An antithrombotic conjugate, a method of preparation and use as a coating

Abstract

This invention relates to an antithrombotic conjugate formed by conjugating a terpolymer with an active anticoagulant fragment, wherein the terpolymer comprises: A) an inert anticoagulant unit; B) a graft unit with reactive groups; and C) a unit that adheres to a substrate. By utilizing the synergistic effect of the active anticoagulant fragment, such as a heparin fragment, and the inert anticoagulant unit simultaneously present in the antithrombotic conjugate, multi-dimensional and multifunctional anticoagulant effects can be achieved, thus matching the different usage scenarios and anticoagulant requirements of blood-contact medical devices in clinical practice. When this antithrombotic conjugate is coated onto a substrate surface, the unit that adheres to the substrate acts as an adhesive layer, achieving excellent adhesion and long-lasting coating performance, maintaining function in a flowing blood environment.

Description

Technical Field

[0001] This invention relates to the field of coating materials, and more specifically to an antithrombotic conjugate formed by subsequent conjugation of terpolymers, its preparation method, and its application as a coating. Background Technology

[0002] In the medical field, surface coating technology for medical devices is one of the key factors in improving device performance and patient safety. Active anticoagulant components (such as heparin) and inert anticoagulant components (such as phosphorycholine and its derivatives, betaine and its derivatives) have been extensively studied and applied to anticoagulant coatings on medical device surfaces to reduce coagulation reactions when blood comes into contact with the device. However, despite the significant anticoagulant properties of these materials, they still face some challenges and problems in practical applications.

[0003] Heparin molecules in heparin coatings effectively inhibit multiple key enzymes in the coagulation cascade by binding to antithrombin III, thereby reducing thrombus formation. However, the stability and durability of the heparin coating are major concerns. Heparin molecules are easily degraded in the body, leading to a decrease in coating effectiveness over time. Furthermore, the immunogenicity of heparin may also cause adverse reactions in patients.

[0004] Phosphorylcholine coatings mimic the surface of natural cell membranes, reducing platelet activation and aggregation. Phosphorylcholine coatings have attracted attention due to their good biocompatibility and anti-inflammatory properties. However, phosphorylcholine coatings primarily function as passive anticoagulants, and their direct effects are not as pronounced as those of heparin.

[0005] The combined use of heparin and phosphorycholine in constructing anticoagulant coatings aims to combine the advantages of both for superior anticoagulant effects. This combined coating strategy improves coating stability and biocompatibility while reducing the limitations of single materials. This is crucial for practical applications in medical devices, enabling adaptive and targeted effects at different stages of thrombosis.

[0006] However, current processes employ a layer-by-layer bonding strategy, resulting in complex coating preparation processes that cannot precisely control the ratio and distribution of the two materials to ensure optimal synergy. Furthermore, the safety and efficacy of the combined substances cannot be clearly assessed. In addition, for blood-contact medical devices, the robust bonding between the coating and the raw material substrate remains a crucial factor in device development, directly impacting the device's function, safety, efficacy, and ultimately, clinical outcomes.

[0007] In conclusion, there is still a need for an antithrombotic coating material that exhibits superior antithrombotic properties compared to previous single-strategy antithrombotic methods, while also possessing strong adhesion to various substrates. Summary of the Invention

[0008] The problem the invention aims to solve To address the shortcomings and deficiencies of existing technologies, this invention designs and synthesizes an antithrombotic conjugate, formed by conjugating a terpolymer with an active anticoagulant fragment. The terpolymer comprises: A) an inert anticoagulant unit; B) a graft unit with reactive groups; and C) a unit that adheres to the substrate. By utilizing the synergistic effect of the active anticoagulant fragment (e.g., heparin) and the inert anticoagulant unit simultaneously present in the antithrombotic conjugate, a multi-dimensional and multifunctional anticoagulant effect can be achieved, thus matching the different usage scenarios and anticoagulant requirements of blood-contact medical devices in clinical practice. This antithrombotic conjugate can be applied as a coating to the surface of a substrate, where the unit that adheres to the substrate acts as an adhesive layer, achieving excellent adhesion and long-lasting coating performance, maintaining function in a flowing blood environment.

[0009] Solution for solving the problem The present invention first provides an antithrombotic conjugate, which is formed by conjugating a terpolymer with an active anticoagulant fragment, wherein the terpolymer comprises: A) an inert anticoagulant unit; B) a graft unit with a reactive group; and C) a unit that acts as an adhesive to the substrate. The inert anticoagulant unit is derived from at least one of polymerizable phosphorylcholine and its derivatives, polymerizable polymethyl polyethylene glycol acrylate and its derivatives, polymerizable polyethylene oxide, polymerizable fluoropolymer, polymerizable sulfonate betaine, and polymerizable carboxylate betaine. The reactive group is selected from active groups that can react with hydroxyl, amino, carboxyl, aldehyde, azide or thiol groups on the active anticoagulant fragment; The unit that acts as an adhesive to the substrate is selected from at least one of polymerizable hydrophobic alkanes, polymerizable silane coupling agents, polymerizable polyphenols, and polymerizable photosensitive molecules. The active anticoagulant fragment is selected from at least one of heparin, heparin derivatives, heparin-like substances, heparin-like derivatives, and hirudin.

[0010] Furthermore, the terpolymer is obtained by free radical polymerization of a polymerizable inert anticoagulant monomer, a graft monomer with reactive groups, and a monomer capable of bonding with the substrate. The polymerizable inert anticoagulant monomer is selected from one or more of 2-methacryloyloxyethyl phosphorylcholine, polyethylene glycol methacrylate, 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate, hydroxyethyl methacrylate, and 2-ethyl-2-azolinium.

[0011] Furthermore, the monomer that acts as an adhesive to the substrate is selected from one or more of aminopropyltriethoxysilane, vinyltriethoxysilane, vinyltrimethoxysilane, vinyltrichlorosilane, vinyltris(2-ethoxyethoxy)silane, and γ-(methacryloyloxy)propyltrimethoxysilane. Alternatively, it is selected from one or more of polyethylene glycol catechols with double bonds and 3-methacryloyldopamine.

[0012] Furthermore, in the terpolymer, the molar percentage of the polymerizable inert anticoagulant monomer is 10-50%, preferably 20-40%; the molar percentage of the monomer capable of bonding with the substrate is 50%-70%; the molar percentage of the grafted monomer with reactive groups is 0.5-10%; and the sum of the molar percentages of the three is 100%.

[0013] Furthermore, the molar ratio of the polymerizable inert anticoagulant monomer to the grafted monomer with reactive groups is (3-20):1.

[0014] Furthermore, the active anticoagulant fragment is covalently bonded to the side chain end of the ternary copolymer.

[0015] Furthermore, the covalent grafting ratio of the active anticoagulant fragment is 10%-70%.

[0016] Furthermore, the number average molecular weight of the terpolymer is 2,000-200,000; preferably 3,000-150,000; more preferably 5,000-100,000.

[0017] Furthermore, the active anticoagulant fragment is a heparin fragment, the fragment being composed of at least 5 glycounits, and at least some of the plurality of fragments containing polysaccharide sequence A:

[0018] The present invention also provides a method for preparing the antithrombotic conjugate, comprising: performing living free radical polymerization on the inert anticoagulant monomer, the monomer that acts as an adhesive to the substrate, and the graft monomer with reactive groups to obtain the ternary random copolymer; subsequently dissolving the obtained ternary random copolymer in a buffer solution and adding an active anticoagulant fragment; reacting the mixed solution at room temperature under the action of a coupling agent to finally obtain the antithrombotic conjugate.

[0019] The present invention further provides a coating composition comprising a solvent and at least one of the aforementioned antithrombotic conjugates.

[0020] Furthermore, the antithrombotic conjugate has a mass fraction of 5%~20% w / w in the coating composition.

[0021] The present invention also provides an antithrombotic coating, which is obtained by curing the above-mentioned coating composition, for example by heat curing, radiation curing, solvent evaporation, etc.

[0022] Finally, the present invention also provides a medical article having at least a portion of its surface covered by the aforementioned antithrombotic coating.

[0023] The effects of the invention 1. Heparin grafted onto the side chain (non-terminal end) can achieve high-density heparin while allowing for easy control of the grafting density. Furthermore, side-chain heparin can maintain the exposure of the active heparin fragment on the surface as much as possible.

[0024] 2. The inert anticoagulant unit can produce a synergistic effect with the heparin unit to enhance anticoagulant performance. Furthermore, even when the heparin unit loses its activity, the inert anticoagulant unit can still maintain excellent anti-protein adsorption properties on its surface, effectively preventing thrombus formation.

[0025] 3. The bonding units that adhere to the substrate are adjustable in both strength and proportion to adapt to the specific requirements of different substrate surfaces. This adjustability ensures excellent adhesion between the coating and the substrate, meeting diverse application needs.

[0026] 4. The antithrombotic conjugate can be directly coated on the substrate surface, which is simple to operate and suitable for industrialization.

[0027] 5. This method does not use harmful chemicals and the reaction is carried out at room temperature or under appropriate heating conditions, reducing energy consumption and potential environmental pollution. Attached Figure Description

[0028] Figure 1 SEM images of antiplatelet adhesion assays for Example 3 and Comparative Example 3. Detailed Implementation

[0029] To better illustrate the invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art will understand that the invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail to highlight the spirit of the invention. Unless otherwise defined, the technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In this specification, the numerical range referred to as "numerical value A to numerical value B" refers to the range including the endpoints A and B. In this specification, the word "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0030] It should be understood that the singular article “a” (corresponding to the English words “a,” “an,” and “the”) used in this specification and appended claims includes plural objects unless otherwise expressly stated herein. In this specification, references to “one or more specific / preferred embodiments / solutions,” “another or more specific / preferred embodiments / solutions,” “one or another embodiment / solution,” “one or another technical solution,” etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one embodiment described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0031] The term "comprising" and any variations thereof in the specification and claims of this invention are intended to cover a non-exclusive inclusion. For example, a process, method, system, product, or apparatus that comprises a series of steps or units is not limited to the steps or units listed, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to such processes, methods, products, or apparatus.

[0032] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of the invention, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified. In the description of the present invention, "several" means at least one, such as one, two, etc., unless otherwise explicitly specified.

[0033] In this invention, the term "functionalization" and related terms include: the process of treating a material to alter its surface properties to meet specific requirements for a particular application, or the process of providing a function that a chemical substance does not normally possess by adding functional groups to it.

[0034] Here, the substrate refers to organic or inorganic materials, generally polymeric or metallic materials. This invention does not specifically limit the material of the substrate; polymeric materials include polyesters, polytetrafluoroethylene, polyurethane, polyether polyurethane, polyamide, vinyl chloride, polycarbonate, polystyrene, polyethylene, polypropylene, polymethyl methacrylate, various synthetic fibers, etc.; metallic materials include stainless steel, titanium and its alloys, cobalt-based alloys, magnesium alloys, shape memory alloys, etc.

[0035] This invention also includes a transition layer between the substrate and the functional surface layer. The presence of the transition layer can provide reactive sites for certain substrates that do not have reactive functional groups, or can provide more uniform and dense reactive sites on the substrate surface, facilitating subsequent reactive covalent bonding. The transition layer is obtained by surface activation treatment and coupling agent treatment. The surface activation treatment includes acid treatment, alkali treatment, chemical reagent treatment, plasma treatment, corona discharge treatment, radiation irradiation treatment, heat treatment, etc.; the coupling agent treatment includes catechol treatment, silane coupling agent treatment, isocyanate treatment, etc.

[0036] The solvent comprises one or more of water, low molecular weight alcohols (methanol, ethanol, isopropanol, butanol, pentanol, ethylene glycol, propylene glycol, glycerol, etc.), N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, acetone, and phenol. Preferably, the solvent is a single or mixed solvent capable of dissolving the polymer of the present invention to form a homogeneous solution. Preferably, the solvent is a mixture of water and isopropanol. Further, in order to promote the polymer to form a coating more quickly, the isopropanol content in the solvent is suitable in the range of 15-70% of the total weight of the solvent, more preferably 20-60%, and even more preferably 30-50%. The mass fraction of the solvent in the coating composition ranges from 60% to 99.9%, preferably from 90-99%.

[0037] The coating composition may also include additives as needed. Additives include one or more of the following: leveling agents, defoamers, film-forming aids, viscosity modifiers, pigments, antibacterial agents, colorants, surfactants, pH adjusters, buffer solutions, preservatives, etc., to optimize and adjust the coating base liquid.

[0038] This invention specifically provides an antithrombotic conjugate, which is formed by conjugating a terpolymer with an active anticoagulant fragment, wherein the terpolymer comprises: A) Inert anticoagulation unit; B) Grafted units with reactive groups; C) Units that act as adhesives to the substrate; Specifically, the inert anticoagulant unit refers to a unit capable of reducing the adsorption and activation of blood components (such as plasma proteins and platelets) on the surface of a medical device. These units provide anticoagulant effects through their physicochemical properties rather than through the release of bioactive molecules. Specifically, they can include polymerizable phosphorylcholine and its derivatives, polymerizable polymethyl polyethylene glycol acrylate and its derivatives, polymerizable polyethylene oxide, polymerizable sulfonate betaine, polymerizable carboxylate betaine, etc. These can be categorized as polyethylene glycol and zwitterionic units, possessing high hydrophilicity, anti-protein adsorption capacity, and anti-platelet adhesion capacity, thereby reducing the risk of thrombosis. Inert anticoagulant units can also include superhydrophobic units such as polymerizable fluoropolymers, which can passivate the surface, reduce friction, reduce ion release, and achieve anticoagulant effects by preferentially adsorbing non-thrombotic proteins such as albumin.

[0039] The grafting unit with reactive groups primarily provides reactive sites for the subsequent conjugation of active anticoagulant fragments. In principle, reactive groups such as hydroxyl, amino, carboxyl, and aldehyde groups on the active anticoagulant fragment (e.g., natural heparin degradation fragments) can serve as the basis for the conjugation reaction. Alternatively, the active anticoagulant fragment can be post-modified, or reactive sites such as azide, mercapto, and maleimide groups can be appropriately introduced during synthesis. For details, please refer to the literature (ISBN: 978-0-12-370501-3, Bioconjugate techniques, 2nd edition, 2008, the entirety of which is incorporated herein by reference). More preferably, the reactive group is selected from at least one of amino, carboxyl, halogen, epoxy, aldehyde, azide, mercapto, and vinyl groups.

[0040] The unit that acts as an adhesive to the substrate specifically refers to a unit that can form a strong bond with the substrate surface through a certain force. For example, it can be a unit that can be tightly bonded to the substrate surface through chemical bonding, physical adsorption, swelling interlocking, or other interaction methods. Specifically, it can include the following types: (1) polymerizable hydrophobic alkanes. Due to their nonpolar properties, hydrophobic alkane molecules tend to avoid contact with water molecules. This hydrophobic effect promotes the aggregation of hydrophobic molecules and their adsorption and fixation to the surface through hydrophobic interactions, van der Waals forces, etc. For example, hydrophobic alkanes have the structure shown in the following formula (I): Equation (I) Each occurrence of Rb is independently selected from alkyl or alkoxy groups having 1 to 10 carbon atoms; preferably, it is a straight chain. Each occurrence of Ra is independently selected from -H or methyl; Each time A appears, it is independently selected from O or NH; (2) Polymerizable silane coupling agents: silane coupling agents are compounds containing two different functional groups. One end is a silanol group (Si-OH) or silanoxy group (Si-OR) that can react with the surface of inorganic materials (such as glass, metal, oxides, etc.), and the other end is an organic functional group (such as amino, epoxy, methacryloyloxy, etc.) that can react with organic materials. They are widely used, especially in metal substrates. (3) Polymerizable polyphenols: such as dopamine, tannic acid, catechol, or polyphenolic compounds. These polyphenolic materials can form a stable bond with the surface of materials through self-polymerization, co-deposition, etc., and have broad substrate applicability. (4) Polymerizable photosensitive molecules: Units containing photosensitive structures can generate active free radicals under UV or visible light irradiation. The active free radicals can abstract hydrogen atoms or other unstable atoms from the polymer to achieve polymerization and curing crosslinking. The unit containing the photosensitive structure does not contain functional groups that would significantly interfere with the cationic polymerization process and is capable of absorbing light in the wavelength range of about 100 to about 600 nanometers (nm). More specifically, the unit containing the photosensitive structure may be derived from, but is not exclusively limited to, the following group, which consists of: substituted or unsubstituted benzophenone, acetophenone, thioxanthone, thioxanthone, fluorenone, anthraquinone acrylone, dibenzocycloheptanone, benzoin, phenylcoumarone, and their derivatives. Preferably, it is derived from substituted or unsubstituted benzophenone, acetophenone, thioxanthone, thioxanthone, fluorenone, and their derivatives. More specifically, the unit containing the photosensitive structure can typically be a substituted or unsubstituted benzophenone. When a benzophenone unit is used as the photosensitive structural unit of the present invention, superior photoinitiation efficiency can be obtained.

[0041] The active anticoagulant fragment is selected from at least one of heparin, heparin derivatives, heparin-like substances, heparin-like derivatives, and hirudin. Heparin is a glycoprotein whose anticoagulant effect mainly depends on antithrombin III (AT-III). Upon binding to AT-III, heparin induces a conformational change in AT-III, thereby increasing its affinity for thrombin and rapidly neutralizing thrombin (FIIa). Furthermore, heparin enhances the inhibitory effect of AT-III on other coagulation factors such as FX1a, FX10a, FX11a, and FX12a. Heparin prevents blood clotting by inhibiting the coagulation cascade. Heparin-like substances generally refer to substances with heparin-like anticoagulant properties and can be natural, synthetic, or semi-synthetic. Some heparin-like substances may exert their effects by mimicking the interaction between heparin and AT-III, while others may affect the coagulation process through different mechanisms. Hirudin is a polypeptide with extremely strong anticoagulant activity. It irreversibly inhibits thrombin activity by directly binding to the active site of thrombin, forming a stable complex. This direct inhibition prevents thrombin from catalyzing the conversion of fibrinogen to fibrin, and from performing other functions of thrombin in the coagulation cascade, such as activating platelets and coagulation factors V and VIII. This action of hirudin is independent of antithrombin, thus providing an anticoagulant mechanism distinct from heparin.

[0042] The ternary copolymer of this invention is obtained by free radical polymerization of a polymerizable inert anticoagulant monomer, a graft monomer with reactive groups, and a monomer capable of bonding with a substrate. This includes, but is not limited to, ordinary free radical polymerization and living controlled free radical polymerization. Preferably, the ternary copolymer is prepared by ordinary free radical polymerization. The ternary copolymer is prepared in a certain medium, including, but not limited to, solution polymerization, emulsion polymerization, reverse emulsion polymerization, suspension polymerization, and bulk polymerization. From the perspective of ease of operation, preferably, the ternary copolymer is completed by solution polymerization. Suitable solvents include polar organic solvents such as methanol, ethanol, acetonitrile, and DMF. In one embodiment of this invention, the polymerizable inert anticoagulant monomer, the graft monomer with reactive groups, and the monomer capable of bonding with a substrate are dissolved in an organic solvent. A free radical initiator is added to the system, nitrogen is purged to remove oxygen, and the reaction is carried out at a specific temperature to obtain the ternary copolymer of this invention.

[0043] In some embodiments of the present invention, the polymerizable inert anticoagulant monomer is selected from one or more of 2-methacryloyloxyethyl phosphorylcholine, polyethylene glycol methacrylate, 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate, hydroxyethyl methacrylate, and 2-ethyl-2-azolinium. The monomer that acts as an adhesive to the substrate is selected from one or more of aminopropyltriethoxysilane, vinyltriethoxysilane, vinyltrimethoxysilane, vinyltrichlorosilane, vinyltri(2-ethoxyethoxy)silane, γ-(methacryloyloxy)propyltrimethoxysilane, polyethylene glycol catechols with double bonds, and 3-methacryloyldopamine; the grafting monomer with reactive groups is selected from N-(2-aminoethyl)methacrylamide hydrochloride, N-(3-aminopropyl)methacrylamide hydrochloride, glycidyl methacrylate, acrylic acid, methacrylic acid, etc.

[0044] The ternary copolymer with the above structure can achieve a strong bond with the substrate surface by bonding the part that acts as an adhesive to the substrate. The inert anticoagulant unit can reduce the adhesion of non-specific biomolecules on the surface, improve the biocompatibility of the material and reduce the possibility of coagulation. On the other hand, the grafting unit with reactive groups can exhibit specific and effective binding to active anticoagulant molecules, improve coverage density and maintain the bioactivity of anticoagulant active molecules.

[0045] In some specific embodiments of the present invention, the molar percentage of the polymerizable inert anticoagulant monomer in the ternary copolymer is 10-50%, more preferably 20%-40%, and exemplaryly, it can be 20%, 25%, 30%, 35%, or 40%. The proportion of inert polymer should not be less than 20% to ensure that the polymer has a significant inert anticoagulant effect. The molar percentage of the monomer capable of bonding with the substrate is 50%-70%, and exemplaryly, it can be 50%, 55%, 60%, 65%, or 70%. If the proportion of the bonding groups with the substrate is less than 50%, it will affect the strength of the bond between the polymer and the substrate. The molar percentage of the grafted monomer with reactive groups is 0.5-10%, preferably 5%-10%, and the sum of the molar percentages of the three is 100%. The proportion of the grafted monomer with reactive groups should be controlled to not exceed 10%. This is because the molecular weight of the active anticoagulant fragments to be grafted later (such as heparin molecules) is relatively large, resulting in a volume repulsion effect. Therefore, increasing the proportion of reactive groups will not significantly increase the grafting ratio of heparin, but may instead lead to the exposure of more active groups.

[0046] In some specific embodiments of the present invention, the molar ratio of the polymerizable inert anticoagulant monomer to the grafted monomer with reactive groups is (3-20):1, more preferably (2-8):1. Exemplary ratios can be 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, etc. Because heparin molecules have a large molecular weight and exhibit volume repulsion, increasing the proportion of reactive groups does not significantly increase the grafting ratio of heparin; on the contrary, it may lead to the exposure of more active groups. A higher proportion of inert monomers ensures that the overall surface maintains a good anticoagulant inert state.

[0047] In some specific embodiments of the present invention, the number average molecular weight of the terpolymer is 2,000-200,000; preferably 3,000-150,000; more preferably 5,000-100,000.

[0048] In some specific embodiments of the present invention, the terpolymer has an exemplary formula as follows: The structural formula of AG: (A) (B) (C) (D) (E) (F) (G) The active anticoagulant fragment can covalently bond with reactive groups such as amino and epoxy groups at the side chain end of the terpolymer to form an antithrombotic conjugate.

[0049] In some specific embodiments of the present invention, the active anticoagulant fragment is covalently bonded to the ternary copolymer. Preferably, the active anticoagulant fragment is a heparin fragment, the fragment being composed of at least 5 sugar units, and at least some of the plurality of fragments containing polysaccharide sequence A:

[0050] This pentasaccharide sequence is the main source of heparin's anticoagulant activity.

[0051] In some specific embodiments of the present invention, the active anticoagulant fragment is covalently bonded to the side chain (non-terminal group) of the ternary copolymer. When an active anticoagulant fragment, such as a heparin fragment, is covalently bonded to the ternary copolymer, it must not disrupt the pentasaccharide sequence and must expose as many active sites as possible. In this invention, grafting units with reactive groups exist in the ternary copolymer as repeating units, and their reactive sites, capable of conjugating with the heparin fragment, are distributed on the side chain of the copolymer, thereby providing an adjustable grafting density for the grafting of the active anticoagulant molecule.

[0052] Furthermore, in this invention, the covalent grafting ratio of the active anticoagulant fragment is 10%-70%. Exemplary values ​​can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%. The covalent grafting ratio refers to the proportion of reactive groups on the polymer chain that can actually attach to heparin molecules. Typically, due to steric hindrance and other reasons, the covalent grafting ratio cannot reach 100%.

[0053] The method for preparing the antithrombotic conjugate of the present invention includes: subjecting the inert anticoagulant monomer, the monomer that acts as an adhesive to the substrate, and the graft monomer with reactive groups to living free radical polymerization to obtain the ternary random copolymer; subsequently dissolving the obtained ternary random copolymer in a buffer solution and adding an active anticoagulant fragment; reacting the mixed solution at room temperature under the action of a coupling agent to finally obtain the antithrombotic conjugate. The coupling agent includes, but is not limited to, carboxyl reactive reagents (EDC / NHS), thiol reactive reagents (such as maleimide and pyridine dimercapto), N-hydroxysuccinimide esters, isocyanates, and other activators and crosslinking agents.

[0054] The present invention also provides a coating composition comprising a solvent and the antithrombotic conjugate described herein. In some embodiments, the antithrombotic conjugate has a mass fraction of 5% to 20% w / w in the coating composition. Optionally, the mass fraction of the antithrombotic conjugate in the coating composition may be, for example, 5%, 10%, 15%, 20%, etc.

[0055] In a preferred embodiment of the present invention, the choice of solvent is not overly limited, and includes, but is not limited to, methanol, ethanol, isopropanol, n-butanol, water, ethylene glycol, diethylene glycol, polyethylene glycol, glycerol, propylene glycol, pentaerythritol, vinyl alcohol, polyvinyl alcohol, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), ethyl acetate, toluene, chloroform, dichloromethane, and combinations thereof.

[0056] The present invention also provides an antithrombotic coating obtained by curing the coating composition.

[0057] In this invention, the curing method of the coating is not limited. It can be selected according to the characteristics of the copolymer, especially the characteristics of the unit that acts as an adhesive to the substrate. For example, hydrophobic alkanes can be cured by solvent evaporation or thermal curing, including but not limited to solvent evaporation; silane coupling agents and polyphenols can be cured by chemical grafting in solution, solvent evaporation, or thermal curing, including but not limited to solvent evaporation; photosensitive molecules can be cured by photocuring or radiation curing, including but not limited to photocuring.

[0058] The antithrombotic coating of the present invention can be used in various articles (e.g., medical or diagnostic articles) and the shape of the articles is not limited, including films, sheets, rods, tubes, molded parts, fibers, fabrics and granules.

[0059] This invention also provides a medical device comprising the aforementioned antithrombotic coating. In this invention, "medical device" should be interpreted broadly, encompassing implantable devices, external devices, and in vitro diagnostic products. Implantable devices can be used temporarily for short periods or permanently implanted. Suitable examples of medical devices include drug delivery stents, other vascular devices (e.g., grafts, catheters, valves, artificial hearts, cardiac assist devices), implantable defibrillators, blood oxygenation devices (e.g., tubing, membranes), surgical devices, cell culture equipment, biosensors, wound care devices, endoscopic devices, orthopedic equipment, dental instruments, urological instruments, colostomy bag attachments, ophthalmic equipment, intraocular lenses, and dialysis equipment.

[0060] In particular, this invention is applicable to catheters / guidewires made of various materials, including polyvinyl chloride, polyethylene, polypropylene, silicone rubber, latex, polytetrafluoroethylene, and perfluoroethylene propylene.

[0061] Example Section Referring to the above implementation content, in order to make the technical solution of this application more specific, clear and easy to understand, examples of the technical solution of this application are now given. However, it should be noted that the following examples are used to illustrate the present invention. Those skilled in the art can understand that the examples are merely exemplary and not exhaustive.

[0062] Preparation Example 1: Preparation of Antithrombotic Conjugates 2-Methacryloxyethylphosphorylcholine (1.19 g, 4 mmol), N-(3-aminopropyl)methacrylamide hydrochloride (0.18 g, 1 mmol), and 3-methacrylamide dopamine (1.11 g, 5 mmol) were dissolved in 50 mL of methanol. A hypoxic environment was created by purging with nitrogen gas. Azobisisobutyronitrile (16.4 mg, 0.1 mmol) was added as an initiator, and the mixture was stirred until dissolved. The reaction was carried out at 65 °C for 8 h. After the reaction was complete, polymer-1 was precipitated by passing it through n-hexane.

[0063] Weigh 0.5 g of heparin and dissolve it in 150 mL of phosphate buffer at pH 5.3. Add 1.4 g of polymer-1, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (0.96 g, 6 mmol) and N-hydroxysuccinimide (0.29 g, 2.5 mmol). Stir at room temperature for 24 h, dialyze for 3 days, and then freeze dry to obtain conjugate-1.

[0064] Preparation Example 2: Preparation of Antithrombotic Conjugates Polyethylene glycol methacrylate (2 g, 4 mmol), N-(3-aminopropyl)methacrylamide hydrochloride (0.18 g, 1 mmol), and 3-(methacryloyloxy)propyltrimethoxysilane (1.24 g, 5 mmol) were dissolved in 50 mL of methanol. A hypoxic environment was created by purging with nitrogen, and the initiator azobisisobutyronitrile (16.4 mg, 0.1 mmol) was added. The mixture was stirred until dissolved and reacted at 65 °C for 8 h. After the reaction, polymer-2 was obtained by precipitation with n-hexane.

[0065] Weigh 0.5 g of heparin and dissolve it in 150 mL of dichloromethane. Add 1.4 g of polymer-2,1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (0.96 g, 6 mmol) and N-hydroxysuccinimide (0.29 g, 2.5 mmol). Stir at room temperature for 24 h, dialyze for 3 days, and freeze dry to obtain conjugate-2.

[0066] Preparation Examples 3-6: Preparation of Antithrombotic Conjugates 2-Methacryloxyethylphosphorylcholine was selected as monomer A, glycidyl methacrylate as monomer B, and 3-(methacryloyloxy)propyltrimethoxysilane as monomer C. The monomers were subjected to free radical polymerization according to the molar ratios shown in Table 1, as described in Example 1. After polymerization, polymer-3—polymer-6 was obtained by precipitation with n-hexane.

[0067] Then, following the method of Preparation Example 1, the synthesized polymer was conjugated with heparin.

[0068] Table 1. Molar ratio of monomers in ternary polymers A C B Copolymer-3 20% 70% 10% Copolymer-4 30% 50% 20% Copolymer-5 40% 50% 10% Copolymer-6 60% 30% 10% Preparation Example 7: Hydroxyethyl methacrylate (0.52 g, 4 mmol), N-(3-aminopropyl)methacrylamide hydrochloride (0.18 g, 1 mmol), and 3-methacrylamide dopamine (1.11 g, 5 mmol) were dissolved in 50 mL of methanol. A hypoxic environment was created by purging with nitrogen gas. Azobisisobutyronitrile (16.4 mg, 0.1 mmol) was added as an initiator, and the mixture was stirred until dissolved. The reaction was carried out at 65 °C for 8 h. After the reaction, polymer 7 was precipitated by passing the precipitate through n-hexane. 0.5 g of heparin was dissolved in 150 mL of dichloromethane. 1.4 g of polymer 7, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (0.96 g, 6 mmol), and N-hydroxysuccinimide (0.29 g, 2.5 mmol) were added. The mixture was stirred at room temperature for 24 h, dialyzed for 3 days, and then lyophilized to obtain conjugate-7.

[0069] Example 1 After cleaning the central venous catheter, immerse it in a 5% conjugate-1 solution (solvent: pH 7.4 phosphate buffer). Stir thoroughly, soak for 30 minutes, and then cure at 70°C for 2 hours. Example

[0070] After cleaning the PVC conduit, immerse it in a 5% conjugate-2 solution (solvent: pH 7.4 phosphate buffer). Stir thoroughly, soak for 30 minutes, and then cure at 70°C for 2 hours. Example

[0071] After cleaning the PVC conduit, immerse it in a 5% conjugate-3 solution (solvent: pH 7.4 phosphate buffer). Stir thoroughly, soak for 30 minutes, and then cure at 70°C for 2 hours.

[0072] Comparative Example 1 After cleaning the central venous catheter, immerse it in a 5% conjugate-4 solution (solvent: pH 7.4 phosphate buffer). Stir thoroughly, soak for 30 minutes, and then cure at 70°C for 2 hours.

[0073] Comparative Example 2 After cleaning the central venous catheter, immerse it in a 5% conjugate-6 solution (solvent: pH 7.4 phosphate buffer). Stir thoroughly, soak for 30 minutes, and then cure at 70°C for 2 hours.

[0074] Comparative Example 3 Untreated central venous catheter.

[0075] Test Example 1: Protein Adsorption Test Protein adsorption on the sample surface was tested using a fibrinogen (Fg) protein detection kit. The test data are shown in Table 2 below. Specifically, the corresponding bare substrate was used as a control sample for the coating of this invention.

[0076] Experimental data show that the conjugate of this invention can significantly reduce the adsorption of non-specific proteins, achieving a good inert anticoagulant effect. However, Comparative Example 1 shows a poorer anti-protein effect because the proportion of grafted units is too high, leading to the exposure of more active groups on the conjugate. These active groups bind to sites on proteins, thus failing to achieve a good anti-protein adhesion effect.

[0077] Table 2 Protein Adsorption Test Data Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 3 Protein adsorption capacity / OD value 0.4611 0.4862 0.4321 0.6908 0.7307 Protein adsorption decreased by % 47.0% 44.1% 50.3% 20.6% 16.1% Test Example 2: Partial Thromboplastin Time Test Fresh anticoagulated whole blood was collected from healthy adult rabbits and centrifuged to obtain platelet-poor plasma (PPP). A certain proportion of PPP was added to the prepared sample, negative control, and positive control. A blank control was prepared by adding only PPP. The control group and sample were placed in a constant-temperature shaker at 37°C and incubated for 15 minutes. After incubation, the extract was transferred to centrifuge tubes, with at least 600 μL of sample extract per tube. One centrifuge tube was placed in an automated coagulation analyzer for testing, and the results are shown in Table 3 below.

[0078] Experimental data show that the antithrombotic conjugate of the present invention can effectively prolong clotting time. In Comparative Example 2, due to the lower binding unit component in the conjugate structure, the amount of heparin molecules bound to the surface was reduced.

[0079] Table 3 shows some of the thromboplastin time (PTT) tests. Example 1 Example 2 Comparative Example 2 Comparative Example 3 Sample solidification time / blank solidification time (%) 298 298 237 121 Test Example 3: Antiplatelet Adhesion Test Fresh anticoagulated whole blood was collected from healthy adult rabbits, and the supernatant was collected by centrifugation to obtain platelet-rich plasma (PRP). The test sample and PRP were mixed in a specific ratio and incubated at 37°C for 60 min. After incubation, the sample was removed from the PRP and washed three times with PBS. After initial air-drying, the sample was fixed with 2.5% glutaraldehyde. A gradient ethanol dehydration process was performed: elution at 20%, 40%, 60%, 80%, and 100% ethanol for 10 min each, followed by air-drying. The sample was then sputter-coated with gold and analyzed by SEM. The test results are shown below. Figure 1 .

[0080] Test Example 4: Coating Adhesion Test After rinsing the tubing in a buffer solution to simulate the real blood flow environment in the human body for 14 days, the tubing was removed and subjected to partial thromboplastin time testing.

[0081] Table 4 shows some of the thromboplastin time (PTT) tests. Example 1 Example 2 Comparative Example 2 Comparative Example 3 Sample solidification time / blank solidification time (%) 298 272 197 106

Claims

1. An antithrombotic conjugate, characterized in that, The antithrombotic conjugate is formed by conjugating a terpolymer with an active anticoagulant fragment, wherein the terpolymer comprises: A) Inert anticoagulation unit; B) Grafted units with reactive groups; C) Units that act as adhesives to the substrate; The inert anticoagulant unit is derived from at least one of polymerizable phosphorylcholine and its derivatives, polymerizable polymethyl polyethylene glycol acrylate and its derivatives, polymerizable polyethylene oxide, polymerizable fluoropolymers, polymerizable sulfonate betaine, and polymerizable carboxylate betaine; the reactive group is selected from active groups capable of reacting with hydroxyl, amino, carboxyl, aldehyde, azide, or thiol groups on the active anticoagulant fragment; the unit that acts as an adhesive to the substrate is selected from at least one of polymerizable hydrophobic alkanes, polymerizable silane coupling agents, polymerizable polyphenols, and polymerizable photosensitive molecules; and the active anticoagulant fragment is selected from at least one of heparin, heparin derivatives, heparin-like substances, heparin-like derivatives, and hirudin.

2. The antithrombotic conjugate according to claim 1, characterized in that, The terpolymer is obtained by free radical polymerization of a polymerizable inert anticoagulant monomer, a graft monomer with reactive groups, and a monomer capable of bonding with a substrate. The polymerizable inert anticoagulant monomer is selected from one or more of 2-methacryloyloxyethyl phosphorylcholine, polyethylene glycol methacrylate, 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium]propane-1-sulfonic acid inner salt, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate, hydroxyethyl methacrylate, and 2-ethyl-2-azolin.

3. The antithrombotic conjugate according to claim 2, characterized in that, The monomer that acts as an adhesive to the substrate is selected from one or more of aminopropyltriethoxysilane, vinyltriethoxysilane, vinyltrimethoxysilane, vinyltrichlorosilane, vinyltri(2-ethoxyethoxy)silane, and γ-(methacryloyloxy)propyltrimethoxysilane.

4. The antithrombotic conjugate according to claim 2, characterized in that, The monomer that acts as an adhesive to the substrate is selected from one or more of polyethylene glycol catechols with double bonds and 3-methacrylamide dopamine.

5. The antithrombotic conjugate according to any one of claims 2-4, characterized in that, The polymerizable inert anticoagulant monomer in the terpolymer accounts for 10-50% molar percentage, preferably 20-40%; the monomer capable of bonding with the substrate accounts for 50%-70% molar percentage; the grafted monomer with reactive groups accounts for 0.5-10% molar percentage; and the sum of the three molar percentages is 100%.

6. The antithrombotic conjugate according to claim 5, characterized in that, in, The molar ratio of the polymerizable inert anticoagulant monomer to the grafted monomer with reactive groups is (3-20):

1.

7. The antithrombotic conjugate according to claim 1, characterized in that, The active anticoagulant fragment is covalently bonded to the side chain end of the ternary copolymer.

8. The antithrombotic conjugate according to claim 7, characterized in that, The covalent grafting ratio of the active anticoagulant fragment is 10%-70%.

9. The antithrombotic conjugate according to claim 1, characterized in that, The number average molecular weight of the terpolymer is 2,000-200,000; preferably 3,000-150,000; more preferably 5,000-100,000.

10. The antithrombotic conjugate according to claim 1, characterized in that, The active anticoagulant fragment is a heparin fragment, the fragment is composed of at least 5 glycounits, and at least some of the plurality of fragments contain polysaccharide sequence A:

11. A method for preparing the antithrombotic conjugate as described in any one of claims 1-10, characterized in that, The inert anticoagulant monomer, the monomer that acts as an adhesive to the substrate, and the grafted monomer with reactive groups are subjected to living free radical polymerization to obtain the ternary random copolymer. The obtained ternary random copolymer is then dissolved in a buffer solution, and an active anticoagulant fragment is added. The mixed solution is reacted under the action of a coupling agent to finally obtain the antithrombotic conjugate.

12. A coating composition, characterized in that, The coating composition comprises a solvent and at least the antithrombotic conjugate as described in any one of claims 1-10.

13. The coating composition according to claim 12, characterized in that, The antithrombotic conjugate has a mass fraction of 5%~20% w / w in the coating composition.

14. An antithrombotic coating, characterized in that, The antithrombotic coating is obtained by curing the coating composition according to claim 12 or 13.

15. A medical product, characterized in that, The medical article has at least a portion of its surface covered by the antithrombotic coating as described in claim 14.

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