Heparin anticoagulant coatings and methods of making and using the same

Abstract

The present application relates to a heparin anticoagulant coating and its preparation method and application. The preparation method of the heparin anticoagulant coating comprises the following steps: modifying a substrate to make the substrate carry a mercapto group; modifying heparin with a double bond-containing carboxylic acid, a double bond-containing anhydride, a double bond-containing epoxy compound or a double bond-containing amide compound to make the heparin carry a double bond; treating the modified substrate with a solution containing the modified heparin and an initiator to make the double bond carried by the modified heparin and the mercapto group carried by the modified substrate undergo a thiol-ene click reaction under the action of the initiator, and form a heparin anticoagulant coating on the surface of the modified substrate. The preparation method of the heparin anticoagulant coating can make the heparin anticoagulant coating stably combined on the surface of the substrate while maintaining biological activity.

Description

Technical Field

[0001] This invention relates to the field of medical devices, and in particular to a heparin anticoagulant coating, its preparation method, and its application. Background Technology

[0002] Medical devices that come into contact with blood often suffer from poor biocompatibility due to the materials they are made of. Upon contact with blood, plasma proteins can rapidly adhere to the material's surface, leading to coagulation and even embolism. Therefore, systemic anticoagulation and antiplatelet therapy are generally required in clinical treatment, especially for patients requiring implanted medical devices, where regular anticoagulant injections are essential. However, this not only complicates the treatment process but also exposes patients to risks of bleeding and inflammation. Therefore, applying an anticoagulant coating to the surface of the medical device to achieve localized anticoagulation has become the simplest and most direct option.

[0003] Heparin is an excellent anticoagulant, and its anticoagulant coatings are among the most researched and widely used anticoagulant coatings. The heparin molecule contains various active groups, which can be fixed to material surfaces through electrostatic adsorption, covalent bonding, and other methods. Currently, most heparin-based anticoagulant coatings used in practice employ covalent bonding, but this method still suffers from problems such as affecting the biological activity of heparin, weak bonding between heparin and the substrate, and poor stability. Summary of the Invention

[0004] Therefore, it is necessary to provide a method for preparing a heparin anticoagulant coating, which enables the heparin anticoagulant coating to maintain its biological activity while having a high heparin content and being stably bound to the substrate surface.

[0005] In addition, it is necessary to provide a heparin anticoagulant coating and its application.

[0006] A method for preparing a heparin anticoagulant coating includes the following steps:

[0007] The substrate is modified to carry thiol groups;

[0008] Heparin is modified with a carboxylic acid containing a double bond, an anhydride containing a double bond, an epoxy compound containing a double bond, or an amide compound containing a double bond, so that the heparin carries a double bond; and

[0009] The modified substrate is treated with a solution containing modified heparin and an initiator, so that the double bonds carried by the modified heparin and the thiol groups carried by the modified substrate undergo a thiol-ene click reaction under the action of the initiator, forming a heparin anticoagulant coating on the surface of the modified substrate.

[0010] In one embodiment, the step of modifying the substrate includes:

[0011] First, the substrate is surface activated to carry carboxyl groups. Then, the substrate carrying carboxyl groups is treated with polyamino substances and thiocarboxylic acids to carry thiol groups.

[0012] In one embodiment, the step of treating the carboxyl-containing substrate with a polyamino substance and a mercaptocarboxylic acid includes:

[0013] The substrate carrying carboxyl groups is immersed in a solution containing the polyamine-like substance for 0.5 h to 3 h to graft the polyamine-like substance onto the substrate. The concentration of the polyamine-like substance in the solution is 1 mg / mL to 5 mg / mL. Then, the substrate grafted with the polyamine-like substance is immersed in a solution containing the mercaptocarboxylic acid for an amide reaction for 1 h to 8 h. The concentration of the mercaptocarboxylic acid in the solution is 1 mg / mL to 5 mg / mL. Alternatively...

[0014] First, the polyamino substance and the mercaptocarboxylic acid are subjected to an amide reaction for 0.5 h to 5 h, wherein the mass ratio of the polyamino substance to the mercaptocarboxylic acid is 1:(0.2 to 1.5). Then, the substrate carrying the carboxyl group is immersed in a solution containing the reaction product and reacted for 0.5 h to 3 h. The concentration of the reaction product in the solution is 1 mg / mL to 5 mg / mL.

[0015] In one embodiment, the step of treating the carboxyl-containing substrate with a polyamino substance and a mercaptocarboxylic acid satisfies at least one of the following conditions:

[0016] 1) The polyamino substance is selected from at least one of polyethyleneimine, chitosan, polyallylamine, polyallylamine hydrochloride, poly-L-arginine, poly-L-arginine hydrochloride, poly-L-lysine, poly-L-lysine hydrobromide, diethylenetriamine, triethylenetetramine, and tetraethylenepentamine;

[0017] 2) The thiocarboxylic acid is selected from at least one of thioacetic acid, thiopropionic acid, thiobutyric acid, thiosuccinic acid, thiovaleric acid, thiooctanoic acid, 3-mercaptoisobutyric acid and ω-mercaptohexanoic acid;

[0018] 3) During the amide reaction, a catalyst was also added, which was selected from at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride / N-hydroxysulfosuccinimide, genipin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride / 1-hydroxybenzotriazole, O-benzotriazole-tetramethylurea hexafluorophosphate, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and 1H-benzotriazole-1-yloxotris(dimethylamino)hexafluorophosphate.

[0019] In one embodiment, the polyamino substance is selected from at least one of polyethyleneimine, chitosan, and poly-L-arginine hydrochloride; the thiocarboxylic acid is selected from at least one of mercaptopropionic acid, mercaptovalerate, mercaptooctanoic acid, mercaptoacetic acid, and 3-mercaptoisobutyric acid; and a catalyst is added during the amide reaction, the catalyst being selected from at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride / N-hydroxysulfosuccinimide, genipin, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride / 1-hydroxybenzotriazole.

[0020] In one embodiment, the step of modifying the substrate includes:

[0021] First, the substrate is surface activated to carry carboxyl groups. Then, the substrate carrying carboxyl groups undergoes an amide reaction with thiolamine or thiolamine hydrochloride.

[0022] In one embodiment, the thioamine is selected from at least one of mercaptoethylamine, mercaptopropylamine, mercaptobutylamine, mercaptopentylamine, and mercaptooctylamine; and / or,

[0023] The substrate carrying the carboxyl group is immersed in a solution containing the mercaptoamine or the hydrochloride of the mercaptoamine and reacted for 1 h to 5 h. The concentration of the mercaptoamine or the hydrochloride of the mercaptoamine in the solution is 1 mg / mL to 5 mg / mL.

[0024] In one embodiment, the method for surface activation of the substrate includes at least one of plasma treatment, ultraviolet irradiation, chemical modification, and photochemical grafting.

[0025] In one embodiment, the step of surface activation of the substrate using plasma treatment includes: purging the substrate with oxygen plasma for 5 to 15 minutes; or...

[0026] The step of surface activation of the substrate using chemical modification includes: treating the substrate in concentrated sulfuric acid for 10 to 20 minutes, wherein the concentrated sulfuric acid has a mass percentage ≥ 70%; or,

[0027] The step of surface activation of the substrate using ultraviolet irradiation includes: irradiating the substrate with ultraviolet light for 10 to 30 minutes; or...

[0028] The step of surface activation of the substrate using photochemical grafting includes: placing the substrate in a solution containing a photoinitiator and a carboxylic acid, and irradiating it with ultraviolet or visible light for 10 minutes to 1 hour. The photoinitiator includes at least one of benzophenone or thioxanthone. The carboxylic acid has the general formula CHX=CY-L-COOZ, where X is hydrogen or phenyl, Y is hydrogen, a C1-C6 alkyl group, or a halogen-substituted C1-C6 alkyl group, L is a divalent linker, and Z is hydrogen or sodium. The concentration of the carboxylic acid is 1 mg / mL to 5 mg / mL, and the mass ratio of the photoinitiator to the carboxylic acid is 1:(0.01-0.1).

[0029] In one embodiment, the step of modifying the substrate includes:

[0030] The substrate surface is treated with ammonia plasma to make the substrate carry amino groups, and then the amino-carrying substrate undergoes an amide reaction with mercaptocarboxylic acid.

[0031] In one embodiment, the step of treating the substrate surface with ammonia plasma includes: purging the substrate with ammonia plasma for 5 to 30 minutes; and / or,

[0032] The step of reacting an amino-containing substrate with a mercaptocarboxylic acid includes: immersing the amino-containing substrate in a solution containing the mercaptocarboxylic acid for 1 h to 8 h, wherein the concentration of the mercaptocarboxylic acid in the solution is 1 mg / mL to 5 mg / mL.

[0033] In one embodiment, in the step of modifying heparin with a carboxylic acid or an anhydride containing a double bond, the heparin is reacted with the carboxylic acid or anhydride containing a double bond in an alkaline solution for 3 to 8 hours, wherein the mass ratio of heparin to the carboxylic acid or anhydride containing a double bond is 1:(0.05 to 0.5); or,

[0034] In the step of modifying heparin with an epoxy compound containing double bonds, the heparin and the epoxy compound containing double bonds are reacted in a solution with a pH of 7-8 for 3-8 days, and the mass ratio of the heparin to the epoxy compound containing double bonds is 1:(0.3-1); or,

[0035] In the step of modifying heparin with an amide compound containing double bonds, the heparin and the amide compound containing double bonds are stirred and reacted at 0℃~4℃ for 3h~8h under the action of an activator and a condensing agent. The mass ratio of the heparin, the amide compound containing double bonds, the activator and the condensing agent is (1~1.2):(0.8~1):(0.8~1):(0.8~1.2).

[0036] In one embodiment, the general formula of the carboxylic acid containing a double bond is CHX=CY-L-COOZ, wherein X is hydrogen or phenyl, Y is hydrogen, C1-C6 alkyl or halogen-substituted C1-C6 alkyl, L is a divalent linker, and Z is hydrogen or sodium.

[0037] The general formula of the acid anhydride containing a double bond is R1-C(=O)OC(=O)-R2, where at least one of R1 and R2 is an alkenyl group of C2 to C6, or R1 and R2 form a 5-7 membered monocyclic ring, wherein the 5-7 membered monocyclic ring contains a carbon-carbon double bond or has a substituent containing a carbon-carbon double bond.

[0038] The double-bonded epoxy compound is selected from at least one of glycidyl methacrylate, allyl glycidyl ether, epoxybutene and its homologues, 1,2-epoxy-4-vinylcyclohexane and 3,4-epoxy-1-cyclohexene.

[0039] The amide compound containing a double bond is N-(3-aminopropyl)methacrylamide or N-(3-aminopropyl)methacrylamide hydrochloride.

[0040] In one embodiment, the solution containing the modified heparin and the initiator comprises at least one of the following:

[0041] 1) The concentration of the modified heparin is 2 mg / mL to 5 mg / mL, and the mass ratio of the modified heparin to the initiator is 1:(0.005 to 0.05);

[0042] 2) The pH of the solution containing the modified heparin and the initiator is 6 to 6.8;

[0043] 3) In the step of treating the modified substrate with a solution containing modified heparin and an initiator, a coating method is used;

[0044] 4) The initiator is selected from at least one of 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone, benzophenone, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, lithium bis(2,4,6-trimethylbenzoyl)phosphinic acid, 2,2'-azo(2-methyl-N-(2-hydroxyethyl)propionamide, riboflavin, and eosin Y.

[0045] In one embodiment, the solution containing modified heparin and an initiator further contains a crosslinking agent selected from at least one of thiol crosslinking agents and double bond crosslinking agents.

[0046] In one embodiment, the solution containing the modified heparin and the initiator includes at least one of the following:

[0047] 1) The concentration of the crosslinking agent is ≤2 mg / mL;

[0048] 2) The thiol crosslinking agent is selected from at least one of dithiothreitol, pentaerythritol tetra(3-mercaptopropionate), ethoxylated pentaerythritol tetra(3-mercaptopropionate), dipentaerythritol hexamercaptoacetate, trimethylolpropane tri(2-mercaptoacetate), pentaerythritol tetra(2-mercaptoacetate), tripentaerythritol octamercaptoacetate, ethylene glycol bis(3-mercaptopropionate), thiol-terminated propoxylated glycerol, and thiol-terminated polyethylene glycol;

[0049] 3) The double bond crosslinking agent is selected from at least one of diol diacrylate, diol divinyl ether, trimethylolpropane triacrylate, pentaerythritol triacrylate, pentaerythritol tetraacrylate, 1,3,5-tricyanate triallyl ester, triallyl isocyanurate and triethylene glycol divinyl ether.

[0050] A heparin anticoagulant coating is formed on a substrate. The heparin anticoagulant coating comprises a plurality of heparin molecules modified with carboxylic acids, anhydrides, epoxy compounds, or amide compounds. The modified heparin molecules are covalently linked to the substrate in the manner of modified heparin molecules-CS-substrate.

[0051] Such as the application of heparin anticoagulant coatings in blood contact medical devices.

[0052] The above-mentioned methods for preparing heparin anticoagulant coatings involve thiolation modification of the substrate to incorporate thiol groups, double bond modification of heparin, and then direct covalent bonding of heparin to the substrate surface via a thiol-ene click reaction under the action of an initiator. This achieves chemical cross-linking and provides highly stable adhesion, ensuring that heparin molecules do not detach during use of medical devices coated with this layer. Furthermore, modifying heparin with carboxylic acids, anhydrides, epoxy compounds, or amide compounds containing double bonds offers several advantages. First, compared to the endpoint fixation method that degrades heparin to generate terminal aldehyde groups and reacts with amino groups, these methods eliminate the need for heparin degradation, preserving its activity. Second, the use of these substances provides more reaction sites for heparin, allowing for the grafting of more heparin molecules onto the substrate. Therefore, the above-mentioned methods for preparing heparin anticoagulant coatings enable the coating to maintain bioactivity while ensuring a high heparin content and stable bonding to the substrate surface. Attached Figure Description

[0053] Figure 1 A process flow diagram of a method for preparing a heparin anticoagulant coating according to one embodiment;

[0054] Figure 2 The graph shows the platelet adhesion experimental data of the heparin anticoagulant coatings prepared in Example 1 and Comparative Examples 1-4. Detailed Implementation

[0055] To facilitate understanding of the present invention, a more comprehensive description of the invention will be provided below in conjunction with specific embodiments. Preferred embodiments of the invention are given in the specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0056] Unless otherwise defined, 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 pertains. 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. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0057] In this article, "amino" refers to -NH2 or -NH-.

[0058] "Double bond" refers to a carbon-carbon double bond.

[0059] "Carrying" refers to having through covalent bonding. For example, making a substrate carry thiol groups means making the substrate have thiol groups through covalent bonding.

[0060] The "multi" in "multi-XX" means ≥3. For example, polyamino substances refer to substances with ≥3 amino groups.

[0061] "Alkyl" refers to a saturated hydrocarbon group containing a primary (normal) carbon atom, or a secondary carbon atom, or a tertiary carbon atom, or a quaternary carbon atom, or a combination thereof. Phrases containing this term, such as "C1-C6 alkyl," refer to alkyl groups containing 1 to 6 carbon atoms, and each time it appears, it can independently be C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl. Suitable examples include, but are not limited to: methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), etc.

[0062] "Alkylene" refers to a hydrocarbon group formed by removing one hydrogen atom from an alkyl group to form a hydrocarbon group with two monovalent groups at its center. The alkyl group can be a saturated branched alkyl group or a saturated straight-chain alkyl group. Suitable examples include, but are not limited to: methylene (-CH2-), 1,1-ethyl (-CH(CH3)-), 1,2-ethyl (-CH2CH2-), 1,1-propyl (-CH(CH2CH3)-), 1,2-propyl (-CH2CH(CH3)-), 1,3-propyl (-CH2CH2CH2-), and 1,4-butyl (-CH2CH2CH2CH2-), etc.

[0063] "Homologues" refer to organic compounds with similar structures and molecular compositions that differ by a few -CH2- groups.

[0064] To address the problems of low heparin activity and weak binding in current covalently bound heparin anticoagulant coatings, this invention provides a method for preparing a heparin anticoagulant coating. The heparin anticoagulant coating prepared by this invention can achieve a strong binding of heparin and maintain its biological activity, thereby enabling medical devices to have continuous anticoagulant capabilities.

[0065] Specifically, to achieve the above objectives, please refer to Figure 1 One embodiment of the method for preparing a heparin anticoagulant coating includes the following steps S110, S120 and S130:

[0066] Step S110: Modify the substrate to carry thiol groups.

[0067] The substrate is either a metal-based biomaterial or a polymer-based biomaterial. Specifically, the metal-based biomaterial is stainless steel, cobalt-based alloy, titanium and titanium alloy, or magnesium and magnesium alloy. The polymer-based biomaterial is polyester (PET), polymethyl methacrylate (PMMA), polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polytetrafluoroethylene (PTFE), polyetheretherketone (PEEK), polyurethane (PU), silicone rubber, polycaprolactone (PCL), polylactic acid (PLA), glycolide-lactide copolymer (PLGA), or polymethylpentene (PMP).

[0068] Specifically, this embodiment provides three methods for modifying the substrate.

[0069] In some embodiments, a first method for modifying the substrate includes:

[0070] First, the substrate is surface activated to enable it to carry carboxyl groups;

[0071] The substrate carrying carboxyl groups is then treated with polyamino substances and thiocarboxylic acids to make the substrate carry thiol groups.

[0072] Specifically, in this method, during the modification of the substrate, the substrate can carry thiol groups as well as amino groups.

[0073] Specifically, methods for surface activation of the substrate include at least one of plasma treatment, ultraviolet irradiation, chemical modification, and photochemical grafting. Surface activation treatment of the substrate imbues it with carboxyl groups. The reaction between carboxyl and amino groups introduces thiol groups onto the substrate, and these thiol groups are chemically bonded to the substrate, resulting in a more stable bond.

[0074] In one embodiment, the step of surface activation of the substrate using plasma treatment includes purging the substrate with oxygen plasma for 5 to 15 minutes. For example, purging the substrate with oxygen plasma for 5 minutes, 8 minutes, 10 minutes, 12 minutes, 15 minutes, or any range of two of these values.

[0075] The steps of surface activation of the substrate using chemical modification methods include: treating the substrate in concentrated sulfuric acid for 10 to 20 minutes, wherein the mass percentage of concentrated sulfuric acid is ≥70%. For example, the substrate is treated in concentrated sulfuric acid for 10 minutes, 12 minutes, 15 minutes, 18 minutes, 20 minutes, or any combination of these values.

[0076] The steps of surface activation of a substrate using ultraviolet irradiation include irradiating the substrate with ultraviolet light for 10 to 30 minutes. For example, irradiating the substrate with ultraviolet light for 10, 12, 15, 18, 20, 22, 25, 28, or 30 minutes, or any combination of these values.

[0077] The steps for surface activation of a substrate using photochemical grafting include: placing the substrate in a solution containing a photoinitiator and a carboxylic acid, and irradiating it with ultraviolet or visible light for 10 minutes to 1 hour. The photoinitiator includes at least one of benzophenone and thioxanthone. The carboxylic acid has the general formula CHX=CY-L-COOZ, where X is hydrogen or phenyl, Y is hydrogen, a C1-C6 alkyl group, or a halogen-substituted C1-C6 alkyl group, L is a divalent linker, and Z is hydrogen or sodium. The concentration of the carboxylic acid is 1 mg / mL to 5 mg / mL, and the mass ratio of the photoinitiator to the carboxylic acid is 1:(0.01 to 0.1).

[0078] In a specific example, in the step of surface activation of the substrate using photochemical grafting, the substrate is irradiated with ultraviolet or visible light for 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, or any combination of these values.

[0079] In a specific example, the carboxylic acid can be acrylic acid, butenoic acid, pentenoic acid, undecenoic acid, oleic acid, methacrylic acid, methylbutenoic acid, sodium acrylate, sodium butenoate, sodium pentenoate, sodium undecenoate, sodium oleate, sodium methacrylate, sodium methylbutenoate, cinnamic acid, trifluoromethacrylic acid, etc.

[0080] In a specific example, the concentration of carboxylic acid is 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, or a range of any two of these values.

[0081] In a specific example, the mass ratio of photoinitiator to carboxylic acid is 1:0.01, 1:0.02, 1:0.03, 1:0.04, 1:0.05, 1:0.06, 1:0.07, 1:0.08, 1:0.09, 1:0.1, or a range of any two of these values.

[0082] In one embodiment, the step of treating a carboxyl-containing substrate with a polyamine and a mercaptocarboxylic acid includes: first reacting the carboxyl-containing substrate with a polyamine to graft the polyamine onto the substrate; and then reacting the substrate grafted with the polyamine with a mercaptocarboxylic acid to undergo an amide reaction.

[0083] Specifically, the polyamino substance is selected from at least one of polyethyleneimine, chitosan, polyallylamine, polyallylamine hydrochloride, poly-L-arginine, poly-L-arginine hydrochloride, poly-L-lysine, and poly-L-lysine hydrobromide. Further, the polyamino substance is selected from at least one of polyethyleneimine, chitosan, and poly-L-arginine hydrochloride.

[0084] Specifically, the step of reacting the carboxyl-containing substrate with the polyamine-based substance includes: reacting the carboxyl-containing substrate with the polyamine-based substance in a first solution for 0.5 h to 3 h, followed by washing and drying. In a specific example, washing with purified water followed by freeze-drying.

[0085] In one specific example, the first solution is pure water, PBS buffer (phosphate buffer), or Tris-HCl buffer (tris(hydroxymethyl)aminomethane-hydrochloric acid buffer). In the first solution, the concentration of the polyamine is 1 mg / mL to 5 mg / mL. In one specific example, the concentration of the polyamine is 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, or a range of any two of these values.

[0086] Specifically, the step of reacting the substrate grafted with polyamines with thiocarboxylic acid includes: reacting the substrate grafted with polyamines with thiocarboxylic acid in a second solution for 1 h to 8 h, followed by washing and drying. In a specific example, the substrate is washed with purified water and then dried.

[0087] In a specific example, the thiocarboxylic acid is selected from at least one of mercaptoacetic acid, mercaptopropionic acid, mercaptobutyric acid, mercaptosuccinic acid, mercaptovalerate, mercaptooctanoic acid, 3-mercaptoisobutyric acid, and ω-mercaptohexanoic acid. Further, the thiocarboxylic acid is selected from at least one of mercaptopropionic acid, mercaptovalerate, mercaptooctanoic acid, mercaptoacetic acid, and 3-mercaptoisobutyric acid.

[0088] In a specific example, the concentration of thiocarboxylic acid in the second solution is between 1 mg / mL and 5 mg / mL. For example, the concentration of thiocarboxylic acid in the second solution is 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, or a range of any two of these values. Further, the concentration ratio of thiocarboxylic acid in the second solution to the concentration of the polyamino substance in the first solution is 1:1.

[0089] In a specific example, the second solution is pure water, PBS buffer, Tris-HCl buffer, or MES buffer (2-(N-morpholine) ethanesulfonic acid buffer).

[0090] Furthermore, a catalyst can be added during the amide reaction. Specifically, the catalyst is selected from at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride / N-hydroxysulfosuccinimide (EDC / NHS), genipin, EDC / HOBT (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride / 1-hydroxybenzotriazole), HBTU (O-benzotriazole-tetramethylurea hexafluorophosphate), HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate), and BOP (1H-benzotriazole-1-yloxotris(dimethylamino)hexafluorophosphate). Furthermore, the catalyst is selected from at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride / N-hydroxysulfosuccinimide, genipin, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride / 1-hydroxybenzotriazole. It is understood that a catalyst may not be added during the amide reaction.

[0091] In another embodiment, the step of treating the carboxyl-containing substrate with a polyamine and a mercaptocarboxylic acid includes: firstly, reacting the polyamine with the mercaptocarboxylic acid with an amide, and then treating the carboxyl-containing substrate with the product of the reaction. Further, the step of treating the carboxyl-containing substrate with the polyamine and mercaptocarboxylic acid includes: firstly, reacting the polyamine with the mercaptocarboxylic acid with an amide for 0.5 h to 5 h, wherein the mass ratio of the polyamine to the mercaptocarboxylic acid is 1:(0.2 to 1.5), and then immersing the carboxyl-containing substrate in a solution containing the product of the reaction for 0.5 h to 3 h, wherein the concentration of the product in the solution is 1 mg / mL to 5 mg / mL. Specifically, the polyamine and mercaptocarboxylic acid are the same as described above and will not be repeated.

[0092] Specifically, the step of reacting the polyamine with a mercaptocarboxylic acid via an amide reaction includes: reacting the polyamine with the mercaptocarboxylic acid in a second solution for 0.5 h to 5 h, followed by washing and drying. Specifically, the mass ratio of the polyamine to the mercaptocarboxylic acid is 1:(0.2–1.5). In one specific example, the mass ratio of the polyamine to the mercaptocarboxylic acid is 1:0.2, 1:0.5, 1:0.8, 1:1, 1:1.2, 1:1.5, or any combination of these values. Further, the concentration of the mercaptocarboxylic acid in the second solution is 1 mg / mL to 5 mg / mL. In one specific example, the concentration of the mercaptocarboxylic acid is 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, or any combination of these values. In one specific example, the mixture is washed with purified water and then freeze-dried.

[0093] Specifically, the step of treating the carboxyl-containing substrate with the reaction product includes: immersing the carboxyl-containing substrate in a first solution containing the reaction product for 0.5 h to 3 h, followed by washing and drying. In a specific example, washing with purified water followed by drying is performed. The concentration of the reaction product in the first solution is 1 mg / mL to 5 mg / mL. In a specific example, the concentration of the reaction product is 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, or a range of any two of these values.

[0094] During substrate modification, grafting polyamines onto the substrate surface can increase the number of active groups, thereby increasing the amount of thiolation and further improving heparin density. Furthermore, the positive charge of polyamines allows them to adsorb more heparin molecules before the thiol-ene click reaction occurs. This is particularly suitable for medical device surfaces that require prolonged contact with blood.

[0095] In other embodiments, a second method for surface modification of the substrate includes: treating the substrate surface with ammonia plasma to imbue the substrate with amino groups, and then reacting the amino-imbued substrate with a mercaptocarboxylic acid via an amide reaction.

[0096] Specifically, the steps of treating the substrate surface with ammonia plasma include: purging the substrate with ammonia plasma for 5 to 30 minutes. For example, purging the substrate with ammonia plasma for 5 minutes, 8 minutes, 10 minutes, 15 minutes, 18 minutes, 20 minutes, 22 minutes, 25 minutes, 28 minutes, 30 minutes, or any combination of these values.

[0097] Specifically, the reaction of the amino-containing substrate with the amide of a mercaptocarboxylic acid is carried out in a second solution, wherein the concentration of the mercaptocarboxylic acid in the second solution is 1 mg / mL to 5 mg / mL. The reaction time is 1 h to 8 h. In a specific example, the concentration of the mercaptocarboxylic acid is 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, or any combination of these values. The reaction time is 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, or any combination of these values.

[0098] The reaction also includes steps of washing with pure water and freeze-drying.

[0099] Similarly, a catalyst may or may not be added during the amide reaction. The catalyst is the same as described above and will not be repeated here.

[0100] In other embodiments, a third method for surface modification of the substrate includes: activating the substrate surface to carry carboxyl groups, and then reacting the carboxyl-carrying substrate with a mercaptoamine or a mercaptoamine hydrochloride via an amide reaction.

[0101] Specifically, the method for surface activation of the substrate is the same as described above and will not be repeated here.

[0102] Specifically, the thioamine is selected from at least one of mercaptoethylamine, mercaptopropylamine, mercaptobutylamine, mercaptopentylamine, and mercaptooctylamine. Further, the thioamine is selected from at least one of mercaptoethylamine and mercaptooctylamine.

[0103] Specifically, the amide reaction of the carboxyl-containing substrate with a thiolamine or its hydrochloride is carried out in a third solution, which may be pure water, PBS buffer, Tris-HCl buffer, MES buffer (2-(N-morpholine)ethanesulfonic acid buffer), methanol, ethanol, acetone, or isopropanol. Further, in the third solution, the concentration of the thiolamine or its hydrochloride is between 1 mg / mL and 5 mg / mL. For example, the concentration of the thiolamine or its hydrochloride may be 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, or a range of any two of these values.

[0104] The reaction time is 1 h to 5 h. In a specific example, the reaction time is 1 h, 2 h, 3 h, 4 h, 5 h, or any range of two of these values.

[0105] The reaction also includes steps of washing with pure water and freeze-drying.

[0106] Similarly, a catalyst may or may not be added during the amide reaction. The catalyst is the same as described above and will not be repeated here.

[0107] The above three methods are used to modify the substrate surface, enabling the substrate to carry thiol groups. Different thiolization methods can be selected based on the substrate and the degree of thiolization. Furthermore, the thiolized groups on the substrate surface are covalently linked to the substrate, exhibiting very stable bonding.

[0108] Step S120: Modify heparin with a carboxylic acid containing a double bond, an anhydride containing a double bond, an epoxy compound containing a double bond, or an amide compound containing a double bond so that heparin carries a double bond.

[0109] In some embodiments, the step of modifying heparin with a carboxylic acid containing a double bond or an anhydride containing a double bond includes: reacting heparin with a carboxylic acid containing a double bond or an anhydride containing a double bond in an alkaline solution for 3 to 8 hours, followed by dialysis and freeze-drying.

[0110] Specifically, the mass ratio of heparin to a carboxylic acid or anhydride containing a double bond is 1:(0.05 to 0.5). In a specific example, the mass ratio of heparin to a carboxylic acid or anhydride containing a double bond is 1:0.05, 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, or a range of any two of these values.

[0111] Specifically, the general formula for carboxylic acids containing double bonds is CHX=CY-(L). n-COOZ, where X is hydrogen or phenyl, Y is hydrogen, a C1-C6 alkyl group, or a halogen-substituted C1-C6 alkyl group, L is a divalent linker, n is 0 or 1, and Z is hydrogen or sodium. Further, Y is hydrogen, methyl, or a halogen-substituted methyl group. For example, a halogen-substituted methyl group can be trifluoromethyl. Preferably, L is an alkylene group. The alkylene group can be straight-chain or branched. For example, the alkylene group can be methylene (-CH2-), 1,1-ethyl (-CH(CH3)-), 1,2-ethyl (-CH2CH2-), 1,1-propyl (-CH(CH2CH3)-), 1,2-propyl (-CH2CH(CH3)-), 1,3-propyl (-CH2CH2CH2-), and 1,4-butyl (-CH2CH2CH2CH2-).

[0112] In one embodiment, the carboxylic acid containing the double bond is CH2=CH-(L). n -COOH, CH2=CH-(L) n -COONa、CH2=C(CH3)-(L) n -COOH, CH2=C(CH3)-(L) n -COONa, CH2=C(CF3)-COOH, or C6H5-CH=CH-COOH, where n is 0 or 1, and L is an alkylene group. In a specific example, the carboxylic acid containing a double bond can be acrylic acid, butenoic acid, pentenoic acid, undecenoic acid, oleic acid, methacrylic acid, methylbutenoic acid, sodium acrylate, sodium butenoate, sodium pentenoate, sodium undecenoate, sodium oleate, sodium methacrylate, sodium methylbutenoate, cinnamic acid, trifluoromethacrylic acid, etc.

[0113] Specifically, the general formula for acid anhydrides containing double bonds is R1-C(=O)OC(=O)-R2, where at least one of R1 and R2 is a C2-C6 alkenyl group; or, R1 and R2 form a 5-7 membered monocyclic ring containing a carbon-carbon double bond or having a substituent containing a carbon-carbon double bond. Further, the acid anhydride containing double bonds is selected from at least one of acrylic anhydride, methacrylic anhydride, and maleic anhydride.

[0114] In one embodiment, the pH of the alkaline solution may be 8. The alkaline solution may be an aqueous solution of sodium hydroxide.

[0115] In other embodiments, the step of modifying heparin with an epoxy compound containing double bonds includes reacting heparin with the epoxy compound containing double bonds in a solution at pH 7-8 for 3-8 days, followed by dialyzing and freeze-drying.

[0116] Specifically, the mass ratio of heparin to the double-bonded epoxide is 1:(0.3 to 1). In a specific example, the mass ratio of heparin to the double-bonded epoxide is 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, or a range of any two of these values.

[0117] Furthermore, the double-bonded epoxy compound is selected from at least one of glycidyl methacrylate, allyl glycidyl ether, 1,2-epoxy-4-vinylcyclohexane, 3,4-epoxy-1-cyclohexene, epoxybutene, and their homologues.

[0118] In one embodiment, the solution with a pH of 7 to 8 may be a phosphate buffer solution.

[0119] In some other embodiments, the step of modifying heparin with an amide compound containing a double bond includes: reacting heparin with an amide compound containing a double bond at 0°C to 4°C for 3 to 8 hours under the action of an activator and a condensing agent, followed by dialysis and freeze-drying.

[0120] Further, the mass ratio of heparin, the double-bonded amide compound, the activator, and the condensing agent is (1-1.2):(0.8-1):(0.8-1):(0.8-1.2). Preferably, the mass ratio of heparin, the double-bonded amide compound, the activator, and the condensing agent is 1.2:1:1:1. In one embodiment, the activator is NHS (N-hydroxysulfosuccinimide), and the condensing agent is EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride).

[0121] In one embodiment, the amide compound containing the double bond is N-(3-aminopropyl)methacrylamide or N-(3-aminopropyl)methacrylamide hydrochloride.

[0122] During the modification of heparin, the reaction sites of heparin can be regulated by different modification methods, such as carboxylic acid / anhydride modification, epoxy modification, amide modification, etc. Furthermore, the amount of heparin double bond can be effectively controlled by adjusting the ratio of heparin to the modifying agent, thereby controlling its crosslinking density while ensuring the biological activity of heparin.

[0123] Double bond modification of heparin can be achieved by adjusting its reaction sites and the number of double bonds through different modification methods, thereby controlling the final cross-linking degree and heparin density while ensuring the biological activity of heparin.

[0124] Step S130: Treat the modified substrate with a solution containing modified heparin and an initiator, so that the double bonds carried by the modified heparin and the thiol groups carried by the modified substrate undergo a thiol-ene click reaction under the action of the initiator, forming a heparin anticoagulant coating on the surface of the modified substrate.

[0125] Specifically, in the solution containing modified heparin and an initiator, the concentration of modified heparin is 2 mg / mL to 5 mg / mL. In a specific example, in the solution containing modified heparin and an initiator, the concentration of modified heparin is 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, or a range of any two of these values.

[0126] Furthermore, in the solution containing modified heparin and an initiator, the mass ratio of modified heparin to initiator is 1:(0.005–0.05). In a specific example, the mass ratio of modified heparin to initiator is 1:0.005, 1:0.01, 1:0.02, 1:0.03, 1:0.04, 1:0.05, or a range of any two of these values.

[0127] Specifically, the initiator is a photoinitiator. The photoinitiator is selected from at least one of Irgacure 2959 (2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone), benzophenone, TPO (diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide), LAP (lithium phenyl(2,4,6-trimethylbenzoyl)phosphate), BAPO-OLi (lithium bis(2,4,6-trimethylbenzoyl)phosphine), VA-086 (2,2'-azo(2-methyl-N-(2-hydroxyethyl)propionamide), riboflavin, and eosin Y. Further, the photoinitiator is selected from at least one of Irgacure 2959, LAP, VA-086, benzophenone, and riboflavin.

[0128] The thiol-ene click reaction has advantages such as insensitivity to oxygen and water, fast reaction rate, controllable crosslinking degree, small initiator dosage, and in-situ reaction, and has been widely used in coating preparation. In this embodiment, the thiol-ene click reaction is used to covalently link heparin and the substrate, which has advantages such as high efficiency, environmental protection, and economy. Furthermore, since the conversion rate of this reaction is almost 100%, the number of residual active functional groups in the entire coating can be easily reduced to a minimum by adjusting the ratio of double bonds to thiols, thereby improving the safety of the coating. In addition, the anticoagulant coating based on the thiol-ene click reaction for immobilizing heparin prepared in this embodiment can effectively ensure that the biological activity of heparin is not significantly affected, while significantly enhancing the bonding force between heparin and the substrate surface, avoiding or reducing the shedding of heparin molecules. Moreover, the thiol-ene click reaction uses photoinitiation, making the entire preparation process more economical and environmentally friendly. This embodiment also provides a variety of photoinitiators to meet the needs of different materials and preparation environments.

[0129] Furthermore, compared to other thiol-ene click reactions, such as those involving thiols with isocyanates or bromine, the bromination and isocyanate modification of heparin are difficult to achieve in this embodiment. This is especially true because isocyanate groups react with water, while heparin is almost entirely soluble in water, making the reaction impossible. Therefore, in this embodiment, the thiol-ene click reaction is chosen to immobilize heparin. Additionally, the inventors found in experiments that while thiolation modification of heparin combined with a double-bonded modified substrate can also achieve heparin immobilization, thiolated heparin is prone to deterioration during purification and storage, which is detrimental to practical applications. Moreover, maleimide is difficult to react with amides, and this embodiment utilizes an amide reaction to connect with heparin. Therefore, the reaction between maleimide and thiols is not applicable in this embodiment.

[0130] In one embodiment, in the step of causing the double bond carried by the modified heparin to undergo a thiol-ene click reaction with the thiol group carried by the modified substrate, the substrate is irradiated with ultraviolet or visible light for 15 minutes to 2 hours.

[0131] Furthermore, in some embodiments, the solution containing modified heparin and the initiator also contains a crosslinking agent selected from at least one of thiol crosslinking agents and double bond crosslinking agents. Further, in the solution containing modified heparin and the initiator, the concentration of the crosslinking agent is ≤2 mg / mL. In a specific example, the concentration of the crosslinking agent is 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, or a range of any two of these values.

[0132] Specifically, the thiol crosslinking agent is selected from at least one of dithiothreitol, pentaerythritol tetra(3-mercaptopropionate), ethoxylated pentaerythritol tetra(3-mercaptopropionate), dipentaerythritol hexamercaptoacetate, trimethylolpropane tri(2-mercaptoacetate), pentaerythritol tetra(2-mercaptoacetate), tripentaerythritol octamercaptoacetate, ethylene glycol bis(3-mercaptopropionate), thiol-terminated propoxylated glycerol sold under Capcure 3-800, and thiol-terminated polyethylene glycol. For example, the thiol crosslinking agent is dithiothreitol.

[0133] The double bond crosslinking agent is selected from at least one of glycol diacrylates, glycol divinyl ethers, trimethylolpropane triacrylate, pentaerythritol triacrylate, pentaerythritol tetraacrylate, triallyl 1,3,5-cyanurate, triallyl isocyanurate, and triethylene glycol divinyl ether. In a specific example, the glycol diacrylate may be ethylene glycol diacrylate, propylene glycol diacrylate, or other glycol diacrylates.

[0134] Depending on the ratio of thiolation and heparin double bond formation in the substrate, a crosslinking agent may or may not be added, the purpose of which is to ensure that the thiols and double bonds react fully.

[0135] In some embodiments, the pH of the solution containing modified heparin and the initiator is 6 to 6.8. In one embodiment, the solution containing modified heparin and the initiator is PBS buffer.

[0136] In some embodiments, the step of treating the modified substrate with a solution containing modified heparin and an initiator is performed by coating. Further, the coating method is dip coating, spray coating, or spin coating.

[0137] In a specific example, in the step of treating the modified substrate with a solution containing modified heparin and an initiator, a spraying method is used, with a spraying amount of 0.05 mL / cm². 2 ~0.08mL / cm 2 .

[0138] In a specific example, in the step of treating the modified substrate with a solution containing modified heparin and an initiator, a dip coating method is used, and the dip coating time is 5 to 15 minutes.

[0139] Furthermore, it also includes steps of washing and drying with pure water.

[0140] The above-mentioned method for preparing the heparin anticoagulant coating has at least the following advantages:

[0141] (1) The above-mentioned methods for preparing heparin anticoagulant coatings involve thiolation modification of the substrate and double bond modification of heparin. Through thiolation-ene click reaction, heparin is directly covalently bonded to the substrate surface under the action of an initiator, thereby forming a chemical crosslink with very stable bonding force, forming a coating with a continuous anticoagulant effect, thus ensuring that heparin molecules do not fall off during the use of medical devices coated with this coating. Moreover, the thiolation groups on the substrate surface are also covalently linked to the substrate, exhibiting very stable bonding force. Modifying heparin with carboxylic acids, anhydrides, epoxy compounds, or amide compounds containing double bonds can control the active sites of heparin. On the one hand, compared with the method of using endpoint fixation to degrade heparin and generate terminal aldehyde groups, which then react with the substrate, there is no need to degrade heparin, thus ensuring the activity of heparin. On the other hand, heparin has more reaction sites, thereby allowing more heparin molecules to be grafted onto the substrate.

[0142] (2) The above-mentioned methods for preparing heparin anticoagulant coatings provide various methods for thiolation modification of the substrate surface, allowing for the selection of different thiolation methods depending on the substrate and the degree of thiolation. Furthermore, the double bond modification of heparin can be achieved by adjusting its reaction sites and the number of double bonds through different modification methods, thereby controlling the final crosslinking degree and heparin density while ensuring the biological activity of heparin.

[0143] (3) In the preparation method of the above-mentioned heparin anticoagulant coating, during the substrate modification process, the substrate surface can be grafted with polyamine substances to increase the number of active groups, thereby increasing the amount of thiolation and further increasing the heparin density. On the other hand, polyamine substances are positively charged and can also adsorb more heparin molecules before the thiol-ene click reaction occurs. This is particularly suitable for the surface of medical devices that need to be in contact with blood for a long time.

[0144] (4) The above-mentioned method for preparing the heparin anticoagulant coating uses a thiol-ene click reaction to graft heparin onto the substrate. The thiol-ene click reaction itself has the characteristics of high conversion rate and high efficiency. This reaction occurs through free radical polymerization under the action of a photoinitiator, giving it the environmentally friendly, economical, and efficient characteristics of photoinitiation. In addition, the photoinitiator can be selected according to the requirements of light wavelength and time, initiator concentration, etc. Furthermore, using the thiol-ene click reaction to connect heparin and the substrate can achieve a very high conversion rate of double bonds and thiols. By adding thiol crosslinking agents or double bond crosslinking agents, the ratio of double bonds and thiols can be further controlled to achieve almost 100% conversion of double bonds and thiols, minimizing the number of residual active functional groups in the entire coating and thus ensuring the safety of the coating.

[0145] The present invention also provides a heparin anticoagulant coating according to one embodiment, the heparin anticoagulant coating being formed on a substrate, the heparin anticoagulant coating comprising a plurality of heparin molecules modified with carboxylic acids, anhydrides, epoxy compounds or amide compounds, the modified heparin molecules being covalently linked to the substrate in the following manner: modified heparin molecule-CS-substrate.

[0146] Specifically, the heparin anticoagulant coating is prepared by the heparin anticoagulant coating preparation method described in the above embodiments.

[0147] The present invention also provides an embodiment of the application of a heparin anticoagulant coating in the preparation of blood contact medical devices.

[0148] To make the objectives and advantages of the present invention clearer, the preparation method and effects of the heparin anticoagulant coating of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and should not be used to limit the present invention.

[0149] Comparative Example 1

[0150] Comparative Example 1 provides a method for preparing a heparin anticoagulant coating, which is obtained through the following steps:

[0151] (1) Modification of the substrate

[0152] After washing and drying the polyethylene film with purified water, it was purged with oxygen plasma for 10 minutes. The film was then placed in a 2 mg / mL PEI aqueous solution and reacted for 1 hour, followed by washing with purified water and drying.

[0153] (2) Double bond modification of heparin

[0154] 1g of heparin was reacted with 0.1g of methacrylic anhydride in an aqueous sodium hydroxide solution with a pH of 8 for 8 hours, followed by dialyzing and freeze-drying to obtain the modified heparin.

[0155] (3) Preparation of heparin solution

[0156] Take 0.8g of the modified heparin prepared in step (2) and mix it with 25mg of Irgacure2959 to prepare PBS buffer with a pH of 6.4. The concentration of the modified heparin is 2mg / mL.

[0157] (4) Coating preparation

[0158] The heparin solution prepared in step (3) was sprayed onto the modified polyethylene film prepared in step (1) (spraying amount: 0.05 mL / cm). 2After spraying, the film was continuously irradiated with 275nm ultraviolet light for 1 hour. Then it was washed with purified water, dried, and finally coated with a heparin anticoagulant coating onto the polyethylene film.

[0159] Comparative Example 2

[0160] Comparative Example 2 provides a method for preparing a heparin anticoagulant coating, which is obtained through the following steps:

[0161] (1) Thiol modification of the substrate

[0162] The polyethylene film was washed sequentially with isopropanol and then with 5M NaOH solution. The film was then placed in a 2 mg / mL PEI aqueous solution for 1 hour to allow the polyethylene film to adsorb PEI. The film was then washed with purified water and dried.

[0163] The polyethylene film adsorbed with PEI was reacted with a 2 mg / mL aqueous solution of mercaptopropionic acid using EDC / NHS as a catalyst for 4 hours. The film was then washed with purified water and dried to obtain the thiol-modified polyethylene film.

[0164] (2) Double bond modification of heparin

[0165] 1 g of heparin was reacted with 0.1 g of methacrylic anhydride in an aqueous sodium hydroxide solution at pH 8 for 8 hours, followed by dialyzing and freeze-drying to obtain heparin with double bond modification.

[0166] (3) Preparation of heparin solution

[0167] Take 0.8g of the double-bonded modified heparin prepared in step (2) and mix it with 25mg of Irgacure2959 to prepare PBS buffer with a pH of 6.4. The concentration of the double-bonded modified heparin is 2mg / mL.

[0168] (4) Coating preparation

[0169] The heparin solution prepared in step (3) was sprayed onto the thiol-modified polyethylene film prepared in step (1) (spraying amount: 0.05 mL / cm). 2 After spraying, the film was continuously irradiated with 275nm ultraviolet light for 1 hour. Then it was washed with purified water, dried, and finally coated with a heparin anticoagulant coating onto the polyethylene film.

[0170] Comparative Example 3

[0171] Comparative Example 3 provides a method for preparing a heparin anticoagulant coating, which is obtained through the following steps:

[0172] (1) Thiol modification of the substrate

[0173] The polyethylene film was washed with purified water, dried, and then purged with oxygen plasma for 10 minutes. The film was then placed in a 2 mg / mL PEI aqueous solution and reacted for 1 hour. Afterward, it was washed with purified water and dried to obtain a PEI-grafted polyethylene film.

[0174] The PEI-grafted polyethylene film was reacted with a 2 mg / mL aqueous solution of mercaptopropionic acid using EDC / NHS as a catalyst for 4 hours. The film was then washed with purified water and dried to obtain the thiol-modified polyethylene film.

[0175] (2) Double bond modification of heparin

[0176] 1g of heparin with aldehyde groups, degraded by nitrite, was reacted with 0.4g of 2-methylallylamine in an aqueous solution for 2 days, and the pH was adjusted to 4 with 4M NaOH. After the reaction was completed, the product was purified by dialysis and freeze-dried to obtain modified heparin with double bonds at the end groups.

[0177] (3) Preparation of heparin solution

[0178] Take 0.8g of the double-bonded modified heparin prepared in step (2) and mix it with 25mg of Irgacure2959 to prepare PBS buffer with a pH of 6.4. The concentration of the double-bonded modified heparin is 2mg / mL.

[0179] (4) Coating preparation

[0180] The heparin solution prepared in step (3) was sprayed onto the thiol-modified polyethylene film prepared in step (1) (spraying amount: 0.05 mL / cm). 2 After spraying, the film was continuously irradiated with 275nm ultraviolet light for 1 hour. Then it was washed with purified water, dried, and finally coated with a heparin anticoagulant coating onto the polyethylene film.

[0181] Comparative Example 4

[0182] Comparative Example 4 provides a method for preparing a heparin anticoagulant coating, which is obtained through the following steps:

[0183] (1) Modify the substrate by double bond formation

[0184] The polyethylene film was washed with purified water, dried, and then purged with ammonia plasma for 10 minutes. The film was then placed in a 2 mg / mL aqueous solution of acrylic acid for 4 hours, followed by washing with purified water and drying to obtain the double-bonded modified polyethylene film.

[0185] (2) Thiolization modification of heparin

[0186] 1g of heparin was reacted with 0.1g of mercaptopropionic acid in an aqueous sodium hydroxide solution at pH 8 for 8 hours, followed by dialyzing and freeze-drying to obtain thiolated modified heparin.

[0187] (3) Preparation of heparin solution

[0188] Take 0.8g of the thiolated heparin prepared in step (2) and mix it with 25mg of Irgacure2959 to prepare PBS buffer with a pH of 6.4 and a concentration of 2mg / mL of the thiolated heparin.

[0189] (4) Coating preparation

[0190] The heparin solution prepared in step (3) was sprayed onto the double-bonded modified polyethylene film prepared in step (1) (spraying amount: 0.05 mL / cm). 2 After spraying, the film was continuously irradiated with 275nm ultraviolet light for 1 hour. Then it was washed with purified water, dried, and finally coated with a heparin anticoagulant coating onto the polyethylene film.

[0191] Examples 1 to 3

[0192] Examples 1 through 3 all provide a method for preparing a heparin anticoagulant coating, which is obtained through the following steps:

[0193] (1) Thiol modification of the substrate

[0194] The substrate was modified by thiolation using the following three methods. The heparin anticoagulant coating obtained by modifying the substrate using method 1 and through subsequent steps (2), (3), and (4) is the heparin anticoagulant coating prepared in Example 1. The heparin anticoagulant coating obtained by modifying the substrate using method 2 and through subsequent steps (2), (3), and (4) is the heparin anticoagulant coating prepared in Example 2. The heparin anticoagulant coating obtained by modifying the substrate using method 3 and through subsequent steps (2), (3), and (4) is the heparin anticoagulant coating prepared in Example 3.

[0195] Specifically, method 1:

[0196] The polyethylene film was washed with purified water, dried, and then purged with oxygen plasma for 10 minutes. The film was then placed in a 2 mg / mL PEI aqueous solution and reacted for 1 hour. Afterward, it was washed with purified water and dried to obtain a PEI-grafted polyethylene film.

[0197] The PEI-grafted polyethylene film was reacted with a 2 mg / mL aqueous solution of mercaptopropionic acid using EDC / NHS as a catalyst for 4 hours. The film was then washed with purified water and dried to obtain the thiol-modified polyethylene film.

[0198] Method 2:

[0199] The polyethylene film was washed with purified water, dried, purged with oxygen plasma for 10 minutes, and then immersed in a 4 mg / mL mercaptoethylamine aqueous solution. A 0.2 mg / mL EDC / NHS catalyst was used as the catalyst, and the reaction time was 4 hours. The film was then washed with purified water and dried to obtain the thiol-modified polyethylene film.

[0200] Method 3:

[0201] The polyethylene film was washed with purified water, dried, and then purged with ammonia plasma for 10 minutes. It was then placed in a 4 mg / mL aqueous solution of mercaptopropionic acid, using 0.2 mg / mL EDC / NHS as a catalyst, and reacted for 4 hours. Afterward, it was washed with purified water and dried to obtain the thiol-modified polyethylene film.

[0202] (2) Double bond modification of heparin

[0203] 1 g of heparin was reacted with 0.1 g of methacrylic anhydride in an aqueous sodium hydroxide solution at pH 8 for 8 hours, followed by dialyzing and freeze-drying to obtain heparin with double bond modification.

[0204] (3) Preparation of heparin solution

[0205] Take 0.8g of the double-bonded modified heparin prepared in step (2) and mix it with 25mg of Irgacure2959 to prepare PBS buffer with a pH of 6.4. The concentration of the double-bonded modified heparin is 2mg / mL.

[0206] (4) Coating preparation

[0207] The heparin solution prepared in step (3) was sprayed onto the thiol-modified polyethylene film prepared in step (1) (spraying amount: 0.05 mL / cm). 2 After spraying, the film was continuously irradiated with 275nm ultraviolet light for 1 hour. Then it was washed with purified water, dried, and finally coated with a heparin anticoagulant coating onto the polyethylene film.

[0208] Examples 4 to 5

[0209] Examples 4 and 5 all provide a method for preparing a heparin anticoagulant coating, which is obtained by the following steps:

[0210] (1) Thiol modification of the substrate

[0211] The polyethylene film was washed with purified water, dried, and then purged with oxygen plasma for 10 minutes. The film was then placed in a 2 mg / mL PEI aqueous solution and reacted for 1 hour. Afterward, it was washed with purified water and dried to obtain a PEI-grafted polyethylene film.

[0212] The PEI-grafted polyethylene film was reacted with a 2 mg / mL aqueous solution of mercaptopropionic acid using EDC / NHS as a catalyst for 4 hours. The film was then washed with purified water and dried to obtain the thiol-modified polyethylene film.

[0213] (2) Double bond modification of heparin

[0214] Three methods were used to modify heparin by double bond conversion. The heparin anticoagulant coating obtained by modifying heparin by method a and subsequent steps (3) and (4) is the heparin anticoagulant coating prepared in Example 1. The heparin anticoagulant coating obtained by modifying heparin by method b and subsequent steps (3) and (4) is the heparin anticoagulant coating prepared in Example 4. The heparin anticoagulant coating obtained by modifying heparin by method c and subsequent steps (3) and (4) is the heparin anticoagulant coating prepared in Example 5.

[0215] Specifically, method a is:

[0216] 1 g of heparin was reacted with 0.1 g of methacrylic anhydride in an aqueous sodium hydroxide solution at pH 8 for 8 hours, followed by dialyzing and freeze-drying to obtain heparin with double bond modification.

[0217] Method b is:

[0218] 1 g of heparin and 0.9 g of N-(3-aminopropyl)methacrylamide hydrochloride were reacted at 0 °C for 6 hours under the action of 0.9 g NHS and 1.2 g EDC. Then, the mixture was dialyzed and freeze-dried to obtain heparin with double bond modification.

[0219] Method c is:

[0220] 1g of heparin and 0.5g of glycidene were reacted in phosphate buffer at pH 7.4 for 4 days, followed by dialysis and freeze-drying to obtain heparin with double bond modification.

[0221] (3) Preparation of heparin solution

[0222] Take 0.8g of the double-bonded modified heparin prepared in step (2) and mix it with 25mg of Irgacure2959 to prepare PBS buffer with a pH of 6.4. The concentration of the double-bonded modified heparin is 2mg / mL.

[0223] (4) Coating preparation

[0224] The heparin solution prepared in step (3) was sprayed onto the thiol-modified polyethylene film prepared in step (1) (spraying amount: 0.05 mL / cm). 2After spraying, the film was continuously irradiated with 275nm ultraviolet light for 1 hour. Then it was washed with purified water, dried, and finally coated with a heparin anticoagulant coating onto the polyethylene film.

[0225] Example 6

[0226] This embodiment provides a method for preparing a heparin anticoagulant coating, which is obtained through the following steps:

[0227] (1) Thiol modification of the substrate

[0228] After washing and drying the polyethylene film with purified water, it was placed in concentrated sulfuric acid solution for 10 minutes, and then placed in 2.5 mg / mL PEI aqueous solution for 1 hour. After washing with purified water and freeze-drying, PEI-grafted polyethylene film was obtained.

[0229] The PEI-grafted polyethylene film was reacted with 2.5 mg / mL mercaptovalerate in MES buffer at pH 6, using EDC / NHS as a catalyst, for 3 hours. The film was then washed with purified water and freeze-dried to obtain the thiol-modified polyethylene film.

[0230] (2) Double bond modification of heparin

[0231] 1 g of heparin and 0.9 g of N-(3-aminopropyl)methacrylamide hydrochloride were reacted at 0 °C for 6 hours under the action of 0.9 g NHS and 1.2 g EDC. Then, the mixture was dialyzed and freeze-dried to obtain heparin with double bond modification.

[0232] (3) Preparation of heparin solution

[0233] Take 0.8g of the double-bonded modified heparin prepared in step (2), mix it with 0.15g of dithiothreitol and 30mg of LAP to prepare PBS buffer with pH value of 6.4 and the concentration of double-bonded modified heparin is 2.5mg / mL.

[0234] (4) Coating preparation

[0235] The heparin solution prepared in step (3) was sprayed onto the thiol-modified polyethylene film prepared in step (1) (spraying amount: 0.05 mL / cm). 2 After spraying, the film was continuously irradiated with 405nm visible light for 1 hour. Then it was washed with purified water, dried, and finally coated with a heparin anticoagulant coating onto the polyethylene film.

[0236] Example 7

[0237] This embodiment provides a method for preparing a heparin anticoagulant coating, which is obtained through the following steps:

[0238] (1) Thiol modification of the substrate

[0239] The polyvinyl chloride (PVC) film was washed with purified water, dried, and then placed in a pure aqueous solution containing 2 mg / mL acrylic acid and 0.08 mg / mL benzophenone, and irradiated with ultraviolet light for 30 minutes. It was then washed with purified water, and the film was placed in a PBS solution of 2 mg / mL chitosan (pH 6.4) using EDC / NHS as a catalyst. After reacting for 2 hours, it was washed with purified water and freeze-dried to obtain the chitosan-grafted PVC film.

[0240] The chitosan-grafted polyvinyl chloride film was placed in a 3 mg / mL aqueous solution of mercaptooctanoic acid and reacted for 8 hours. After washing with purified water and freeze-drying, the thiolated modified polyvinyl chloride film was obtained.

[0241] (2) Double bond modification of heparin

[0242] 1 g of heparin was reacted with 0.1 g of methacrylic anhydride in an aqueous sodium hydroxide solution at pH 8 for 8 hours, followed by dialyzing and freeze-drying to obtain double-bonded modified heparin.

[0243] (3) Preparation of heparin solution

[0244] Take 0.8g of the double-bonded modified heparin prepared in step (2) and mix it with 30mg of Irgacure2959 to prepare PBS buffer with a pH of 6.5 and a double-bonded modified heparin concentration of 3mg / mL.

[0245] (4) Coating preparation

[0246] The thiol-modified polyvinyl chloride film prepared in step (1) was immersed in the heparin solution prepared in step (3) for 5 minutes, then lifted out and continuously irradiated with 275 nm ultraviolet light for 45 minutes. Then it was washed with purified water and dried, and finally a heparin anticoagulant coating was coated on the surface of the polyvinyl chloride film.

[0247] Example 8

[0248] This embodiment provides a method for preparing a heparin anticoagulant coating, which is obtained through the following steps:

[0249] (1) Thiol modification of the substrate surface

[0250] The polyurethane film was washed with purified water, purged with oxygen plasma for 15 minutes, and then immersed in a pure aqueous solution of 4 mg / mL mercaptoethylamine. A 0.2 mg / mL genipin catalyst was used as the catalyst, and the reaction time was 4 hours. The film was then washed with purified water and freeze-dried to obtain the thiolated modified polyurethane film.

[0251] (2) Double bond modification of heparin

[0252] 1 g of heparin was reacted with 0.5 g of 1,2-epoxy-4-vinylcyclohexane in phosphate buffer at pH 7.4 for 5 days, followed by dialyzing and freeze-drying to obtain double-bonded modified heparin.

[0253] (3) Preparation of heparin solution

[0254] Take 0.8g of the double-bonded modified heparin prepared in step (2) and mix it with 20mg VA-086 to prepare PBS buffer with a pH of 6.4. The concentration of the double-bonded modified heparin is 4mg / mL.

[0255] (4) Coating preparation

[0256] The heparin solution prepared in step (3) was sprayed onto the thiol-modified polyurethane film prepared in step (1) (spraying amount: 0.05 mL / cm). 2 After spraying, the film was continuously irradiated with 405nm visible light for 1 hour. Then it was washed with purified water and dried, finally obtaining a heparin anticoagulant coating on the surface of the polyurethane film.

[0257] Example 9

[0258] This embodiment provides a method for preparing a heparin anticoagulant coating, which is obtained through the following steps:

[0259] (1) Thiol modification of the substrate surface

[0260] The polypropylene film was washed with purified water, dried, and then irradiated with 250 nm ultraviolet light for 20 minutes. It was then treated in a 5 mg / mL PEI aqueous solution for 2 hours, followed by washing with purified water. Finally, it was treated in a 5 mg / mL mercaptopropionic acid aqueous solution for 3 hours, washed with purified water, and freeze-dried to obtain the thiol-modified polypropylene film.

[0261] (2) Double bond modification of heparin

[0262] 1 g of heparin was reacted with 0.1 g of methacrylic anhydride in an aqueous sodium hydroxide solution at pH 8 for 8 hours, followed by dialyzing and freeze-drying to obtain double-bonded modified heparin.

[0263] (3) Preparation of heparin solution

[0264] Take 0.8g of the double-bonded modified heparin prepared in step (2) and mix it with 30mg of Irgacure2959 to prepare PBS buffer with a pH of 6.4 and a concentration of 5mg / mL of double-bonded modified heparin.

[0265] (4) Coating preparation

[0266] The thiol-modified polypropylene film prepared in step (1) was immersed in the heparin solution prepared in step (3) for 5 minutes, then lifted out and continuously irradiated with 275 nm ultraviolet light for 1.5 hours. It was then washed with purified water and dried, and finally a heparin anticoagulant coating was coated on the surface of the polypropylene film.

[0267] Example 10

[0268] This embodiment provides a method for preparing a heparin anticoagulant coating, which is obtained through the following steps:

[0269] (1) Thiol modification of the substrate surface

[0270] The polycaprolactone film was washed with purified water, dried, purged with ammonia plasma for 10 minutes, and then treated in a 5 mg / mL mercaptopropionic acid aqueous solution for 3 hours. It was then washed with purified water and freeze-dried to obtain the thiolated modified polycaprolactone film.

[0271] (2) Double bond modification of heparin

[0272] 1 g of heparin was reacted with 0.5 g of 1,2-epoxy-4-vinylcyclohexane in phosphate buffer at pH 7.4 for 5 days, followed by dialyzing and freeze-drying to obtain double-bonded modified heparin.

[0273] (3) Preparation of heparin solution

[0274] Take 0.8g of the double-bonded modified heparin prepared in step (2) and mix it with 30mg of Irgacure2959 to prepare PBS buffer with a pH of 6.4 and a concentration of 5mg / mL of double-bonded modified heparin.

[0275] (4) Coating preparation

[0276] The thiolated modified polycaprolactone film prepared in step (1) was immersed in the heparin solution prepared in step (3) for 5 minutes, then lifted out and continuously irradiated with 275 nm ultraviolet light for 1.5 hours. It was then washed with purified water and dried, and finally a heparin anticoagulant coating was coated on the surface of the polycaprolactone film.

[0277] Example 11

[0278] This embodiment provides a method for preparing a heparin anticoagulant coating, which is obtained through the following steps:

[0279] (1) Thiol modification of the substrate surface

[0280] The polyester PET pipe was washed with purified water, dried, and then treated with oxygen plasma for 15 minutes. It was then immersed in a 3 mg / mL mercaptooctylamine aqueous solution for 1.5 hours, washed with purified water, and dried to obtain the thiol-modified PET pipe.

[0281] (2) Double bond modification of heparin

[0282] 1 g heparin, 0.3 g N-(3-aminopropyl)methacrylamide hydrochloride, 0.3 g NHS and 0.3 g EDC were reacted in PBS buffer at pH 7.4 at 0 °C for 5 hours, followed by dialyzing and freeze-drying to obtain double-bonded modified heparin.

[0283] (3) Preparation of heparin solution

[0284] Take 0.8g of the double-bonded modified heparin prepared in step (2) and mix it with 35mg of Irgacure2959 to prepare PBS buffer with a pH of 6.4 and a concentration of 3mg / mL of double-bonded modified heparin.

[0285] (4) Coating preparation

[0286] The heparin solution prepared in step (3) was sprayed onto the surface of the thiol-modified PET pipe prepared in step (1) (spraying amount: 0.05 mL / cm). 2 After spraying, the material is continuously irradiated with 275nm ultraviolet light for 2 hours, then washed with purified water and dried to obtain a heparin anticoagulant coating on the surface of the PET pipe.

[0287] Example 12

[0288] This embodiment provides a method for preparing a heparin anticoagulant coating, which is obtained through the following steps:

[0289] (1) Thiol modification of the substrate surface

[0290] Prepare a 1.5 mg / mL PEI aqueous solution, add 0.02 mg / mL EDC / NHS for activation, then add a 1.5 mg / mL mercaptopropionic acid aqueous solution. After reacting for 2 hours, wash with purified water and freeze-dry to obtain thiol-modified PEI.

[0291] After washing and drying, the polypropylene pipe was treated with oxygen plasma for 15 minutes. Then, it was placed in PBS buffer of the thiol-modified PEI at a concentration of 1.5 mg / mL and reacted for 1 hour. After that, it was taken out, washed with purified water, and dried to obtain the thiol-modified polypropylene pipe.

[0292] (2) Double bond modification of heparin

[0293] 1 g heparin, 0.3 g N-(3-aminopropyl)methacrylamide hydrochloride, 0.3 g NHS and 0.3 g EDC were reacted in PBS buffer at pH 7.4 at 0 °C for 5 hours, followed by dialyzing and freeze-drying to obtain double-bonded modified heparin.

[0294] (3) Preparation of heparin solution

[0295] Take 0.8g of the double-bonded modified heparin prepared in step (2) and mix it with 35mg of Irgacure2959 to prepare PBS buffer with a pH of 6.4 and a concentration of 3mg / mL of double-bonded modified heparin.

[0296] (4) Coating preparation

[0297] The heparin solution prepared in step (3) was sprayed onto the surface of the thiol-modified PP pipe prepared in step (1) (spraying amount: 0.05 mL / cm). 2 After spraying, the material is continuously irradiated with 275nm ultraviolet light for 2 hours, then washed with purified water and dried to obtain a heparin anticoagulant coating on the surface of the PP pipe.

[0298] Example 13

[0299] This embodiment provides a method for preparing a heparin anticoagulant coating, which is obtained through the following steps:

[0300] (1) Thiol modification of the substrate surface

[0301] The 316L SS scaffold was washed with purified water and then treated in concentrated sulfuric acid for 10 minutes. The scaffold was then immersed in a 2 mg / mL chitosan aqueous solution for 2.5 hours, washed with purified water, and dried to obtain the chitosan-grafted 316L SS scaffold.

[0302] The chitosan-grafted 316L SS scaffold was immersed in a 2 mg / mL aqueous solution of mercaptoacetic acid and reacted for 3 hours. After washing with purified water and drying, the thiol-modified 316L SS scaffold was obtained.

[0303] (2) Double bond modification of heparin

[0304] 1 g of heparin was reacted with 0.1 g of methacrylic anhydride in an aqueous sodium hydroxide solution at pH 8 for 6 hours, followed by dialyzing and freeze-drying to obtain double-bonded modified heparin.

[0305] (3) Preparation of heparin solution

[0306] Take 0.8g of the double-bonded modified heparin prepared in step (2), and mix it with 25mg Irgacure2959 and 15mg benzophenone to prepare PBS buffer with a pH of 6.4 and a concentration of 2mg / mL of double-bonded modified heparin.

[0307] (4) Coating preparation

[0308] The thiolated modified 316L SS scaffold prepared in step (1) was immersed in the heparin solution prepared in step (3) for 5 minutes, then lifted out and continuously irradiated with 295nm ultraviolet light for 2 hours. It was then washed with purified water and dried, and finally a heparin anticoagulant coating was coated on the surface of the 316L SS scaffold.

[0309] Example 14

[0310] This embodiment provides a method for preparing a heparin anticoagulant coating, which is obtained through the following steps:

[0311] (1) Thiol modification of the substrate surface

[0312] The cobalt-chromium alloy scaffold was washed with purified water, dried, and then purged with oxygen plasma for 15 minutes. It was then immersed in a Tris-HCl buffer solution of 2.5 mg / mL poly-L-arginine hydrochloride for 2 hours, followed by washing with purified water and drying.

[0313] The cobalt-chromium alloy scaffold treated with poly-L-arginine hydrochloride was immersed in a 2.5 mg / mL aqueous solution of 3-mercaptoisobutyric acid, using EDC / HOBT as a catalyst, for 4 hours. After the reaction, it was washed with purified water and dried to obtain the thiol-modified cobalt-chromium alloy scaffold.

[0314] (2) Double bond modification of heparin

[0315] 1 g of heparin and 0.5 g of glycidene were reacted in phosphate buffer at pH 7.4 for 4 days, followed by dialysis and freeze-drying to obtain double-bonded modified heparin.

[0316] (3) Preparation of heparin solution

[0317] Take 0.8g of the double-bonded modified heparin prepared in step (2) and mix it with 40mg of riboflavin to prepare a PBS buffer with a pH of 6.4 and a double-bonded modified heparin concentration of 2mg / mL.

[0318] (4) Coating preparation

[0319] The heparin solution prepared in step (3) was sprayed onto the surface of the thiolated modified cobalt-chromium alloy scaffold prepared in step (1) (spraying amount: 0.05 mL / cm). 2 The sample was irradiated with 425nm visible light for 2 hours. Then it was washed with purified water, dried, and finally coated with a heparin anticoagulant coating.

[0320] The following is the specific test section:

[0321] (1) Heparin content determination

[0322] The heparin content of Comparative Examples 1-4 and Examples 1-14 was determined by the toluidine blue colorimetric method, and the results are shown in Table 1.

[0323] Table 1. Heparin content in the heparin anticoagulant coatings of each embodiment.

[0324]

[0325]

[0326] As shown in Table 1, the materials with heparin-containing anticoagulant coatings prepared in Comparative Examples 1-4 and Examples 1-14 all exhibited high heparin content, indicating that heparin was well fixed to the substrate surface. Meanwhile, the coatings prepared in Examples 1, 4-7, 9, and 12-14, which had their substrates pre-treated with polyamines, all showed even higher heparin content, demonstrating that the polyamines indeed had a scale-up effect, increasing the heparin content of the coating.

[0327] (2) Coating stability test

[0328] The polyethylene films prepared in Comparative Examples 1-4 and Examples 1-3 were cut into 5mm × 5mm fragments and immersed in hydrochloric acid solution (pH 1), sodium hydroxide solution (pH 12), and physiological saline solution for 1 hour, 6 hours, and 24 hours, respectively. After removal, they were washed with purified water, and the heparin content was measured using the toluidine blue colorimetric method. The results are shown in Table 2.

[0329] Table 2 Coating stability test results

[0330]

[0331]

[0332] As shown in Table 2, in both acidic and alkaline environments, Comparative Example 1 showed significant heparin detachment, and some heparin molecules also detached in physiological saline. This indicates that the substrate, without thiolation modification, directly bonds heparin, leading to heparin detachment during use. Comparative Example 2 showed even more significant heparin detachment because the substrate and thiolated PEI were fixed solely by physical adsorption, resulting in the entire coating detaching during use. Furthermore, Comparative Example 3 exhibited good stability, but the overall heparin content was low and showed a significant decrease. Comparative Example 4, through thiolation of heparin and covalent bonding with the double-bonded substrate, also showed good stability, but its thiolated heparin was highly susceptible to deterioration during purification and storage. The heparin anticoagulant coatings prepared in Examples 1-3, regardless of whether in physiological saline, acidic, or alkaline conditions, only showed a slight decrease during initial immersion, maintaining a stable heparin content throughout. This indicates that the heparin anticoagulant coatings prepared through the above examples have strong binding force and are not prone to heparin detachment.

[0333] (3) Long-term stability test

[0334] The polyethylene films prepared in Comparative Examples 1-4 and Examples 1-3 were placed in PBS buffer (pH 7.4) for 7 days, washed with purified water, and then the heparin content was determined by the toluidine blue colorimetric method. The results are shown in Table 3 below.

[0335] Table 3. Results of Long-Term Stability Tests for Coatings

[0336]

[0337] As shown in Table 3, after prolonged PBS immersion, the heparin content in Comparative Examples 1 and 2 decreased significantly, with severe heparin molecule shedding. Comparative Example 3 also showed a certain decrease. However, the polyethylene films with heparin-containing anticoagulant coatings prepared based on the thiol-ene click reaction in Comparative Examples 4 and 1-3 still maintained a high heparin content on their surface, showing only a slight decrease compared to the initial heparin content, demonstrating excellent long-lasting anticoagulant ability.

[0338] (4) Antiplatelet adhesion test

[0339] Polyethylene films without anticoagulant coating (blank group), comparative examples 1-4, and polyethylene films with heparin anticoagulant coating prepared in Example 1 were cut into 4cm × 4cm squares. Blood was centrifuged to obtain platelet-containing plasma (PRP). These three samples were then completely immersed in PRP and rotated at 37°C for 2 hours, followed by washing five times with physiological saline. Subsequently, the platelet content adhering to the sample surface was semi-quantitatively analyzed using a lactate dehydrogenase (LDH) kit. The results are as follows: Figure 2 As shown.

[0340] like Figure 2 As shown, the polyethylene films prepared in Comparative Examples 1-4 and Example 1 all exhibited a significant decrease in platelet adhesion, indicating that this type of heparin-containing anticoagulant coating has excellent anticoagulant effects. Furthermore, the effect of Example 1 is more pronounced, demonstrating that the anticoagulant effect is more significant after covalently fixing heparin to the substrate using a thiol-ene click reaction.

[0341] The experimental data above show that the heparin anticoagulant coating prepared in the above embodiments has a high heparin content, good stability, and excellent anticoagulant effect. Furthermore, the above embodiments demonstrate that the preparation method of the heparin anticoagulant coating is applicable to various substrates, such as polymer films, polymer pipes, and metal supports, and can form a heparin anticoagulant coating on various substrates, thus having a wide range of applications.

[0342] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0343] The above-described embodiments are merely illustrative of several implementation methods of the present invention, facilitating a detailed understanding of the technical solutions of the present invention, but should not be construed as limiting the scope of protection of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. It should be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided by the present invention through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this invention patent should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for preparing a heparin anticoagulant coating, characterized in that, Includes the following steps: The substrate is modified to carry thiol groups, resulting in a modified substrate; Heparin is modified by using a carboxylic acid containing a double bond, an anhydride containing a double bond, an epoxy compound containing a double bond, or an amide compound containing a double bond to carry a double bond, thereby obtaining modified heparin. and The modified substrate is treated with a solution containing modified heparin and an initiator, so that the double bond carried by the modified heparin and the thiol group carried by the modified substrate undergo a thiol-ene click reaction under the action of the initiator, forming a heparin anticoagulant coating on the surface of the modified substrate. The term "carrying" refers to having something through covalent bonding; The step of modifying the substrate includes: firstly, surface-activating the substrate to imbue it with carboxyl groups, and then treating the carboxyl-containing substrate with polyamino substances and thiocarboxylic acids to imbue it with thiol groups; or, The step of modifying the substrate includes: first, surface-activating the substrate to make it carry carboxyl groups, and then reacting the carboxyl-carrying substrate with a mercaptoamine or a mercaptoamine hydrochloride in an amide reaction; or... The step of modifying the substrate includes: treating the surface of the substrate with ammonia plasma to make the substrate carry amino groups, and then reacting the amino-carrying substrate with mercaptocarboxylic acid to form an amide reaction.

2. The method for preparing the heparin anticoagulant coating according to claim 1, characterized in that, The step of treating the carboxyl-containing substrate with polyamino substances and mercaptocarboxylic acids includes: The substrate carrying carboxyl groups is immersed in a solution containing the polyamine-like substance for 0.5 h to 3 h to graft the polyamine-like substance onto the substrate. The concentration of the polyamine-like substance in the solution is 1 mg / mL to 5 mg / mL. Then, the substrate grafted with the polyamine-like substance is immersed in a solution containing the mercaptocarboxylic acid for an amide reaction for 1 h to 8 h. The concentration of the mercaptocarboxylic acid in the solution is 1 mg / mL to 5 mg / mL. Alternatively... First, the polyamino substance and the mercaptocarboxylic acid are subjected to an amide reaction for 0.5 h to 5 h, wherein the mass ratio of the polyamino substance to the mercaptocarboxylic acid is 1:(0.2~1.5). Then, the substrate carrying the carboxyl group is immersed in a solution containing the reaction product and reacted for 0.5 h to 3 h. The concentration of the reaction product in the solution is 1 mg / mL to 5 mg / mL.

3. The method for preparing the heparin anticoagulant coating according to claim 1, characterized in that, The step of treating the carboxyl-containing substrate with polyamino substances and mercaptocarboxylic acids shall satisfy at least one of the following conditions: 1) The polyamino substance is selected from at least one of polyethyleneimine, chitosan, polyallylamine, polyallylamine hydrochloride, poly-L-arginine, poly-L-arginine hydrochloride, poly-L-lysine, poly-L-lysine hydrobromide, diethylenetriamine, triethylenetetramine, and tetraethylenepentamine; 2) The thiocarboxylic acid is selected from at least one of thioacetic acid, thiopropionic acid, thiobutyric acid, thiosuccinic acid, thiovaleric acid, thiooctanoic acid, 3-mercaptoisobutyric acid and ω-mercaptohexanoic acid; 3) During the amide reaction, a catalyst was also added, which was selected from at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride / N-hydroxysulfosuccinimide, genipin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride / 1-hydroxybenzotriazole, O-benzotriazole-tetramethylurea hexafluorophosphate, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and 1H-benzotriazole-1-yloxotris(dimethylamino)hexafluorophosphate.

4. The method for preparing the heparin anticoagulant coating according to claim 3, characterized in that, The polyamino substance is selected from at least one of polyethyleneimine, chitosan, and poly-L-arginine hydrochloride; the thiocarboxylic acid is selected from at least one of mercaptopropionic acid, mercaptovalerate, mercaptooctanoic acid, mercaptoacetic acid, and 3-mercaptoisobutyric acid; a catalyst is also added during the amide reaction, and the catalyst is selected from at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride / N-hydroxysulfosuccinimide, genipin, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride / 1-hydroxybenzotriazole.

5. The method for preparing the heparin anticoagulant coating according to claim 1, characterized in that, The thiol amine is selected from at least one of mercaptoethylamine, mercaptopropylamine, mercaptobutylamine, mercaptopentylamine, and mercaptooctylamine; and / or, The substrate carrying the carboxyl group is immersed in a solution containing the mercaptoamine or the hydrochloride of the mercaptoamine and reacted for 1 h to 5 h. The concentration of the mercaptoamine or the hydrochloride of the mercaptoamine in the solution is 1 mg / mL to 5 mg / mL.

6. The method for preparing the heparin anticoagulant coating according to any one of claims 2 to 5, characterized in that, The methods for surface activation of the substrate include at least one of plasma treatment, ultraviolet irradiation, concentrated sulfuric acid chemical modification, and photochemical grafting.

7. The method for preparing the heparin anticoagulant coating according to claim 5, characterized in that, The step of surface activation of the substrate using plasma treatment includes: purging the substrate with oxygen plasma for 5 to 15 minutes; or, The step of surface activation of the substrate using concentrated sulfuric acid chemical modification includes: treating the substrate in concentrated sulfuric acid for 10 to 20 minutes, wherein the concentrated sulfuric acid has a mass percentage ≥ 70%; or, The step of surface activation of the substrate by ultraviolet irradiation includes: irradiating the substrate with ultraviolet light for 10 to 30 minutes; or, The step of surface activation of the substrate using photochemical grafting includes: placing the substrate in a solution containing a photoinitiator and a carboxylic acid, and irradiating it with ultraviolet or visible light for 10 minutes to 1 hour. The photoinitiator includes at least one of benzophenone and thioxanthone, and the carboxylic acid has the general formula CHX=CY-L-COOZ, where X is hydrogen or phenyl, Y is hydrogen, and C is phenyl. 1~ C6 alkyl or halogen-substituted C 1~ The carboxylic acid is a C6 alkyl group, L is a divalent linker, Z is hydrogen or sodium, the concentration of the carboxylic acid is 1 mg / mL to 5 mg / mL, and the mass ratio of the photoinitiator to the carboxylic acid is 1:(0.01~0.1).

8. The method for preparing the heparin anticoagulant coating according to claim 1, characterized in that, The step of treating the substrate surface with ammonia plasma includes: purging the substrate with ammonia plasma for 5 to 30 minutes; and / or The step of reacting an amino-containing substrate with a mercaptocarboxylic acid includes: immersing the amino-containing substrate in a solution containing the mercaptocarboxylic acid for 1 h to 8 h, wherein the concentration of the mercaptocarboxylic acid in the solution is 1 mg / mL to 5 mg / mL.

9. The method for preparing the heparin anticoagulant coating according to claim 1, characterized in that, In the step of modifying heparin with a carboxylic acid or anhydride containing a double bond, the heparin is reacted with the carboxylic acid or anhydride containing a double bond in an alkaline solution for 3-8 hours, wherein the mass ratio of heparin to the carboxylic acid or anhydride containing a double bond is 1:(0.05-0.5); or, In the step of modifying heparin with an epoxy compound containing double bonds, the heparin and the epoxy compound containing double bonds are reacted in a solution with a pH of 7-8 for 3-8 days, and the mass ratio of the heparin to the epoxy compound containing double bonds is 1:(0.3-1); or, In the step of modifying heparin with an amide compound containing double bonds, the heparin and the amide compound containing double bonds are stirred and reacted at 0℃~4℃ for 3h~8h under the action of an activator and a condensing agent. The mass ratio of the heparin, the amide compound containing double bonds, the activator and the condensing agent is (1~1.2):(0.8~1):(0.8~1):(0.8~1.2).

10. The method for preparing the heparin anticoagulant coating according to claim 1 or 9, characterized in that, The general formula of the carboxylic acid containing a double bond is CHX=CY-L-COOZ, where X is hydrogen or phenyl, and Y is hydrogen or C. 1~ C6 alkyl or halogen-substituted C 1~ C6 alkyl group, L is a divalent linker, and Z is hydrogen or sodium; The general formula of the acid anhydride containing double bonds is R1-C(=O)OC(=O)-R2, where at least one of R1 and R2 is an alkenyl group of C2 to C6, or R1 and R2 form a 5 to 7 membered monocyclic ring, wherein the 5 to 7 membered monocyclic ring contains a carbon-carbon double bond or has a substituent containing a carbon-carbon double bond. The double-bonded epoxy compound is selected from at least one of glycidyl methacrylate, allyl glycidyl ether, epoxybutene and its homologues, 1,2-epoxy-4-vinylcyclohexane and 3,4-epoxy-1-cyclohexene. The amide compound containing a double bond is N-(3-aminopropyl)methacrylamide or N-(3-aminopropyl)methacrylamide hydrochloride.

11. The method for preparing the heparin anticoagulant coating according to claim 1, characterized in that, The solution containing the modified heparin and the initiator contains at least one of the following: 1) The concentration of the modified heparin is 2 mg / mL to 5 mg / mL, and the mass ratio of the modified heparin to the initiator is 1:(0.005~0.05). 2) The pH of the solution containing the modified heparin and the initiator is 6 to 6.8; 3) In the step of treating the modified substrate with a solution containing modified heparin and an initiator, a coating method is used; 4) The initiator is selected from at least one of 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone, benzophenone, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, lithium bis(2,4,6-trimethylbenzoyl)phosphinic acid, 2,2'-azo(2-methyl-N-(2-hydroxyethyl)propionamide), riboflavin, and eosin Y.

12. The method for preparing the heparin anticoagulant coating according to claim 1 or 11, characterized in that, The solution containing modified heparin and an initiator also contains a crosslinking agent selected from at least one of thiol crosslinking agents and double bond crosslinking agents.

13. The method for preparing the heparin anticoagulant coating according to claim 12, characterized in that, The solution containing modified heparin and an initiator contains at least one of the following: 1) The concentration of the crosslinking agent is ≤2 mg / mL; 2) The thiol crosslinking agent is selected from at least one of dithiothreitol, pentaerythritol tetra(3-mercaptopropionate), ethoxylated pentaerythritol tetra(3-mercaptopropionate), dipentaerythritol hexamercaptoacetate, trimethylolpropane tri(2-mercaptoacetate), pentaerythritol tetra(2-mercaptoacetate), tripentaerythritol octamercaptoacetate, ethylene glycol bis(3-mercaptopropionate), thiol-terminated propoxylated glycerol, and thiol-terminated polyethylene glycol; 3) The double bond crosslinking agent is selected from at least one of diol diacrylate, diol divinyl ether, trimethylolpropane triacrylate, pentaerythritol triacrylate, pentaerythritol tetraacrylate, 1,3,5-tricyanate triallyl ester, triallyl isocyanurate and triethylene glycol divinyl ether.

14. A heparin anticoagulant coating, characterized in that, The heparin anticoagulant coating is prepared by the preparation method described in claim 1. The heparin anticoagulant coating is formed on a substrate and comprises a plurality of heparin molecules modified with a carboxylic acid containing a double bond, an anhydride containing a double bond, an epoxy compound containing a double bond, or an amide compound containing a double bond. The modified heparin molecules are covalently linked to the substrate in the following manner: modified heparin molecule-CS-substrate.

15. The application of the heparin anticoagulant coating prepared by any one of claims 1 to 13 or the heparin anticoagulant coating of claim 14 in the preparation of blood contact medical devices.

Images