阅读说明：本技术 一种可降解封堵器涂层及其制备方法 (Degradable occluder coating and preparation method thereof ) 是由 王云兵 郭高阳 杨立 陈娟 许贤春 胡金鹏 于 2021-08-26 设计创作，主要内容包括：本发明公开了一种可降解封堵器涂层及其制备方法。制备方法为：首先,将封堵器浸泡在含有至少两个氨基的化合物的溶液中,取出清洗,将封堵器浸泡在甲基丙烯酸酐水溶液中,取出,使用去离子水和乙醇清洗；然后在封堵器表面浸涂或喷涂明胶甲基丙烯酰胺溶液和引发剂溶液,或者将封堵器浸泡在两性离子单体、丙烯酸单体和引发剂的混合溶液中,在加热或光照条件下引发聚合,聚合后用去离子水清洗。本发明首先将明胶固定在封堵器表面,明胶是细胞外基质的组成成分之一,其中含有大量细胞黏附位点,可以促进细胞黏附,然后在明胶表面共价修饰两性离子聚合物,可以阻止血浆蛋白的非特异性吸附,从而防止血小板黏附和激活,最终同时实现内皮化和抗凝血功能。(The invention discloses a degradable occluder coating and a preparation method thereof. The preparation method comprises the following steps: firstly, soaking a stopper in a solution of a compound containing at least two amino groups, taking out and cleaning, soaking the stopper in a methacrylic anhydride aqueous solution, taking out, and cleaning with deionized water and ethanol; then dipping or spraying gelatin methacrylamide solution and initiator solution on the surface of the stopper, or soaking the stopper in mixed solution of zwitterion monomer, acrylic acid monomer and initiator, initiating polymerization under heating or illumination condition, and cleaning with deionized water after polymerization. According to the invention, firstly, gelatin is fixed on the surface of the occluder, wherein the gelatin is one of the components of extracellular matrix, contains a large number of cell adhesion sites and can promote cell adhesion, and then a zwitterionic polymer is covalently modified on the surface of the gelatin, so that the nonspecific adsorption of plasma protein can be prevented, thus preventing platelet adhesion and activation, and finally, the endothelialization and anticoagulation functions are realized simultaneously.)

1. A preparation method of a degradable occluder coating is provided, wherein the surface of the occluder is provided with the degradable coating, and is characterized by firstly comprising the following steps of 1):

step 1): soaking the stopper in a solution of a compound containing at least two amino groups, and then taking out and cleaning; then soaking the stopper in methacrylic anhydride aqueous solution, taking out, and cleaning with deionized water and ethanol;

then one of the steps 2) and 4) is also included, or the steps 2) and 4) are included in sequence):

step 2): dipping or spraying gelatin methacrylamide solution and initiator solution on the surface of the stopper, initiating polymerization under the condition of heating or illumination, and cleaning with deionized water after polymerization;

step 4): soaking the stopper in a mixed solution of a zwitterion monomer, an acrylic acid monomer and an initiator, initiating polymerization under the condition of heating or illumination, and cleaning with deionized water after polymerization.

2. The method for preparing a degradable occluder coating according to claim 1, wherein when the steps 2) and 4) are included in sequence, a step 3) is further included between the steps 2) and 4):

and (3) soaking the stopper obtained in the step 2) in an N-3-dimethylaminopropyl-N' -ethylcarbodiimide solution, taking out the stopper, soaking the stopper in an N- (3-aminopropyl) methacrylamide hydrochloride solution, and finally washing the stopper with deionized water.

3. The method for preparing a degradable occluder coating according to claim 1 or 2, further comprising step 5) when step 2) is included and step 4) is not included or when step 4) is included:

soaking the stopper obtained in the step 2) or the step 4) in an N-3-dimethylaminopropyl-N' -ethylcarbodiimide solution, taking out the stopper, soaking the stopper in a 4-amine-2, 2,6, 6-tetramethyldiphenoxylate solution, taking out the stopper, and washing the stopper with deionized water.

4. A degradable occluder coating prepared by the method of preparing a degradable occluder coating according to any one of claims 1 to 3.

5. The degradable occluding device coating of claim 4, wherein the occluding device comprises a framework and a flow-blocking membrane, wherein the degradable coating is disposed on the framework or on the flow-blocking membrane, or wherein both the framework and the flow-blocking membrane are disposed with the degradable coating.

6. The degradable occluder coating of claim 5, wherein said scaffold is woven from filaments of degradable material.

7. The degradable occluder coating of claim 6, wherein said degradable material wire is made of at least one of polylactic acid, polyglycolic acid, polylactic-co-glycolic acid, polyhydroxyalkanoates, polydioxanone, polycaprolactone, polyamide, polyanhydride, polyphosphoester, polyurethane and polycarbonate.

8. The degradable occluder coating of claim 5, wherein said flow blocking membrane is sewn to the scaffold and is made of degradable polymeric material.

9. The degradable occluder coating of claim 8, wherein said degradable polymeric material is at least one of polylactic acid, polyglycolic acid, polylactic-co-glycolic acid, polyhydroxyalkanoate, polydioxanone, polycaprolactone, polyamide, polyanhydride, polyphosphate, polyurethane and polycarbonate.

Technical Field

The invention relates to a degradable occluder coating and a preparation method thereof, belonging to the technical field of surgical instruments.

Background

The existing degradable occluder has the following defects:

1. most of the existing biocompatible coatings are combined in a non-covalent form and are easy to fall off.

2. After the degradable occluder is implanted into a heart part, because the occluder is in direct contact with blood, thrombus is easily formed on the surface of the occluder, and the thrombus falls off to cause stroke, so the existing occluder needs to improve the anticoagulation performance.

3. The degradable occluder can be gradually degraded after being implanted into a human body, and loses mechanical property, thereby having the risk of falling off. Therefore, endothelialization of the occluder needs to be achieved as soon as possible after the occluder is implanted into a human body, so that tissue and the occluder are combined as far as possible before the occluder loses mechanical properties, and fragments in the occluder are prevented from falling off.

4. The surface modification of the zwitterionic polymer used in the existing coating can prevent the nonspecific adsorption of plasma protein on the surface of the implant, thereby improving the blood compatibility, but also can prevent the growth of cells on the surface of the implant, thereby delaying the endothelialization.

5. After the implant is implanted into a human body, oxidative stress reaction is caused, severe inflammatory reaction is caused, and tissue repair is hindered.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: how to improve the firmness of the surface coating of the degradable occluder, improve the anticoagulation performance of the occluder, and how to accelerate the endothelialization of the occluder.

In order to solve the technical problems, the invention provides a preparation method of a degradable occluder coating, wherein the surface of the occluder is provided with the degradable coating, and the preparation method is characterized by firstly comprising the following steps of 1):

step 1): soaking the stopper in a solution of a compound containing at least two amino groups, and then taking out and cleaning; then soaking the stopper in methacrylic anhydride aqueous solution, taking out, and cleaning with deionized water and ethanol;

then one of the steps 2) and 4) is also included, or the steps 2) and 4) are included in sequence):

step 2): dipping or spraying gelatin methacrylamide solution and initiator solution on the surface of the stopper, initiating polymerization under the condition of heating or illumination, and cleaning with deionized water after polymerization;

step 4): soaking the stopper in a mixed solution of a zwitterion monomer, an acrylic acid monomer and an initiator, initiating polymerization under the condition of heating or illumination, and cleaning with deionized water after polymerization.

Preferably, when the step 2) and the step 4) are sequentially included, a step 3) is further included between the step 2) and the step 4):

and (3) soaking the stopper obtained in the step 2) in an N-3-dimethylaminopropyl-N' -ethylcarbodiimide solution, taking out the stopper, soaking the stopper in an N- (3-aminopropyl) methacrylamide hydrochloride solution, and finally washing the stopper with deionized water.

Preferably, when step 2) is included and step 4) is not included or when step 4) is included, step 5) is further included:

soaking the stopper obtained in the step 2) or the step 4) in an N-3-dimethylaminopropyl-N' -ethylcarbodiimide solution, taking out the stopper, soaking the stopper in a 4-amine-2, 2,6, 6-tetramethyldiphenoxylate solution, taking out the stopper, and washing the stopper with deionized water.

The invention also provides a degradable occluder coating prepared by the preparation method of the degradable occluder coating.

Preferably, the occluder comprises a framework and a flow-blocking film, wherein a degradable coating is arranged on the framework or on the flow-blocking film, or the framework and the flow-blocking film are both provided with the degradable coating.

More preferably, the skeleton is woven by degradable material wires.

Further, the degradable material wire adopts at least one of polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, polyhydroxyalkanoate, polydioxanone, polycaprolactone, polyamide, polyanhydride, polyphosphate, polyurethane and polycarbonate.

More preferably, the flow-resisting film is sewn on the framework and made of degradable high polymer materials.

Further, the degradable high polymer material is at least one of polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, polyhydroxyalkanoate, polydioxanone, polycaprolactone, polyamide, polyanhydride, polyphosphate, polyurethane and polycarbonate.

Compared with the prior art, the invention has the beneficial effects that:

1. most of the existing biocompatible coatings are combined in a non-covalent form and are easy to fall off, and the components of the coating and the substrate are connected by covalent bonds and are not easy to fall off.

2. After the degradable occluder is implanted into a heart part, because the occluder is in direct contact with blood, thrombus is easily formed on the surface of the occluder, and the thrombus falls off to cause stroke, so the existing occluder needs to improve the anticoagulation performance. The surface of the coating is modified with the zwitterionic polymer, so that the blood compatibility can be improved.

3. The degradable occluder can be gradually degraded after being implanted into a human body, and loses mechanical property, thereby having the risk of falling off. Therefore, endothelialization of the occluder needs to be achieved as soon as possible after the occluder is implanted into a human body, so that tissue and the occluder are combined as far as possible before the occluder loses mechanical properties, and fragments in the occluder are prevented from falling off. The coating of the invention comprises a gelatin coating, wherein the gelatin contains a sequence for promoting cell adhesion, so that endothelialization can be promoted.

4. The surface modification of the zwitterionic polymer used in the existing coating can prevent the nonspecific adsorption of plasma protein on the surface of the implant, thereby improving the blood compatibility, but also can prevent the growth of cells on the surface of the implant, thereby delaying the endothelialization. Therefore, according to the technical scheme, firstly, gelatin is fixed on the surface of the occluder, is one of the components of extracellular matrix and contains a large number of cell adhesion sites, cell adhesion can be promoted, and then the surface of the gelatin is covalently modified with a zwitterionic polymer, so that nonspecific adsorption of plasma protein can be prevented, platelet adhesion and activation are prevented, and endothelialization and anticoagulation functions are finally realized at the same time.

5. After the implant is implanted into a human body, oxidative stress reaction can be caused, severe inflammatory reaction can be caused, and tissue repair is hindered.

6. The coating of the present invention can be degraded in vivo.

Drawings

FIG. 1 is a graph of absorbance data after endothelial cells have been grown on a sample;

FIG. 2 shows the results of quantification of platelet adsorption on a sample;

fig. 3 is the antioxidant results of the samples.

Detailed Description

In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings.

Example 1

A preparation method of a degradable occluder coating comprises the following steps:

step 1): methylacrylamization treatment on surface of stopper skeleton

The occluder skeleton woven from polydioxanone filaments was placed in a solution of 1, 6-hexanediamine in isopropanol (0.08g/mL), heated at 50 ℃ for 10 minutes, and then ultrasonically cleaned using deionized water. Then soaking the aminated skeleton in a newly-prepared 0.5 wt% methacrylic anhydride aqueous solution, adjusting the pH to 8.0 by using a sodium hydroxide solution, carrying out reaction at room temperature for 3 hours, then carrying out ultrasonic cleaning by using deionized water, and then carrying out ultrasonic cleaning by using ethanol.

The purpose of the step is to covalently introduce a double bond group on the surface of the stopper, so that subsequent free radical polymerization reaction is facilitated, and the modified substance is introduced to the surface of the stopper in a covalent manner through free radical polymerization.

Step 2): gelatin coating fixed on surface of plugging device framework

Soaking the plugging device framework obtained in the step 1) in an aqueous solution of 2 wt% of gelatin methacrylamide and 0.5 wt% of Igracure 2959, taking out after 2 minutes, irradiating for 10 minutes by ultraviolet light to completely crosslink the gelatin on the surfaces of all parts of the framework, and washing with deionized water after crosslinking.

The purpose of the step is to covalently introduce gelatin molecules on the surface of the stopper, and double bond groups contained in the gelatin acrylamide molecules can perform free radical polymerization reaction with the double bond groups introduced on the surface of the stopper, so that the gelatin is introduced to the surface of the stopper in a covalent manner.

Example 2

A preparation method of a degradable occluder coating comprises the following steps:

step 1): methylacrylamization treatment on surface of stopper skeleton

The occluder skeleton woven from polydioxanone filaments was placed in a solution of 1, 6-hexanediamine in isopropanol (0.08g/mL), heated at 50 ℃ for 10 minutes, and then ultrasonically cleaned using deionized water. Then soaking the aminated skeleton in a newly-prepared 0.5 wt% methacrylic anhydride aqueous solution, adjusting the pH to 8.0 by using a sodium hydroxide solution, carrying out reaction at room temperature for 3 hours, then carrying out ultrasonic cleaning by using deionized water, and then carrying out ultrasonic cleaning by using ethanol.

The purpose of the step is to covalently introduce a double bond group on the surface of the stopper, so that subsequent free radical polymerization reaction is facilitated, and the modified substance is introduced to the surface of the stopper in a covalent manner through free radical polymerization.

Step 2): anticoagulation functionalization of gelatin coating on surface of occluder framework

The stopper was immersed in a mixed aqueous solution of a sulfonic acid betaine monomer (500mM), an acrylic acid monomer (100mM), ammonium persulfate (10mM), and sodium hydrogen sulfite (10mM), polymerization was initiated at room temperature for 3 hours, and then washed with deionized water.

The purpose of the step is to covalently introduce a zwitterionic polymer on the surface of the stopper, wherein the zwitterionic monomer is subjected to polymerization reaction under the action of an initiator and simultaneously subjected to free radical polymerization reaction with a double bond group introduced on the surface of the stopper or a double bond group in gelatin on the surface of the stopper, so that the amphoteric polymer is covalently introduced on the surface of the stopper.

Example 3

A preparation method of a degradable occluder coating comprises the following steps:

step 1): methylacrylamization treatment on surface of stopper skeleton

The occluder skeleton woven from polydioxanone filaments was placed in a solution of 1, 6-hexanediamine in isopropanol (0.08g/mL), heated at 50 ℃ for 10 minutes, and then ultrasonically cleaned using deionized water. Then soaking the aminated skeleton in a newly-prepared 0.5 wt% methacrylic anhydride aqueous solution, adjusting the pH to 8.0 by using a sodium hydroxide solution, carrying out reaction at room temperature for 3 hours, then carrying out ultrasonic cleaning by using deionized water, and then carrying out ultrasonic cleaning by using ethanol.

The purpose of the step is to covalently introduce a double bond group on the surface of the stopper, so that subsequent free radical polymerization reaction is facilitated, and the modified substance is introduced to the surface of the stopper in a covalent manner through free radical polymerization.

Step 2): gelatin coating fixed on surface of plugging device framework

Soaking the plugging device framework obtained in the step 1) in an aqueous solution of 2 wt% of gelatin methacrylamide and 0.5 wt% of Igracure 2959, taking out after 2 minutes, irradiating for 10 minutes by ultraviolet light to completely crosslink the gelatin on the surfaces of all parts of the framework, and washing with deionized water after crosslinking.

The purpose of the step is to covalently introduce gelatin molecules on the surface of the stopper, and double bond groups contained in the gelatin acrylamide molecules can perform free radical polymerization reaction with the double bond groups introduced on the surface of the stopper, so that the gelatin is introduced to the surface of the stopper in a covalent manner.

Step 3): double bond functionalization of gelatin coating on surface of occluder framework

The stopper was placed in an N-3-dimethylaminopropyl-N' -ethylcarbodiimide solution (10mM, pH 5.0) for 1 hour, taken out and immersed in an N- (3-aminopropyl) methacrylamide hydrochloride (50mM, pH 7.0) solution for 6 hours, and washed with deionized water.

The purpose of the step is to introduce double bond groups into gelatin molecules after the surface of the stopper is modified, and the double bond groups are consumed after the gelatin acrylamide and the double bonds on the surface of the stopper are subjected to free radical polymerization reaction, so that double bond molecules can be introduced into residual carboxyl groups of the gelatin through carbodiimide coupling reaction in the step, and subsequent free radical polymerization reaction on the surface of the gelatin molecules is facilitated.

Step 4): anticoagulation functionalization of gelatin coating on surface of occluder framework

The stopper was immersed in a mixed aqueous solution of a sulfonic acid betaine monomer (500mM), an acrylic acid monomer (100mM), ammonium persulfate (10mM), and sodium hydrogen sulfite (10mM), polymerization was initiated at room temperature for 3 hours, and then washed with deionized water.

The purpose of the step is to covalently introduce a zwitterionic polymer on the surface of the stopper, wherein the zwitterionic monomer is subjected to polymerization reaction under the action of an initiator and simultaneously subjected to free radical polymerization reaction with a double bond group introduced on the surface of the stopper or a double bond group in gelatin on the surface of the stopper, so that the amphoteric polymer is covalently introduced on the surface of the stopper.

Example 4

A preparation method of a degradable occluder coating comprises the following steps:

step 1): methylacrylamization treatment on surface of stopper skeleton

The occluder skeleton woven from polydioxanone filaments was placed in a solution of 1, 6-hexanediamine in isopropanol (0.08g/mL), heated at 50 ℃ for 10 minutes, and then ultrasonically cleaned using deionized water. Then soaking the aminated skeleton in a newly-prepared 0.5 wt% methacrylic anhydride aqueous solution, adjusting the pH to 8.0 by using a sodium hydroxide solution, carrying out reaction at room temperature for 3 hours, then carrying out ultrasonic cleaning by using deionized water, and then carrying out ultrasonic cleaning by using ethanol.

The purpose of the step is to covalently introduce a double bond group on the surface of the stopper, so that subsequent free radical polymerization reaction is facilitated, and the modified substance is introduced to the surface of the stopper in a covalent manner through free radical polymerization.

Step 2): gelatin coating fixed on surface of plugging device framework

Soaking the plugging device framework obtained in the step 1) in an aqueous solution of 2 wt% of gelatin methacrylamide and 0.5 wt% of Igracure 2959, taking out after 2 minutes, irradiating for 10 minutes by ultraviolet light to completely crosslink the gelatin on the surfaces of all parts of the framework, and washing with deionized water after crosslinking.

The purpose of the step is to covalently introduce gelatin molecules on the surface of the stopper, and double bond groups contained in the gelatin acrylamide molecules can perform free radical polymerization reaction with the double bond groups introduced on the surface of the stopper, so that the gelatin is introduced to the surface of the stopper in a covalent manner.

Step 3): oxidation resistance functionalization of gelatin coating on surface of plugging device framework

The cleaned stopper was put into a N-3-dimethylaminopropyl-N' -ethylcarbodiimide solution (10mM, pH 5.0) for 1 hour, taken out, immersed in an aqueous solution of 4-amine-2, 2,6, 6-tetramethyldiphenoyl (50mM, pH 7.0) for 6 hours, and washed with deionized water.

The purpose of the step is to covalently introduce an antioxidant group on the modified carboxyl group in the surface of the stopper through carbodiimide coupling reaction, thereby endowing the stopper with antioxidant function.

Example 5

A preparation method of a degradable occluder coating comprises the following steps:

step 1): methylacrylamization treatment on surface of stopper skeleton

The occluder skeleton woven from polydioxanone filaments was placed in a solution of 1, 6-hexanediamine in isopropanol (0.08g/mL), heated at 50 ℃ for 10 minutes, and then ultrasonically cleaned using deionized water. Then soaking the aminated skeleton in a newly-prepared 0.5 wt% methacrylic anhydride aqueous solution, adjusting the pH to 8.0 by using a sodium hydroxide solution, carrying out reaction at room temperature for 3 hours, then carrying out ultrasonic cleaning by using deionized water, and then carrying out ultrasonic cleaning by using ethanol.

The purpose of the step is to covalently introduce a double bond group on the surface of the stopper, so that subsequent free radical polymerization reaction is facilitated, and the modified substance is introduced to the surface of the stopper in a covalent manner through free radical polymerization.

Step 2): anticoagulation functionalization of gelatin coating on surface of occluder framework

The stopper was immersed in a mixed aqueous solution of a sulfonic acid betaine monomer (500mM), an acrylic acid monomer (100mM), ammonium persulfate (10mM), and sodium hydrogen sulfite (10mM), polymerization was initiated at room temperature for 3 hours, and then washed with deionized water.

The purpose of the step is to covalently introduce a zwitterionic polymer on the surface of the stopper, wherein the zwitterionic monomer is subjected to polymerization reaction under the action of an initiator and simultaneously subjected to free radical polymerization reaction with a double bond group introduced on the surface of the stopper or a double bond group in gelatin on the surface of the stopper, so that the amphoteric polymer is covalently introduced on the surface of the stopper.

Step 3): oxidation resistance functionalization of gelatin coating on surface of plugging device framework

The cleaned stopper was put into a N-3-dimethylaminopropyl-N' -ethylcarbodiimide solution (10mM, pH 5.0) for 1 hour, taken out, immersed in an aqueous solution of 4-amine-2, 2,6, 6-tetramethyldiphenoyl (50mM, pH 7.0) for 6 hours, and washed with deionized water.

The purpose of the step is to covalently introduce an antioxidant group on the modified carboxyl group in the surface of the stopper through carbodiimide coupling reaction, thereby endowing the stopper with antioxidant function.

Example 6

A preparation method of a degradable occluder coating comprises the following steps:

step 1): methylacrylamization treatment on surface of stopper skeleton

The occluder skeleton woven from polydioxanone filaments was placed in a solution of 1, 6-hexanediamine in isopropanol (0.08g/mL), heated at 50 ℃ for 10 minutes, and then ultrasonically cleaned using deionized water. Then soaking the aminated skeleton in a newly-prepared 0.5 wt% methacrylic anhydride aqueous solution, adjusting the pH to 8.0 by using a sodium hydroxide solution, carrying out reaction at room temperature for 3 hours, then carrying out ultrasonic cleaning by using deionized water, and then carrying out ultrasonic cleaning by using ethanol.

The purpose of the step is to covalently introduce a double bond group on the surface of the stopper, so that subsequent free radical polymerization reaction is facilitated, and the modified substance is introduced to the surface of the stopper in a covalent manner through free radical polymerization.

Step 2): gelatin coating fixed on surface of plugging device framework

Soaking the plugging device framework obtained in the step 1) in an aqueous solution of 2 wt% of gelatin methacrylamide and 0.5 wt% of Igracure 2959, taking out after 2 minutes, irradiating for 10 minutes by ultraviolet light to completely crosslink the gelatin on the surfaces of all parts of the framework, and washing with deionized water after crosslinking.

The purpose of the step is to covalently introduce gelatin molecules on the surface of the stopper, and double bond groups contained in the gelatin acrylamide molecules can perform free radical polymerization reaction with the double bond groups introduced on the surface of the stopper, so that the gelatin is introduced to the surface of the stopper in a covalent manner.

Step 3): double bond functionalization of gelatin coating on surface of occluder framework

The stopper was placed in an N-3-dimethylaminopropyl-N' -ethylcarbodiimide solution (10mM, pH 5.0) for 1 hour, taken out and immersed in an N- (3-aminopropyl) methacrylamide hydrochloride (50mM, pH 7.0) solution for 6 hours, and washed with deionized water.

The purpose of the step is to introduce double bond groups into gelatin molecules after the surface of the stopper is modified, and the double bond groups are consumed after the gelatin acrylamide and the double bonds on the surface of the stopper are subjected to free radical polymerization reaction, so that double bond molecules can be introduced into residual carboxyl groups of the gelatin through carbodiimide coupling reaction in the step, and subsequent free radical polymerization reaction on the surface of the gelatin molecules is facilitated.

Step 4): anticoagulation functionalization of gelatin coating on surface of occluder framework

The stopper was immersed in a mixed aqueous solution of a sulfonic acid betaine monomer (500mM), an acrylic acid monomer (100mM), ammonium persulfate (10mM), and sodium hydrogen sulfite (10mM), polymerization was initiated at room temperature for 3 hours, and then washed with deionized water.

The purpose of the step is to covalently introduce a zwitterionic polymer on the surface of the stopper, wherein the zwitterionic monomer is subjected to polymerization reaction under the action of an initiator and simultaneously subjected to free radical polymerization reaction with a double bond group introduced on the surface of the stopper or a double bond group in gelatin on the surface of the stopper, so that the amphoteric polymer is covalently introduced on the surface of the stopper.

Step 5): oxidation resistance functionalization of gelatin coating on surface of plugging device framework

The cleaned stopper was put into a N-3-dimethylaminopropyl-N' -ethylcarbodiimide solution (10mM, pH 5.0) for 1 hour, taken out, immersed in an aqueous solution of 4-amine-2, 2,6, 6-tetramethyldiphenoyl (50mM, pH 7.0) for 6 hours, and washed with deionized water.

The purpose of the step is to covalently introduce an antioxidant group on the modified carboxyl group in the surface of the stopper through carbodiimide coupling reaction, thereby endowing the stopper with antioxidant function.

The functions of the samples 1 to 6 obtained in examples 1 to 6 are shown in Table 1.

TABLE 1

Samples 1 to 6 obtained in accordance with examples 1 to 6 were subjected to the relevant tests:

first, endothelial cell growth test and results

The occluder samples prepared in examples 1-6, and the occluder sample without a coating (control) were sterilized with ethylene oxide and placed in a 96-well plate, and human umbilical vein endothelial cells were seeded on the surface of the material at a density of: 10000 per well, 3 days of culture. The samples were then removed and transferred to new 96-well plates, 100. mu.l of CCK-8 working solution was added to each well, and after incubation for 1 hour, the suspension was tested for absorbance at 450 nm. The results of the experiments (fig. 1) show that the endothelial cells of samples 2 and 5 grow poorly, and the endothelial cells of samples 1, 3, 4 and 6 grow well.

Second, thrombosis test and results

The occluder samples prepared in examples 1-6, and the occluder sample without a coating (control group) were placed in a 96-well plate and washed with deionized water, 100 μ l of fresh platelet plasma PRP was added to the sample, incubated at 37 ℃ for 1 hour, aspirated plasma, and washed with PBS. A1 wt% Triton-X PBS solution was added to the sample, incubated at 37 ℃ for 1 hour, and the supernatant was mixed with a lactate dehydrogenase working solution to measure the absorbance at 490 nm. The experimental results show (fig. 2) that samples 2, 3, 5, and 6 have significant anticoagulant effect, and the anticoagulant ability of the control sample is poor.

Third, antioxidant ability test and results

The following concentrations of solutions were prepared: ferrous sulfate 2mM (0.304mg/mL), safranin O (0.36mg/mL), H₂O₂Solution: 6 wt%. The reagents were added in the order shown in table 2, wherein no sample was added to the blank control group and the negative control group, and the stopper samples prepared in examples 1 to 6, and the stopper sample without coating were added to the experimental group.

TABLE 2

	Blank control	Negative control	Experimental group
				Water (W)	50 microliter	130 microliter	50 microliter
Sample (I)	——	——	5mg
				Ferrous sulfate	60 microliter	60 microliter	60 microliter
Safranin O	50 microliter	50 microliter	50 microliter
				Hydrogen peroxide	80 microliter	——	80 microliter

Reacting at room temperature for 10min, and heating in water bath at 55 deg.C for 30 min. The absorbance at 492nm was measured with a microplate reader. The scavenging effect on hydrogen peroxide radicals was calculated according to the following formula:

degree of oxidation (%) ═ a_{Experimental group}-A_{Blank group})/(A_{Negative control}-A_{Blank group})×100

The experimental results show (fig. 3) that samples 4, 5, and 6 have significant antioxidant effects, and the control sample has poor antioxidant ability.