A hydrophilic anticoagulant coating and its preparation method and application

Abstract

The invention discloses a method for preparing a hydrophilic anticoagulant coating: S1, preparing a bottom solution: taking raw materials PEI and KH560, adding them to deionized water, allowing the raw materials to fully react, and obtaining a bottom solution; S2, preparing an oxidized heparin solution: dissolving an oxidant in deionized water, adjusting the solution pH to 5.5, and then adding heparin sodium, after the raw materials fully react, rotary evaporating the solution and freeze-drying, purifying the freeze-dried powder, and dissolving the obtained oxidized heparin powder in deionized water to obtain an oxidized heparin solution; S3, applying an anticoagulant coating: allowing the medical material to be treated to fully contact the bottom solution and wash; taking the oxidized heparin solution, allowing the medical material to contact the oxidized heparin solution for 1-3h, adding a reducing agent to the oxidized heparin solution, fully contacting for 8-18h, washing, and drying to obtain a hydrophilic anticoagulant coating. The hydrophilic anticoagulant coating of the present invention significantly improves the anticoagulant performance of the PVC pipeline.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and in particular to a hydrophilic anticoagulant coating and a preparation method and application thereof. Background Art

[0002] In the medical field, medical polymer materials and medical devices are mainly used for diagnosis and treatment. Every day, thousands of patients use blood-contact devices, such as hemodialysis circulation lines, central venous catheters, heart stents, vascular grafts, heart valves, etc. When such devices come into direct contact with blood, a series of reactions will occur. Plasma proteins will first be instantly adsorbed on the surface of the material to form plasma proteins, and then platelets will adhere and aggregate on the surface of plasma proteins, thereby activating platelets, coagulation cascades and complement, and ultimately leading to thrombosis. According to statistics from the U.S. Food and Drug Administration (FDA), the market sales of such medical devices in the United States in 2016 were approximately US$9 billion, but the mortality rate caused by thrombosis during the use of heart stents was as high as 80%, which seriously threatened human health and life safety. Therefore, improving the biocompatibility of blood-contact devices, especially blood compatibility, is the key to solving this problem.

[0003] At present, the existing technical solutions to solve this technical problem are:

[0004] 1. Inject anticoagulants during treatment to inhibit thrombosis on the device surface.

[0005] 2. Surface coating technology, which includes coating or grafting another blood-compatible material on the surface of the material, can be roughly divided into three categories according to its principle:

[0006] A. Constructing inert surfaces of biomaterials: Since many components in the blood (such as hemoglobin, platelets, some plasma proteins, etc.) are electronegative in the blood environment, and the inner wall of the blood vessel is also electronegative, the electrostatic repulsion can hinder the adsorption of substances such as plasma proteins and platelets, which is beneficial to anticoagulation. The hydrophilic anionic polymer is covalently bonded to the surface of the material to improve the hydrophilicity of the material surface. By constructing a biologically inert layer on the surface of the material, the interfacial free energy can be reduced, and the interaction between the material surface and plasma proteins and blood cells can be weakened, thereby maintaining the normal conformation and morphology of plasma proteins and blood cells, so that the material shows good anticoagulation performance. There are currently two main construction methods: ① Constructing hydrophilic brushes or hydrogels such as polyethylene glycol (PEG) or zwitterionic polymers to inhibit the adsorption of proteins, platelets, etc. ② Constructing a slip surface to inhibit water or blood wetting and thus hinder the adsorption of proteins, platelets, etc. on the surface of the material / device.

[0007] B. Biomimetic material surface: There are three main technologies for simulating the functions of biological membranes: modifying the material surface with phosphorylcholine, amphiphilic molecules or liposomes; simulating the structure and morphology of biological membranes by connecting long flexible chains or natural phospholipids on the material surface; simulating biological membrane functions such as transport, catalysis and molecular recognition. These coatings increase the charge polarity and hydrophilicity of the biomaterial surface and reduce the surface energy of the biomaterial by attracting water molecules. The polar groups on the side chains of long-chain molecules can aggregate water molecules and form a water molecule layer on the contact surface between the material and the blood to organize the adsorption of proteins and platelets. Such coatings mainly include phosphorylcholine coatings, glycosaminoglycan coatings, etc.

[0008] C. Bioactive coating: Bioactive substances such as heparin, nitric oxide (NO), anticoagulant drugs, endothelial cell adhesion substances, plasminogen adsorbents, etc. are introduced on the surface of polymer materials to form bioactive coatings to prevent coagulation and thrombosis. These active substances can selectively adsorb or bind to specific biomacromolecules to interact with blood and reduce the generation of thrombin. Currently, the most common bioactive coatings are: heparin sodium salt coating, anticoagulant drug coating and nitric oxide coating.

[0009] However, the current anticoagulation treatment methods of these technologies have the following disadvantages: 1) Systemic heparinization may destroy the body's coagulation regulation ability, leading to complications such as spontaneous bleeding, coagulation difficulties and hypersensitivity reactions. In addition, the dosage of systemic heparinization is difficult to control. Low-dose heparin cannot achieve the anticoagulant effect, and high-dose anticoagulants will cause certain side effects, such as high-dose heparin can cause thrombocytopenia (HIT), bleeding, pulmonary embolism and thrombophlebitis. 2) Some coatings involve complex synthesis processes, which are costly. Some toxic reagents are used in the synthesis process, which easily lead to toxic substances. 3) The anticoagulant coating of the existing technology needs to pre-treat the surface of the interventional medical device before applying heparin, such as pre-treatment with a strong alkali or plasma, so that the anticoagulant coating substrate can be stably coated on the surface of the device. However, this may cause certain damage to the surface, further increasing the risk of coagulation. 4) The traditional heparin modification method is to modify multiple sites of the molecular chain such as the carboxyl group and sulfonic acid group of heparin sodium, which easily leads to the inactivation of heparin and reduces the anticoagulant properties of heparin. 5) At present, most coatings on the market are coated by immersion, which will lead to uneven coating. Summary of the invention

[0010] One of the purposes of the present invention is to provide a method for preparing a hydrophilic anticoagulant coating in view of the above problems, comprising the following steps:

[0011] S1. Prepare the bottom solution: take raw materials PEI and KH560, add them into deionized water, the concentration of PEI is 0.001-0.02 g / mL, the concentration of KH560 is 0.01-0.05 g / mL, stir to dissolve the raw materials, adjust the pH value to 8.5, stir to make the raw materials fully react, and obtain the bottom solution;

[0012] S2, preparing an oxidized heparin solution: dissolving an oxidant in deionized water, adjusting the solution pH to 5.5, then adding heparin sodium, wherein the concentration of heparin sodium in the solution is 0.15-0.2 g / mL, and the concentration of the oxidant is 0.005-0.05 g / mL, reacting in the dark at room temperature for 10-24 h until the raw materials are fully reacted, adding terminator ethylene glycol to the reaction solution to terminate the reaction, then rotary evaporating the solution and freeze-drying, purifying the freeze-dried powder with an organic solvent aqueous solution to obtain purified oxidized heparin powder, dissolving the obtained oxidized heparin powder in deionized water to obtain an oxidized heparin solution with a concentration of 0.08-1 g / mL; the oxidant is selected from sodium periodate, hydrogen peroxide, and peracetic acid, and the organic solvent is selected from ethanol, acetone, and methanol;

[0013] S3, applying an anticoagulant coating: taking the bottom solution prepared in step S1, allowing the medical material to be treated to be in full contact with the bottom solution for 3-6 hours, and then washing it with ultrapure water; taking the oxidized heparin solution prepared in step S2, allowing the medical material treated with the bottom solution to be in full contact with the oxidized heparin solution for 1-3 hours, adding a reducing agent to the oxidized heparin solution, allowing the medical material to continue to be in full contact with the oxidized heparin solution for 8-18 hours, and after the reaction is completed, washing it with ultrapure water and drying it, so as to prepare a hydrophilic anticoagulant coating on the surface of the medical material; the reducing agent is selected from sodium borohydride, ascorbic acid, and sodium cyanoborohydride; the concentration of the reducing agent in the oxidized heparin solution is 0.005-0.01 g / mL.

[0014] Preferably, the reaction in step S1 is carried out at 25-70° C. and stirred for 2-6 hours; the reaction in step S2 is carried out at room temperature in the dark for 10-24 hours, and the concentration of the oxidized heparin solution in step 2) is 0.08-0.5 g / mL or 0.09-0.3 g / mL or 0.09-0.2 g / mL or 0.09-0.15 g / mL.

[0015] Preferably, in steps S1 and S2, 5-10% sodium hydroxide solution is used to adjust the pH value.

[0016] Preferably, in step S2, the volume ratio of the terminator to the reaction solution is 1:16-24, and the volume ratio of the organic solvent aqueous solution to the reaction solution is 3-7:1;

[0017] The rotary evaporation temperature is 30-70°C, the rotary evaporation time is 3-5 hours, and the rotary evaporation is followed by freeze drying for 20-28 hours. The freeze-dried powder is then added to an organic solvent to precipitate the oxidized heparin and then centrifuged. The supernatant is discarded and freeze-dried to obtain purified oxidized heparin powder.

[0018] The preferred organic solvent is ethanol, preferably an ethanol aqueous solution with a concentration of 60-85%.

[0019] Preferably, in step S3, the bottom solution and the oxidized heparin solution circulate and flow in contact on the surface of the medical material to be treated.

[0020] Preferably, the medical material is a pipeline. S3, coating an anticoagulant coating: take the bottom solution prepared in step S1, pass it into the pipeline to be coated, let the bottom solution circulate in the pipeline for 3-6 hours, pass ultrapure water for washing 1-3 times after the reaction is completed, and then pass oxidized heparin solution into the pipeline. After the oxidized heparin solution circulates for 1-3 hours, a reducing agent is added to continue the reaction for 8-18 hours. After the reaction is completed, pass ultrapure water for washing 1-3 times, and then dry the pipeline at 45-55°C for 3-8 hours. The inner surface of the pipeline is coated with a hydrophilic anticoagulant coating.

[0021] Preferably, the material of the medical material is selected from acrylonitrile-butadiene-styrene copolymer, polypropylene, polyvinyl chloride (PVC), and polyethylene.

[0022] Another object of the present invention is to provide a hydrophilic anticoagulant coating prepared by any of the preparation methods described above.

[0023] The last object of the present invention is to provide the use of the above-mentioned hydrophilic anticoagulant coating in the preparation of blood-contacting medical devices.

[0024] In the application technology scheme, the blood-contact medical devices include hemodialysis circulation circuits, central venous catheters, heart stents, vascular grafts, and heart valves.

[0025] The beneficial effects of the present invention are as follows: the hydrophilic anticoagulant coating of the present invention is a membrane structure composed of two layers, the bottom layer is silane with amino groups, the top layer is a heparin anticoagulant layer with aldehyde groups, and the top layer and the bottom layer are covalently bonded through aldehyde-amine condensation. Compared with the prior art, the anticoagulant coating of the present invention has the following advantages:

[0026] 1. Systemic heparinization may destroy the body's coagulation regulation ability, leading to complications such as spontaneous bleeding, coagulation difficulties and hypersensitivity reactions. In addition, the dosage of systemic heparinization is difficult to control. Low-dose heparin cannot achieve the anticoagulant effect, and high-dose anticoagulants can cause certain side effects, such as high-dose heparin can cause thrombocytopenia (HIT), bleeding, pulmonary embolism and thrombophlebitis. The present invention adopts a heparin coating method to avoid the hidden dangers of systemic heparinization, and can effectively reduce the amount of heparin used and reduce costs.

[0027] 2. Some coatings involve complex synthesis processes, which are costly and use some toxic reagents in the synthesis process, which easily lead to toxic residues. The present invention requires fewer types of materials, is simple to synthesize, does not contain any toxic or harmful substances, and is safer.

[0028] 3. The anticoagulant coating of the prior art needs to pre-treat the surface of the interventional medical device before applying heparin, such as using a strong alkali or plasma pre-treatment, so that the anticoagulant coating base can be stably coated on the device surface. However, this may cause certain damage to the surface, further increasing the risk of coagulation. The present invention does not require surface pre-treatment, and the base material selected for the anticoagulant coating of the present invention has good adhesion and does not require surface pre-treatment.

[0029] 4. The traditional heparin modification method is to modify multiple sites of the molecular chain of sodium heparin, such as the carboxyl group and sulfonic acid group, which easily leads to the inactivation of heparin and reduces the anticoagulant properties of heparin. The present invention, by oxidizing the non-reducing end group, makes the heparin molecule have an aldehyde group, and reacts with the amino group in the amino compound with reactive groups in the bottom layer, so as to stably fix the heparin molecule on the surface of various coated substrates, avoid the problem that the carboxyl active group of heparin directly reacts with the amino group, thereby reducing the anticoagulant activity of heparin, and fully ensures the anticoagulant properties of the grafted heparin after modification.

[0030] 5. Currently, most coatings on the market are applied by immersion, which will result in uneven coating. Coating by solution flow method will result in more uniform coating. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The water contact angle data of the hydrophilic anticoagulation coatings obtained in Examples 1-3 and Comparative Examples 1-3.

[0032] Figure 2 The water contact angle data of each layer during the coating process of the hydrophilic anticoagulation coating obtained in Example 1 are shown.

[0033] Figure 3 Cytotoxicity test results of the hydrophilic anticoagulant coating material obtained in Example 1.

[0034] Figure 4Blood compatibility results of the hydrophilic anticoagulant coating material obtained in Example 1. DETAILED DESCRIPTION

[0035] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0036] The experimental methods in the following examples are all conventional methods unless otherwise specified; the materials and reagents used are all conventional materials and reagents in the art and are commercially available unless otherwise specified.

[0037] The main reagents are as follows:

[0038] 3-Aminopropyltrimethoxysilane (γ-APS): CAS No.: 13822-56-5;

[0039] 1,2-Bis(triethoxysilyl)ethane (BTSE): CAS No.: 16068-37-4;

[0040] Polyethyleneimine (PEI): CAS No.: 9002-98-6;

[0041] γ-Glycidyl ether propyl trimethoxysilane (KH560): CAS No.: 2530-83-8;

[0042] γ-Aminopropyltrimethoxysilane (KH540): CAS No.: 13822-56-5;

[0043] Heparin sodium: CAS number: 9041-08-1.

[0044] The anticoagulation coating of Table 1 was prepared according to the method of the present invention:

[0045] Table 1

[0046]

[0047] Example 1: Preparation of the hydrophilic anticoagulant coating of the present invention

[0048] (1) Preparation of bottom solution: Weigh 1 g PEI, 1 g KH560 and 50 mL deionized water, add into a 250 mL three-necked flask, add 10% sodium hydroxide solution to adjust the pH to 8.5. Stir in a 65°C water bath. React for 2 h to obtain KH560 / PEI solution (Solution 1).

[0049] The epoxy group in KH560 reacts with the amino group of PEI after ring opening to generate siloxane with amino groups. 3Hydrolysis will occur in aqueous solution to form silicon ions, and the hydroxyl group (-OH) in the medical material pipeline PVC will react with the silicon ions to form a Si-O bond, allowing the amino-containing siloxane to successfully adhere to the surface of the pipeline.

[0050] (2) Preparation of anticoagulant layer solution: 5 g of sodium periodate was dissolved in 100 mL of deionized water, and the pH was adjusted to 5.5 with 5% sodium hydroxide solution, and then 20 g of sodium heparin was added while stirring. The reaction was carried out at room temperature in the dark for 10 h. After the reaction was completed, 5 mL of ethylene glycol was added to terminate the reaction. Then, the aqueous solution was evaporated by rotary evaporation at 30°C for 4 h, and then lyophilized at 30°C for 24 h. 500 mL of ethanol aqueous solution (i.e., 80% alcohol) was prepared in a volume ratio of water: anhydrous ethanol = 1:4, and the powder obtained after lyophilization was purified: the powder obtained after lyophilization was added to 80% alcohol to precipitate the oxidized heparin, and then centrifuged. The supernatant was discarded and freeze-dried to obtain purified oxidized heparin powder. The purified oxidized heparin powder was dissolved in deionized water to prepare an oxidized heparin coating solution (Solution 2) with a concentration of 0.1 g / mL.

[0051] (3) Tube wall coating: After the medical PVC tube is ultrasonically cleaned with purified water and air-dried, a peristaltic pump is used to circulate solution 1 in the PVC tube at room temperature for 6 hours. Then, the inner wall of the PVC tube is rinsed with pure water once, and solution 2 is driven to circulate in the PVC tube for 1 hour at room temperature, so that the amino group in the reagent reacts with the aldehyde group to form an N=C double bond; then 0.5g of reducing agent sodium borohydride is dissolved in 1ml of deionized water to form a solution and added to the circulating solution 2, and the cyclic reaction is continued for 12 hours, wherein the reducing agent reduces the N=C double bond to a NC single bond. After rinsing with purified water three times, it is placed in a 50°C oven for cross-linking, drying and fixing for 5 hours to obtain an anticoagulant coating.

[0052] Example 2: Preparation of the hydrophilic anticoagulant coating of the present invention

[0053] (1) Weigh 0.05 g PEI, 2.5 g KH560 and 50 mL deionized water, add into a 250 mL three-necked flask, add 10% sodium hydroxide solution, adjust the pH to 8.5. Stir in a 65°C water bath. React for 2 h to obtain KH560 / PEI solution (Solution 1);

[0054] (2) Take 0.5g of hydrogen peroxide and dissolve it in 100mL of deionized water. Adjust the pH to 5.5 with 5% sodium hydroxide solution, and add 15g of sodium heparin while stirring. React at room temperature in the dark for 10 hours. After the reaction is completed, add 5mL of ethylene glycol to terminate the reaction. Rotary evaporate at 30°C and freeze-dry. Prepare 500mL of solution in a volume ratio of water: ethanol = 1:4, and purify the powder obtained after freeze-drying. After discarding the liquid, freeze-dry the sample to obtain oxidized heparin powder. Dissolve the oxidized heparin powder in deionized water to prepare an oxidized heparin coating solution with a concentration of 0.1g / mL (Solution 2);

[0055] (3) After the medical PVC pipe was ultrasonically cleaned with purified water and air-dried, a peristaltic pump was used to circulate solution 1 in the PVC pipe for 6 hours. Then, the inner wall of the PVC pipe was rinsed with pure water once, and solution 2 was circulated in the PVC pipe for 1 hour with a peristaltic pump. 0.8 g of sodium borohydride was added and reacted for 12 hours. After being rinsed with purified water three times, the pipe was placed in a 50°C oven for cross-linking, drying and fixing for 5 hours to obtain an anticoagulant coating.

[0056] Example 3: Preparation of the hydrophilic anticoagulant coating of the present invention

[0057] (1) Weigh 0.3 g PEI, 0.5 g KH560 and 50 mL deionized water, add into a 250 mL three-necked flask, add 10% sodium hydroxide solution, adjust the pH to 8.5. Stir in a 65°C water bath. React for 2 h to obtain KH560 / PEI solution (Solution 1);

[0058] (2) Take 3g of peracetic acid and dissolve it in 100mL of deionized water. Adjust the pH to 5.5 with 5% sodium hydroxide solution, and add 15g of sodium heparin while stirring. React at room temperature in the dark for 10 hours. After the reaction, add 5mL of ethylene glycol to terminate the reaction. Rotary evaporate at 30°C and freeze-dry. Prepare 500mL of solution in a volume ratio of water: ethanol = 1:4, and purify the powder obtained after freeze-drying. After discarding the liquid, freeze-dry the sample to obtain oxidized heparin powder. Dissolve the oxidized heparin powder in deionized water to prepare an oxidized heparin coating solution (solution 2) with a concentration of 0.1g / mL;

[0059] (3) After the medical PVC pipe was ultrasonically cleaned with purified water and air-dried, a peristaltic pump was used to circulate solution 1 in the PVC pipe for 6 hours. Then, the inner wall of the PVC pipe was rinsed with pure water once, and solution 2 was circulated in the PVC pipe for 1 hour with a peristaltic pump. Then, 1 g of ascorbic acid was added and reacted for 12 hours. After being rinsed with purified water three times, the pipe was placed in a 50°C oven for cross-linking, drying and fixing for 5 hours to obtain an anticoagulant coating.

[0060] Comparative Example 1

[0061] (1) Weigh 1 g of γ-APS and dissolve it in 50 mL of deionized water. Stir for 1 h to fully hydrolyze the γ-APS hydrolyzed solution (Solution 1).

[0062] (2) Take 5g of sodium periodate and dissolve it in 100mL of deionized water. Adjust the pH to 5.5 with 5% sodium hydroxide solution, and add 20g of sodium heparin while stirring. React at room temperature in the dark for 10 hours. After the reaction is completed, add 5mL of ethylene glycol to terminate the reaction. Rotary evaporate at 30°C and freeze-dry. Prepare 500mL of solution in a volume ratio of water: ethanol = 1:4, and purify the powder obtained after freeze-drying. After discarding the liquid, freeze-dry the sample to obtain oxidized heparin powder. Dissolve the oxidized heparin powder in deionized water to prepare an oxidized heparin coating solution (solution 2) with a concentration of 0.1g / mL;

[0063] (3) After the medical PVC pipe was ultrasonically cleaned with purified water and air-dried, a peristaltic pump was used to circulate solution 1 in the PVC pipe for 6 hours. Then, the inner wall of the PVC pipe was rinsed with pure water once, and solution 2 was circulated in the PVC pipe for 1 hour with a peristaltic pump. 0.5 g of sodium cyanoborohydride was added and reacted for 12 hours. After being rinsed with purified water three times, the pipe was placed in a 50°C oven for cross-linking, drying and fixing for 5 hours to obtain an anticoagulant coating.

[0064] Comparative Example 2

[0065] (1) Weigh 1 g of γ-APS and 0.5 g of BTSE and dissolve them in 50 mL of deionized water. Stir and react for 1 h to obtain a γ-APS / BTSE solution (solution 1).

[0066] (2) Take 5g of sodium periodate and dissolve it in 100mL of deionized water. Adjust the pH to 5.5 with 5% sodium hydroxide solution, and add 20g of sodium heparin while stirring. React at room temperature in the dark for 10 hours. After the reaction is completed, add 5mL of ethylene glycol to terminate the reaction. Rotary evaporate at 30°C and freeze-dry. Prepare 500mL of solution in a volume ratio of water: ethanol = 1:4, and purify the powder obtained after freeze-drying. After discarding the liquid, freeze-dry the sample to obtain oxidized heparin powder. Dissolve the oxidized heparin powder in deionized water to prepare an oxidized heparin coating solution (solution 2) with a concentration of 0.1g / mL;

[0067] (3) After the medical PVC pipe was ultrasonically cleaned with purified water and air-dried, a peristaltic pump was used to circulate solution 1 in the PVC pipe for 6 hours. Then, the inner wall of the PVC pipe was rinsed with pure water once, and solution 2 was circulated in the PVC pipe for 1 hour with a peristaltic pump. 0.5 g of sodium cyanoborohydride was added and reacted for 12 hours. After being rinsed with purified water three times, the pipe was placed in a 50°C oven for cross-linking, drying and fixing for 5 hours to obtain an anticoagulant coating.

[0068] Comparative Example 3

[0069] Since KH560 does not have an amino group in its structure and cannot directly bind to heparin, KH540, which has a similar structure to KH560, is used in this comparative example to prepare the bottom layer solution. KH540 has an amino group in its structure and can bind to heparin.

[0070] (1) Weigh 1 g of KH540 and dissolve it in 50 mL of deionized water. Stir thoroughly for 1 h to obtain a KH540 solution (solution 1);

[0071] (2) Take 5g of sodium periodate and dissolve it in 100mL of deionized water. Adjust the pH to 5.5 with 5% sodium hydroxide solution, and add 20g of sodium heparin while stirring. React at room temperature in the dark for 10 hours. After the reaction is completed, add 5mL of ethylene glycol to terminate the reaction. Rotary evaporate at 30°C and freeze-dry. Prepare 500mL of solution in a volume ratio of water: ethanol = 1:4, and purify the powder obtained after freeze-drying. After discarding the liquid, freeze-dry the sample to obtain oxidized heparin powder. Dissolve the oxidized heparin powder in deionized water to prepare an oxidized heparin coating solution (solution 2) with a concentration of 0.1g / mL;

[0072] (3) After the medical PVC pipe was ultrasonically cleaned with purified water and air-dried, a peristaltic pump was used to circulate solution 1 in the PVC pipe for 6 hours. Then, the inner wall of the PVC pipe was rinsed with pure water once, and solution 2 was circulated in the PVC pipe for 1 hour with a peristaltic pump. 0.5 g of ascorbic acid was added and reacted for 12 hours. After being rinsed with purified water three times, the pipe was placed in a 50°C oven for cross-linking, drying and fixing for 5 hours to obtain an anticoagulant coating.

[0073] Example 4 Performance Test

[0074] 1. Hydrophilicity test

[0075] The hydrophilic and hydrophobic properties of the blank PVC pipe wall, the pipe wall coated with the anticoagulation coating obtained in Examples 1-3, and Comparative Examples 1-3 were analyzed using an interfacial tension meter (DSA25). The coated pipe was cut open longitudinally, and the contact angle was tested using an interfacial tension meter (DSA25) and the data was recorded.

[0076] Test results( Figure 1 ) showed that after coating the hydrophilic anticoagulation coating obtained in Examples 1-3, the contact angle of the material surface was reduced the most, which significantly improved the hydrophilicity of the PVC pipe, while the hydrophilicity improvement effect of Comparative Examples 1-3 was significantly inferior to that of Examples 1-3.

[0077] 2. Hydrophilic and hydrophobic properties test of the tube wall before and after coating with solution 1 and solution 2

[0078] The hydrophilic and hydrophobic properties of the PVC pipe wall before and after coating with solution 1 and solution 2 were tested, and the effects of solution 1 and solution 2 on the hydrophilic and hydrophobic properties of the pipe wall were analyzed.

[0079] The hydrophilic and hydrophobic properties of the blank PVC tube wall, the tube wall after coating with solution 1 in step (3) of Example 1, and the tube wall after coating with solution 2 at the end of step (3) of Example 1 were analyzed using an interfacial tension meter (DSA25).

[0080] Test results( Figure 2 ) showed that compared with the blank PVC pipe wall, the contact angle of the material surface after coating with solution 1 was significantly reduced, significantly improving the hydrophilicity of the PVC pipe; and after coating with solution 2, the contact angle of the material surface was further significantly reduced, further significantly improving the hydrophilicity of the PVC pipe.

[0081] 3. Cytotoxicity Experiment

[0082] Mouse fibroblast L929 cells were used to test the cytotoxicity of the hydrophilic anticoagulant coating materials obtained in Examples 1-3.

[0083] Preparation of extracts for tubing with hydrophilic anticoagulant coating: After sterilization, the tubing samples treated in Examples 1-3 were immersed in a serum-containing cell culture medium at an extraction ratio of 0.2 g / mL. The extraction container was a sterile, chemically inert sealed glass test tube. The extraction temperature was (37±1)°C and the extraction time was (24±2) h. Extracts of different concentrations (25%, 50%, 75%, 100%) were prepared.

[0084] The L929 cells that are growing vigorously were prepared with MEM medium at a concentration of 1*10 5 / mL cell suspension, and then plated in a 96-well plate (100μL / well). After the cells have grown all over the well plate, 100μL of cell culture medium is added to the control group, and 100μL of different concentrations of extracts are added to the experimental group in turn. After 24h of culture, the original culture medium and extract are discarded, and MEM culture medium containing 10% MTT reagent is added for staining, 100μL per well, placed in an incubator for 4h, all culture medium is discarded, and 150μL of DMSO solution is added to each well, and oscillated in the dark at room temperature for 10min to fully dissolve the purple crystals. The OD value of each well is measured at a wavelength of 490nm using an enzyme marker, and the cell proliferation rate of each group is calculated:

[0085] P% = (OD value of experimental group / OD value of negative control group) * 100

[0086] The test results show that the cell survival rate of the hydrophilic anticoagulant coating materials obtained in Examples 1-3 gradually decreases with the increase of the concentration of the extract, but the survival rate is greater than 75%, indicating that the hydrophilic anticoagulant coating materials obtained in Examples 1-3 have no cytotoxicity. Figure 3 This is a cytotoxicity experiment of the extract of the pipeline after treatment in exemplary embodiment 1.

[0087] 4. Hemolytic performance

[0088] Fill the tubing coated with the hydrophilic anticoagulant coating of the present invention with normal saline, extract for 24 hours under irradiation conditions, and prepare an extract. Move 200 μL of red blood cell suspension (2%) to a test tube and add 200 μL of extract. The group with 100 μL Triton X-100 (10%) added was used as a positive control, and the group with fresh normal saline solution added was used as a negative control. The samples were incubated at 37°C for 3 hours. Finally, the absorbance of the supernatant at 540 nm was measured using an RT 6000 microplate reader. The hemolysis rate of each sample was calculated by the following formula:

[0089] Hemolysis rate (%) = (sample OD value - negative control OD value) / (positive control OD value - negative control OD value) * 100

[0090] The experimental results show that the hemolysis rate of the hydrophilic anticoagulant coating material prepared in Example 1 is 1.8%, which has good hemolysis performance.

[0091] 5. Anticoagulant performance

[0092] The whole blood coagulation time of the tubing coated with the anticoagulant coating obtained in Examples 1-3 and Comparative Examples 1-3 was measured with reference to the whole blood coagulation time determination part of Heparin Bioassay Method in Part IV, 1208 of the 2020 edition of the Chinese Pharmacopoeia.

[0093] Specifically, 4.5 mL of fresh rabbit blood was collected into a test tube containing 0.5 mL of citric acid (109 mmol / L). The blood was centrifuged at 1500 r / min for 15 min, and the plasma was aspirated for later use. The activator in the APTT test kit was added to the pre-warmed plasma containing the sample block and incubated at 37°C for 5 min. Then, pre-warmed CaCl 2 Reagents are used to measure the coagulation time using a coagulation analyzer.

[0094] The whole blood coagulation time of the blank PVC pipe and the pipe coated with the anticoagulant coating obtained in Example 1-3 and Comparative Example 1-3 are 4min, 93min, 88min, 84min, 54min, 76min and 61min respectively. The hydrophilic anticoagulant coating obtained in Example 1-3 significantly improves the anticoagulant performance of the PVC pipe.

Claims

1. A method for preparing a hydrophilic anticoagulant coating, It is characterized in that The steps include: S1. Prepare the bottom solution: take raw materials PEI and KH560, add them into deionized water, the concentration of PEI is 0.001-0.02 g / mL, the concentration of KH560 is 0.01-0.05 g / mL, stir to dissolve the raw materials, adjust the pH value to 7.0-10.0, stir to make the raw materials fully react, and obtain the bottom solution; the reaction is carried out at 25-70°C; S2, preparing an oxidized heparin solution: dissolving an oxidant in deionized water, adjusting the solution pH to 5.0-6.8, then adding heparin sodium, wherein the concentration of heparin sodium in the solution is 0.15-0.2 g / mL, and the concentration of the oxidant is 0.005-0.05 g / mL, reacting in the dark at room temperature for 10-24 hours until the raw materials are fully reacted, adding terminator ethylene glycol to the reaction solution to terminate the reaction, then rotary evaporating the solution and freeze-drying, purifying the freeze-dried powder with an organic solvent aqueous solution to obtain purified oxidized heparin powder, dissolving the obtained oxidized heparin powder in deionized water to obtain an oxidized heparin solution with a concentration of 0.08-1 g / mL; the oxidant is selected from sodium periodate, hydrogen peroxide, and peracetic acid, and the organic solvent is selected from ethanol, acetone, and methanol; S3, coating an anticoagulant coating: taking the bottom solution prepared in step S1, allowing the medical material to be treated to be in full contact with the bottom solution for 3-6 hours, and then washing it with ultrapure water; taking the oxidized heparin solution prepared in step S2, allowing the medical material treated with the bottom solution to be in full contact with the oxidized heparin solution for 1-3 hours, adding a reducing agent to the oxidized heparin solution, allowing the medical material to continue to be in full contact with the oxidized heparin solution for 8-18 hours, and after the reaction is completed, washing it with ultrapure water and drying it, so as to prepare a hydrophilic anticoagulant coating on the surface of the medical material; the reducing agent is selected from sodium borohydride, ascorbic acid, and sodium cyanoborohydride; the concentration of the reducing agent in the oxidized heparin solution is 0.005-0.01 g / mL; the material of the medical material is selected from acrylonitrile-butadiene-styrene copolymer, polypropylene, polyvinyl chloride, and polyethylene.

2. The preparation method according to claim 1, Features: In the step S1, the reaction is stirred for 2-6 hours; in the step S2, the reaction is carried out at room temperature in the dark for 10-24 hours, and the concentration of the oxidized heparin solution in step 2) is 0.08-0.5 g / mL.

3. The preparation method according to claim 1, Features: In the steps S1 and S2, 5-10% sodium hydroxide solution is used to adjust the pH value.

4. The preparation method according to claim 1, Features: In step S2, the volume ratio of the terminator to the reaction solution is 1:16-24, and the volume ratio of the organic solvent aqueous solution to the reaction solution is 3-7:1; The rotary evaporation temperature is 30-70°C, the rotary evaporation time is 3-5 h, and the rotary evaporation is followed by freeze drying for 20-28 h. The freeze-dried powder is then added to an organic solvent to precipitate the oxidized heparin and centrifuged. The supernatant is discarded and freeze-dried to obtain purified oxidized heparin powder.

5. The preparation method according to claim 1, Features: The organic solvent is ethanol, which is an ethanol aqueous solution with a concentration of 60-85%.

6. The preparation method according to claim 1, Features: In step S3, the bottom solution and the oxidized heparin solution circulate and flow in contact with the surface of the medical material to be treated.

7. The preparation method according to claim 1, Features: The medical material is a pipeline. S3, coating an anticoagulant coating: taking the bottom solution prepared in step S1, passing it into the pipeline to be coated, allowing the bottom solution to circulate in the pipeline for 3-6 hours, passing ultrapure water for washing 1-3 times after the reaction is completed, and then passing an oxidized heparin solution into the pipeline. After the oxidized heparin solution circulates for 1-3 hours, a reducing agent is added to continue the reaction for 8-18 hours. After the reaction is completed, passing ultrapure water for washing 1-3 times, and then drying the pipeline at 45-55° C. for 3-8 hours, and the inner surface of the pipeline is coated with a hydrophilic anticoagulant coating.

8. The hydrophilic anticoagulant coating prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the hydrophilic anticoagulant coating according to claim 8 in the preparation of blood-contact medical devices.

10. The use according to claim 9, Features: The blood-contact medical devices include hemodialysis circulation circuits, central venous catheters, heart stents, vascular grafts, and heart valves.

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