CN111544646A

CN111544646A - Small-caliber artificial blood vessel with surface grafted with heparin coating and preparation method thereof

Info

Publication number: CN111544646A
Application number: CN202010239004.0A
Authority: CN
Inventors: 洪枫; 胡高铨; 李格丽; 陈琳; 欧阳晨曦; 李佳荣; 王趁红
Original assignee: Donghua University
Current assignee: Donghua University; National Dong Hwa University
Priority date: 2020-03-30
Filing date: 2020-03-30
Publication date: 2020-08-18

Abstract

The invention relates to a small-caliber artificial blood vessel with a surface grafted with a heparin coating and a preparation method thereof, wherein the small-caliber blood vessel is taken as a substrate, the surface of the substrate is firstly soaked in a dopamine solution for reaction, a plurality of layers of polydopamine coatings are coated, then the polydopamine coatings and polyethyleneimine are combined and grafted through chemical bonds, finally, 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) are utilized to activate heparin, and the heparinized surface is obtained through amide bond combination. The invention is also provided withThe water-soluble polymer has the characteristics of small caliber, good mechanical property, hydrophilicity and long-acting anticoagulation; the preparation method is green and environment-friendly, the compounding is efficient, the prepared heparin coating is uniform, the grafting amount of the heparin is higher, and the grafting amount can reach 4.9 mu g/cm²Has good market application prospect.

Description

Small-caliber artificial blood vessel with surface grafted with heparin coating and preparation method thereof

Technical Field

The invention belongs to the field of artificial blood vessels and preparation thereof, and particularly relates to a small-caliber artificial blood vessel with a surface grafted with a heparin coating and a preparation method thereof.

Background

Cardiovascular diseases are common diseases that endanger human life and health, and atherosclerosis and other cardiovascular diseases cause high morbidity and mortality worldwide each year. When the blood vessel can not maintain the normal function due to atherosclerosis, breakage or aging, the blood vessel needs to be replaced by blood vessel transplantation.

The development and research of artificial blood vessels solve the problem of blood vessel transplantation to a certain extent. In the field of medium and large caliber blood vessel transplantation, both terylene Polyester (PET) blood vessels and expanded polytetrafluoroethylene (ePTFE) blood vessels have better antithrombotic capacity and better long-term patency rate, and are only two clinical application artificial blood vessels at present. However, the chemical structural units of the two materials are hydrophobic structures, which do not support cell adhesion, the adhesion and proliferation performance of endothelial cells is poor, and the endothelialization capability is insufficient. In the field of small-bore vessels (the inner diameter is less than 6mm) with slower blood flow speed, because the endothelialization degree is low, the long-term patency rate of the small-bore vessels and the long-term patency rate of the small-bore vessels are low, and the requirements of clinical use cannot be met.

In recent years, Polyurethane (PU) has been widely studied in the field of biomaterials due to its good biocompatibility, controllable structure, strong processability, and abrasion resistance. Compared with polyester and ePTFE, polyurethane has better processing characteristics, surface modification performance and more excellent compliance. Research shows that the compliance of the polyurethane material is closer to the natural vascular tissue than dacron polyester and expanded polytetrafluoroethylene. However, like polyester and ePTFE, the polyurethane surface does not have an anticoagulant substance, so that a long-lasting anticoagulant effect cannot be achieved. In addition, the polyurethane material may adsorb nonspecific protein after being implanted into a human body, and the surface of the polyurethane material is hydrophobic, so that the polyurethane material is not beneficial to endothelialization of endothelial cells. The above problems have no doubt restricted the development and application of polyurethane materials in the field of small-bore blood vessels.

Chinese patent CN104629058A discloses a method for preparing a heparinized polyurethane film, which takes a polyurethane film with carboxyl as a substrate, and prepares the heparinized polyurethane film by grafting heparin after activating the surface by 1-ethyl-3- (dimethylpropylamine) carbodiimide (WSC). However, the carboxylated polyurethane film has a limited carboxyl content, a small number of reactive groups, and WSC has a low coupling efficiency and a long reaction time, resulting in a low grafting efficiency of heparin. CN105949492A discloses a preparation method of a polyester material for resisting platelet adhesion without affecting platelet function, which comprises the steps of charging positive electricity on the surface of polyurethane by ammonolysis or polydopamine coating method, and then adsorbing the negatively charged biomacromolecules by electrostatic self-assembly. However, in the ammonolysis method, the ammonolysis agent can cause the polyurethane material to age, so that the polyurethane substrate is damaged, the material performance is deteriorated, and the mechanical property is influenced; the method for fixing the biomacromolecule by electrostatic self-assembly is physical adsorption, and the biomacromolecule is easily influenced by external charges, falls off or is lost, so that the long-term use effect of the biomacromolecule is influenced; in addition, the technology of coating chitosan or polydopamine on heparin by electrostatic self-assembly layer by layer has the following problems: chitosan is a natural hemostatic material and cannot be used for anticoagulation, and the anticoagulation function of heparin is shielded after polydopamine covers the heparin. Chinese patent CN106755027A, a non-viral gene vector for gene delivery, a preparation method and application thereof, discloses a method for grafting polyethyleneimine (PEI, molecular weight 1800) to heparin. According to the method, PEI dissolved by chloroform is connected with cell-penetrating peptide R8, then MES solution containing EDCI and NHS is used for activating heparin, and then the activated heparin is added into mixed solution of PEI and PEI-cell-penetrating peptide R8 for reaction to prepare the heparin compound. The method has the defects that the heparin and the PEI can not be directly coupled, the heparin still needs to be subjected to activation pretreatment, and the method has the defects of long and complicated reaction process and high cost.

Disclosure of Invention

The invention aims to solve the technical problem of providing a small-caliber artificial blood vessel with a surface grafted with a heparin coating and a preparation method thereof, overcoming the defects that the content of active groups of an artificial blood vessel polymer material is low, the heparin coupling grafting efficiency is low, active substances such as electrostatic adsorption biomacromolecules and the like are easy to fall off in the prior art, causing no damage to the mechanical property of the material, and having mild reaction conditions. The substrate of the small-caliber polyurethane artificial blood vessel is prepared by an electrostatic spinning or solvent solidification method, the substrate of the small-caliber polyester artificial blood vessel is prepared by a weaving method or an electrostatic spinning method, and the substrate of the small-caliber polytetrafluoroethylene artificial blood vessel is prepared by a bulking method or an electrostatic spinning method. The artificial blood vessel is placed in a dopamine solution to obtain a polydopamine coating through a dopamine self-polymerization process, then the polydopamine coating is placed in a 60 ℃ polyethyleneimine solution to react and graft polyethyleneimine, finally the polydopamine coating is added into a heparin solution of EDC/NHS, and the small-caliber polyurethane blood vessel, the dacron polyester artificial blood vessel and the polytetrafluoroethylene artificial blood vessel with the surface grafted with the heparin coating are prepared after reaction, so that the problems that the surface of the small-caliber artificial blood vessel is hydrophobic and does not have anticoagulation property are solved.

The small-caliber polyurethane artificial blood vessel with the surface grafted with the heparin coating is obtained by taking the small-caliber polyurethane blood vessel as a substrate, sequentially coating a polydopamine coating on the surface of the substrate, grafting polyethyleneimine and finally grafting heparin.

The caliber of the small-caliber polyurethane blood vessel is 0.5-6mm, and the molecular weight of polyethyleneimine is 10000.

The invention relates to a preparation method of a small-caliber artificial blood vessel with a surface grafted with a heparin coating, which comprises the following steps:

(1) placing the small-caliber blood vessel in a dopamine solution, carrying out oscillation reaction, ultrasonic cleaning and drying to obtain the small-caliber artificial blood vessel coated with the polydopamine coating;

(2) placing the small-caliber artificial blood vessel coated with the polydopamine coating in a polyethyleneimine solution, reacting, cleaning, and vacuum-drying to obtain a small-caliber blood vessel grafted with polyethyleneimine;

(3) and (3) placing the small-caliber blood vessel grafted with the polyethyleneimine into a heparin solution containing EDC/NHS, and reacting to obtain the small-caliber artificial blood vessel with the surface grafted with the heparin coating.

The preferred mode of the above preparation method is as follows:

ultrasonically cleaning the small-caliber artificial blood vessel in the step (1), and drying in vacuum; the solvent of the dopamine solution is a Tris buffer solution with the pH value of 8-9, and the concentration of the dopamine solution is 0.1-2 g/L.

The preparation method of the small-caliber polyurethane blood vessel prepared by the electrostatic spinning or solvent solidification method comprises the following steps:

electrostatic spinning preparation of small-caliber polyurethane blood vessels: dissolving polyurethane materials by using solvents which can dissolve polyurethane such as tetrahydrofuran, Dimethylformamide (DMF), Dimethylacetamide (DMA) and the like or mixtures thereof to prepare spinning solution, wherein the mass concentration of polyurethane in the spinning solution is 10-15%. The voltage of electrostatic spinning is 16kV, the flow rate is 1.5mL/h, the spinning time is 24h, and the silk threads are collected by a rod-shaped roller collector. And after spinning, thoroughly cleaning the obtained product with deionized water, and drying the obtained product to obtain the electrostatic spinning small-caliber polyurethane artificial blood vessel.

The preparation method of the small-caliber polyurethane blood vessel by the solvent coagulation method comprises the following steps: dissolving polyurethane materials by using a solvent or a mixture of solvents which can dissolve polyurethane, such as tetrahydrofuran, Dimethylformamide (DMF), Dimethylacetamide (DMA) and the like, to prepare a polyurethane solution, wherein the mass concentration of polyurethane in the solution is 10-15%. And (3) dip-coating the polyurethane solution on a glass shaft, putting the glass shaft into water, removing the glass shaft after 24 hours, repeatedly cleaning the glass shaft by using absolute ethyl alcohol and deionized water, and drying to obtain the small-caliber polyurethane artificial blood vessel by using the solvent coagulation method.

The polyurethane is

AL polycarbonate-based ether-free polyurethane and liquid polyurethane

AR、Carbothane^TM，Tecoflex^TM，

Tecothane^TMOne or more of thermoplastic polyurethane.

The dacron polyester artificial blood vessel is purchased from Shanghai Testet medical science and technology company; the polytetrafluoroethylene vascular prostheses are commercially available and include PTFE tubing of the Boston SCIENTIFIC (BOSTON SCIENTIFIC EXXCEL), Goll (GORE-TEX) ePTFE vascular prostheses and ePTFE tubing of Shanghai Sookang medical materials, Inc.

In the step (1), the oscillation reaction is oscillation reaction at 30 ℃ for 1d, and the coating reaction times are 1-5.

The molecular weight of the polyethyleneimine in the step (2) is 10000; the polyethyleneimine solution is a polyethyleneimine water solution, and the concentration is 1-6 g/L; the reaction is carried out for 1-6h at 60 ℃.

The pH value of the heparin solution containing EDC/NHS in the step (3) is 5-6, and the concentration of the heparin solution is 1 g/L; the molar ratio of EDC to NHS is 0.4-6.

Further, the EDC/NHS-containing heparin solution in the step (3) is specifically as follows: preparing 0.05M morpholine ethanesulfonic acid MES monohydrate solution, controlling the pH to be 5.5 +/-0.5, and then adding heparin, EDC and NHS; wherein the concentration of heparin in the heparin solution is 1 g/L. The reaction in the step (3) is carried out at 37 ℃ for 12-24h in a dark place.

The invention provides a small-caliber polyurethane artificial blood vessel with a surface grafted with a heparin coating, a small-caliber polyester artificial blood vessel with a surface grafted with a heparin coating and a small-caliber polytetrafluoroethylene artificial blood vessel with a surface grafted with a heparin coating, which are prepared by the method.

The invention provides an application of a small-caliber polyurethane artificial blood vessel, a small-caliber polyester artificial blood vessel and a small-caliber polytetrafluoroethylene artificial blood vessel with the surfaces grafted with a heparin coating.

Advantageous effects

(1) The polydopamine coating for coating the small-caliber blood vessel, which is prepared by taking polyurethane as a substrate, can graft polyethyleneimine by utilizing surface active groups of the polydopamine coating and improve the hydrophilicity of the surface of the material, the introduction of the polyethyleneimine further improves the hydrophilicity of the surface of the material, increases binding sites grafted with heparin, improves the grafting amount of the heparin, and improves the anticoagulation performance of the small-caliber polyurethane artificial blood vessel;

(2) the heparin grafted polyurethane small-caliber artificial blood vessel takes a polyurethane small-caliber blood vessel prepared by an electrostatic spinning method and a solvent coagulation method as a substrate, is grafted with polyethyleneimine after a polydopamine coating is polymerized and coated on the surface of the blood vessel, and is grafted with heparin finally, so that the heparin grafted polyurethane small-caliber artificial blood vessel has good hydrophilic performance, a large heparin grafting amount and a good anticoagulation effect;

(3) according to the invention, by utilizing the characteristics that the dopamine monomer is easy to undergo oxidative autopolymerization and adhere in an alkaline solution, no additional reagent is required to be introduced in the reaction, the prepared polydopamine coating is firm and stable, a plurality of surface active groups are provided, and the preparation method is simple and rapid;

(4) according to the invention, through the characteristic that a large number of active groups on the surface of the polydopamine coating can perform a grafting reaction with amino groups, polyethyleneimine is grafted, and the prepared material has the characteristics of high surface amino group content, mild and controllable preparation conditions;

(5) the invention has the characteristics of small caliber, good mechanical property and long-acting anticoagulation;

(6) the preparation method is simple and feasible, the prepared coating is stable, the preparation conditions are mild and controllable, the environment is protected, and the method has good market application prospect.

(7) The technology of the invention can be used for carrying out expression modification on other biological materials contacting with blood except for small-caliber artificial blood vessels, and has broad-spectrum applicability.

Drawings

FIG. 1 is a schematic flow chart of the production process of the present invention;

FIG. 2 is an XPS energy spectrum of the inner layer of a small-bore polyurethane blood vessel by the solvent coagulation method in example 1;

FIG. 3 is an electron micrograph of the small-caliber polyurethane vessel obtained by the electrospinning method in example 2 at different magnifications;

FIG. 4 is a graph showing platelet adhesion of the electrospun small-caliber polyurethane blood vessel of example 2;

FIG. 5 is a water contact angle of the electrospun small-caliber polyurethane blood vessel of example 2;

FIG. 6 is the cell viability data of human umbilical vein endothelial cells of small caliber polyurethane blood vessels by solvent coagulation in example 4;

FIG. 7 is a graph showing the hemolysis rate of the small-caliber polyurethane blood vessel obtained by the electrospinning method in example 5;

FIG. 8 is the plasma recalcification kinetic curve of the small-caliber polyurethane blood vessel by the electrospinning method in example 5;

FIG. 9 shows the data of the whole blood coagulation test of the small-diameter polyurethane blood vessel by the electrospinning method in example 5.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Example 1

(1) The preparation method of the small-caliber polyurethane artificial blood vessel with the inner diameter of 4mm by the solvent coagulation method comprises the following steps: dissolving the polyurethane particulate material with tetrahydrofuran

AL polycarbonate-based ether-free polyurethane available from HnG medical technology, ca), a PU solution was prepared in which the mass concentration of polyurethane was 15% or a liquid polyurethane obtained from HnG medical technology, ca, using DMF as a solvent

AR (solid content: 22%) was diluted to a mass concentration of 15% in the polyurethane. Dip-coating polyurethane solution on a glass shaft with a diameter of 4mm, putting the glass shaft into water, and removing the glass shaft after 24hAnd repeatedly cleaning the artificial blood vessel by using absolute ethyl alcohol and deionized water, and drying to obtain the small-caliber polyurethane artificial blood vessel by using the solvent coagulation method.

(2) The small-caliber artificial blood vessel in the step (1) is taken as a substrate, ultrasonic cleaning is carried out for 3 times, each time is carried out for 5min, after vacuum drying, the small-caliber artificial blood vessel is placed in Tris (Tris) buffer solution containing 2g/L of dopamine monomer (dopamine hydrochloride, 98 percent, 25g, purchased from Shanghai Aladdin Biotechnology Co., Ltd., the same below) and pH value of 8.5 +/-0.5, and oscillation reaction is carried out for 24h at 30 ℃. Subsequently, the mixture was ultrasonically cleaned 3 times and vacuum dried. This procedure was repeated 3 times to obtain 3-layer polydopamine-coated small-bore polyurethane blood vessels (PODA-PU).

(3) Preparing 4g/L aqueous solution of polyethyleneimine (M.W.10000, 99 percent and 25g, which is purchased from Shanghai Aladdin Biotechnology Co., Ltd., the same below), placing the small-caliber polyurethane blood vessel with the polydopamine coating obtained in the step (2) into the 4g/L aqueous solution of polyethyleneimine, and reacting for 6 hours at the temperature of 60 ℃. After the reaction is finished, a large amount of ultrapure water is used for cleaning, and vacuum drying is carried out, so that the polyethyleneimine grafted small-caliber polyurethane blood vessel (PEI-PU) is obtained.

(4) MES solution with a molar concentration of 0.05M was prepared, pH was controlled to 5.5 ± 0.5, and then heparin (heparin sodium, BR grade, 1g, available from national drug group chemical agents ltd., the same below), EDC and NHS were added to obtain heparin solution. Wherein the concentration of heparin is controlled to be 1g/L, the concentration of NHS is controlled to be 0.03M, and the molar ratio of EDC/NHS is controlled to be 2.

(5) And (4) placing the small-caliber polyurethane artificial blood vessel grafted with polyethyleneimine obtained in the step (3) into the heparin solution in the step (4), and reacting for 24 hours at 37 ℃ in a dark place. After the reaction is finished, the mixture is cleaned by a large amount of ultrapure water and then dried by a vacuum drying oven to obtain the small-caliber polyurethane artificial blood vessel (HEP-PU) with the heparin coating.

Table 1 shows XPS data of both the inside and outside of heparinized small-diameter polyurethane blood vessels prepared by the solvent coagulation method in example 1.

TABLE 1

Table 1 shows XPS data of the inside and outside of heparinized small-caliber polyurethane blood vessels obtained by the solvent coagulation method. As can be seen from the table, sulfur elements exist on the inner surface and the outer surface of the HEP-PU, which proves that the heparin is successfully grafted, and the sulfur content of the outer layer is higher than that of the inner layer, which proves that the difference of the material forms of the inner layer and the outer layer can influence the grafting content of the heparin.

As shown in FIG. 2, the XPS spectrum of the solvent coagulation polyurethane intravascular layer of example 1 is shown.

As shown in an XPS energy spectrum of figure 2, sulfur is a characteristic element of heparin, and after heparin grafting modification, an S2 p characteristic peak appears on the inner surface of the material, and the appearance of the sulfur characteristic peak proves the successful grafting of the heparin.

Example 2

(1) The preparation method of the electrostatic spinning small-caliber polyurethane artificial blood vessel with the inner diameter of 4mm comprises the following steps: using DMF to dissolve the polyurethane particulate material (

AL polycarbonate-based ether-free polyurethane available from HnG medical technology limited, canada), a spinning dope was prepared in which the mass concentration of polyurethane was 10%. Or liquid polyurethane prepared from HnG medical technology of Canada by using DMF as solvent

AR (solid content: 22%) was diluted to a polyurethane mass concentration of 10%. The voltage of electrostatic spinning was 16kV, the flow rate was 1.5mL/h, the spinning time was 24h, and the filaments were collected with a 4mm diameter rod-like collector. And after spinning, thoroughly cleaning the obtained product with deionized water, and drying the obtained product to obtain the electrostatic spinning small-caliber polyurethane artificial blood vessel.

(2) And (2) taking the small-caliber polyurethane artificial blood vessel in the step (1) as a substrate, ultrasonically cleaning for 3 times, each time for 5min, drying in vacuum, placing in a Tris buffer solution containing 2g/L of dopamine monomer and having the pH value of 8.5 +/-0.5, and carrying out oscillation reaction for 24h at the temperature of 30 ℃. Subsequently, the mixture was ultrasonically cleaned 3 times and vacuum dried. This procedure was repeated 3 times to obtain 3-layer polydopamine-coated small-bore polyurethane blood vessels (PODA-PU).

(3) And (3) placing the small-caliber polyurethane blood vessel with the polydopamine coating obtained in the step (2) into a polyethyleneimine water solution with the concentration of 4g/L, and reacting for 5 hours at the temperature of 60 ℃. After the reaction is finished, cleaning the mixture by using a large amount of ultrapure water, and drying the mixture in vacuum to obtain small-caliber polyurethane blood vessels (PEI-PU) grafted with polyethyleneimine; wherein the polyethyleneimine is M.W.10000.

(4) MES solution with a molar concentration of 0.05M was prepared, pH was controlled to 5.5 ± 0.5, and then heparin, EDC and NHS were added to obtain heparin solution. Wherein the concentration of heparin is controlled to be 1g/L, the concentration of NHS is controlled to be 0.03M, and the molar ratio of EDC/NHS is controlled to be 2.

(5) And (4) placing the small-caliber polyurethane artificial blood vessel grafted with polyethyleneimine obtained in the step (3) into the heparin solution in the step (4), and reacting for 24 hours at 37 ℃ in a dark place. After the reaction is finished, the mixture is cleaned by a large amount of ultrapure water and then dried by a vacuum drying oven to obtain the small-caliber polyurethane artificial blood vessel (HEP-PU) with the heparin coating. The heparin graft amount of the obtained HEP-PU was 4.149. mu.g/cm²。

FIG. 3 is an electron micrograph of the small-caliber polyurethane vessel obtained by the electrospinning method in example 2 at different magnifications; FIG. 4 is a graph showing platelet adhesion of the electrospun small-caliber polyurethane blood vessel of example 2; FIG. 5 is a water contact angle of the electrospun small-caliber polyurethane blood vessel in example 2.

As can be seen from the electron microscope image shown in FIG. 3, the surface of the electrospun pure PU small-caliber polyurethane artificial blood vessel is smooth, and the surfaces of PODA-PU, PEI-PU and HEP-PU are quite rough and have a plurality of particles due to modified coating, which proves that the surface of PU is successfully coated and modified.

As can be seen from the platelet adhesion electron microscope image shown in fig. 4, the electrospun pure PU has a large platelet adhesion, and after the coating of the poly-dopamine coating (PODA-PU), particles appear on the surface of the material, so that the platelet adhesion number is reduced; after grafting polyethyleneimine (PEI-PU), the number of platelet adhesion increases due to its positive amino charge; after further grafting of heparin (HEP-PU), the material surface was rougher, but no platelet adhesion was seen.

As shown in fig. 5, the water contact angles of polyurethane blood vessels with different small calibers prepared by the electrostatic spinning method are shown, it can be seen that the contact angle of pure PU is larger than 90 °, the material surface is extremely hydrophobic, and the contact angle is reduced after the polydopamine coating is coated; after the polyethyleneimine is grafted, the contact angle is further reduced, and the surface hydrophilic performance of the material is further improved; after heparin grafting, the contact angle is 0 degrees, and the surface of the material is extremely hydrophilic. The result proves that the hydrophobic surface of the small-caliber polyurethane artificial blood vessel is changed into the hydrophilic surface after the modification treatment, and the hydrophilic performance is obviously improved.

Example 3

(1) The preparation method of the electrostatic spinning small-caliber polyurethane artificial blood vessel with the inner diameter of 3mm comprises the following steps: dissolving polyurethane particulate material (Carbothane) with mixed solvent of tetrahydrofuran and Dimethylformamide (DMF)^TM,Tecoflex^TM,

Or Tecothane^TMPolymers in the production of Thermoplastic Polyurethane (TPU) are available from Lubrizol limited, usa. Carbothane^TMIs an aliphatic, aromatic, polycarbonate-based thermoplastic polyurethane. Tecoflex^TMIs aliphatic polyether-based thermoplastic polyurethane.

Thermoplastic polyurethane medical grade polymers are aromatic polyether and polyester products. Tecothane^TMThermoplastic polyurethanes are available both as aromatic polyether-based products and as polyester products. ) Preparing spinning solution, wherein the volume ratio of tetrahydrofuran to DMF in the spinning solution is 1:1, and the mass concentration of polyurethane is 20%. The voltage of electrostatic spinning was 16kV, the flow rate was 1.5mL/h, the spinning time was 24h, and the filaments were collected with a collector of a rod-like roller with a diameter of 3 mm. And after spinning, thoroughly cleaning the obtained product with deionized water, and drying the obtained product to obtain the electrostatic spinning small-caliber polyurethane artificial blood vessel.

(2) And (2) taking the electrostatic spinning small-caliber polyurethane artificial blood vessel in the step (1) as a substrate, ultrasonically cleaning for 3 times, 5min each time, drying in vacuum, placing in a Tris buffer solution containing 2g/L of dopamine monomer and having the pH value of 8.5 +/-0.5, and carrying out oscillation reaction for 24h at the temperature of 30 ℃. Subsequently, the mixture was ultrasonically cleaned 3 times and vacuum dried. This procedure was repeated 3 times to obtain 3-layer polydopamine-coated small-bore polyurethane blood vessels (PODA-PU).

(4) MES solution with a molar concentration of 0.05M was prepared, pH was controlled to 5.5 ± 0.5, and then heparin, EDC and NHS were added to obtain heparin solution. Wherein the concentration of heparin is controlled to be 1g/L, the concentration of NHS is controlled to be 0.03M, and the molar ratio of EDC/NHS is respectively 0.4, 0.6, 0.8, 1, 2, 4 and 6.

Table 2 shows the grafting amount of heparin in the polyurethane vessels of the small-caliber polyurethane vessel obtained by the electrospinning method in example 3 under the condition of different EDC/NHS ratios.

TABLE 2

EDC/NHS molar ratio	0.4	0.6	0.8	1	2	4	6
								Heparin concentration (. mu.g/cm)²)	0.100	0.748	1.136	4.923	4.149	3.899	3.786

Table 2 shows that the grafting amount of heparin is more under different EDC/NHS molar ratio conditions, compared with the CN105949492A patent, the grafting amount of heparin is as high as 4.923 mu g/cm²。

Example 4

(1) The preparation method of the small-caliber polyurethane artificial blood vessel with the inner diameter of 4mm by the solvent coagulation method comprises the following steps: dissolving the polyurethane particle material by using a mixed solvent of tetrahydrofuran and DMA

AL polycarbonate-based ether-free polyurethane available from HnG medical technology ltd, canada) to prepare a PU solution in which the volume ratio of tetrahydrofuran to DMA is 1:1 and the mass concentration of polyurethane is 15%. Or liquid polyurethane prepared from HnG medical technology of Canada by using DMA as solvent

AR (solid content 22%) Diluted to a mass concentration of 15% of polyurethane. And (3) dip-coating the polyurethane solution on a glass shaft with the diameter of 4mm, putting the glass shaft into water, removing the glass shaft after 24 hours, repeatedly cleaning the glass shaft by using absolute ethyl alcohol and deionized water, and drying to obtain the small-caliber polyurethane artificial blood vessel by using the solvent coagulation method.

(2) And (2) taking the small-caliber artificial blood vessel in the step (1) as a substrate, ultrasonically cleaning for 3 times, each time for 5min, drying in vacuum, placing in a Tris (Tris) buffer solution containing 2g/L of dopamine monomer and having the pH value of 8.5 +/-0.5, and carrying out oscillation reaction for 24h at the temperature of 30 ℃. Subsequently, the mixture was ultrasonically cleaned 3 times and vacuum dried. This procedure was repeated 3 times to obtain 3-layer polydopamine-coated small-bore polyurethane blood vessels (PODA-PU).

(3) And (3) placing the small-caliber polyurethane blood vessel with the polydopamine coating obtained in the step (2) into a polyethyleneimine water solution with the concentration of 4g/L, and reacting for 6 hours at the temperature of 60 ℃. After the reaction is finished, cleaning the mixture by using a large amount of ultrapure water, and drying the mixture in vacuum to obtain small-caliber polyurethane blood vessels (PEI-PU) grafted with polyethyleneimine; wherein the polyethyleneimine is M.W.10000.

(4) MES solution with a molar concentration of 0.05M was prepared, pH was controlled to 5.5 ± 0.5, and then heparin, EDC and NHS were added to obtain heparin solution. Wherein the concentration of heparin is controlled to be 1g/L, the concentration of NHS is controlled to be 0.03M, and the molar ratio of EDC/NHS is controlled to be 1.

(6) Cleaning the small-caliber blood vessels obtained in the steps (1), (2), (3) and (5) by 75% (w/v) ethanol, placing the small-caliber blood vessels into a 24-hole plate, planting Human Umbilical Vein Endothelial Cells (HUVEC) on the surface of each small-caliber blood vessel (10000 HUVEC per hole), adding a proper amount of cell culture medium (containing 1% of penicillin-streptomycin mixed solution, 10% of fetal bovine serum FBS and 89% of high-sugar DMEM medium), and testing the cell viability by using a cck-8 kit (Shanghai Biyun Tian biotechnology limited company) when culturing

days

1, 3 and 5.

(7) The cck-8 cell viability experiment procedure at day 1 in step (6) is as follows: the medium in each well was discarded, washed 3 times with PBS buffer, then DMEM (cck-8: DEEM ═ 1: 9, v/v) containing cck-8 at a concentration of 10% was added in the dark, and after incubation for 2 hours in a cell incubator at 37 ℃, 200. mu.L of the resulting solution was aspirated in each well, transferred to a 96-well plate, and the absorbance at 450nm was measured with a microplate reader, and each material was repeated 5 times.

(8) The cck-8 cell viability experiment procedure on day 3 and day 5 is the same as that in step (7).

FIG. 6 is the cell viability data of human umbilical vein endothelial cells of small caliber polyurethane blood vessels by solvent coagulation in example 4.

As shown in FIG. 6, the cell viability data of different small-caliber polyurethane blood vessels prepared by the solvent coagulation method shows that the higher the absorbance at 450nm, the better the cell proliferation condition, and it can be seen that after 5 days of culture, the HUVEC on the surfaces of pure PU and HEP-PU are proliferated, and the cell proliferation condition of HEP-PU on the fifth day is better than that of pure PU, which proves that HEP-PU is favorable for the proliferation of HUVEC.

Example 5

(1) Referring to the method of example 2, electrospun small-caliber PU pipes, namely PU, PODA-PU and PEI-PU, were prepared. Subsequently, a MES solution having a molar concentration of 0.05M was prepared, the pH thereof was controlled to 5.5 ± 0.5, and then heparin, EDC and NHS were added to obtain a heparin solution. Wherein the concentration of heparin is controlled to be 1g/L, the concentration of NHS is controlled to be 0.03M, and the molar ratio of EDC/NHS is controlled to be 1. PEI-PU prepared in advance is placed in a heparin solution and is reacted for 24 hours at 37 ℃ in the absence of light. After the reaction is finished, the mixture is cleaned by a large amount of ultrapure water and then dried by a vacuum drying oven to obtain the small-caliber polyurethane artificial blood vessel (HEP-PU) with the heparin coating.

(2) And (3) performing a hemolysis rate test, a plasma recalcification test and a whole blood coagulation test to evaluate the blood compatibility of the electrostatic spinning small-caliber PU tube. Red blood cells and Platelet Poor Plasma (PPP) were obtained as follows: 5mL of New Zealand white rabbit blood is obtained by using a 5mL sodium citrate vacuum blood collection tube, the blood is centrifuged for 5min under 2000g, the supernatant is collected to obtain Platelet Poor Plasma (PPP), and the centrifuged lower layer sediment is collected to obtain erythrocytes.

(3) The hemolysis rate test method comprises cutting PU tube, PODA-PU, PEI-PU and HEP-PU prepared by electrostatic spinning into 1 × 1cm²The membrane of (1) with the inner surface facing upward, soaking in physiological saline for 4h, removing the physiological saline, and adding 2mL of physiological saline and 1.5 × 10 of red blood cells obtained in step (1)⁸And adding 2mL of physiological saline and erythrocytes into the negative control without adding materials, adding 2mL of ultrapure water and erythrocytes into the positive control, incubating for 1h at 37 ℃, transferring into an EP (ethylene propylene) tube, centrifuging for 5min under the condition of 750g, absorbing 200 mu L of supernatant into a 96-well plate, measuring the light absorption value at the wavelength of 540nm by using an enzyme-labeling instrument, and calculating the hemolysis rate.

(4) The plasma calcium-supplementing experimental method comprises the following steps of cutting the PU tube, PODA-PU, PEI-PU and HEP-PU prepared by electrostatic spinning into 1 × 1cm²The inner surface of the membrane is upward, the membrane is soaked in normal saline for 4h, then the normal saline is removed, 500 mu L of the platelet poor plasma obtained in the step (1) is added, shaking culture is carried out for 1h at the condition of 37 ℃ and 100 mu L of the incubated plasma is absorbed into a 96-well plate, 100 mu L of calcium chloride solution with the concentration of 0.01M is rapidly added, the membrane is placed into a microplate reader at the temperature of 37 ℃ for continuous incubation, and the light absorption value at the position of 405nm is measured every 30s, so that the plasma recalcification kinetic curve is obtained. Sucking 100 mu L of platelet poor plasma cultured with the blank hole, placing the platelet poor plasma in a 96-well plate, adding 100 mu L of physiological saline to measure the absorbance value, and taking the absorbance value as negative control; mu.L of platelet poor plasma incubated with blank wells was pipetted into a 96-well plate, and 100. mu.L of 0.01M calcium chloride solution was added to measure absorbance as a positive control.

(5) The whole blood coagulation experiment method comprises the following steps of cutting a PU tube, PODA-PU, PEI-PU and HEP-PU prepared by electrostatic spinning into 1 × 1cm²The membrane of (2) with the inner surface facing upwards is placed in a 24-pore plate, and the physiological saline is removed after being soaked for 4 hours by the physiological saline. Adding 500 μ L of 0.025M calcium chloride solution into 5mL rabbit whole blood, mixing, rapidly transferring 100 μ L blood to the surface of the membrane, incubating in a water bath at 37 deg.C for 5min, 15min, 25min, 35min, 45min and 55min, adding 2.5mL ultrapure water into the well, mixing, incubating for 5min, sucking 200 μ L into 96-well plate,the absorbance was measured with a microplate reader at 540nm, with 3 replicates set at each time.

The anticoagulation performance of the material can be visually evaluated by a whole blood coagulation experiment. In the process of forming thrombus after the blood coagulation is excited, red blood cells are wrapped in the blood cells, and after ultrapure water is added, the red blood cells wrapped by the thrombus are not broken, so that the release of hemoglobin is reduced, and the red blood cells which are not subjected to the blood coagulation are broken by absorbing water to release the hemoglobin. Therefore, in the whole blood coagulation test, the higher the degree of coagulation of the material, the lower the OD value.

FIG. 7 shows the hemolysis rate of the small-caliber polyurethane blood vessel obtained by the electrospinning method in example 5; FIG. 8 is the plasma recalcification kinetic curve of the small-caliber polyurethane blood vessel by the electrospinning method in example 5; FIG. 9 shows the data of the whole blood coagulation test of the small-diameter polyurethane blood vessel by the electrospinning method in example 5.

Fig. 7 shows the hemolysis rate of different small-caliber polyurethane blood vessels prepared by the electrospinning method. It is known that the hemolysis rates of PU, PODA-PU, PEI-PU and HEP-PU are all lower than 1%, and no hemolysis occurs.

Fig. 8 shows plasma recalcification dynamic curves of different small-caliber polyurethane blood vessels prepared by the electrospinning method. Plasma recalcification time is defined as the time corresponding to half the maximum OD value. As can be seen from the figure, after the PU tube is added with the calcium chloride solution for 30min, the OD value is accelerated obviously and slowly, and almost reaches the maximum value at 45 min; HEP-PU still does not reach a maximum value at 50min, which proves that the endogenous coagulation activation degree of the HEP-PU is far lower than that of pure PU, and the blood compatibility of the pure PU is obviously improved after HEP grafting.

FIG. 9 shows the whole blood coagulation time of different small-bore polyurethane blood vessels prepared by the electrospinning method. The OD value in whole blood coagulation is the OD value resulting from rupture of erythrocytes in non-coagulated blood, i.e., the higher the OD value, the more non-coagulated blood. As can be seen from the figure, the OD value of the PU tube is reduced linearly from 1.1 to 0.15, while the OD value of the HEP-PU tube is reduced from 1.2 to 0.8, the difference is obvious, which proves that the anticoagulation performance of the PU tube is obviously improved by the grafting of HEP.

Example 6

(1) Preparation of the inner diameterThe preparation method of the electrostatic spinning small-caliber polyurethane artificial blood vessel with the diameter of 4mm comprises the following steps: using DMF to dissolve the polyurethane particulate material (

AL polycarbonate-based ether-free polyurethane available from HnG medical technology limited, canada), a spinning dope was prepared in which the mass concentration of polyurethane was 20%. Or liquid polyurethane prepared from HnG medical technology of Canada by using DMF as solvent

AR (solid content: 22%) was diluted to a mass concentration of 20% in the polyurethane. The voltage of electrostatic spinning was 16kV, the flow rate was 1.5mL/h, the spinning time was 24h, and the filaments were collected with a 4mm diameter rod-like collector. And after spinning, thoroughly cleaning the obtained product with deionized water, and drying the obtained product to obtain the electrostatic spinning small-caliber polyurethane artificial blood vessel.

(3) And (3) placing the small-caliber polyurethane blood vessel with the polydopamine coating obtained in the step (2) into a polyethyleneimine water solution with the concentration of 4g/L, and reacting for 5 hours at the temperature of 60 ℃. After the reaction is finished, cleaning the mixture by using a large amount of ultrapure water, and drying the mixture in vacuum to obtain small-caliber polyurethane blood vessels (PEI-PU) grafted with polyethyleneimine; polyethyleneimine, m.w.10000.

(6) The blood collection method, the hemolysis rate test method, the plasma recalcification test method, and the whole blood coagulation test method were the same as in example 5.

In example 6, the results of the experiments on hemolysis rate, plasma recalcification, and whole blood coagulation were not statistically different from those of example 5.

Comparative example 1

AR (solid content: 22%) was diluted to a mass concentration of 15% in the polyurethane. And (3) dip-coating the polyurethane solution on a glass shaft with the diameter of 4mm, putting the glass shaft into water, removing the glass shaft after 24 hours, repeatedly cleaning the glass shaft by using absolute ethyl alcohol and deionized water, and drying to obtain the small-caliber polyurethane artificial blood vessel by using the solvent coagulation method.

(2) And (2) taking the small-caliber artificial blood vessel in the step (1) as a substrate, ultrasonically cleaning for 3 times, each time for 5min, drying in vacuum, placing in a Tris buffer solution containing 2g/L of dopamine monomer and having the pH value of 8.5 +/-0.5, and carrying out oscillation reaction for 24h at the temperature of 30 ℃. Subsequently, the mixture was ultrasonically cleaned 3 times and vacuum dried. This procedure was repeated 3 times to obtain 3-layer polydopamine-coated small-bore polyurethane blood vessels (PODA-PU).

(3) Preparing 4g/L aqueous solution of polyethyleneimine (M.W.1800, 99 percent and 25g of polyethyleneimine selected from Chinese patent CN106755027A, purchased from Shanghai Aladdin Biochemical technology Co., Ltd.), placing the small-caliber polyurethane blood vessel with the polydopamine coating obtained in the step (2) in the 4g/L aqueous solution of polyethyleneimine, and reacting for 6h at 60 ℃. After the reaction is finished, a large amount of ultrapure water is used for cleaning, and vacuum drying is carried out, so that the polyethyleneimine grafted small-caliber polyurethane blood vessel (PEI-PU) is obtained.

(4) According to the procedure (4) of example 4, a MES solution having a molar concentration of 0.05M was prepared, the pH thereof was controlled to 5.5 ± 0.5, and then heparin, EDC and NHS were added to obtain a heparin solution. Wherein the concentration of heparin is controlled to be 1g/L, the concentration of NHS is controlled to be 0.03M, and the molar ratio of EDC/NHS is controlled to be 1.

Comparative example 2

(3) Preparing 4g/L aqueous solution of polyethyleneimine (selected from polyethyleneimine in CN106755027A, M.W.1800, 99%, 25g, available from Shanghai Aladdin Biochemical technology Co., Ltd.), placing the small-caliber polyurethane blood vessel with the polydopamine coating obtained in the step (2) in the 4g/L aqueous solution of polyethyleneimine, and reacting for 6h at 60 ℃. After the reaction is finished, a large amount of ultrapure water is used for cleaning, and vacuum drying is carried out, so that the polyethyleneimine grafted small-caliber polyurethane blood vessel (PEI-PU) is obtained.

(4) According to the preferred process [0033] of patent CN106755027A, 50mg of heparin sodium is dissolved in 100mL of MES solution (pH 5.5 + -0.5, 0.05M), 30mg of EDC and 30mg of NHS are added, that is, EDC is added at a molar ratio of EDC/NHS of 0.6, and the reaction is vigorously stirred for 3h to activate the carboxyl groups on the heparin.

(5) And (4) placing the small-caliber polyurethane artificial blood vessel grafted with the polyethyleneimine obtained in the step (3) into the pre-activated heparin solution obtained in the step (4), and reacting for 24 hours at 37 ℃ in a dark place. After the reaction is finished, the mixture is cleaned by a large amount of ultrapure water and then dried by a vacuum drying oven to obtain the small-caliber polyurethane artificial blood vessel (HEP-PU) with the heparin coating.

Table 3 shows the amount of heparin grafted on the surface of small-diameter polyurethane blood vessels (HEP-PU) obtained in example 4, comparative example 1 and comparative example 2.

TABLE 3

Example numbering	Example 4	Comparative example 1	Comparative example 2
				Amount of heparin grafted (. mu.g/cm)²)	2.092	2.003	1.412

Table 3 shows the grafting amounts of heparin in the conditions of example 4, comparative example 1 and comparative example 2, which are 2.092, 2.003 and 1.4123. mu.g/cm²Wherein, the comparative example 1 is PEI with molecular weight of 1800 in Chinese patent CN106755027A and is implemented by adopting the process of the embodiment 4 of the invention, and the comparative example 2 is an embodiment based on the preferred process in Chinese patent CN 106755027A. The only difference between comparative example 1 and comparative example 2 is step (4), with comparative example 2 requiring the activation of heparin first. From the results, the heparin grafting process based on the invention is much better than the comparative example, and even though PEI in CN106755027A with low molecular weight is adopted, the heparin grafting amount is better than that in CN 106755027A.

Example 7

(1) Referring to the experimental step (1) of example 4, a solvent coagulation method small-caliber polyurethane artificial blood vessel with an inner diameter of 4mm is prepared and recorded as a sample 1; referring to experimental steps (1) - (4) of example 4, a small-bore polyurethane artificial blood vessel (HEP-PU) with a heparin coating was prepared and noted as sample 2 (example 4).

(2) A small-bore polyurethane artificial blood vessel (HEP-PU) with a heparin coating was prepared using polyethyleneimine (M.W.1800, 99%, 25g, available from Shanghai Arlatin Biotech Co., Ltd.) from Chinese patent CN106755027A, according to the experimental procedure of comparative example 1, and is designated as sample 3 (comparative example 1).

(3) A small-bore polyurethane artificial blood vessel (HEP-PU) with a heparin coating was prepared according to the preferred process [0033] of patent CN106755027A, with reference to the experimental procedure of comparative example 2, and is designated as sample 4 (comparative example 2).

(4) The hemocompatibility of sample 1, sample 2 (example 4), sample 3 (comparative example 1) and sample 4 (comparative example 2) was evaluated by referring to the evaluation method of hemocompatibility in example 5 using the whole blood coagulation test as an evaluation standard, and the initial OD value and the terminal OD value thereof were as shown in Table 4 below.

TABLE 4

Example numbering	Sample	1	Sample 2 (example 4)	Sample 3 (comparative example 1)	Sample 4 (comparative example 2)
						Initial OD value	1.121	1.264	1.257	1.186
End point OD value	0.142	0.768	0.712	0.586

Table 4 shows the data of the whole blood coagulation test for sample 1, sample 2 (example 4), sample 3 (comparative example 1) and sample 4 (comparative example 2) in example 7. The higher the OD value, the less blood volume is in the amount of coagulated blood. Sample 1 was an unmodified PU vessel with an initial OD value lower than the remaining three heparin-grafted vessels, demonstrating that grafted heparin can improve the coagulation status from the initial state. The endpoint OD values for sample 1, sample 2 (example 4), sample 3 (comparative example 1), and sample 4 (comparative example 2) were 0.142, 0.768, 0.712, and 0.586, respectively. The grafted heparin can effectively improve the anticoagulation performance of the polyurethane artificial blood vessel, and the grafting amount of the heparin is directly related to the anticoagulation effect, so that the higher the grafting amount of the heparin, the better the anticoagulation performance. The whole blood coagulation data shows that the anticoagulation property of the blood vessel prepared by the heparin grafting process is better than that of the blood vessel prepared by the CN106755027A patent.

Example 8

1) The preparation method of the small-caliber polyurethane artificial blood vessel with the inner diameter of 4mm by the solvent coagulation method comprises the following steps: dissolving the polyurethane particle material by using a mixed solvent of tetrahydrofuran and DMA

(3) And (3) placing the small-caliber polyurethane blood vessel with the polydopamine coating obtained in the step (2) into a 4g/L aqueous solution of polyethyleneimine (polyethyleneimine, M.W.10000, 99 percent and 25g, which is purchased from Shanghai Allantin Biotechnology Co., Ltd.) and reacting for 6 hours at the temperature of 60 ℃. After the reaction is finished, a large amount of ultrapure water is used for cleaning, and vacuum drying is carried out, so that the polyethyleneimine grafted small-caliber polyurethane blood vessel (PEI-PU) is obtained.

(4) MES solution with a molar concentration of 0.05M was prepared, pH was controlled to 5.5 ± 0.5, and then heparin, EDC and NHS were added to obtain heparin solution. Wherein the concentration of heparin is controlled to be 1g/L, the concentration of NHS is controlled to be 0.03M, and the molar ratio of EDC/NHS is controlled to be 1. The carboxyl on the heparin is activated by stirring vigorously for 3 hours according to the process requirements of patent CN 106755027A.

Example 9

Diluting AR (solid content: 22%) to the mass of polyurethaneThe concentration was 15%. And (3) dip-coating the polyurethane solution on a glass shaft with the diameter of 4mm, putting the glass shaft into water, removing the glass shaft after 24 hours, repeatedly cleaning the glass shaft by using absolute ethyl alcohol and deionized water, and drying to obtain the small-caliber polyurethane artificial blood vessel by using the solvent coagulation method.

(3) And (3) placing the small-caliber polyurethane blood vessel with the polydopamine coating obtained in the step (2) in a 4g/L aqueous solution of polyethyleneimine (selected from polyethyleneimine in Chinese patent CN106755027A, M.W.1800, 99 percent and 25g, purchased from Shanghai Allandin Biotechnology Co., Ltd.) and reacting for 6 hours at the temperature of 60 ℃. After the reaction is finished, a large amount of ultrapure water is used for cleaning, and vacuum drying is carried out, so that the polyethyleneimine grafted small-caliber polyurethane blood vessel (PEI-PU) is obtained.

Table 5 shows the amount of heparin grafted on the surface of small-diameter polyurethane blood vessels (HEP-PU) obtained in example 4, example 8, comparative example 1 and example 9.

TABLE 5

Example numbering	Example 4	Example 8	Comparative example 1	Example 9
					Amount of heparin grafted (. mu.g/cm)²)	2.092	2.086	2.003	1.997

Table 5 shows the amount of heparin grafted on the surface of small-diameter polyurethane blood vessels (HEP-PU) obtained in example 4, example 8, comparative example 1 and example 9. Examples 8 and 9 refer to the process of Chinese patent CN106755027A, and the carboxyl on heparin is activated for 3h in advance. However, the actual results show that the heparin grafting amount of the four blood vessels is not different greatly. This proves that compared with the process of Chinese patent CN106755027A, the process saves the time for activating heparin, and has the advantage of saving time and cost in industrial production.

Example 10

(1) Using dacron polyester artificial blood vessel (PET, purchased from Shanghai Testet medical science and technology company) as a substrate, ultrasonically cleaning for 3 times, 5min each time, drying in vacuum, placing in Tris buffer solution containing 2g/L dopamine monomer and having pH of 8.5 +/-0.5, and carrying out shaking reaction for 24h at 30 ℃. Subsequently, the mixture was ultrasonically cleaned 3 times and vacuum dried. The procedure was repeated 3 times according to the procedure to obtain 3-layer polydopamine-coated dacron polyester artificial blood vessel (PODA-PET).

(2) And (2) placing the polyester artificial blood vessel with the polydopamine coating obtained in the step (1) in a polyethyleneimine water solution with the concentration of 4g/L, and reacting for 5 hours at the temperature of 60 ℃. After the reaction is finished, washing with a large amount of ultrapure water, and drying in vacuum to obtain the polyester artificial blood vessel (PEI-PET) grafted with polyethyleneimine; wherein the polyethyleneimine is M.W.10000.

(3) MES solution with a molar concentration of 0.05M was prepared, pH was controlled to 5.5 ± 0.5, and then heparin, EDC and NHS were added to obtain heparin solution. Wherein the concentration of heparin is controlled to be 1g/L, the concentration of NHS is controlled to be 0.03M, and the molar ratio of EDC/NHS is controlled to be 1.

(4) And (3) placing the polyester artificial blood vessel grafted with polyethyleneimine obtained in the step (2) in the heparin solution obtained in the step (3), and reacting for 24 hours at 37 ℃ in a dark place. After the reaction is finished, the polyester artificial blood vessel is cleaned by a large amount of ultrapure water and then dried by a vacuum drying oven to obtain the polyester artificial blood vessel (HEP-PET) with the heparin coating.

Example 11

(1) An expanded polytetrafluoroethylene artificial blood vessel (ePTFE, available from Shanghai Sookang medical materials Co., Ltd.) was used as a substrate, and the vessel was ultrasonically cleaned 3 times for 5 minutes each, vacuum-dried, and then placed in a Tris buffer solution containing 2g/L of dopamine monomer at a pH of 8.5. + -. 0.5, and subjected to a shaking reaction at 30 ℃ for 24 hours. Subsequently, the mixture was ultrasonically cleaned 3 times and vacuum dried. This procedure was repeated 3 times to obtain 3-layer polydopamine-coated expanded polytetrafluoroethylene artificial blood vessels (PODA-ePTFE).

(2) And (2) placing the expanded polytetrafluoroethylene artificial blood vessel with the polydopamine coating obtained in the step (1) in a polyethyleneimine water solution with the concentration of 4g/L, and reacting for 5 hours at the temperature of 60 ℃. After the reaction is finished, cleaning the mixture by using a large amount of ultrapure water, and drying the mixture in vacuum to obtain the expanded polytetrafluoroethylene artificial blood vessel (PEI-ePTFE) grafted with polyethyleneimine; wherein the polyethyleneimine is M.W.10000.

(4) And (3) placing the expanded polytetrafluoroethylene artificial blood vessel grafted with polyethyleneimine obtained in the step (2) in the heparin solution obtained in the step (3), and reacting for 24 hours at 37 ℃ in the dark. After the reaction is finished, the vessel is cleaned by a large amount of ultrapure water and then dried by a vacuum drying oven to obtain the expanded polytetrafluoroethylene artificial blood vessel (HEP-ePTFE) with the heparin coating.

Compared with the polyurethane artificial blood vessel in example 4, the grafting amount of heparin on the dacron polyester artificial blood vessel and the expanded polytetrafluoroethylene artificial blood vessel in example 10 and example 11 has no difference in statistical significance, and the preparation process is proved to have the characteristic of universal applicability. In conclusion, the small-caliber artificial blood vessel with the heparin coating is prepared, the preparation method is simple and rapid, green and environment-friendly, the heparin is successfully grafted on the surface of the material, the hydrophilic property and the anticoagulant property are obviously improved, and the small-caliber artificial blood vessel has the potential of being clinically applied as a small-caliber blood vessel. Besides being used for small-caliber artificial blood vessels, the technology of the invention can be used for carrying out performance modification on other biological materials contacting with blood.

Claims

1. The small-caliber artificial blood vessel with the surface grafted with the heparin coating is characterized in that the artificial blood vessel takes the small-caliber blood vessel as a substrate, the surface of the substrate is sequentially coated with the polydopamine coating, the grafted polyethyleneimine and the grafted heparin.

2. The artificial blood vessel according to claim 1, wherein the small-caliber blood vessel has a caliber of 0.5-6 mm; the molecular weight of polyethyleneimine is 10000.

3. The artificial blood vessel of claim 1, wherein the material of the small-caliber artificial blood vessel is one or more of polyurethane, polyester, and polytetrafluoroethylene.

4. A method for preparing a small-caliber artificial blood vessel with a heparin coating grafted on the surface comprises the following steps:

5. The preparation method according to claim 4, wherein the small-caliber artificial blood vessel in the step (1) is subjected to ultrasonic cleaning and vacuum drying; the solvent of the dopamine solution is a Tris buffer solution with the pH value of 8-9, and the concentration of the dopamine solution is 0.1-2 g/L.

6. The preparation method according to claim 4, wherein the small-caliber artificial blood vessel in the step (1) is a small-caliber polyurethane blood vessel prepared by electrostatic spinning or solvent coagulation; the small-caliber artificial blood vessel in the step (1) is a small-caliber polyester blood vessel prepared by a weaving method or an electrostatic spinning method; the small-caliber artificial blood vessel in the step (1) is a small-caliber expanded polytetrafluoroethylene blood vessel prepared by an expansion method or a small-caliber polytetrafluoroethylene blood vessel prepared by an electrostatic spinning method.

7. The production method according to claim 4, wherein the shaking reaction in the step (1) is a shaking reaction at 30 ℃ for 1d, and the number of coating reactions is 1 to 5.

8. The method according to claim 4, wherein the polyethyleneimine in the step (2) has a molecular weight of 10000; the polyethyleneimine solution is a polyethyleneimine water solution, and the concentration is 1-6 g/L; the reaction is carried out for 1-6h at 60 ℃.

9. The method according to claim 4, wherein the pH of the EDC/NHS-containing heparin solution in the step (3) is 5-6, and the concentration of the heparin solution is 1 g/L; the molar ratio of EDC to NHS is 0.4-6.

10. The preparation method according to claim 4, wherein the reaction in the step (3) is carried out at 37 ℃ for 12 to 24 hours in the absence of light.

11. A small-caliber polyurethane artificial blood vessel with a surface grafted with a heparin coating, a small-caliber polyester artificial blood vessel with a surface grafted with a heparin coating and a small-caliber polytetrafluoroethylene artificial blood vessel with a surface grafted with a heparin coating, which are prepared by the method of claim 4.

12. The use of the small-caliber polyurethane artificial blood vessel, the small-caliber polyester artificial blood vessel and the small-caliber polytetrafluoroethylene artificial blood vessel with the surface grafted with the heparin coating according to claim 1.