Method for preparing article having anticoagulant coating and article

Abstract

The invention discloses a method for preparing a product with an anticoagulant coating and the product. The method comprises: providing a substrate material of the article, bonding an active ester, comprising contacting the amino-functionalized target surface with a first organic solution comprising an active ester and a first organic base catalyst, the amino-functionalized target surface is subjected to a reaction with part of ester groups in the active ester under the catalysis of a first organic base catalyst to generate covalent bonds, the active ester is a polymer, the weight-average molecular weight of the active ester is 1000-250000, and the active ester comprises a structural unit represented by a formula (1); a step of bonding polyethyleneimine; and a heparin bonding step.

Description

Technical Field

[0001] The present application belongs to the technical field of implantable biomaterials, and in particular relates to a method and a product for preparing a product with an anticoagulant coating. Background Art

[0002] Anticoagulant biomaterials are an important biocompatible material, which are widely used in medical materials that come into contact with human blood and tissues, such as hemodialysis materials, in vitro and in vivo circulation materials (artificial blood vessels, vascular stents and blood circulation system catheters), interventional heart valves, central venous catheters, membrane oxygenators, etc. In order to improve the anticoagulability of the material, the surface of the base material needs to be modified. There are many purposes for surface modification, such as improving the hydrophilicity of the surface, making the surface negatively charged, and grafting zwitterionic polymers on the surface. It can also be modified to have good anticoagulant function and blood compatibility.

[0003] Heparin is a natural anticoagulant drug and is usually used to prepare biomaterials with anticoagulant properties. The heparinized coating formed on the surface of the base material can prevent platelet adhesion and reduce its activity, thereby improving the blood compatibility of the material. Heparin is generally attached to the surface of the material by physical coating or chemical grafting. The use of physical coating to fix heparin on the surface of the material has the disadvantages of poor stability and easy detachment, which greatly limits the use time. Chemical grafting usually uses plasma treatment, covalent bond bonding and other methods to make the material surface contain heparin, but the operation steps are very complicated, and there are defects such as waste of heparin raw materials or low anticoagulant activity rate of heparin after grafting. Summary of the invention

[0004] In view of this, the present application provides a method for preparing a product with an anticoagulant coating and a product with an anticoagulant coating, aiming to make full use of heparin as a raw material and increase the content and anticoagulant activity of heparin in the anticoagulant coating.

[0005] In a first aspect, the present invention provides a method for preparing a product having an anticoagulant coating, comprising:

[0006] providing a substrate material of an article, the substrate material comprising an amino-functionalized target surface;

[0007] The step of bonding active esters comprises contacting the amino-functionalized target surface with a first organic solution containing active esters and a first organic base catalyst, so that the amino groups on the amino-functionalized target surface react with part of the ester groups in the active ester under the catalysis of the first organic base catalyst to form covalent bonds, wherein the active ester is a polymer having a weight average molecular weight of 1000-250000, and the active ester is obtained by polymerizing a monomer as shown in formula (1).

[0008]

[0009] R is selected from unsubstituted or substituted CH3 (CH=CH) k -, (CH2=CH)-, CH2=CH(CH2) m C6H4-, CH3(CH=CH) n Any one of C6H4-, substituents include -OH, -COOH, -COOCH3, C1-C 10 At least one of alkyl, phenyl, -NH2; k, m represent natural numbers of 1-10; optionally, the number of monomers is 2-1000;

[0010] The step of bonding polyethyleneimine comprises contacting the target surface for bonding active ester with a second organic solution containing polyethyleneimine and a second organic base catalyst, and reacting at least part of the amino groups of polyethyleneimine with at least part of the ester groups in the active ester under the catalytic action of the second organic base catalyst to form a covalent bond;

[0011] The heparin bonding step comprises reacting heparin containing hydroxyl groups with the reaction product of the polyethyleneimine bonding step, so that the amino groups in the reaction product originating from the polyethyleneimine react with the hydroxyl groups in the heparin to obtain a product with an anticoagulant coating.

[0012] According to an embodiment of one aspect of the present application, the step of bonding active esters satisfies at least one of the following conditions:

[0013] The first organic solution includes one or more of trifluoroethanol, ethanol, propylene glycol, and toluene;

[0014] The reaction temperature is 20-38°C and the reaction time is 1-48h;

[0015] The concentration of active ester is 10-100 mg / ml.

[0016] According to an embodiment of one aspect of the present application, the active ester comprises a cyclic lactamyl group, and the active ester is obtained by polymerizing any monomer as shown in formula (1).

[0017]

[0018] R is selected from unsubstituted or substituted CH3 (CH=CH) k -, (CH2=CH)-, the substituents include -OH, -COOH, -COOCH3, C1-C 10 At least one of alkyl, phenyl, and -NH2; k, m, and n represent a natural number of 1-10.

[0019] According to an embodiment of one aspect of the present application, the step of bonding polyethyleneimine satisfies at least one of the following conditions:

[0020] The second organic solution includes one or more of trifluoroethanol, ethanol, propylene glycol, and toluene;

[0021] The temperature for forming covalent bonds is 20 to 38°C, and the time is 1 to 48 hours.

[0022] According to an embodiment of one aspect of the present application, the step of bonding heparin includes contacting and reacting the reaction product of the step of bonding polyethyleneimine with heparin containing hydroxyl groups in a biological buffer under the action of a catalyst and a condensing agent.

[0023] According to an embodiment of one aspect of the present application, the biological buffer comprises one or more of 2-(N-morpholino)ethanesulfonic acid solution, sodium chloride solution, and phosphate buffer solution.

[0024] According to an embodiment of one aspect of the present application, the pH of the biological buffer is 4.5-4.8.

[0025] According to an embodiment of one aspect of the present application, the catalyst is N-hydroxysuccinimide sulfonic acid sodium salt; and the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0026] According to an embodiment of one aspect of the present application, the method satisfies at least one of the following conditions:

[0027] The base material includes one or more of polyether block polyamide, polyethylene, polypropylene, and polyurethane;

[0028] The first organic base catalyst and the second organic base catalyst respectively include one or more of triethylamine, dimethylamine, diethylamine, diisopropylethylamine, trimethylamine, Downs base, and pyridine.

[0029] According to an embodiment of one aspect of the present application, the method for preparing the substrate material comprising an amino-functionalized target surface comprises: activating the substrate material with a plasma device, and then placing it in a 3-(trimethoxysilyl)propylamine solution so that the target surface of the substrate material has amino groups.

[0030] According to an embodiment of one aspect of the present application, the surface of the substrate material is placed in an oxygen atmosphere of a plasma device, and the plasma device is operated at a power of 60 to 100 W for 3 to 10 minutes.

[0031] According to an embodiment of one aspect of the present application, in the 3-(trimethoxysilyl)propylamine solution, the volume concentration of 3-(trimethoxysilyl)propylamine is 0.1% to 20%.

[0032] According to an embodiment of one aspect of the present application, the 3-(trimethoxysilyl)propylamine solution includes a solvent, and the solvent includes ethanol.

[0033] According to an embodiment of one aspect of the present application, the solvent also includes water.

[0034] According to an embodiment of one aspect of the present application, the volume concentration of ethanol in the 3-(trimethoxysilyl)propylamine solution is 70%-98%.

[0035] According to an embodiment of one aspect of the present application, the method satisfies at least one of the following conditions:

[0036] The active ester bonding step includes: removing unreacted components with ethanol and deionized water in sequence;

[0037] After the polyethyleneimine bonding step, unreacted components were removed with ethanol and deionized water in sequence.

[0038] After the heparin bonding step, the article with the anticoagulant coating is rinsed with deionized water and dried.

[0039] In a second aspect, an embodiment of the present application provides a product having an anti-coagulation coating, which is prepared by the method of the first aspect.

[0040] According to an embodiment of one aspect of the present application, the product includes at least one of a ventricular assist device and an extracorporeal blood circulation device.

[0041] The method of the present application includes at least the following beneficial effects:

[0042] The method of the present application is to first bond an active ester step and then bond polyethyleneimine before bonding heparin on a substrate material having an amino-functionalized target surface. By adopting this process method, on the one hand, the heparin raw material can be fully utilized and the heparin utilization rate can be improved; and by bonding the active ester step, at least part of the ester groups in the active ester react and form covalent bonds, so that the unit mass of the product with the anticoagulant coating has more heparin, and the anticoagulant activity of the heparin in the product with the anticoagulant coating is also improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solution of the implementation regulations of this application, the following is a brief introduction to the drawings required for use in the embodiments of this application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0044] Figure 1 The color development of the heparin-grafted pebax catheters of the examples and comparative examples of the present application and the raw pebax catheters without heparin grafted thereon are shown. DETAILED DESCRIPTION

[0045] In order to make the application purpose, technical solution and beneficial technical effect of this application clearer, the application is further described in detail below in conjunction with the embodiments. It should be understood that the implementation regulations described in this specification are only for explaining this application, not for limiting this application.

[0046] For simplicity, the present application only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an unclearly recorded range; and any lower limit can be combined with other lower limits to form an unclearly recorded range, and any upper limit can be combined with any other upper limit to form an unclearly recorded range. In addition, although not clearly recorded, each point or single value between the range endpoints is included in the range. Thus, each point or single value can be combined with any other point or single value as its own lower limit or upper limit or with other lower limits or upper limits to form an unclearly recorded range.

[0047] In the description of the present application, it should be noted that, unless otherwise specified, “above” and “below” are inclusive of the number, and “several” in “one or several” means two or more.

[0048] The above application content of the present application is not intended to describe each disclosed embodiment or each implementation in the present application. The following description more specifically illustrates exemplary embodiments. In many places throughout the application, guidance is provided through a series of embodiments, and these implementation regulations can be used in various combinations. In each example, enumeration is only used as a representative group and should not be interpreted as exhaustive.

[0049] Products with anticoagulant coatings are widely used in medical materials that come into contact with human blood and tissues, such as hemodialysis systems, extracorporeal circulation systems, artificial heart valves, pacemakers, artificial blood vessels, vascular stents, surgical lines and catheters, etc. In order to improve the anticoagulant properties of the material, the surface of the base material needs to be modified. There are many methods for modifying the surface of the base material, such as improving the hydrophilicity of the surface, making the surface negatively charged, grafting zwitterionic polymers on the surface, designing microphase separation structures, surface heparinization, and fixing biological macromolecules such as phosphates on the surface, so that it has good anticoagulant function and blood compatibility, which has been proven to effectively reduce the risk of thrombosis on the surface of blood-contact medical devices.

[0050] In the related art, the amino-containing substrate material is reacted with aminopropyltrimethoxysilane (APTMS) under certain conditions, and then the heparin molecules are directly reacted to make the heparin molecules grafted to the substrate material. Studies have found that the effect is poor, which may be due to the steric hindrance effect of heparin during the grafting process, the reaction efficiency is low, and the amount of heparin grafted on the surface of the substrate material is limited, which cannot achieve a good anticoagulant effect.

[0051] In the related technology, the heparin molecule is modified by aldehyde group so that the end of the heparin molecule has an aldehyde group; the aldehyde group is used to react with the amino group on the substrate material to covalently fix the heparin to achieve heparin grafting. Studies have found that the effect is poor, which may be due to the fact that the heparin treated with aldehyde group will also cause a certain loss of the anticoagulant activity of the heparin molecule; the covalent fixation of heparin improves the stability, but will greatly reduce the assembly amount and activity of heparin, thereby limiting its clinical long-term anticoagulant effect to a certain extent.

[0052] In the related art, the base material grafted with heparin molecules is immersed in a polyethyleneimine (PEI) solution to make the heparin react with the PEI molecules. At the same time, a new amino group is formed on the surface, which reacts with the heparin molecules and re-grafts the heparin molecules. The operation of grafting PEI → grafting heparin is repeated 4 times, and then immersed in a glutaraldehyde solution to form a cross-linked microstructure. Although it has a certain effect, the heparin raw material used is more wasted, resulting in an increase in cost, and the operation is cumbersome.

[0053] Based on this, an embodiment of the present application provides a method for preparing a product with an anticoagulant coating, which can graft heparin to the surface of a base material with good stability, and the heparin is not easy to fall off, and has a good anticoagulant effect.

[0054] Method for preparing an article having an anticoagulant coating

[0055] In a first aspect, the present invention provides a method for preparing a product having an anticoagulant coating, comprising:

[0056] providing a substrate material of an article, the substrate material comprising an amino-functionalized target surface;

[0057] The step of bonding active esters comprises contacting the amino-functionalized target surface with a first organic solution containing active esters and a first organic base catalyst, so that the amino groups on the amino-functionalized target surface react with part of the ester groups in the active ester under the catalysis of the first organic base catalyst to form covalent bonds, wherein the active ester is a polymer having a weight average molecular weight of 1000-250000, and the active ester is obtained by polymerizing a monomer as shown in formula (1).

[0058]

[0059] R is selected from unsubstituted or substituted CH3 (CH=CH) k -, (CH2=CH)-, CH2=CH(CH2) m C6H4-, CH3(CH=CH) nAny one of C6H4-, substituents include -OH, -COOH, -COOCH3, C1-C 10 At least one of alkyl, phenyl, -NH2; k, m represent natural numbers of 1-10; optionally, the number of monomers is 2-1000;

[0060] The step of bonding polyethyleneimine comprises contacting the target surface for bonding active ester with a second organic solution containing polyethyleneimine and a second organic base catalyst, and reacting at least part of the amino groups of polyethyleneimine with at least part of the ester groups in the active ester under the catalytic action of the second organic base catalyst to form a covalent bond;

[0061] The heparin bonding step comprises reacting heparin containing hydroxyl groups with the reaction product of the polyethyleneimine bonding step, so that the amino groups in the reaction product originating from the polyethyleneimine react with the hydroxyl groups in the heparin to obtain a product with an anticoagulant coating.

[0062] According to the method of the embodiment of the present application, before bonding heparin, active esters are first grafted onto the target surface of the amino group of the base material, so that the amino groups on the target surface react with the active esters to form covalent bonds; then polyethyleneimine is bonded, so that the amino groups of polyethyleneimine react with at least part of the ester groups in the active esters to form covalent bonds, so that the active esters are fixed to the target surface of the base material through chemical bonds, and then polyethyleneimine is fixed; finally, the unreacted amino groups from polyethyleneimine react with the hydroxyl groups in the heparin to achieve the purpose of fixing heparin on the target surface of the base material. The method of the present application makes full use of the heparin raw materials, improves the utilization rate of heparin, and has more heparin per unit mass in the product with the anticoagulant coating, which improves the anticoagulant activity of heparin in the product with the anticoagulant coating.

[0063] In the examples of the present application, "-C6H4-" means

[0064] In the embodiments of the present application, the above covalent bond can be understood as "-C=ONH-".

[0065] The method of the present application introduces active esters, which have more active sites. The ester groups therein can react with amino groups derived from the base material and polyethyleneimine to generate -C=ONH-, which is conducive to the occurrence of the reaction.

[0066] According to the method of the embodiment of the present application, due to the introduction of active esters, the direct reaction of the hydroxyl groups in heparin with the amino groups of the substrate material in the related art is avoided, thereby avoiding the low reaction efficiency and low heparin grafting amount under such conditions due to the steric effect.

[0067] According to the embodiments, heparin can be broadly understood as a heparin macromolecule, which can be heparin sodium molecules and other heparin, heparin salts and heparin derivatives with coagulation function, which have hydroxyl groups. Heparin is a natural polysaccharide anticoagulant derived from heparin sulfate. It is mainly composed of glucosamine and ketose and has a highly complex polysaccharide structure. It has anticoagulant effects and blood vessel containing effects.

[0068] According to the embodiments of the present application, the first organic base is usually used as a catalyst to promote the reaction.

[0069] The amino groups (amines) on the surface of the substrate material undergo a nucleophilic substitution reaction with some ester groups in the active ester polymer, and the active ester polymer is grafted onto the surface of the substrate material.

[0070] In some optional embodiments, the method satisfies at least one of the following conditions:

[0071] The base material includes one or more of polyether block polyamide, polyethylene, polypropylene, and polyurethane;

[0072] The first organic base and the second organic base include one or more of triethylamine, dimethylamine, diethylamine, trimethylamine, Downs base, and pyridine.

[0073] According to the embodiments of the present application, polyether block polyamide can be used as a base material for biomedical materials. Take the Pebax catheter as an example, which is made of polyether block polyamide (Polyether Block Amide, Pebax). Pebax is a thermoplastic elastomer composed of polyether segments and polyamide segments, with excellent mechanical properties and biocompatibility. The Pebax catheter has excellent elasticity and flexibility, can adapt to the tortuosity and bends of blood vessels, and provide a comfortable patient experience. It also has a low coefficient of friction, which helps to reduce discomfort during catheter insertion and extraction, and improves the patency of the catheter. Pebax catheters are widely used in the medical field, such as vascular interventional surgery, catheter placement and medical drainage. It has applications in cardiovascular, interventional radiology, urology, gastrointestinal fields, etc.

[0074] In some optional embodiments, the step of bonding the active ester satisfies at least one of the following conditions:

[0075] The pH of the first organic solution is 7.5 to 9;

[0076] The first organic solution includes one or more of trifluoroethanol, ethanol, and toluene;

[0077] The reaction temperature is 20-38°C and the reaction time is 1-48h;

[0078] The concentration of active ester is 10-100 mg / ml.

[0079] According to an embodiment of the present application, by controlling the reaction conditions in the active ester bonding step, the active ester reacts with the amino group on the target surface of the substrate material while ensuring the reaction rate, so that the active ester is chemically bonded to the target surface of the substrate material.

[0080] In some optional embodiments, the active ester comprises a cyclic lactamyl group, and the active ester is obtained by polymerizing any monomer as shown in formula (1),

[0081]

[0082] R is selected from unsubstituted or substituted CH3 (CH=CH) k -, (CH2=CH)-, CH2=CH(CH2) m C6H4-, CH3(CH=CH) n Any one of C6H4-, substituents include -OH, -COOH, -COOCH3, C1-C 10 At least one of alkyl, phenyl, and -NH2; k, m, and n represent a natural number of 1-10.

[0083] According to an embodiment of the present application, the structural monomer containing the active ester of the cyclic lactam may be (N-hydroxysuccinimide) methacrylate, which is polymerized by free radicals to obtain the corresponding polymer, poly (N-hydroxysuccinimide) methacrylate (pNHSMA).

[0084] N-hydroxysuccinimide (NHS) is an important lactam compound, which contains active nitrogen hydroxyl groups in its structure and can be covalently bound to amino, carboxyl or carbonyl groups of antigens, antibodies, proteins and nucleic acids without affecting their biological activity. As an example, the active ester containing cyclic lactams can be polymethacrylate (N-hydroxysuccinimide) ester (pNHSMA). As its high molecular polymer, pNHSMA maintains more active sites, which is conducive to the occurrence and progress of the reaction. Introducing pNHSMA to the surface of the substrate material can make the reaction process easier in the subsequent process of grafting heparin. In some optional embodiments, the mass concentration of pNHSMA is 20 to 100 mg / mL.

[0085] In some optional embodiments, the structural unit of the active ester includes at least one of formula (2) to formula (7):

[0086]

[0087] In some optional embodiments, the step of bonding polyethyleneimine satisfies at least one of the following conditions:

[0088] The second organic solution includes one or more of trifluoroethanol, ethanol, and toluene;

[0089] The temperature for forming covalent bonds is 20 to 38°C, and the time is 1 to 48 hours.

[0090] In some optional embodiments, the mass concentration of polyethyleneimine is 20-100 mg / mL.

[0091] According to the embodiments of the present application, polyethylene ethylene (PEI) is a polyamino polymer containing primary amines, secondary amines and tertiary amines, has high activity, is soluble in water and ethanol, a low-toxic solvent, and can undergo a variety of chemical modifications.

[0092] According to the embodiment of the present application, by controlling the reaction conditions in the polyethyleneimine step, the active ester is reacted with the amino group of the polyethyleneimine while ensuring the reaction rate, so that the polyethyleneimine is fixed on the base material through the active ester.

[0093] In some optional embodiments, the heparin bonding step comprises contacting and reacting the reaction product of the polyethyleneimine bonding step with heparin containing a hydroxyl group in a biological buffer in the presence of a catalyst and a condensing agent.

[0094] In some optional embodiments, the biological buffer comprises one or more of 2-(N-morpholino)ethanesulfonic acid solution (MES), sodium chloride solution, and phosphate buffer solution.

[0095] In some optional embodiments, the pH of the biological buffer is 4.5-4.8.

[0096] In some optional embodiments, the catalyst is N-hydroxysuccinimide sulfonic acid sodium salt; and the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0097] In some optional embodiments, the concentration of the MES solution is 8-13 mg / mL, optionally 11 mg / mL. The pH of the MES solution is 4.5-4.8, the concentration of N-hydroxysuccinimide sulfonic acid sodium salt is 0-10 mg / mL, and the concentration of the condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 0-10 mg / mL. In some optional embodiments, the concentration of heparin sodium is 0-125 mg / mL, optionally 2-25 mg / mL, and optionally 5-20 mg / mL.

[0098] According to the embodiments of the present application, by selecting appropriate biological buffers, catalysts, condensing agents and concentrations, heparin can be fixed on the base material through polyethyleneimine and active esters. On the one hand, this is beneficial for accelerating the reaction rate, and on the other hand, it is beneficial for increasing the grafting amount of heparin and improving the activity of heparin after fixation.

[0099] In some optional embodiments, the method for preparing the substrate material comprising an amino-functionalized target surface comprises:

[0100] The substrate material is activated by a plasma device and then placed in a mixed solution of 3-(trimethoxysilyl)propylamine and an alcohol / water solution, so that the target surface of the substrate material has amino groups.

[0101] In some embodiments, activating the substrate material comprises: activating the substrate material with a plasma device, and then placing it in a solution containing 3-(trimethoxysilyl)propylamine so that the target surface of the substrate material has amino groups, which can provide active sites to facilitate subsequent reactions.

[0102] According to the embodiment of the present application, the base material can be made to have hydroxyl groups by using plasma equipment for activation, and then the above-mentioned compound can be used in a solution to make the hydroxyl groups react so that the base material has amino groups.

[0103] In some optional embodiments, the surface of the substrate material is placed in an oxygen atmosphere of a plasma device, and the plasma device is operated at a power of 60 to 100 W for 3 to 10 minutes.

[0104] According to an embodiment of the present application, under the above parameters, the target surface of the substrate material is placed in a plasma device, so that the target surface of the substrate material can have a certain amount of hydroxyl functional groups while saving resources and ensuring output.

[0105] In some optional embodiments, in the 3-(trimethoxysilyl)propylamine solution, the volume concentration of 3-(trimethoxysilyl)propylamine is 0.1% to 20%.

[0106] In some alternative embodiments, the 3-(trimethoxysilyl)propylamine solution comprises a solvent, and the solvent comprises ethanol.

[0107] In some optional embodiments, the solvent further comprises water.

[0108] In some optional embodiments, the volume concentration of ethanol in the 3-(trimethoxysilyl)propylamine solution is 70%-98%.

[0109] According to the embodiments of the present application, the concentration of the above-mentioned reagents is advantageous in ensuring that the target surface of the substrate material has a certain amount of amino groups while ensuring the reaction efficiency.

[0110] In some embodiments, the substrate material is activated in a water bath at 25-60° C., optionally 37° C., so that the target surface of the substrate material has amino groups. In some embodiments, the substrate material is activated in a water bath for 0.5-12 hours, optionally 2-5 hours.

[0111] In some optional embodiments, the method satisfies at least one of the following conditions:

[0112] The active ester bonding step includes: removing unreacted components with ethanol and deionized water in sequence;

[0113] After the polyethyleneimine bonding step, unreacted components were removed using ethanol and deionized water in sequence;

[0114] After the heparin bonding step, the article with the anticoagulant coating is rinsed with deionized water and dried.

[0115] According to the embodiment of the present application, by removing the unreacted components with the above reagents, the substances fixed on the base material by chemical bonding are retained, and the reaction products are purified.

[0116] In a second aspect, an embodiment of the present application provides a product having an anti-coagulation coating, which is prepared by the method of the first aspect.

[0117] In some optional embodiments, the product includes at least one of a ventricular assist device and an extracorporeal blood circulation device.

[0118] In some optional embodiments, the article having the anti-coagulation coating includes at least one of an artificial blood vessel, an artificial blood vessel stent, an artificial heart valve, a cardiac pacemaker, and a surgical line.

[0119] According to the embodiments of the present application, the grafting amount of heparin fixed by the method of the present application on the surface of the substrate material is greatly increased, and its activity is better, thereby improving its clinical long-term anticoagulant effect to a certain extent.

[0120] Example

[0121] The following examples describe the disclosure of the present application in more detail, and these examples are intended for illustrative purposes only, as it will be apparent to those skilled in the art that various modifications and variations may be made within the scope of the disclosure of the present application. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are by weight, and all reagents used in the examples are commercially available or synthesized according to conventional methods and can be used directly without further processing, and the instruments used in the examples are commercially available.

[0122] Example 1

[0123] This embodiment provides a method for preparing a catheter comprising a product having an anticoagulant coating, comprising the following steps:

[0124] Step 1: Pretreatment of the substrate material: Activate the pebax catheter (raw material pebax catheter, whose material is polyether block polyamide) using plasma equipment (O2 atmosphere, 80W, 5min, PLAUX-PR-5L, Kunshan Pules), and then immerse it in a 2% volume ratio of aminopropyltrimethoxysilane (APTMS) solution, the solvent is 98% ethanol aqueous solution. The reaction is carried out for 2 to 5 hours under the condition of a constant temperature of 37°C in a water bath.

[0125] Step 2: Bonding poly (N-hydroxysuccinimide methacrylate) (pNHSMA)

[0126] The active ester monomer, N-hydroxysuccinimide methacrylate, was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. The corresponding polymer pNHSMA was obtained by polymerization of the monomer: initiator azobisisobutyl cyanide (AIBN), the molar ratio of which to the monomer was 0.005:1, the solvent was N-dimethylformamide (DMF), and the reaction was carried out at 75°C in a nitrogen atmosphere for 24 hours. It was precipitated with methanol and vacuum dried at room temperature to obtain pNHSMA.

[0127] The reaction product obtained in step 1 was immersed in a trifluoroethanol solution of pNHSMA, 50 mg / mL of pNHSMA, and a weight average molecular weight of pNHSMA of 100,000-110,000, and 2-3 drops of triethylamine catalyst were added to react at room temperature for 24 hours. After the reaction was completed, it was cleaned with ethanol and deionized water in turn.

[0128] Step 3: Bonding Polyethyleneimine (PEI)

[0129] Immerse the tube obtained in the above steps into a trifluoroethanol solution of polyethyleneimine, add a certain amount of triethylamine as a catalyst, 20-100 mg / mL of polyethyleneimine, and adjust the solution pH to a weak alkalinity of 7.5-9.0. React at room temperature for 0-48 hours. This embodiment uses 24 hours. Wash with ethanol and deionized water in turn.

[0130] Step 4: Bonding Heparin

[0131] Prepare 2-(N-morpholino)ethanesulfonic acid (MES) solution (11 mg / mL, adjust pH 4.5 to 4.8), dilute to 100 mL, add 0.5 g of sulfo-NHS, EDC-HCl, and 0.5 g of sodium heparin from Hepalink Pharmaceutical Group Co., Ltd., and the concentration of sodium heparin is 5 mg / mL. Soak the reaction product obtained in step 3 in 2-(N-morpholino)ethanesulfonic acid (MES) solution and react at room temperature for 1 h to 48 h. After the reaction is completed, wash with deionized water and vacuum dry to obtain an anticoagulant catheter.

[0132] Example 2-1 to Example 2-2

[0133] The difference between this embodiment and embodiment 1 is that the concentration of aminopropyltrimethoxysilane in step 1 is different. In the aminopropyltrimethoxysilane solution in embodiment 2-1, the volume ratio of aminopropyltrimethoxysilane is 5%. In the aminopropyltrimethoxysilane solution in embodiment 2-2, the volume ratio of aminopropyltrimethoxysilane is 7%.

[0134] Example 3-1 to Example 3-2

[0135] The difference between this embodiment and embodiment 1 is: in step 2, the concentration of pNHSMA is different. In embodiment 3-1, the concentration of pNHSMA is 10 mg / mL; in embodiment 3-2, the concentration of pNHSMA is 100 mg / mL.

[0136] Example 4-1 to Example 4-2

[0137] The difference between this embodiment and embodiment 1 is that in step 2, the weight average molecular weight of pNHSMA is different. In embodiment 4-1, the weight average molecular weight of pNHSMA is 170,000-180,000; in embodiment 4-2, the weight average molecular weight of pNHSMA is 240,000-250,000.

[0138] Example 5-1 to Example 5-2

[0139] The difference between this embodiment and embodiment 1 is that in step 4, the concentration of heparin is different. In embodiment 5-1, the concentration of heparin is 25 mg / mL. In embodiment 5-2, the concentration of heparin is 125 mg / mL.

[0140] Comparative Example 1

[0141] Compared with Example 1, step 1, the pretreatment of the substrate material in this comparative example is the same as that in Example 1, except that step 2 is not performed, that is, active ester is not introduced for reaction, and steps 1, 3, and 4 in Example 1 of the present application are directly used to prepare the anticoagulant catheter. The operation process of step 1, step 3, and step 4 is the same as that in Example 1.

[0142] Test Section

[0143] 1) Contact angle test:

[0144] The base material in the step of Example 1 was subjected to a contact angle test after each reaction. The test results of the water contact angles of the examples and comparative examples in each step are shown in Table 1. The water contact angle values ​​on the surface of the pebax catheter during the entire experiment were measured using a SZ-CAMB3 static water contact angle tester (Shanghai Xuanzhun Instrument Co., Ltd.). The Sessiledrop method was used for measurement, and 2.5 μL of distilled water was used each time. The test results were taken as the average of 5 tests.

[0145] Table 1

[0146]

[0147] The contact angle is an important means to characterize the wettability of the material surface and can be used to study the analysis of the surface characteristics of the material. The initial contact angle of the substrate material surface is about 80°. In each step of the grafting reaction, when new groups are formed on the surface of the substrate material, the surface contact angle will change accordingly, indicating that new groups are formed on the surface of the substrate material in each step of the reaction. When the hydrophilic heparin molecules are grafted, the lower the contact angle, the more heparin molecules are grafted. The final contact angle in Example 1 is as low as 26°. Figure 1 Comparing the results, the best experimental results can be obtained by increasing the concentration of active ester pNHSMA in step 2 in Example 3-2 and increasing the heparin concentration in step 4 in Example 5-2. The contact angle of the catheter product with an anticoagulant surface layer in the example is lower than that of the initial base material (raw material pebax catheter), indicating that a certain amount of heparin molecules are grafted. In addition, the concentration and weight average molecular weight of the active ester pNHSMA and the concentration of heparin in the example have a certain effect on the amount of grafting. The greater the concentration of active ester pNHSMA within a certain range and the greater the weight average molecular weight within a certain range, the greater the concentration of heparin within a certain range, and the more heparin molecules are grafted.

[0148] Compared with Example 1, in Comparative Example 1, the active ester pNHSMA reaction in step 2 was not performed, and the contact angle after the reaction with polyethyleneimine in step 3 and the reaction with heparin in step 4 was 78°, which was the same as the contact angle of the amino functionalized material surface in step 1. The preparation process lacked the participation of active ester pNHSMA, and polyethyleneimine and heparin could not be grafted onto the material surface.

[0149] 2) Identification of heparin components using toluidine blue staining:

[0150] Heparin can be stained by toluidine blue solution, and the integrity of the heparin coating can be evaluated through the stained samples. Soak the catheters grafted with heparin in a 1% toluidine blue / water solution for 30 minutes and rinse them with running water for 2 minutes. Compare the staining results: the blank sample is not stained, and the outer wall of the catheter grafted with heparin is completely blue. Figure 1 As shown, it is illustrated that the anticoagulant catheter grafted with heparin can be prepared in the embodiment, while the staining effect of comparative example 1 is poor, and it is difficult to distinguish whether there is heparin grafted with the naked eye, indicating that the amount of grafting is small.

[0151] 3) In vitro coagulation test:

[0152] The partial thromboplastin time (PTT) is a commonly used method to determine the activation of the intrinsic coagulation pathway. The risk of the medical device to cause thrombosis is assessed by measuring the clotting time of plasma exposed to the medical device to be tested.

[0153] Collect fresh anticoagulated rabbit whole blood within 4 hours and centrifuge to obtain the upper plasma. Use rabbit whole blood that has not been in contact with any material as a blank control. Add the prepared fresh rabbit plasma to test tube group A and test tube group B for packaging, take blank catheters and heparin-grafted catheters and immerse them in test tube group A and test tube group B respectively, and mix them at 37°C. Each tube of plasma is tested twice, and the coefficient of variation is ≤15%, otherwise the test is repeated. After the incubation is completed, the plasma in the test tube is transferred to a new test tube, and the PTT value of the plasma in each test tube is determined using a fully automatic coagulation analyzer.

[0154] The BC value represents the ratio of the clotting time of the coated catheter to the clotting time of the blank sample, and can be used to analyze and determine the anticoagulant properties of the sample.

[0155] Table 2 Coagulation test of anticoagulant catheters and pebax catheters without heparin grafting in some examples.

[0156]

[0157]

[0158] Compared with the plasma in the blank group that was not in contact with any material, the PTT value of the blank sample was 30.2s, which did not change much. The PTT values ​​of the samples grafted with heparin in different embodiments all increased to varying degrees, indicating that the heparin coating has a significant delay effect on coagulation. The thrombin time of the catheter grafted with heparin has a certain correlation with its contact angle. The smaller the contact angle, the longer the thrombin time and the better the anticoagulation effect. Among them, the heparin coatings prepared in Examples 3-2 and 5-2 have the best anticoagulation effect.

[0159] Compared with the blank group, the contact angle, PTT and BC value of the embodiment are all improved, which shows that the catheter containing heparin prepared by the method of the present application has a better coagulation effect.

[0160] In summary, toluidine blue staining and in vitro coagulation experiment tests confirmed that the anticoagulant coating prepared by this technical route has good anticoagulant properties.

[0161] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed in the present application, and these modifications or replacements should be included in the protection scope of the present application. Therefore, the protection scope of the present application shall be based on the protection scope of the claims.

Claims

1. A method for preparing a product having an anticoagulant coating, characterized in that: include: providing a substrate material of an article, the substrate material comprising an amino-functionalized target surface; The step of bonding active esters comprises contacting the amino-functionalized target surface with a first organic solution containing active esters and a first organic base catalyst, so that the amino groups on the amino-functionalized target surface react with part of the ester groups in the active ester under the catalysis of the first organic base catalyst to form covalent bonds, wherein the active ester is a polymer having a weight average molecular weight of 1000-250000; the active ester is obtained by polymerizing a monomer as shown in formula (1), R is selected from unsubstituted or substituted CH3 (CH=CH) k -, (CH2=CH)-, CH2=CH(CH2) m C6H4-, CH3(CH=CH) n Any one of C6H4-, substituents include -OH, -COOH, -COOCH3, C1-C 10 At least one of alkyl, phenyl, -NH2; k, m, n represent natural numbers of 1-10; optionally, the number of monomers is 2-1000; The step of bonding polyethyleneimine comprises contacting the target surface for bonding active ester with a second organic solution containing polyethyleneimine and a second organic base catalyst, and reacting at least part of the amino groups of polyethyleneimine with at least part of the ester groups in the active ester under the catalytic action of the second organic base catalyst to form a covalent bond; The heparin bonding step comprises reacting heparin containing hydroxyl groups with the reaction product of the polyethyleneimine bonding step, so that the amino groups in the reaction product originating from the polyethyleneimine react with the hydroxyl groups in the heparin to obtain a product with an anticoagulant coating.

2. The method according to claim 1, characterized in that The step of bonding active esters satisfies at least one of the following conditions: The first organic solution includes one or more of trifluoroethanol, ethanol, toluene, and propylene glycol; The reaction temperature is 20-38°C and the reaction time is 1-48h; The concentration of the active ester is 10-100 mg / ml.

3. The method according to claim 1 or 2, characterized in that: The active ester comprises a cyclic lactamyl group, and the active ester is obtained by polymerizing any monomer as shown in formula (1). R is selected from unsubstituted or substituted CH3 (CH=CH) k -, (CH2=CH)-, the substituents include -OH, -COOH, -COOCH3, C1-C 10 At least one of alkyl, phenyl, and -NH2; k, m, and n represent a natural number of 1-10.

4. The method according to claim 1, characterized in that The step of bonding polyethyleneimine satisfies at least one of the following conditions: The second organic solution includes one or more of trifluoroethanol and ethanol; The temperature for generating the covalent bond is 20 to 38° C., and the time is 1 to 48 hours.

5. The method according to claim 1, characterized in that The heparin bonding step comprises contacting and reacting the reaction product of the polyethyleneimine bonding step with heparin containing hydroxyl groups in a biological buffer under the action of a catalyst and a condensing agent; Optionally, the biological buffer comprises one or more of 2-(N-morpholino)ethanesulfonic acid solution, tris(hydroxymethyl)aminomethane solution, and phosphate buffer solution; Optionally, the pH of the biological buffer is 4.5 to 4.8; Optionally, the catalyst is N-hydroxysuccinimide sulfonic acid sodium salt, and the condensation agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

6. The method according to claim 1, characterized in that The method satisfies at least one of the following conditions: The base material includes one or more of polyether block polyamide (pebax), polyethylene, polypropylene, and polyurethane; The first organic base catalyst and the second organic base catalyst respectively include one or more of triethylamine, dimethylamine, diethylamine, diisopropylethylamine, trimethylamine, Downs base, and pyridine.

7. The method according to claim 1, characterized in that The method for preparing the substrate material comprising an amino-functionalized target surface comprises: activating the substrate material with a plasma device, and then placing the substrate material in a 3-(trimethoxysilyl)propylamine solution so that the surface of the substrate material has amino groups; Optionally, placing the surface of the substrate material in an oxygen atmosphere of a plasma device, and operating the plasma device at a power of 60 to 100 W for 3 to 10 minutes; Optionally, in the 3-(trimethoxysilyl)propylamine solution, the volume concentration of 3-(trimethoxysilyl)propylamine is 0.1% to 20%; Optionally, the 3-(trimethoxysilyl)propylamine solution includes a solvent, and the solvent includes ethanol; optionally, the solvent also includes water, and optionally, the volume concentration of ethanol in the 3-(trimethoxysilyl)propylamine solution is 70%-98%.

8. The method according to claim 1, characterized in that: The method satisfies at least one of the following conditions: After the step of bonding active esters, the following steps are included: removing unreacted components with ethanol and deionized water in sequence; After the step of bonding polyethyleneimine, unreacted components are removed with ethanol and deionized water in sequence; After the heparin bonding step, the article with the anticoagulant coating is rinsed with deionized water and dried.

9. A product having an anticoagulant coating, characterized in that: It is prepared by the method according to any one of claims 1 to 8.

10. The product according to claim 9, characterized in that The product comprises at least one of a ventricular assist device and an extracorporeal blood circulation device.

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