CN115998963B - Anticoagulation coating and preparation method and application thereof - Google Patents

Abstract

The invention relates to an anticoagulant coating, a preparation method and application thereof, and a blood contact medical instrument. The preparation method of the anticoagulation coating comprises the following steps: obtaining heparin molecules with aldehyde groups at non-reducing terminal groups; carrying out an amine-aldehyde condensation reaction on a heparin molecule with aldehyde groups at non-reducing terminal groups and an amino compound under a reducing condition to prepare modified heparin, wherein the amino compound also contains a reactive group; mixing the modified heparin with a reaction monomer, and polymerizing a reactive group contained in the modified heparin with an unsaturated double bond contained in the reaction monomer to prepare a polymer; coating a solution containing a polymer on the surface of a substrate, and then crosslinking and solidifying the crosslinking group to form an anticoagulant coating on the surface of the substrate. The preparation method of the anticoagulation coating can ensure the anticoagulation effect of heparin and has universality to medical substrates.

Description

Anticoagulation coating and preparation method and application thereof

Technical Field

The invention relates to the field of medical instruments, in particular to an anticoagulant coating, and a preparation method and application thereof.

Background

In clinical use, the blood contact surface of the blood contact implantation and intervention medical instrument is extremely easy to trigger a plurality of coagulation mechanisms such as coagulation factor activation, platelet adhesion aggregation, erythrocyte adhesion, complement activation and the like, and causes complications such as coagulation, thrombus formation and the like. The biological medical raw materials are modified by the anticoagulation coating, so that the safety and effectiveness of medical instruments can be greatly improved.

Heparin anticoagulation coating is the most widely studied and commercialized mature type of anticoagulation coating at present, and in addition, inert anticoagulation coating such as phosphorylcholine, albumin and the like also form the very successful medical instrument product application. The heparin molecular chain has carboxyl, sulfonic group and other electronegative active groups. The heparin anticoagulation coating grafting technology can generally utilize the active groups to carry out covalent reaction to directly graft on the surface of the substrate, or form new target active groups such as azido, amino, double bond and the like, and then further connect on the surface of the substrate. However, the covalent connection based on the multiple points of the heparin molecule chains or the covalent connection after the multiple points of the modification is not beneficial to the outward extension of the heparin molecule chains, and the anticoagulation active sites such as five-membered sugar rings of heparin are easy to hide, so that the activity of the heparin molecule chains is greatly inhibited, and even the heparin molecule chains are inactivated after grafted to the surface of a substrate.

The Carmeda heparin anticoagulation coating adopts an end grafting method, is favorable for outward extension and exposure of heparin molecule chain anticoagulation active sites, and can realize a trace amount of heparin molecules and an excellent anticoagulation effect. However, the Carmeda heparin anticoagulant coating requires extremely complex specific targeted modification of the substrate surface, thereby greatly limiting its applicability to substrates.

Disclosure of Invention

Based on this, it is necessary to provide an anticoagulation coating layer capable of ensuring the anticoagulation activity of heparin and having universality to a substrate, and a preparation method thereof.

Furthermore, it is necessary to provide an application of an anticoagulant coating.

A method for preparing an anticoagulant coating, comprising the steps of:

obtaining heparin molecules with aldehyde groups at non-reducing terminal groups;

carrying out an amine-aldehyde condensation reaction on the heparin molecule with aldehyde groups at the non-reducing terminal groups and an amino compound under a reducing condition to prepare modified heparin, wherein the amino compound also contains a reactive group;

mixing the modified heparin with a reaction monomer, wherein the reaction monomer is selected from any one or more of acrylic ester monomers containing crosslinking groups and olefin monomers containing crosslinking groups, and polymerizing a reactive group contained in the modified heparin and an unsaturated double bond contained in the reaction monomer to prepare a polymer containing the crosslinking groups; a kind of electronic device with high-pressure air-conditioning system

Coating a solution containing the polymer on the surface of a substrate, and then crosslinking and solidifying the crosslinking group to form an anticoagulant coating on the surface of the substrate.

In one embodiment, the reactive group comprises any one or a combination of several of a carbon-carbon double bond, a carbon-carbon triple bond, a methacryloxy group, an epoxy group, a cycloalkyl group, a carbon-nitrogen double bond, and a carbon-nitrogen triple bond.

In one embodiment, the step of subjecting the heparin molecule with aldehyde groups at the non-reducing end groups to an amine aldehyde condensation reaction with an amino compound satisfies at least one of the following conditions:

(1) The amino compound is selected from any one or a combination of a plurality of propenyl amine, methacrylamide, cyclopropylamine, p-propenyl aniline, p-methyl propenyl phenethylamine, isocyanic acid ethylamine and isocyanic acid aniline;

(2) The mass ratio of the amino compound to the heparin molecule with aldehyde group at the non-reducing terminal group is 1: (0.5-100);

(3) In the process of the amine aldehyde condensation reaction, a reducing agent is also added, and the mass ratio of the reducing agent to the amino compound is 1: (1-50); and

(4) The temperature of the amine aldehyde condensation reaction is 30-60 ℃ and the time is 0.5-3 h.

In one embodiment, the crosslinking group includes any one or a combination of a hydroxyl group, a carbon-carbon double bond, a carbon-carbon triple bond, and a siloxyl group.

In one embodiment, the step of polymerizing the reactive group contained in the modified heparin with the unsaturated double bond contained in the reactive monomer satisfies at least one of the following conditions:

(1) The reaction monomer is selected from any one or a combination of a plurality of 4-hydroxy butyl acrylate, 2-hydroxy ethyl acrylate, 3- (trimethoxysilyl) propyl acrylate, [3- (methacryloxy) propyl ] trimethoxy silane, triethoxy vinyl silane and propynyl acrylate;

(2) The mass ratio of the modified heparin to the reaction monomer is 1: (0.2-3);

(3) The temperature of the polymerization reaction is 60-90 ℃, and the time of the polymerization reaction is 12-48 h;

(4) The modified heparin, the double-bonded phosphorylcholine and the reaction monomer are added with the mass ratio of (0.2-1): 1: (0.2 to 0.6); and

(5) An initiator is also added, and the ratio of the total mass of the raw materials participating in the reaction to the mass of the initiator is (10-40): 1.

in one embodiment, the step of crosslinking curing the crosslinking group comprises: heating and crosslinking the base material coated with the polymer solution at 50-90 ℃ for 12-48 hours; or alternatively, the process may be performed,

spraying 1, 4-butanediol glycidyl ether on the surface of a substrate coated with the polymer solution, and then crosslinking and curing at 25-50 ℃ for 12-24 hours; or alternatively, the process may be performed,

and (3) irradiating and curing the base material coated with the polymer solution for 1-2 hours under a UV illumination lamp with the wavelength of 254 nm.

In one embodiment, the step of applying a solution containing the polymer to the surface of the substrate comprises: and immersing the substrate in a solution containing the polymer for 1-5 min, and taking out the substrate, wherein the concentration of the solution containing the polymer is 2-15%.

In one embodiment, the step of obtaining a heparin molecule having aldehyde groups at the non-reducing end groups comprises: and mixing heparin sodium and an oxidant for reaction at the temperature of 0-20 ℃ and the pH of 2.5-4 to obtain the heparin molecule with aldehyde groups at the non-reducing terminal groups.

In one embodiment, the step of obtaining a heparin molecule with aldehyde groups at the non-reducing end groups satisfies at least one of the following conditions:

(1) The mass ratio of the heparin sodium to the oxidant is 100: (0.1 to 5);

(2) The oxidant is selected from any one or more of sodium nitrite, nitrous acid, periodic acid and sodium periodate; and

(3) The reaction time is 1 h-3 h.

The anticoagulation coating is prepared by the preparation method of the anticoagulation coating.

The application of the anticoagulation coating in preparing blood contact medical devices.

The preparation method of the anticoagulation coating utilizes the single point reaction of aldehyde groups in heparin molecules with aldehyde groups at non-reducing end groups and amino groups in amino compounds with reactive groups to form heparin end group single active sites with the reactive groups, and then the heparin end group single active sites with the reactive groups are polymerized with reactive monomers containing unsaturated double bonds and crosslinking groups to enable the reactive groups carried by heparin and the unsaturated double bonds in the reactive monomers to react covalently to form polymers containing the crosslinking groups, and then the solution containing the polymers is coated on the surface of a substrate to realize uniform physical adsorption on the surface of the substrate. Finally, crosslinking and solidifying crosslinking groups of the polymer to realize stable grafting of an interconnected network structure on the surface of the substrate, so that the method can stably fix heparin molecules on the surfaces of various substrates to be coated, the problem of high selectivity of the conventional end-point-attached heparin coating to the substrate is solved, and the method has universality to the substrate. In addition, different from the traditional modification of the multi-site molecular chains such as heparin carboxyl, sulfonic acid group and the like, the preparation method utilizes the single-site reaction of aldehyde group and amino group to carry out single-site specific modification on the end point of heparin and realize single-site connection with a substrate, thereby fully ensuring the anticoagulation activity of the modified heparin, and obviously strengthening the anticoagulation test such as the whole blood coagulation time and the like compared with other modified heparin coatings.

Drawings

FIG. 1 is a process flow diagram of a method of preparing an anticoagulant coating according to an embodiment of the invention;

FIG. 2 is a graph showing the change of heparin graft density of the anticoagulation coating prepared in examples 2-4 and comparative example 3 over time;

FIG. 3 is a graph showing the whole blood coagulation time and the anti-platelet adhesion test results of the anti-coagulation coatings prepared in examples 5 to 12, comparative examples 1 and 2.

Detailed Description

In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to specific embodiments that are now described. Preferred embodiments of the invention are given in the detailed description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Conventional non-endpoint based modified or linked heparin anticoagulation coatings often come at the expense of heparin anticoagulation efficacy; the fixation method of the heparin anticoagulation coating based on the end points has high selectivity to the substrate to be coated. These problems have greatly limited the development of heparin anticoagulant coatings.

Based on the above, the invention provides an anticoagulation coating and a preparation method thereof, wherein the method is based on the single-point specific modification of heparin end points, and the connection between heparin and a substrate is realized by utilizing the specific modification end points, so that the anticoagulation activity of the anticoagulation coating is ensured; meanwhile, the heparin modified by the endpoint unit point is polymerized and crosslinked with the reaction monomer to be fixed on the surface of the nonspecific substrate, so that the substrate universality of the coating technology is realized.

Specifically, referring to fig. 1, a method for preparing an anticoagulant coating according to an embodiment includes the following steps:

step S110: obtaining heparin molecules with aldehyde groups at non-reducing terminal groups.

In some embodiments, step S110 includes: and mixing heparin sodium and an oxidant for reaction at the temperature of 0-20 ℃ and the pH of 2.5-4 to prepare the heparin molecule with aldehyde groups at the non-reducing terminal groups.

Wherein the non-reducing end refers to one end of the heparin sodium, which is oxidized to form aldehyde group after oxidative cleavage.

In some embodiments, the mass ratio of heparin sodium to the oxidizing agent is 100 (0.1-5). For example, the mass ratio of heparin sodium to oxidizing agent is 100:0.1, 100:0.2, 100:0.3, 100:0.4, 100:0.5, 100:0.6, 100:0.7, 100:0.8, 100:0.9, 100:1, 100:1.5, 100:2, 100:2.5, 100:3, 100:3.5, 100:4, 100:4.5, 100:5, or a range of values consisting of any two of these values. Further, the mass ratio of heparin sodium to the oxidant is 100 (0.1-1).

In some embodiments, the reaction time is 1h to 3h. For example, the reaction time may be 1h, 1.5h, 2h, 2.5h, 3h, or a range of any two of these values.

In some embodiments, after the reaction is complete, further comprising: the pH is adjusted to be neutral to quench the reaction, and then the heparin molecule with aldehyde groups at the non-reducing terminal end group is prepared by dialysis and purification.

In some embodiments, the oxidizing agent is selected from any one or more of sodium nitrite, nitrous acid, periodic acid, and sodium periodate.

In some embodiments, in step S110, heparin sodium is reacted with an oxidizing agent in the form of an aqueous solution. The mass volume fraction of the heparin sodium aqueous solution is 0.1% -10%. For example, the mass volume fraction of the aqueous heparin sodium solution is 0.1%, 0.2%, 0.5%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2%, 3%, 4%, 5%, 10% or a range consisting of any two of these values.

In some embodiments, step S110 includes: and adding an oxidant into the aqueous solution of heparin sodium at the temperature of 0-20 ℃, adjusting the pH value to 2.5-4, enabling the heparin sodium to react with the oxidant for 1-3 hours, adjusting the pH value to be neutral, dialyzing and purifying to prepare the heparin molecule with aldehyde groups at the non-reducing terminal end groups, wherein the mass ratio of the heparin sodium to the oxidant is 100 (0.1-5). Further, the mass ratio of heparin sodium to the oxidant is 100 (0.1-1).

Through the steps, the heparin sodium is partially oxidized and degraded to obtain the heparin molecule with aldehyde groups at the non-reducing terminal end groups.

Step S120: the heparin molecule with aldehyde group at the non-reducing terminal group and amino compound are subjected to amine aldehyde condensation reaction under the reducing condition to prepare modified heparin, wherein the amino compound also contains a reactive group.

In some embodiments, the reactive group comprises any one or a combination of carbon-carbon double bonds, carbon-carbon triple bonds, methacryloxy groups, epoxy groups, cycloalkyl groups, carbon-nitrogen double bonds, and carbon-nitrogen triple bonds.

Further, the amino compound is selected from propenyl amine) Methacrylamide) Cyclopropylamine (++>) Para-propenylaniline ()>) Para-methylpropenyl phenethylamine (/ -methyl)>) Ethylamine isocyanate (+)>) Aniline isocyanate (+)>) Any one or a combination of a plurality of them.

In some embodiments, in the process of the amine aldehyde condensation reaction, the mass ratio of the amino compound to the reducing agent is (1-50): 1. for example, the mass ratio of amino compound to reducing agent is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1 or a range of any two of these values. Further, the mass ratio of the amino compound to the reducing agent is preferably (1 to 5): 1.

in one embodiment, the reducing agent is selected from any one or more of sodium borohydride, sodium cyanoborohydride, and sodium triacetylborohydride.

In some embodiments, the mass ratio of amino compound to heparin molecule with aldehyde groups on the non-reducing end groups is 1: (0.5 to 100). In one embodiment, the mass ratio of amino compound to heparin molecule with aldehyde group at the non-reducing end group is 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:10 or a range consisting of any two of these values. Further, the mass ratio of the amino compound to the heparin molecule having aldehyde groups at the non-reducing terminal group is preferably 1 (1 to 5).

In some embodiments, the amino compound is directly added into the aqueous solution containing the heparin molecule with the aldehyde group at the non-reducing end group obtained in the step S110, so that the mass percentage of the amino compound is 0.05% -1%.

In some embodiments, the temperature of the aldol condensation reaction is 30-60 ℃ and the time is 0.5-3 hours.

Through the reaction, the non-reducing terminal group of the heparin molecule is provided with a reactive group by utilizing the amine aldehyde condensation reaction of aldehyde groups and amino groups. The reaction of the carboxyl group and the amino group contained in the heparin molecule is required to be carried out under specific reaction conditions, and under the above conditions, the amino group and the carboxyl group are only bonded by unstable ionic bonds and are easily broken. Thus, in the manner described above, the non-reducing end groups of the heparin molecule are reacted with the amino groups in single sites.

In some embodiments, step S120 includes: adding an amino compound and a reducing agent into the aqueous solution containing the heparin molecules with aldehyde groups at the non-reducing end groups, which are obtained in the step S110, wherein the mass volume percentages of the amino compound and the reducing agent are respectively 0.05% -1% and 0.01% -0.1%, and carrying out an amine-aldehyde condensation reaction for 0.5 h-3 h at the temperature of 30 ℃ -60 ℃, wherein the mass ratio of the amino compound to the heparin molecules with aldehyde groups at the non-reducing end groups is 1: (0.5-100), wherein the mass ratio of the amino compound to the reducing agent is (1-50): 1. further, the mass ratio of the amino compound to the heparin molecule with aldehyde group at the non-reducing terminal group is 1: (1-5), wherein the mass ratio of the amino compound to the reducing agent is (1-5): 1.

step S130: mixing the modified heparin with a reaction monomer, wherein the reaction monomer is selected from any one or more of acrylic ester monomers containing crosslinking groups and olefin monomers containing crosslinking groups, and polymerizing a reactive group contained in the modified heparin and an unsaturated double bond contained in the reaction monomer to prepare a polymer containing the crosslinking groups.

Specifically, a crosslinking group refers to a group capable of undergoing crosslinking under certain conditions, such as heat, light, etc., to form a network structure on a polymer chain. In some implementations, the crosslinking group includes any one or a combination of several of hydroxyl groups, carbon-carbon double bonds, carbon-carbon triple bonds, and siloxyl groups.

Further, the reaction monomer is selected from any one or a combination of a plurality of 4-hydroxy butyl acrylate, 2-hydroxy ethyl acrylate, 3- (trimethoxysilyl) acrylic acid propyl ester, [3- (methacryloxy) propyl ] trimethoxy silane, triethoxy vinyl silane and acrylic acid propynyl ester.

In some embodiments, the mass ratio of modified heparin to reactive monomer is 1: (0.2-3). In a specific example, the mass ratio of modified heparin to reactive monomer is 1:0.2, 1:0.3, 1:0.5, 1:0.8, 1:1, 1:1.2, 1:1.5, 1:1.8, 1:2, 1:2.2, 1:2.5, 1:2.8, 1:3, or a range of any two of these values.

In some embodiments, the polymerization reaction is carried out at a temperature of 60 ℃ to 90 ℃ for a time of 12 hours to 48 hours.

In some embodiments, in the step of polymerizing the reactive group contained in the modified heparin and the unsaturated double bond contained in the reactive monomer, double-bonded phosphorylcholine is further added, and the mass ratio of the modified heparin, the double-bonded phosphorylcholine and the reactive monomer is (0.2-1): 1: (0.2 to 0.6). In one specific example, the mass ratio of modified heparin, doubly linked phosphorylcholine, and reactive monomer is 0.2:1:0.2, 0.3:1:0.5, 0.5:1:0.2, 0.5:1:0.4, and 0.5:1:0.6, 0.8:1:0.4, etc.

In one embodiment, the doubly linked phosphorylcholine is 2-Methacryloyloxyethyl Phosphorylcholine (MPC).

In some embodiments, an initiator is also added, and the ratio of the total mass of the raw materials participating in the reaction to the mass of the initiator is (10-40): 1. wherein, the raw materials participating in the reaction comprise modified heparin, reaction monomers and double-bonded phosphorylcholine. In one specific example, the ratio of the total mass of the starting materials involved in the reaction to the mass of the initiator is 10:1, 12:1, 14:1, 22:1.5, 15:1, 16:1, 18:1, 20:1, 25:1, 30:1, 35:1, 40:1 or a range consisting of any two of these values.

In one embodiment, the initiator is azobisisobutyronitrile. It will be appreciated that the initiator is not limited to azobisisobutyronitrile, but may be other initiators capable of initiating free radical polymerization as are commonly used in the art.

In some embodiments, the step of mixing the modified heparin with the reactive monomer with an unsaturated double bond is performed in an organic solvent. In a specific example, the organic solvent is isoamyl alcohol.

In some embodiments, step S130 includes: the modified heparin, double-bonded phosphorylcholine and reaction monomers are mixed according to the mass ratio of (0.2-1): 1: (0.2-0.6) is dissolved in an organic solvent, an initiator is added, and polymerization is initiated for 12-48 h at 60-90 ℃. In an organic solvent, the total mass volume percentage of the raw materials participating in the reaction is 20% -40%, the mass volume percentage of the initiator is 1% -5%, and the ratio of the total mass of the raw materials participating in the reaction to the mass of the initiator is (10-40): 1.

step S140: coating a solution containing a polymer on the surface of a substrate, and then crosslinking and solidifying the crosslinking group to form an anticoagulant coating on the surface of the substrate.

In some embodiments, the step of applying a solution comprising a polymer to the surface of the substrate comprises: immersing the substrate in a solution containing a polymer, standing for 1-5 min, and taking out. In one embodiment, the withdrawal speed is 0.5cm/s to 2cm/s. The removal rate affects the coating amount to some extent, and is too fast, with less coating solution loading.

In one embodiment, the solvent used for the polymer-containing solution is a polar solvent. For example, the solvent used for the polymer-containing solution is ethanol, methanol, or the like.

In one embodiment, the concentration of the polymer in the solution containing the polymer is 2% -15%. The thickness of the coating is controlled by adjusting the concentration of the solution containing the polymer.

In one embodiment, prior to the step of applying the polymer-containing solution to the surface of the substrate, the method further comprises subjecting the substrate to purified water ultrasonic cleaning and drying.

In some embodiments, the manner of crosslinking curing includes: and (3) heating and shrinking covalent crosslinking among siloxanes, ultraviolet photosensitive activated free radical crosslinking of double bonds and crosslinking between hydroxyl and epoxy groups.

Further, the step of crosslinking the crosslinking group includes: heating and crosslinking the base material coated with the polymer solution at 50-90 ℃ for 12-48 hours; or alternatively, the process may be performed,

spraying 1, 4-butanediol glycidyl ether on the surface of a substrate coated with the polymer solution, and then crosslinking and curing at 25-50 ℃ for 12-24 hours; or alternatively, the process may be performed,

and (3) irradiating and curing the base material coated with the polymer solution for 1-2 hours under a UV illumination lamp with the wavelength of 254 nm.

In some embodiments, the substrate may be a commonly used medical substrate, such as PVC tubing, PE sheet, TPU tubing, and the like.

Further crosslinking and fixing are carried out by the modes of heating, ultraviolet irradiation or chemical covalent bonding and the like, so that a compact and stable crosslinked reticular coating substance is formed and attached on the surface of the substrate, and the coating is firmly attached on the surface of the substrate.

The preparation method of the anticoagulation coating has at least the following advantages:

(1) The preparation method of the anticoagulation coating comprises the steps of reacting an aldehyde group in a heparin molecule with an aldehyde group at a non-reducing end group with a single site of an amino group in an amino compound with a reactive group to form a heparin end single-active site with the reactive group, polymerizing the heparin end single-active site with a reactive monomer containing an unsaturated double bond and a crosslinking group to enable the reactive group carried by heparin and the unsaturated double bond in the reactive monomer to react covalently to form a polymer containing the crosslinking group, coating a solution containing the polymer on the surface of a substrate to realize uniform physical adsorption on the surface of the substrate, and finally crosslinking and curing the crosslinking group of the polymer to finally realize single-site connection of the heparin molecule and the substrate. Compared with the traditional modification of the multi-site molecular chains such as the carboxyl group and the sulfonic group of heparin sodium, the preparation method utilizes the single-site reaction of aldehyde groups and amino groups to carry out single-site specific modification on the end points of heparin and realize single-site connection with a substrate, so that the anticoagulation effect of the modified grafted heparin is fully ensured, and compared with other modified heparin coatings, the anticoagulation test such as the whole blood coagulation time is obviously enhanced.

(2) According to the preparation method, the reactive group is introduced into the heparin non-reducing terminal end group, the modified heparin is copolymerized with other reactive monomers, and the crosslinking group of the reactive monomers is utilized to further crosslink on the surface of the substrate, so that heparin molecules can be stably fixed on the surfaces of various substrates to be coated, the problem of high selectivity of the conventional terminal-attached heparin coating on the substrate is solved, and the method has universality on the substrate.

(3) According to the preparation method of the anticoagulation coating, the crosslinking degree of the copolymerization coating on the surface of the substrate can be controlled by adjusting the copolymerization proportion of the modified heparin and other monomers, so that the accurate regulation and control of the timeliness of the coating are realized.

(4) According to the preparation method of the anticoagulation coating, the heparin after end point modification and the double-bonded phosphorylcholine monomer are grafted together, so that the multifunctional composite coating with the ultra-efficient end point fixed heparin anticoagulation and the protein cell adsorption inert surface inhibition is prepared.

The invention also provides an anticoagulant coating of an embodiment, which comprises polymer molecules with a network structure, wherein the polymer molecules are obtained by polymerizing, crosslinking and solidifying a heparin molecule with a non-reducing terminal group and a reactive group, and a reactive monomer containing an unsaturated double bond and a crosslinking group.

In some examples, the above-described anti-coagulant coating is prepared by the preparation method of the anti-coagulant coating of the above-described embodiments.

The invention also provides application of the anticoagulation coating in preparation of a blood contact medical device.

The invention also provides a blood contact medical device in an embodiment, which comprises the anticoagulation coating.

In one embodiment, the blood contacting medical device may be tubing, sheet, film, or the like.

In order to make the objects and advantages of the present invention more apparent, the following more particular description of the method for preparing an anticoagulant coating and the effects thereof will be given by way of example only, and it should be understood that the examples are not intended to be limiting. The following examples are not specifically described but do not include other components than the unavoidable impurities. The drugs and apparatus used in the examples are all routine choices in the art, unless specifically indicated. The experimental methods without specific conditions noted in the examples were carried out according to conventional conditions, such as those described in the literature, books, or recommended by the manufacturer.

Example 1

The embodiment provides an anticoagulant coating, which is obtained by the following steps:

(1) 1L of 0.1% (w/v) aqueous heparin sodium solution was prepared, cooled to 4℃with an ice-water bath, and 10mg of sodium nitrite powder was rapidly weighed into the solution system, and the pH of the solution was adjusted to 2.5. After 1h of reaction, the pH was adjusted to 7 and the reaction quenched. And (3) dialyzing and purifying the reaction solution to obtain heparin molecule solution with aldehyde groups at the non-reducing terminal groups. Then, 2g of propenyl amine and 0.2g of sodium cyanoborohydride are added into the heparin molecule solution system with aldehyde group at the non-reducing terminal group, and amine-aldehyde condensation reaction is carried out for 0.5h at 60 ℃ to lead the terminal of the heparin molecule to carry double bond active groups. Then, dialysis and freeze-drying were carried out to obtain 0.935g of modified heparin powder.

(2) The modified heparin, 2-Methacryloxyethyl Phosphorylcholine (MPC) and triethoxyvinylsilane monomers were weighed by 0.3g, 1g and 0.5g respectively, dissolved in 5g of isoamyl alcohol, and added with 0.1g of azobisisobutyronitrile, and the monomers were initiated to polymerize at 60℃for 24 hours to prepare a polymer.

(3) And adding 1L of ethanol into the obtained viscous solution containing the polymer to dilute and stir uniformly, and preparing the coating solution. And (3) carrying out ultrasonic cleaning on the medical TPU pipe by using purified water, air-drying, immersing the medical TPU pipe in the coating solution, standing for 2min, and then slowly taking out. And then the TPU pipe with the coating solution adsorbed is placed into a 50 ℃ oven for crosslinking and fixing for 48 hours, and an anticoagulation coating is obtained.

Example 2

The present embodiment provides an anticoagulant coating obtained by the steps of:

(1) 1L of 1% (w/v) aqueous heparin sodium solution is prepared, cooled to 10 ℃ by using an ice water bath, and 0.2g of sodium nitrite powder is quickly weighed and added into a solution system, and the pH value of the solution is regulated to 3.2. After 1h of reaction, the pH was adjusted to 7 and the reaction quenched. And (3) dialyzing and purifying the reaction solution to obtain heparin molecule solution with aldehyde groups at the non-reducing terminal groups. Next, 5g of methacrylamide and 0.5g of sodium cyanoborohydride are added into the heparin molecule solution system with aldehyde groups at the non-reducing end groups, and an amine aldehyde condensation reaction is carried out for 1h at 52 ℃ so that the end points of the heparin molecules are provided with acrylate active groups. Then dialyzing and freeze-drying to obtain 9.017g of modified heparin flocculent powder.

(2) The end point modified heparin, 2-Methacryloxyethyl Phosphorylcholine (MPC) and triethoxyvinylsilane monomers are respectively weighed and evenly mixed according to the mass ratio of 0.5:1:0.2, 3g of the mixture is dissolved in 10g of isoamyl alcohol, 0.3g of azodiisobutyronitrile is added, and the polymerization of the monomers is initiated for 48 hours at 60 ℃ to prepare the polymer.

(3) Adding 1L of ethanol into the viscous solution containing the polymer obtained by the polymerization reaction, diluting and uniformly stirring to prepare a dip-coating solution. And (3) carrying out ultrasonic cleaning on the medical PVC pipe with purified water, air-drying, immersing the medical PVC pipe in the coating solution, standing for 3min, and then slowly taking out. Spreading in a constant temperature incubator, spraying 1, 4-butanediol glycidyl ether on the surface of the PVC pipe, drying at 25 ℃ for 12h for curing, and then placing the PVC pipe adsorbed with the coating solution in a 50 ℃ oven for crosslinking, drying and fixing for 48h to obtain the anticoagulation coating of the embodiment.

Example 3 to example 4

Examples 3 to 4 each provide an anticoagulant coating, which is similar to example 2 in specific preparation steps, except that the modified heparin, 2-methacryloyloxyethyl phosphorylcholine and triethoxyvinylsilane monomers in step (2) have different mass ratios, and in examples 3 and 4, the mass ratios are 0.5:1:0.4 and 0.5:1:0.6, respectively.

Example 5

The present embodiment provides an anticoagulant coating, which is specifically obtained by the following steps:

(1) 100mL of a 2% (w/v) aqueous heparin sodium solution was prepared and cooled to 0℃with an ice-water bath, and 2mg of sodium nitrite powder was rapidly weighed into the solution system and the pH of the solution was adjusted to 4. After 2.5h of reaction, the pH was adjusted to 7 and the reaction was quenched. And (3) dialyzing and purifying the reaction solution to obtain heparin molecule solution with aldehyde groups at the non-reducing terminal groups. Then, 0.1g of cyclopropylamine and 0.05g of sodium cyanoborohydride are added into the heparin molecule solution system with aldehyde groups at the non-reducing end groups, and an amine-aldehyde condensation reaction is carried out at 40 ℃ for 0.5h, so that the end points of the heparin molecules are provided with cycloalkyl unsaturated active groups. Then, dialysis and freeze-drying were carried out to obtain 1.736g of a terminal group-modified heparin flocculent powder.

(2) The above-mentioned end-modified heparin, 2-Methacryloxyethyl Phosphorylcholine (MPC) and triethoxyvinylsilane monomers were weighed by 0.80g, 1g and 0.4g, respectively, in 5g of isoamyl alcohol, and 0.15g of azobisisobutyronitrile was added to initiate polymerization of the monomers at 60℃for 24 hours to prepare a polymer.

(3) Adding 1L of ethanol into the viscous solution containing the polymer obtained by the polymerization reaction, diluting and uniformly stirring to prepare a dip-coating solution. After the medical PE sheet is subjected to ultrasonic cleaning by purified water and air-dried, the medical PE sheet is immersed in the coating solution for standing for 5min and then is taken out slowly. And then placing the sheet material adsorbed with the coating solution into a 60 ℃ oven for crosslinking, drying and fixing for 12 hours to obtain the anticoagulation coating of the embodiment.

Example 6

This example provides an anticoagulant coating, which is prepared in a similar manner to example 5, except that 2-methacryloyloxyethyl phosphorylcholine is not added in step (2).

Example 7

This example provides an anticoagulant coating, which is prepared in a similar manner to example 5, except that the phosphorylcholine used in step (2) is different from the phosphorylcholine used in this example, which is L-alpha-phosphatidylcholine.

Example 8

The present example provides an anticoagulant coating, the specific preparation steps of which are similar to those of example 5, except that step (2) and step (3) are different, and step (2) and step (3) of the present example are as follows:

(2) The end point modified heparin, 2-Methacryloxyethyl Phosphorylcholine (MPC) and 4-hydroxybutyl acrylate monomers were weighed 0.8g, 1g and 0.4g respectively, dissolved in 5g of isoamyl alcohol, and added with 0.15g of azobisisobutyronitrile, and the monomers were initiated to polymerize at 60℃for 24 hours to prepare a polymer.

(3) Adding 1L of ethanol into the viscous solution containing the polymer obtained by the polymerization reaction, diluting and uniformly stirring to prepare a dip-coating solution. After the medical PE sheet is subjected to ultrasonic cleaning by purified water and air-dried, the medical PE sheet is immersed in the coating solution for standing for 5min and then is taken out slowly. Spreading in a constant temperature incubator, spraying 1, 4-butanediol glycidyl ether on the surface, and drying at 25deg.C for 18 hr for solidification to obtain the anticoagulant coating of this example.

Example 9

The present example provides an anticoagulant coating, the specific preparation steps of which are similar to those of example 5, except that step (2) and step (3) are different, and step (2) and step (3) of the present example are as follows:

(2) The end point modified heparin, 2-Methacryloxyethyl Phosphorylcholine (MPC) and propynylacrylate monomers were weighed 0.8g, 1g and 0.4g respectively, dissolved in 5g of isoamyl alcohol, and charged with 0.15g of azobisisobutyronitrile, and the monomers were initiated to polymerize at 60℃for 24 hours to prepare a polymer.

(3) Adding 1L of ethanol into the viscous solution containing the polymer obtained by the polymerization reaction, diluting and uniformly stirring to prepare a dip-coating solution. After the medical PE sheet is subjected to ultrasonic cleaning by purified water and air-dried, the medical PE sheet is immersed in the coating solution for standing for 5min and then is taken out slowly. And then placing the sheet material adsorbed with the coating solution into a 60 ℃ oven for crosslinking, drying and fixing for 12 hours to obtain the anticoagulation coating of the embodiment.

Example 10

This example provides an anticoagulant coating, which is prepared in a manner similar to example 5, except that the amino compound used in step (1) is different, and 0.25g of ethylamine isocyanate is used in step (1) of this example to replace 0.1g of cyclopropylamine in example 5.

Example 11

This example provides an anticoagulant coating, which is prepared in a manner similar to that of example 5, except that the amino compound used in step (1) is different, and 0.2g of p-methylpropenyl phenethylamine is used in step (1) of this example instead of 0.1g of cyclopropylamine in example 5.

Example 12

This example provides an anticoagulant coating, which is prepared in a similar manner to example 9, except that the propynylacrylate monomer is used in an amount of 0.08g.

Comparative example 1

Comparative example 1 provides an anticoagulant coating, the specific preparation steps of which are similar to those of example 1, except that the modified heparin of step (1) is prepared in a different process, and steps (2) and (3) are the same as those of example 1. The procedure for the preparation of the modified heparin of step (1) of comparative example 1 is as follows: 1g of heparin sodium and 0.9g N- (3-aminopropyl) methacrylamide hydrochloride are stirred and reacted for 6 hours at 0 ℃ under the action of 0.9g of NHS and 1.2g of EDC, and then dialyzed and freeze-dried to obtain the modified heparin.

Comparative example 2

Comparative example 2 provides an anticoagulant coating which is prepared by acidifying a PE sheet and grafting polyethyleneimine by using a method of Carmeda end point attaching coating (specifically realized by O.Larm literature report method in 1983), and then forming a polyamino surface and end point aldehyde-forming heparin for covalent connection of end point attaching heparin coating by amine aldehyde condensation. Wherein the preparation of the end-point-hydroformylation heparin was the same as in example 5.

Comparative example 3

Comparative example 3 provides an anticoagulant coating, which is similar to example 5 in specific preparation steps, except that step (2) and step (3) are different, and step (2) and step (3) of comparative example 3 are specifically as follows:

(2) The end point modified heparin, 2-Methacryloxyethyl Phosphorylcholine (MPC) and dodecyl methacrylate monomers were weighed 0.8g, 1g and 0.4g respectively, dissolved in 5g of isoamyl alcohol, and added with 0.15g of azobisisobutyronitrile, and the monomers were initiated to polymerize at 60℃for 24 hours to prepare a polymer.

(3) Adding 1L of ethanol into the viscous solution containing the polymer obtained by the polymerization reaction, diluting and uniformly stirring to prepare a dip-coating solution. And (3) carrying out ultrasonic cleaning on the medical PVC pipe with purified water, air-drying, immersing the medical PVC pipe in the coating solution, standing for 5min, and then slowly taking out. And then the sheet material adsorbed with the coating solution is placed into a 60 ℃ oven for crosslinking, drying and fixing for 12 hours, so as to obtain the anticoagulation coating of the comparative example 3.

The following are specific test parts:

(1) Heparin Activity test

And (3) performing potency comparison test evaluation on the end point modified heparin sodium prepared in the step (1) of the example 1 and the molecular chain multi-site double bond modified heparin sodium prepared in the step (1) of the comparative example 1. The specific test method refers to the potency activity determination part of four 1208 heparin bioassays in Chinese pharmacopoeia 2020 edition.

Valence results example 1 and comparative example 1 were 165IU and 132IU, respectively. Whereas unmodified plain heparin sodium has an activity titer of 188IU. It can be seen from this: the end point single site modification in example 1 had less effect on heparin activity, in such a way that heparin activity was well maintained.

The two anticoagulation coatings prepared in example 1 and comparative example 1 were tested for total hemagglutination time of 78min and 23min, respectively, and the uncoated TPU tubing for total hemagglutination time of 8min. The method for testing the whole blood agglutination time comprises the following steps: the whole blood agglutination time measurement part of the four 1208 heparin bioassay is referred to in the Chinese pharmacopoeia 2020 edition.

From this, it can be seen that the anticoagulation performance of the heparin coating attached by single-site modification and single-end fixation is significantly better than that of the heparin coating attached by multi-site modification and cross-linking fixation. The clotting time was significantly longer for both coatings compared to uncoated TPU tubing.

(2) Heparin density testing of anticoagulant coatings

PVC pipes made of the polymers with the three different crosslinking monomer ratios in examples 2-4 and heparin density on the surfaces of the PVC pipes prepared in comparative example 3 are tested. Meanwhile, after simulating a blood circulation transfer machine with PBS buffer (pH 7.4) for 3 days, 7 days and 14 days, the heparin graft density of the anticoagulated coated PVC tubing was tested. The results are shown in table 1 and fig. 2 below:

TABLE 1

As can be seen from Table 1 and FIG. 2, the anticoagulation coatings prepared in examples 2-4 all have higher grafting heparin density, and the stable retention time of the prepared anticoagulation coating on the substrate is prolonged along with the increase of the proportion of the reaction monomers containing the crosslinking groups. Therefore, in the actual process, the proportion of the reaction monomers can be regulated and controlled through design, and the anti-coagulation coating with different grafting timeliness can be developed according to the needs of different actual products. While when no crosslinking groups were present (comparative example 3), the coating heparin graft density was essentially unsuccessful.

(3) Time of whole blood coagulation and anti-platelet adhesion

The anticoagulation coatings prepared in examples 5 to 12 and the anticoagulation coatings prepared in comparative examples 1 to 2 were sampled respectively for whole blood coagulation time and platelet adhesion resistance test. The results are shown in FIG. 3:

in FIG. 3, PE-Blank represents an uncoated PE sheet. As can be seen from fig. 3, the anticoagulation coating prepared in example 5, example 8, example 9, example 10 and example 11 effectively inhibited the adhesion of platelets in addition to significantly prolonging the whole blood coagulation time, compared to the heparin coating attached to the end point of comparative example 2 carbomada. Compared with the non-end group modified heparin and phosphorylcholine composite coating prepared in the comparative example 1, the anticoagulation coating prepared in the example 5 has excellent platelet adhesion inhibition property and has very obvious inhibition effect on the aggregation of whole blood; in example 6, phosphorylcholine was not added, but in example 7, phosphorylcholine was added, but the phosphorylcholine did not have a double bond covalently intercalated by copolymerization with other monomers, resulting in a difference in the inhibition of platelet adhesion between the coatings of the two processes compared with example 5.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples merely represent a few embodiments of the present invention, which facilitate a specific and detailed understanding of the technical solutions of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. It should be understood that, based on the technical solutions provided by the present invention, those skilled in the art can obtain technical solutions through logical analysis, reasoning or limited experiments, which are all within the protection scope of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.

Claims (11)

1. The preparation method of the anticoagulation coating is characterized by comprising the following steps of:

obtaining heparin molecules with aldehyde groups at non-reducing terminal groups;

carrying out an amine-aldehyde condensation reaction on the heparin molecule with aldehyde groups at the non-reducing terminal groups and an amino compound under a reducing condition to prepare modified heparin, wherein the amino compound also contains a reactive group;

mixing the modified heparin with a reaction monomer, wherein the reaction monomer is selected from any one or more of acrylic ester monomers containing crosslinking groups and olefin monomers containing crosslinking groups, and polymerizing a reactive group contained in the modified heparin and an unsaturated double bond contained in the reaction monomer to prepare a polymer containing the crosslinking groups; a kind of electronic device with high-pressure air-conditioning system

Coating a solution containing the polymer on the surface of a substrate, and then crosslinking and solidifying the crosslinking group to form an anticoagulant coating on the surface of the substrate;

wherein the reactive group comprises a carbon-carbon double bond;

the crosslinking group comprises any one or a combination of a plurality of hydroxyl groups and silica groups;

the amino compound is selected from any one or a combination of a plurality of propenyl amine, methacrylamide, para-propenyl aniline and para-methyl propenyl phenethylamine;

the reaction monomer is selected from any one or a combination of a plurality of 4-hydroxy butyl acrylate, 2-hydroxy ethyl acrylate, 3- (trimethoxysilyl) acrylic acid propyl ester, [3- (methacryloxy) propyl ] trimethoxy silane and triethoxy vinyl silane.

2. The method for preparing an anticoagulant coating according to claim 1, wherein the mass ratio of the amino compound to the heparin molecule with aldehyde group at the non-reducing terminal group is 1: (0.5 to 100).

3. The method for preparing an anticoagulant coating according to claim 1 or 2, wherein the step of subjecting the heparin molecule having an aldehyde group at the non-reducing terminal group to an amine-aldehyde condensation reaction with an amino compound satisfies at least one of the following conditions:

(1) In the process of the amine aldehyde condensation reaction, a reducing agent is also added, and the mass ratio of the reducing agent to the amino compound is 1: (1-50); and

(2) The temperature of the amine aldehyde condensation reaction is 30-60 ℃ and the time is 0.5-3 h.

4. The method for preparing an anticoagulant coating according to claim 1, wherein the mass ratio of the modified heparin to the reactive monomer is 1: (0.2-3).

5. The method for producing an anticoagulant coating according to claim 1 or 4, wherein the step of polymerizing the reactive group contained in the modified heparin and the unsaturated double bond contained in the reactive monomer satisfies at least one of the following conditions:

(1) The temperature of the polymerization reaction is 60-90 ℃, and the time of the polymerization reaction is 12-48 h;

(2) The modified heparin, the double-bonded phosphorylcholine and the reaction monomer are added with the mass ratio of (0.2-1): 1: (0.2 to 0.6); and

(3) An initiator is also added, and the ratio of the total mass of the raw materials participating in the reaction to the mass of the initiator is (10-40): 1.

6. the method of producing an anticoagulant coating according to claim 1 or 4, wherein the crosslinking group includes a siloxane group, and the step of crosslinking and curing the crosslinking group includes: heating and crosslinking the base material coated with the polymer solution at 50-90 ℃ for 12-48 hours; or alternatively, the process may be performed,

and the crosslinking group comprises hydroxyl, spraying 1, 4-butanediol glycidyl ether on the surface of the substrate coated with the solution of the polymer, and then crosslinking and curing for 12-24 hours at the temperature of 25-50 ℃.

7. The method of producing an anticoagulant coating according to claim 1, wherein the step of applying a solution containing the polymer on the surface of the substrate comprises: and immersing the substrate in a solution containing the polymer for 1-5 min, and taking out the substrate, wherein the concentration of the solution containing the polymer is 2-15%.

8. The method for preparing an anticoagulant coating according to any one of claims 1 to 2, 4 and 7, wherein the step of obtaining heparin molecules having aldehyde groups at non-reducing end groups comprises: and mixing heparin sodium and an oxidant for reaction at the temperature of 0-20 ℃ and the pH of 2.5-4 to obtain the heparin molecule with aldehyde groups at the non-reducing terminal groups.

9. The method of preparing an anticoagulant coating according to claim 8, wherein the step of obtaining heparin molecules with aldehyde groups at non-reducing end groups satisfies at least one of the following conditions:

(1) The mass ratio of the heparin sodium to the oxidant is 100: (0.1 to 5);

(2) The oxidant is selected from any one or more of sodium nitrite, nitrous acid, periodic acid and sodium periodate; and

(3) The reaction time is 1 h-3 h.

10. An anticoagulant coating, characterized in that the anticoagulant coating is prepared by the preparation method of the anticoagulant coating according to any one of claims 1 to 9.

11. Use of an anticoagulant coating according to claim 10 for the preparation of a blood contact medical device.