS ═ 1:6: 4. The reaction was carried out at 25 ℃ for 2 hours. And cleaning with pure water to remove soluble micromolecule substances, and drying to finally obtain the polyurethane material containing the coating.

(3) Determination of the material dissolution after surface modification

Taking 50mL of leaching solution, and detecting results show that the concentration of diosgenin in the solution is less than 1 mu g/mL, the concentration of vitamin E in the solution is less than 1 mu g/mL, and the concentrations are all lower than the detection limit of 1 mu g/mL.

(4) Surface adsorption of the surface-modified material after contact with blood

According to the platelet adhesion experiment method, a partial morphological structure of the material surface is obtained, and compared with the aggregation and deposition of a large amount of red blood cells, platelets and proteins on the surface of an unmodified polyurethane material (shown in fig. 4), the material surface modified according to the embodiment has no red blood cell adhesion, and only individual platelets and a small amount of proteins are adhered (shown in fig. 5).

Example 2

A copolymer suitable for modifying small-caliber blood vessels is prepared by the following steps:

(1) preparation of anticoagulant copolymers

The proportions of the monomers in this example are as follows:

the molar ratio of sodium p-styrenesulfonate (A), N-vinylpyrrolidone (B), vinyltriethoxysilane (C) and azobisisobutyronitrile (D) is A (B + C), D is 25:75:1.5, wherein B: and C is 75: 25.

Weighing an appropriate amount of A, B, C according to the proportion, dissolving the A, B, C in 200mL of DMAc solution, stirring for 30 minutes under the protection of nitrogen, adding an initiator of azobisisobutyronitrile, and reacting for 20 hours at 80 ℃ to ensure that 3 monomers containing double bonds are polymerized into macromolecules.

Heating the polymerized high molecular polymer in an aqueous solution of 5% of sodium hydroxide and N, N-dimethylacetamide (the mass ratio of the N, N-dimethylacetamide to the water is 1:1) for 1h, so that a pyrrolidone five-membered ring on a side chain of the polymer is opened, and an amino group and a carboxyl group are generated.

Titrating with 1% hydrochloric acid solution to change the solution from alkalinity to acidity, controlling the pH value to 6, removing water and soluble inorganic ions in the solution by dialysis, adding diosgenin under the catalysis and dehydration of concentrated sulfuric acid, reacting at 70 ℃ for 2 hours, adding vitamin E, reacting at 70 ℃ for 4 hours, and fully completing the esterification reaction of carboxyl and alcohol; wherein the addition amount of diosgenin and vitamin E is 10% of monomer B.

(2) Surface modification of polyurethanes

(3) Determination of the material dissolution after surface modification

According to the platelet adhesion experiment method, a partial morphological structure of the material surface is obtained, and compared with the aggregation and deposition of a large amount of red blood cells, platelets and proteins on the surface of an unmodified polyurethane material (shown in figure 4), the material surface modified according to the embodiment has no red blood cells, platelets and a small amount of proteins (shown in figure 6).

The present invention may be better understood and appreciated by those skilled in the art with reference to the following examples. However, the protection of the invention and the scope of the claims are not limited to the examples provided. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Claims

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

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;

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:

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.