Coating for surface of medical material, preparation method and application of coating and medical material

Abstract

The invention provides a coating for the surface of a medical material, a preparation method and application of the coating and the medical material.The preparation method comprises the following steps that dopamine and a polyamino compound are co-deposited on the surface of the medical material, and an adhesive layer with amino is formed on the surface of the medical material; heparin and hyaluronic acid are mixed, and carboxyl groups of heparin and hyaluronic acid are activated; and contacting the aminated medical material with the carboxyl activated heparin and hyaluronic acid, so that the amino group on the surface of the medical material and the carboxyl activated by the heparin and the hyaluronic acid are subjected to a covalent grafting reaction, and the coating for the surface of the medical material is prepared. The coating has high-efficiency anticoagulation and anti-inflammatory dual biological activities, and has excellent stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of surface modification of medical materials, in particular to a coating for the surface of a medical material and a preparation method, application and medical material thereof. BACKGROUND

[0002] When blood or tissue fluid contacts the surface of a medical device implanted in the body (such as a vascular stent, a heart valve, an artificial blood vessel, an indwelling catheter, a hemodialysis membrane, an artificial organ, etc.), a cascade of biological reactions will be triggered rapidly: on the one hand, the non-specific adsorption of plasma proteins (such as fibrinogen, von Willebrand factor, etc.) on the material surface will promote platelet adhesion, activation and aggregation, activate the coagulation cascade, and ultimately lead to thrombosis; on the other hand, the material as a "foreign body" will also activate the complement system, induce the recruitment and adhesion of immune cells (such as neutrophils, monocytes / macrophages) to the implant site, release inflammatory factors (such as IL-1β, TNF-α), and trigger acute and chronic inflammatory reactions, and even lead to tissue fibrosis or implant failure. Thrombosis and inflammation promote each other to form a vicious cycle, which constitutes the main challenge for the clinical application of medical devices (especially blood-contacting devices and implants). Therefore, improving the hemocompatibility (anticoagulant property) and biocompatibility (anti-inflammatory property) of medical materials is the core goal of the field of surface engineering.

[0003] To improve the surface properties of materials, surface modification techniques have been widely studied; in particular, forming a coating on the surface of a medical material is widely used for surface modification of medical materials. The stability of the coating has a great influence on the efficacy and service life of the medical material.

[0004] Therefore, there is an urgent need to provide a coating for the surface of a medical material that has both efficient anticoagulant and anti-inflammatory dual biological activities and excellent stability. SUMMARY

[0005] Based on this, the present application provides a coating for the surface of a medical material that has both efficient anticoagulant and anti-inflammatory dual biological activities and excellent stability, and a preparation method, application and medical material thereof.

[0006] The first aspect of the present application provides a preparation method of a coating for the surface of a medical material, comprising the following steps:

[0007] Dopamine and a polyamino compound are co-deposited on the surface of a medical material to form an adhesion layer with amino groups on the surface of the medical material;

[0008] Heparin and hyaluronic acid are mixed, and the carboxyl groups of the heparin and the hyaluronic acid are activated;

[0009] contacting the adhesion layer having the amino group with the heparin and the hyaluronic acid after the carboxyl group of the heparin and the hyaluronic acid is activated to make a covalent grafting reaction between the amino group and the carboxyl group of the heparin and the hyaluronic acid after the activation, to obtain a coating for a medical material surface.

[0010] In some embodiments, the mass ratio of the dopamine and the polyamino compound is (1-2):1.

[0011] In some embodiments, the polyamino compound comprises one or more of polylysine, polyethyleneimine, and lysozyme.

[0012] In some embodiments, the step of performing co-deposition of the dopamine and the polyamino compound on the surface of the medical material to perform an amino group treatment on the surface of the medical material comprises:

[0013] dissolving the dopamine and the polyamino compound in a weak alkaline buffer solution with a pH value of 8-9 to obtain a first mixed solution, and immersing the medical material in the first mixed solution to make the dopamine and the polyamino compound co-deposit on the surface of the medical material.

[0014] In some embodiments, the sum of the mass ratios of the dopamine and the polyamino compound in the first mixed solution is 1%-5%.

[0015] In some embodiments, the co-deposition time is 2 hours-24 hours.

[0016] In some embodiments, the mass ratio of the heparin and the hyaluronic acid is (1-16):4.

[0017] In some embodiments, the step of mixing heparin and hyaluronic acid and performing activation treatment on the carboxyl group of the heparin and the hyaluronic acid comprises:

[0018] dissolving the heparin and the hyaluronic acid in a weak acid buffer solution with a pH value of 5-6 to obtain a second mixed solution, and then adding a carboxyl group activator and a catalyst to the second mixed solution to perform activation treatment on the carboxyl group of the heparin and the hyaluronic acid.

[0019] In some embodiments, the sum of the mass ratios of the heparin and the hyaluronic acid in the second mixed solution is 0.5%-3%.

[0020] In some embodiments, the carboxyl group activator comprises one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, and N,N'-diisopropylcarbodiimide.

[0021] In some embodiments, the molar concentration of the carboxyl activating agent in the second mixed solution after the carboxyl activating agent and the catalyst are added is 2 mM-20 mM.

[0022] In some embodiments, the catalyst comprises one or more of 1-hydroxy-2,5-pyrrolidinedione, 1-hydroxy-7-azabenzotriazole, and 1,1'-carbonyldiimidazole.

[0023] In some embodiments, the molar concentration of the catalyst in the second mixed solution after the carboxyl activating agent and the catalyst are added is 2 mM-20 mM.

[0024] In some embodiments, the temperature of the activation treatment is 20°C-25°C and the time is 30 minutes-120 minutes.

[0025] In some embodiments, the temperature of the covalent grafting reaction is 35°C-40°C and the time is 8 hours-12 hours.

[0026] A second aspect of the present application provides a coating for a medical material surface, comprising an adhesion layer and heparin and hyaluronic acid fixed on the surface of the adhesion layer, wherein the adhesion layer comprises a polymer of dopamine and a polyamino compound, and the heparin and hyaluronic acid are fixed on the surface of the adhesion layer through an amide bond.

[0027] In some embodiments, the mass ratio of the heparin to the hyaluronic acid is (1-16):4.

[0028] In some embodiments, there are protrusions on the surface of the coating, and the height of the protrusions is 20 nm-30 nm.

[0029] In some embodiments, the surface roughness of the coating is 12 nm-80 nm.

[0030] In some embodiments, the water contact angle of the surface of the coating is 30°-45°.

[0031] A third aspect of the present application provides use of the coating prepared by the preparation method of the first aspect of the present application or the coating of the second aspect of the present application in the preparation of a medical material having both anticoagulant and anti-inflammatory effects.

[0032] A fourth aspect of the present application provides a medical material, wherein the surface of the medical material has the coating prepared by the preparation method of the first aspect of the present application or the coating of the second aspect of the present application.

[0033] In some embodiments, the medical material comprises one or more of a blood-contacting medical device and an implant.

[0034] In some embodiments, the medical material comprises one or more of a vascular stent, a heart valve, an artificial blood vessel, an indwelling catheter, a hemodialysis membrane, and an extracorporeal membrane oxygenator.

[0035] The method for preparing the coating on the surface of the medical material has at least the following beneficial effects:

[0036] (1) The method innovatively realizes the chemical co-fixation of heparin and hyaluronic acid in the same coating, and endows the surface of the medical material with synchronous and persistent anticoagulation and anti-inflammatory dual biological activities. Meanwhile, heparin and hyaluronic acid can produce a synergistic effect in the coating (for example, the steric hindrance of hyaluronic acid enhances the accessibility of the anticoagulation activity of heparin, and the binding of heparin to antithrombin III also indirectly regulates the inflammation pathway).

[0037] (2) The method connects the amino-functionalized surface and the pre-activated carboxyl group (heparin / hyaluronic acid) through a covalent amide bond, and has significantly improved chemical stability and mechanical stability compared with traditional physical adsorption, ionic bonding or single-component modification. The coating is difficult to fall off in a body fluid environment, and can maintain its biological activity for a long time (several weeks to several months), and is particularly suitable for medical devices that need to be implanted in the body for a long time.

[0038] (3) The method co-deposits dopamine and a polyamino compound, which can form an adhesion layer through self-polymerization / copolymerization of dopamine on one hand, and introduce a high density of polyamino compounds on the other hand, thereby significantly enhancing the primary amino group density and stability of the surface, and providing sufficient active sites for subsequent covalent fixation. The preparation method has clear and controllable preparation steps, mild conditions, and a wide range of suitable substrates, such as metal, polymer, inorganic material and other commonly used materials of medical devices.

[0039] (4) The hydrophilic hyaluronic acid molecules provide a large hydration layer space barrier, combined with the negative charge effect of heparin, to effectively resist the non-specific adsorption of blood cells and inflammatory cells on the material surface, forming a key starting point for inhibiting subsequent thrombosis and inflammatory response. The coating can effectively solve the two common complications of thrombosis and inflammation after the implantation or use of medical materials (such as vascular stents, catheters, heart valves, extracorporeal circulation pipelines, etc.), and is expected to greatly improve the biocompatibility and clinical safety / effectiveness of medical devices. BRIEF DESCRIPTION OF DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the embodiments of the present application, more completely understand the present application and its beneficial effects, the following will briefly introduce the drawings needed to be used in the embodiment description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creating any creative labor on the basis of these drawings.

[0041] Figure 1 Microscopic morphology of the surface coating of the medical material prepared in Example 1.

[0042] Figure 2 Microscopic morphology of the medical polyurethane material employed in Comparative Example 1.

[0043] Figure 3 Surface water contact angle of the surface coating of the medical material prepared in Example 1.

[0044] Figure 4 Surface water contact angle of the medical polyurethane material employed in Comparative Example 1.

[0045] Figure 5 Blood cell adhesion of the surface coating of the medical material prepared in Example 1.

[0046] Figure 6 Blood cell adhesion of the medical polyurethane material employed in Comparative Example 1.

[0047] Figure 7 Inflammatory cell adhesion of the surface coating of the medical material prepared in Example 1.

[0048] Figure 8 Inflammatory cell adhesion of the medical polyurethane material employed in Comparative Example 1.

[0049] Figure 9 Inflammatory factor secretion data after co-culturing the surface coating of the medical material prepared in Example 1 and the medical polyurethane material employed in Comparative Example 1 with inflammatory phagocytes, respectively. DETAILED DESCRIPTION

[0050] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the accompanying examples. The following gives the preferred embodiments of the present application. However, the present application can be realized in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the disclosure of the present application will be thorough and complete, and will fully convey the scope of the present application to those skilled in the art.

[0051] 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 application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application.

[0052] The selection scope of the terms "and / or", "or / and", "and / or" used in the present application includes any one of two or more relevant listed items, and also includes any and all combinations of the relevant listed items, including any two relevant listed items, any more relevant listed items, or all relevant listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", "and / or", it should be understood that in the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or".

[0053] In the present application, the technical features described in an open manner include both the closed technical solution consisting of the listed features and the open technical solution containing the listed features.

[0054] In the present application, if no special description is provided, the numerical interval is considered to be continuous, and includes the minimum value and the maximum value of the range, as well as every value between the minimum value and the maximum value. Further, when the range is an integer, every integer between the minimum value and the maximum value of the range is included. In addition, when multiple ranges are provided to describe a feature or a characteristic, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges therein.

[0055] Only some numerical ranges are specifically disclosed herein. However, any lower limit can be combined with any upper limit to form a range not explicitly recited; and any lower limit can be combined with other lower limits to form a range not explicitly recited, and likewise any upper limit can be combined with any other upper limit to form a range not explicitly recited. In addition, each individual disclosed point or single numerical value can itself be combined as a lower limit or an upper limit with any other point or single numerical value or with other lower limits or upper limits to form a range not explicitly recited.

[0056] In the present application, the temperature parameter, if not specifically limited, allows for constant temperature treatment, and also allows for treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuation within a range such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C is allowed.

[0057] In the present application, "suitable" in "suitable combination", "suitable manner", "any suitable manner" and the like means that the technical solution of the present application can be implemented, the technical problems of the present application can be solved, and the expected technical effects of the present application can be achieved.

[0058] In the present application, “further”, “furthermore”, “in particular” and the like are used to describe purposes and represent differences in content, but should not be understood as limiting the scope of protection of the present application.

[0059] In the present application, “optionally”, “optional” and “optional” mean optional, that is, selected from either of the two parallel schemes of “yes” or “no”. If there are multiple “options” in a technical solution, unless otherwise specified, and there is no contradictory relationship or mutual restriction, each “option” is independent.

[0060] In the description of the application, “multiple” means at least two, for example, two, three, and the like, unless otherwise specifically limited.

[0061] If not specifically stated, all embodiments and optional embodiments of the present application can be combined to form new technical solutions. If not specifically stated, all technical features and optional technical features of the present application can be combined to form new technical solutions.

[0062] If not specifically stated, all steps of the present application can be performed in sequence or randomly, preferably in sequence.

[0063] In order to improve the surface properties of materials, surface modification technology has been widely studied. Among them, heparin (Heparin) has become the most recognized anticoagulant with the longest application history due to its excellent anticoagulation effect. Heparin binds to antithrombin III (ATIII) through its specific pentasaccharide sequence with high affinity, accelerates the inactivation of ATIII to thrombin (Thrombin) and coagulation factor Xa, and thus effectively blocks the coagulation pathway. Physical adsorption, ionic bonding or covalent immobilization of heparin is a common strategy to improve the anticoagulant properties of materials. However, the existing heparin coating has significant limitations, for example, in patent applications CN113384758A and CN106730051A, heparin and other positively charged compounds are adsorbed on the surface of medical materials by electrostatic force and van der Waals force. The heparin anticoagulant coating prepared has the following limitations:

[0064] 1. Single function: Heparin is mainly directed against the coagulation pathway, and lacks direct regulation of inflammatory response, making it difficult to meet the complex needs of multiple biological reactions after implantation of devices;

[0065] 2. Stability defects: physically adsorbed or ionically bonded heparin is easily detached and lost under the action of body fluid flushing, mechanical friction or biological enzymes (such as heparinase), resulting in a significant decrease in anticoagulant activity over time after implantation;

[0066] 3. Potential inflammation risk: heparin coating is insufficient to inhibit the inflammatory cascade (such as complement activation, leukocyte infiltration) at the early stage of implantation while preventing blood clotting, and even some studies have found that improper heparin immobilization may trigger adverse reactions through the contact activation pathway.

[0067] To make up for the shortcomings of heparin coating in biocompatibility and anti-inflammatory, a natural glycosaminoglycan, hyaluronic acid (HA), has attracted attention due to its unique properties: HA has excellent biocompatibility, high hydrophilicity, lubricity, non-immunogenicity, and can exert active anti-inflammatory effects by regulating macrophage phenotype (such as promoting the polarization of anti-inflammatory M2 type). The dense hydration layer formed by its highly hydrophilic long-chain molecules can effectively shield protein adsorption ("non-thrombogenic surface"), thereby indirectly inhibiting cell adhesion. However, there are obvious shortcomings in using hyaluronic acid alone as a coating, for example, in the patent applications CN107976472A and CN109825835A, which prepare a hyaluronic acid coating on the surface of medical materials, there are the following shortcomings: weak anticoagulant ability: HA lacks a direct and efficient targeted anticoagulant mechanism (such as activating ATIII) similar to heparin, and cannot provide sufficient antithrombotic protection strength, especially in high thrombosis risk environments (such as arterial blood flow, low shear stress area), it is difficult to meet the requirements alone.

[0068] Given the natural complementarity of heparin and hyaluronic acid in function (heparin strong anticoagulation + hyaluronic acid active anti-inflammatory / passive antifouling), researchers have begun to explore the use of both in recent years. However, related technologies (such as simple physical mixing, alternating layer-by-layer self-assembly) still have key bottlenecks:

[0069] Poor functional synergy: unoptimized proportions or non-stable combined components are prone to non-synergistic release in the body environment, making it difficult to ensure the formation of a durable, uniform, and functionally coordinated dual-active interface on the material surface;

[0070] Low stability: physically bonded coatings (such as electrostatic, van der Waals forces) have weak resistance to elution and poor mechanical stability, and component dissociation during long-term use leads to uncontrollable efficacy;

[0071] Uncontrollable structure: key parameters such as molecular density, orientation, and spatial distribution that affect function are difficult to accurately control, resulting in uneven biological activity of the coating and poor reproducibility.

[0072] Therefore, developing a complex coating technology that can stably, densely, and controllably co-immobilize heparin and hyaluronic acid on the surface of medical materials, fully exerting its "anticoagulant-anti-inflammatory" dual synergistic effect, and having excellent stability and biological safety, is of great significance for breaking through the thrombosis and inflammation complication bottleneck of high-end medical devices (especially long-term / permanent implant devices), and improving overall clinical safety and efficiency.

[0073] Based on the above problems, the present application first modifies the surface of the medical material to be aminated, constructs a high-density primary amino interface, activates the carboxyl groups of heparin and hyaluronic acid, and then carries out an amidation reaction between the amino groups on the surface of the medical material and the activated carboxyl groups of heparin and hyaluronic acid, so as to covalently graft heparin and hyaluronic acid on the surface of the medical material. The coating has the strong anticoagulation of heparin and the active anti-inflammatory / space shielding function of hyaluronic acid, and the high hydrophilicity of hyaluronic acid cooperates with the blood cell adhesion reduction of heparin to significantly inhibit the adhesion of inflammatory cells and the release of inflammatory factors. The covalent bonding ensures long-term stability and can be widely used for the surface functionalization of cardiovascular implant devices and extracorporeal circulation pipeline.

[0074] One or more embodiments of the present application provide a preparation method of a coating for the surface of a medical material, comprising the following steps: co-depositing dopamine and a polyamino compound on the surface of the medical material to form an adhesive layer with amino groups on the surface of the medical material; mixing heparin and hyaluronic acid and activating the carboxyl groups of heparin and hyaluronic acid; and contacting the adhesive layer with amino groups with heparin and hyaluronic acid after the carboxyl group activation treatment, so that the amino groups and the activated carboxyl groups of heparin and hyaluronic acid covalently graft to prepare a coating for the surface of a medical material.

[0075] It should be noted that when dopamine and a polyamino compound are co-deposited on the surface of the medical material, a polymerization reaction occurs, including self-polymerization of dopamine and co-polymerization of dopamine and the polyamino compound.

[0076] After the covalent grafting reaction of the amino groups and the activated carboxyl groups of heparin and hyaluronic acid, heparin and hyaluronic acid are fixed on the surface of the adhesive layer through amide bonds.

[0077] Understandably, the preparation method of the coating for the surface of a medical material of the present application at least has the following beneficial effects:

[0078] (1) The preparation method innovatively realizes the chemical co-fixing of heparin and hyaluronic acid in the same coating, and endows the surface of the medical material with simultaneous and persistent anticoagulation and anti-inflammatory dual biological activity. At the same time, heparin and hyaluronic acid can produce a synergistic effect in the coating (for example, the steric hindrance of hyaluronic acid enhances the accessibility of the anticoagulation activity of heparin, and heparin combined with antithrombin III also indirectly regulates the inflammatory pathway).

[0079] (2) The preparation method connects the aminated surface and the pre-activated carboxyl groups (heparin / hyaluronic acid) through covalent amide bonds, which has significantly improved chemical stability and mechanical stability compared with traditional physical adsorption, ionic bonding or single component modification. The coating is difficult to fall off in a body fluid environment, and can maintain its biological activity for a long time (several weeks to several months), and is especially suitable for medical devices that need to be implanted in the body for a long time.

[0080] (3) The preparation method can significantly enhance the primary amino group density and stability on the surface by co-deposition of dopamine and polyamino compounds, which can form an adhesion layer by self-polymerization / copolymerization of dopamine and introduce a high density of polyamino compounds, thereby providing sufficient active sites for subsequent covalent immobilization. The preparation method has clear and controllable preparation steps, mild conditions, and a wide range of suitable substrates, such as medical device commonly used materials such as metals, polymers, inorganic materials, etc.

[0081] (4) The hydrophilic hyaluronic acid molecules provide a large hydration layer space barrier, combined with the negative charge effect of heparin, which effectively resists the non-specific adsorption of blood cells and inflammatory cells on the material surface, forming a key starting point for inhibiting subsequent thrombosis and inflammatory response. The coating can effectively solve the two common complications of medical devices (such as vascular stents, catheters, heart valves, extracorporeal circulation pipelines, etc.) after implantation or use, namely thrombosis and inflammatory response, and is expected to greatly improve the biocompatibility and clinical safety / effectiveness of medical devices.

[0082] As a possible implementation, the mass ratio of dopamine and polyamino compound is (1-2):1; for example, it can be but is not limited to 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, or a range between any two of the above mass ratios, etc. Thus, it is beneficial to improve the efficiency of co-deposition reaction of dopamine and polyamino compound on the surface of medical device, thereby improving the amino activity on the surface of medical device.

[0083] In some embodiments, the polyamino compound includes one or more of polylysine, polyethyleneimine, and lysozyme.

[0084] In some optional implementations, the step of performing amino treatment on the surface of the medical device by co-deposition of dopamine and polyamino compound on the surface of the medical device includes: dissolving dopamine and polyamino compound in a weak alkaline buffer solution with a pH value of 8-9 to obtain a first mixed solution, and immersing the medical device in the first mixed solution to allow co-deposition of dopamine and polyamino compound on the surface of the medical device.

[0085] It should be noted that the "first mixed solution", "second mixed solution", etc. mentioned above and below are only for description purposes and cannot be understood as indicating or implying relative importance or quantity, nor can it be understood as implying the importance or quantity of the indicated technical features. Moreover, "first", "second", etc. only serve the purpose of non-exhaustive enumeration description and should be understood as not constituting a closed limitation on the quantity.

[0086] As an example, the pH value of the weakly basic buffer can be, but is not limited to, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, or a range between any two of the above pH values. Alternatively, the weakly basic buffer can be Tris-HCl with a pH of 8.5.

[0087] In some exemplary embodiments, the sum of the mass fractions of dopamine and polyamino compound in the first mixed solution is 1%-5%. For example, it can be, but is not limited to, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or a range between any two of the above values. In this way, dopamine self-polymerizes on the surface of the medical material to form polydopamine with strong adhesion, and dopamine and polyamino compound copolymerize to realize the co-deposition of both on the surface of the medical material to form a stable composite film.

[0088] In some alternative embodiments, the co-deposition time is 2-24 hours; for example, it can be, but is not limited to, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, or a range between any two of the above times. When the co-deposition time is in the above range, the thickness, uniformity, hydrophilicity, chemical composition, and functionality of the prepared coating can be most suitable for subsequent applications.

[0089] As a possible embodiment, the mass ratio of heparin and hyaluronic acid is (1-16):4; for example, it can be, but is not limited to, 1:4, 2:4, 3:4, 4:4, 5:4, 6:4, 7:4, 8:4, 9:4, 10:4, 11:4, 12:4, 13:4, 14:4, 15:4, 16:4, or a range between any two of the above mass ratios. When the mass ratio of heparin and hyaluronic acid is in the above range, it is beneficial to balance the strong anticoagulant activity of heparin and the anti-inflammatory, hydrophilic, and steric hindrance barrier effects of hyaluronic acid.

[0090] In some embodiments, the step of mixing heparin and hyaluronic acid and activating the carboxyl groups of heparin and hyaluronic acid includes: dissolving heparin and hyaluronic acid in a weakly acidic buffer with a pH of 5-6 to obtain a second mixed solution, and then adding a carboxyl activator and a catalyst to the second mixed solution to activate the carboxyl groups of heparin and hyaluronic acid. In this way, the free carboxyl groups on the molecular chains of heparin and hyaluronic acid are efficiently converted into highly reactive esters.

[0091] As an example, the pH of the weakly acidic buffer can be, but is not limited to, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, or a range between any two of the above pH values, etc. Alternatively, the weakly acidic buffer is a 2-(N-morpholino)ethanesulfonic acid buffer with a pH of 6.

[0092] In some alternative embodiments, the sum of the mass ratios of heparin and hyaluronic acid in the second mixed solution is 0.5%-3%. For example, it can be, but is not limited to, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, or a range between any two of the above values, etc. In this way, heparin and hyaluronic acid have a better dissolution state in the second mixed solution, and the solution viscosity range is lower, which is beneficial to subsequent reactions.

[0093] As a possible embodiment, the carboxyl activating agent includes one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, and N,N'-diisopropylcarbodiimide.

[0094] In some embodiments, the molar concentration of the carboxyl activating agent in the second mixed solution after the carboxyl activating agent and the catalyst are added is 2mM-20mM; for example, it can be, but is not limited to, 2mM, 4mM, 6mM, 8mM, 10mM, 12mM, 14mM, 16mM, 18mM, 20mM, or a range between any two of the above molar concentrations, etc. In this way, the carboxyl activating agent is in a more suitable concentration range, which helps to activate the carboxyl functional groups on the heparin and hyaluronic acid molecules.

[0095] It should be noted that "mM" mentioned above and below means millimoles per liter.

[0096] As a possible embodiment, the catalyst includes one or more of 1-hydroxy-2,5-pyrrolidinedione, 1-hydroxy-7-azabenzotriazole, and 1,1'-carbonyldiimidazole.

[0097] In some alternative embodiments, the molar concentration of the catalyst in the second mixed solution after the carboxyl activating agent and the catalyst are added is 2mM-20mM; for example, it can be, but is not limited to, 2mM, 4mM, 6mM, 8mM, 10mM, 12mM, 14mM, 16mM, 18mM, 20mM, or a range between any two of the above molar concentrations, etc. In this way, the catalyst and the carboxyl activating agent work together to catalyze the reaction of the carboxyl functional groups on the heparin and hyaluronic acid with the amino groups on the surface of the medical material.

[0098] In some alternative embodiments, the temperature of the activation treatment is 20-25°C; for example, but not limited to, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, or a range between any two of the aforementioned temperatures.

[0099] In some embodiments, the time of the activation treatment is 30-120 minutes; for example, but not limited to, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, or a range between any two of the aforementioned times.

[0100] In some embodiments, the temperature of the covalent grafting reaction is 35-40°C; for example, but not limited to, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, or a range between any two of the aforementioned temperatures.

[0101] As a possible embodiment, the time of the covalent grafting reaction is 8-12 hours; for example, but not limited to, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or a range between any two of the aforementioned times.

[0102] When the temperature and time of the covalent grafting reaction are within the aforementioned ranges, respectively, the activated carboxyl groups can efficiently react with the primary amino groups on the surface of the medical material to form stable covalent amide bonds, thereby firmly covalently grafting heparin and hyaluronic acid onto the surface of the material.

[0103] As a non-limiting example, after the covalent grafting reaction is completed, the application material is removed and the surface is thoroughly washed with deionized water (3-5 times) to completely remove the physically adsorbed or unreacted components.

[0104] One or more embodiments of the present application provide a coating for the surface of a medical material, comprising an adhesion layer and heparin and hyaluronic acid fixed on the surface of the adhesion layer, the adhesion layer comprising a polymer of dopamine and a polyamino compound, and the heparin and hyaluronic acid being fixed on the surface of the adhesion layer through amide bonds.

[0105] It should be noted that the polymer of dopamine and a polyamino compound is formed by copolymerization of dopamine and a polyamino compound. Further, the adhesion layer can further comprise a polymer obtained by self-polymerization of dopamine.

[0106] Understandably, the heparin and hyaluronic acid are stably fixed on the surface of the adhesion layer in the coating of the medical material surface of the present application through amide bonds, forming a coating with synchronous and persistent anticoagulant and anti-inflammatory dual biological activity. The hydrophilic hyaluronic acid molecules in the coating provide a large hydration layer space barrier, combined with the negative charge effect of heparin, which effectively resists the non-specific adsorption of blood cells and inflammatory cells on the material surface, forming the key starting point of inhibiting subsequent thrombosis and inflammatory response. The coating can effectively solve the two common complications of thrombosis and inflammation after the implantation or use of medical materials (such as vascular stents, catheters, heart valves, extracorporeal circulation pipelines, etc.), and is expected to greatly improve the biocompatibility and clinical safety / effectiveness of medical devices.

[0107] The coating of the present application applied to the surface of the medical material has at least the following beneficial effects: 1. significantly reducing blood cell adhesion: especially effectively reducing the adhesion and activation of blood cells such as platelets on the material surface; 2. enhancing coagulation: significantly prolonging the coagulation time of the material surface and inhibiting the formation of thrombosis; 3. effectively reducing inflammatory cell adhesion: significantly reducing the adhesion of inflammatory cells such as neutrophils and macrophages on the material surface; 4. enhancing anti-inflammatory effect: reducing the inflammatory response of the tissue around the material after implantation or use.

[0108] In some embodiments, the mass ratio of heparin and hyaluronic acid is (1-16):4; for example, it can be but is not limited to 1:4, 2:4, 3:4, 4:4, 5:4, 6:4, 7:4, 8:4, 9:4, 10:4, 11:4, 12:4, 13:4, 14:4, 15:4, 16:4 or a range between any two of the above mass ratios. When the mass ratio of heparin and hyaluronic acid is in the above range, it is beneficial to balance the strong anticoagulant activity of heparin and the anti-inflammatory, hydrophilic, steric hindrance barrier effect of hyaluronic acid.

[0109] As a possible embodiment, in the coating, the heparin and hyaluronic acid are in a protruding form, and the height of the protrusion is 20nm-30nm; for example, it can be but is not limited to 20nm, 21nm, 22nm, 23nm, 24nm, 25nm, 26nm, 27nm, 28nm, 29nm, 30nm or a range between any two of the above heights.

[0110] It should be noted that the "height of the protrusion" mentioned above and below refers to the maximum vertical distance of the protrusion to the adhesion layer in the direction perpendicular to the adhesion layer.

[0111] In some embodiments, the coating has a surface roughness of 12 nm to 80 nm. For example, it can be, but is not limited to, 12 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, or a range between any two of the aforementioned values.

[0112] As one possible embodiment, the coating surface has a water contact angle of 30° to 45°. For example, it can be, but is not limited to, 30°, 32°, 34°, 36°, 38°, 40°, 42°, 44°, 45°, or a range between any two of the aforementioned water contact angles.

[0113] One or more embodiments of the present application provide a use of the coating prepared by the above-mentioned method or the coating as described above in the preparation of a medical material having both anti-coagulation and anti-inflammatory effects.

[0114] It should be noted that the anti-coagulation function mentioned above and below includes inhibiting platelet adhesion and activation, prolonging coagulation time, and inhibiting thrombosis. The anti-inflammatory function includes inhibiting inflammatory cell adhesion and reducing tissue inflammatory response.

[0115] One or more embodiments of the present application provide a medical material, which has a surface with the coating prepared by the above-mentioned method or the coating as described above.

[0116] In some embodiments, the medical material includes one or more of blood-contacting medical devices and implants.

[0117] In some alternative embodiments, the medical material includes one or more of vascular stents, heart valves, artificial blood vessels, indwelling catheters, hemodialysis membranes, and extracorporeal membrane oxygenators.

[0118] The technical solutions of the present application are described in detail below in connection with specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application. In the following examples, the experimental methods not specified in the specific conditions can be preferably referred to the instructions given in the present application, or can be performed according to the experimental manuals or conventional conditions in the art, or can be referred to the known experimental methods in the art.

[0119] In the following specific examples, the measurement parameters of the raw material components may, unless otherwise specified, have slight deviations within the weighing accuracy range. The temperature and time parameters allow for acceptable deviations caused by the instrument testing accuracy or operation accuracy.

[0120] In the following examples and comparative examples, all raw materials are commercially available or prepared by conventional methods, unless otherwise specified.

[0121] The coatings prepared in the following examples and comparative examples were evaluated, mainly including surface micro-morphology, water contact angle test, surface blood cell adhesion test, surface inflammatory cell adhesion test, and surface inflammatory factor secretion test. The test methods are as follows:

[0122] (1) Surface micro-morphology test: The surface micro-morphology of the material was analyzed and tested by using the Dimension Icon atomic force microscope of the American Burker company, and the tapping mode was selected as the test mode. The material was fixed on a glass slide and placed in the test position of the instrument. The surface of the material was scanned according to the instrument test method, and the scanning range was 20x20 μm. Three different areas of each sample were scanned. The surface micro-roughness was analyzed and the average value was taken.

[0123] (2) Water contact angle test of material surface: The hydrophilic and hydrophobic properties of the obtained material surface were analyzed and tested by using the OCA25 optical contact angle measuring instrument of the German Dataphysics company. The material was fixed on a glass slide and placed on the test platform for testing. The contact time of the droplet with the material surface was set to 30 s. After stabilization, each sample was tested 5 times, and the average value was taken.

[0124] (3) Blood cell adhesion test of material surface: After the surface of the medical material is contacted with blood, blood cells will adhere to the material surface, and activate the blood clotting effect. To verify the adhesion properties of the material surface to blood cells, the material was incubated with rat blood for co-culture, and the material surface after co-culture with blood was analyzed and tested by SEM. The material was incubated with fresh rat anticoagulant whole blood (1:9 sodium citrate anticoagulant) at 37°C for 2 hours, then fixed with 2.5% glutaraldehyde, dehydrated, gold sprayed, and observed by SEM for 3 fields and counted the blood cell adhesion density.

[0125] (4) Inflammatory cell adhesion test of material surface: After the surface of the medical material is contacted with blood, inflammatory cells in the blood will adhere to the material surface, causing further inflammatory reaction. To verify the adhesion properties of the material surface to inflammatory cells, the material was co-cultured with rat inflammatory macrophages. The material was cut into 1x1 cm size and placed in a culture dish, and the inflammatory macrophages were cultured on the material for 2 days. Then the cells were washed once with PBS, fixed and stained, and then observed by laser scanning confocal microscope, photographed 3 fields and counted the inflammatory cell adhesion density.

[0126] (5) Material surface inflammation factor test: after the medical material contacts with blood, it can stimulate the inflammatory cells in the blood to release inflammatory cytokines and trigger inflammatory response. In order to verify the anti-inflammatory properties of the material surface, the content of inflammatory factors in the blood is tested after the material is co-cultured with the blood. After the material is co-cultured with the inflammatory cells for 24 hours, the supernatant is obtained by centrifugation, and the inflammatory factor test is carried out by enzyme-linked immunosorbent assay.

[0127] Example 1

[0128] Step S1, modifying the surface of the polyurethane material to build a reaction active platform rich in primary amino groups (-NH2): select a medical polyurethane (PU) material as the substrate, and ultrasonically clean the medical polyurethane material with ethanol and deionized water for 15 minutes, and then dry it with nitrogen. Mix dopamine and polylysine in a weak alkaline buffer solution (Tris-HCl, pH≈8.5), and after fully dissolving, obtain a first mixed solution, the mass fraction of dopamine in the first mixed solution is 2%, and the mass fraction of polylysine in the first mixed solution is 1%. Submerge the medical polyurethane material in the first mixed solution, and react for 8 hours under constant temperature oscillation (25℃, 120 rpm) in the dark. Take out the material, ultrasonically rinse it with deionized water for 3 times to remove the physically adsorbed substances, and dry it with nitrogen to obtain an aminated polyurethane material.

[0129] Step S2, mixing and carboxyl activation of heparin and hyaluronic acid: mix heparin and hyaluronic acid, and dissolve the mixture in a (2-(N-morpholino) ethanesulfonic acid buffer, MES, pH≈6.0) buffer solution to obtain a second mixed solution; the mass fraction of heparin in the second mixed solution is 0.8%, and the mass fraction of hyaluronic acid in the second mixed solution is 0.2%. Add carboxyl activator 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and catalyst 1-hydroxy-2,5-pyrrolidinedione (NHS) to the second mixed solution to obtain a third mixed solution, the molar concentration of the carboxyl activator in the third mixed solution is 2mM, and the molar concentration of the catalyst in the third mixed solution is 10mM. Place the third mixed solution in a room temperature environment (25℃) for about 30 minutes of carboxyl activation reaction, and efficiently convert the free carboxyl groups (-COOH) on the molecular chains of heparin and hyaluronic acid into highly reactive esters (such as NHS ester).

[0130] Step S3, covalent grafting of heparin and hyaluronic acid: the aminated medical material prepared in step S1 was moved into the third mixed solution containing carboxyl-activated heparin and hyaluronic acid described in step S2. The reaction was carried out at 37°C under constant temperature oscillation (120 rpm) for 8 hours in the dark. After the reaction was completed, the material was taken out and sequentially washed with NaCl solution and deionized water for 3 times each by ultrasonic washing to completely remove the physically adsorbed or unreacted components. After drying, the polyurethane material with a surface covalently modified with a heparin and hyaluronic acid composite coating was obtained.

[0131] The test results are shown in the following table: Figure 1 The coating surface showed strip-shaped protrusions with a height of 20-30 nm, corresponding to the covalently immobilized heparin and hyaluronic acid molecules, and the surface roughness was 15.8±2.1 nm. The coating surface water contact angle is shown in the following table: Figure 3 The coating surface showed a high hydrophilicity with a surface water contact angle of 38±6°. The coating surface blood cell adhesion is shown in the following table: Figure 5 The coating surface showed a small number of blood cell adhesion, and the blood cell adhesion density was ≤200 cells / mm 2 The coating surface inflammation cell adhesion is shown in the following table: Figure 7 The coating surface showed a small number of inflammation cell adhesion. The coating surface inflammation factor release amount is shown in the following table: Figure 9 The coating surface showed a small number of inflammation cell adhesion. The coating surface inflammation factor release amount is shown in the following table:

[0132] Example 2

[0133] Step S1, modification of the surface of the polyurethane material to construct a reaction active platform rich in primary amino groups (-NH2): a medical polyurethane (PU) material was selected as the substrate, and the medical polyurethane material was sequentially ultrasonically cleaned with ethanol and deionized water for 15 minutes and dried with nitrogen. Dopamine and polyethyleneimine were mixed in a weak alkaline buffer solution (Tris-HCl, pH≈8.5), and after being fully dissolved, a first mixed solution was obtained, in which the mass fraction of dopamine was 2% and the mass fraction of polyethyleneimine was 1%. The medical polyurethane material was immersed in the first mixed solution, and the reaction was carried out under constant temperature oscillation (25°C, 120 rpm) for 8 hours in the dark. The material was taken out, washed with deionized water for 3 times to remove the physically adsorbed substances, and dried with nitrogen to obtain an aminated polyurethane material.

[0134] Step S2, mixing of heparin and hyaluronic acid and carboxyl activation: the heparin and hyaluronic acid are mixed, and the mixture is dissolved in a (2-(N-morpholino)ethanesulfonic acid buffer, MES, pH≈6.0) buffer solution to obtain a second mixed solution; the mass fraction of heparin in the second mixed solution is 0.5%, and the mass fraction of hyaluronic acid in the second mixed solution is 0.5%. The carboxyl activator 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and the catalyst 1-hydroxy-2,5-pyrrolidinedione (NHS) are added to the second mixed solution to obtain a third mixed solution, and the molar concentration of the carboxyl activator in the third mixed solution is 20 mM, and the molar concentration of the catalyst in the third mixed solution is 10 mM. The third mixed solution is placed in a room temperature environment (25°C) for about 30 minutes of carboxyl activation reaction, and the free carboxyl groups (-COOH) on the molecular chains of heparin and hyaluronic acid are efficiently converted into highly reactive esters (such as NHS esters).

[0135] Step S3, covalent grafting of heparin and hyaluronic acid: the medical material treated with amino groups prepared in step S1 is moved into the third mixed solution containing carboxyl-activated heparin and hyaluronic acid described in step S2. The reaction is carried out at 37°C under constant temperature oscillation (120 rpm) for 8 hours in the dark. After the reaction is completed, the material is taken out and ultrasonically washed with NaCl solution and deionized water for 3 times respectively, and the physically adsorbed or unreacted components are completely removed. After drying, the polyurethane material with a surface covalently modified with a heparin and hyaluronic acid composite coating is obtained.

[0136] It is tested that the coating surface appears strip-shaped protrusions with a height of 20-30 nm, corresponding to the covalently fixed heparin and hyaluronic acid molecules, and the surface roughness is 13.2±1.9 nm. The water contact angle of the coating surface is 32±4°, and the coating surface shows high hydrophilicity. The number of blood cell adhesion on the coating surface is small, and the blood cell adhesion density is ≤280 cells / mm 2 . The number of inflammatory cell adhesion on the coating surface is small, and the surface inflammatory factor release amount is 980±210 pg / mL.

[0137] Example 3

[0138] Step S1, modifying the surface of the polyurethane material to construct a reaction active platform rich in primary amino groups (-NH2): Selecting medical polyurethane (PU) material as the substrate, and then ultrasonic cleaning the medical polyurethane material with ethanol and deionized water for 15 minutes, and blowing dry with nitrogen. Mixing dopamine and lysozyme in a weak alkaline buffer solution (Tris-HCl, pH≈8.5), and then obtaining a first mixed solution after fully dissolving, the mass fraction of dopamine in the first mixed solution is 2%, and the mass fraction of lysozyme in the first mixed solution is 1%. Immersing the medical polyurethane material in the first mixed solution, and then constant temperature oscillation (25℃, 120rpm) reaction for 8 hours in the dark. Taking out the material, and then ultrasonic washing with deionized water for 3 times to remove the physical adsorption, and then drying with nitrogen, and then obtaining the aminated polyurethane material.

[0139] Step S2, mixing and carboxyl activation of heparin and hyaluronic acid: mixing heparin and hyaluronic acid, and then dissolving the mixture in a (2-(N-morpholino) ethanesulfonic acid buffer, MES, pH≈6.0) buffer solution to obtain a second mixed solution; the mass fraction of heparin in the second mixed solution is 0.2%, and the mass fraction of hyaluronic acid in the second mixed solution is 0.8%. Adding carboxyl activator 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC) and catalyst 1-hydroxy-2,5-pyrrolidinedione (NHS) to the second mixed solution to obtain a third mixed solution, the molar concentration of the carboxyl activator in the third mixed solution is 20mM, and the molar concentration of the catalyst in the third mixed solution is 10mM. Placing the third mixed solution in a room temperature environment (25℃) for about 30 minutes of carboxyl activation reaction, and then efficiently converting the free carboxyl groups (-COOH) on the molecular chains of heparin and hyaluronic acid into highly reactive esters (such as NHS ester).

[0140] Step S3, covalent grafting of heparin and hyaluronic acid: moving the aminated medical material prepared in step S1 into the third mixed solution containing carboxyl activated heparin and hyaluronic acid described in step S2. Constant temperature oscillation (120 rpm) reaction for 8 hours in the dark at 37℃. After the reaction is completed, taking out the material, and then ultrasonic washing with NaCl solution and deionized water for 3 times respectively, and then thoroughly removing the physically adsorbed or unreacted components. After drying, the polyurethane material with heparin and hyaluronic acid composite coating covalently modified on the surface is obtained.

[0141] After testing, the coating surface appears strip-shaped protrusions with a height of 20-30nm, corresponding to the covalently fixed heparin and hyaluronic acid molecules, and the surface roughness is 25.3±3.1nm. The water contact angle of the coating surface is 31±6°, and the coating surface presents high hydrophilicity. The number of blood cell adhesion on the coating surface is less, and the blood cell adhesion density is ≤310 cells / mm 2The number of inflammatory cells adhering to the surface of the coating is small, and the amount of inflammatory factor released on the surface is 890 ± 175 pg / mL.

[0142] Example 4

[0143] The preparation method of Example 4 is similar to that of Example 1, except that the mass percentage of dopamine in the first mixed solution in step S1 of Example 4 is 1.8%, and the mass percentage of polylysine in the first mixed solution is 1.2%; the others are the same.

[0144] It is tested that the coating surface appears strip-shaped protrusions with a height of 20-30 nm, corresponding to covalently immobilized heparin and hyaluronic acid molecules, and the surface roughness is 28.2 ± 5.3 nm. The water contact angle of the coating surface is 34 ± 2°, and the coating surface presents high hydrophilicity. The number of blood cells adhering to the surface of the coating is small, and the blood cell adhesion density is ≤390 cells / mm 2 The number of inflammatory cells adhering to the surface of the coating is small, and the amount of inflammatory factor released on the surface is 910 ± 162 pg / mL.

[0145] Example 5

[0146] The preparation method of Example 5 is similar to that of Example 1, except that the mass percentage of dopamine in the first mixed solution in step S1 of Example 5 is 1.5%, and the mass percentage of polylysine in the first mixed solution is 1.5%; the others are the same.

[0147] It is tested that the coating surface appears strip-shaped protrusions with a height of 20-30 nm, corresponding to covalently immobilized heparin and hyaluronic acid molecules, and the surface roughness is 32.1 ± 4.3 nm. The water contact angle of the coating surface is 36 ± 4°, and the coating surface presents high hydrophilicity. The number of blood cells adhering to the surface of the coating is small, and the blood cell adhesion density is ≤410 cells / mm 2 The number of inflammatory cells adhering to the surface of the coating is small, and the amount of inflammatory factor released on the surface is 1020 ± 131 pg / mL.

[0148] Comparative Example 1

[0149] The medical polyurethane (PU) material is sequentially cleaned with ethanol and deionized water for 15 minutes, and dried with nitrogen, as Comparative Example 1. That is, the polyurethane material without surface modification is used as Comparative Example 1.

[0150] It is tested that the surface of the polyurethane material is flat and smooth without obvious characteristics, and the surface roughness is 3.2 ± 0.5 nm. The water contact angle of the polyurethane surface is as shown in FIG. 6, and the surface water contact angle is 102 ± 3°, and the surface presents high hydrophobicity. The blood cell adhesion of the polyurethane surface is as shown in FIG. 7, and the blood cell adhesion density is 1200 ± 100 cells / mm Figure 2 The surface of the polyurethane material is flat and smooth without obvious characteristics, and the surface roughness is 3.2 ± 0.5 nm. The water contact angle of the polyurethane surface is as shown in FIG. 6, and the surface water contact angle is 102 ± 3°, and the surface presents high hydrophobicity. The blood cell adhesion of the polyurethane surface is as shown in FIG. 7, and the blood cell adhesion density is 1200 ± 100 cells / mm Figure 4 The surface of the polyurethane material is flat and smooth without obvious characteristics, and the surface roughness is 3.2 ± 0.5 nm. The water contact angle of the polyurethane surface is as shown in FIG. 6, and the surface water contact angle is 102 ± 3°, and the surface presents high hydrophobicity. The blood cell adhesion of the polyurethane surface is as shown in FIG. 7, and the blood cell adhesion density is 1200 ± 100 cells / mm Figure 6, a large number of blood cells adhere to the surface, and the blood cell adhesion density is >1200 cells / mm 2 . The polyurethane surface inflammatory cell adhesion is shown in Figure 2. Figure 8 , a large number of inflammatory cells adhere to the surface. Figure 3 shows the polyurethane surface inflammatory cell adhesion. Figure 9 The amount of inflammatory factor released from the surface of the polyurethane material in Example 1 is 3410 ± 290 pg / mL.

[0151] Comparative Example 2

[0152] The preparation method of Comparative Example 2 is similar to the preparation method of Example 1, except that only heparin is used in step S2 of Comparative Example 2, and the prepared amino-modified medical material is moved into the mixed solution containing carboxyl-activated heparin described in step S2 for reaction in step S3, to obtain a polyurethane material with only a covalently modified heparin coating on the surface.

[0153] Tests show that the polyurethane material with only a covalently modified heparin coating on the surface can better inhibit blood cell adhesion, but has no obvious inhibitory effect on inflammatory cell adhesion. The coating only has an anticoagulant effect, but has poor anti-inflammatory effect.

[0154] Comparative Example 3

[0155] The preparation method of Comparative Example 3 is similar to the preparation method of Example 1, except that only hyaluronic acid is used in step S2 of Comparative Example 3, and the prepared amino-modified medical material is moved into the mixed solution containing carboxyl-activated hyaluronic acid described in step S2 for reaction in step S3, to obtain a polyurethane material with only a covalently modified hyaluronic acid coating on the surface.

[0156] Tests show that the polyurethane material with only a covalently modified hyaluronic acid coating on the surface has general inhibitory effect on blood cell adhesion, and the coating has poor anticoagulant effect.

[0157] Comparative Example 4

[0158] The preparation method of Comparative Example 4 is similar to the preparation method of Example 1, except that only dopamine is used for surface modification of the polyurethane material in step S1 of Comparative Example 4, and the mass fraction of dopamine in the first mixed solution is 3%; the others are the same. Step S1 of Comparative Example 4 is as follows:

[0159] Step S1, modifying the surface of the polyurethane material to construct a reactive platform containing primary amino groups (-NH2): Select a medical polyurethane (PU) material as the substrate, and ultrasonically clean the medical polyurethane material with ethanol and deionized water for 15 minutes, and then dry it with nitrogen. Mix dopamine in a weak alkaline buffer solution (Tris-HCl, pH ≈ 8.5), and after fully dissolving, obtain a first mixed solution, and the mass fraction of dopamine in the first mixed solution is 3%. Immerse the medical polyurethane material in the first mixed solution, and react for 8 hours under constant temperature oscillation (25°C, 120 rpm) in the dark. Remove the material, wash it with deionized water for 3 times to remove the physically adsorbed substances, dry it with nitrogen, and obtain an aminated polyurethane material.

[0160] After testing, a small amount of 20-30 nm high strip-shaped protrusions appeared on the surface of the coating, indicating that the covalently immobilized heparin and hyaluronic acid molecules were less, and the surface roughness was 28.1±2.3 nm. The water contact angle of the coating surface was 54±4°, and the hydrophilicity of the coating surface was weakened. The number of blood cell adhesion on the coating surface was more, and the blood cell adhesion density was ≥620 cells / mm 2 . The number of inflammatory cell adhesion on the coating surface was more, and the amount of surface inflammatory factor release was 1420±139 pg / mL. Only through the adhesion of the active amino group in the intermediate layer deposited on the material surface by dopamine, the amount of subsequent surface modification of heparin and hyaluronic acid was less, which affected the subsequent surface modification of heparin and hyaluronic acid.

[0161] Comparative Example 5

[0162] The preparation method of Comparative Example 5 is similar to that of Example 1, except that in Step S1 of Comparative Example 5, only polylysine is used to modify the surface of the polyurethane material, and the mass fraction of polylysine in the first mixed solution is 3%; the others are the same. Step S1 of Comparative Example 5 is as follows:

[0163] Step S1, modifying the surface of the polyurethane material to construct a reactive platform containing primary amino groups (-NH2): Select a medical polyurethane (PU) material as the substrate, and ultrasonically clean the medical polyurethane material with ethanol and deionized water for 15 minutes, and then dry it with nitrogen. Mix dopamine in a weak alkaline buffer solution (Tris-HCl, pH ≈ 8.5), and after fully dissolving, obtain a first mixed solution, and the mass fraction of dopamine in the first mixed solution is 3%. Immerse the medical polyurethane material in the first mixed solution, and react for 8 hours under constant temperature oscillation (25°C, 120 rpm) in the dark. Remove the material, wash it with deionized water for 3 times to remove the physically adsorbed substances, dry it with nitrogen, and obtain an aminated polyurethane material.

[0164] The coating surface did not appear more significant strip-shaped protrusions, indicating that the covalently immobilized heparin and hyaluronic acid molecules were few, and the surface roughness was 12.2±1.9nm. The water contact angle of the coating surface was 61±5°, and the hydrophilicity of the coating surface was weakened. The number of blood cell adhesion on the coating surface was more, and the blood cell adhesion density was ≥820 cells / mm 2 The number of inflammatory cell adhesion on the coating surface was more, and the release amount of surface inflammatory factor was 1730±146pg / mL. Only through the electrostatic adsorption of polylysine, a stable modification layer cannot be formed on the surface.

[0165] Comparative Example 6

[0166] In Comparative Example 6, only heparin and hyaluronic acid were used for electrostatic adsorption to modify the surface of the polyurethane material. Comparative Example 6 is as follows:

[0167] Step S1, modifying the surface of the polyurethane material, selecting a medical polyurethane (PU) material as the substrate, and ultrasonically cleaning the medical polyurethane material with ethanol and deionized water for 15 minutes, and then blowing dry with nitrogen.

[0168] Step S2, mixing heparin and hyaluronic acid, and dissolving the mixture in a (2-(N-morpholino) ethanesulfonic acid buffer, MES, pH≈6.0) buffer solution to obtain a mixed solution; the mass fraction of heparin in the mixed solution is 0.8%, and the mass fraction of hyaluronic acid in the mixed solution is 0.2%.

[0169] Step S3, placing the polyurethane material in the mixed solution, avoiding light, and constant temperature oscillation (37℃, 120 rpm) for 8 hours. Take out the material, ultrasonically rinse with deionized water for 3 times to remove the physical adsorbent, and dry with nitrogen to obtain a polyurethane material with heparin and hyaluronic acid electrostatically adsorbed on the surface.

[0170] The main difference points of the above examples and comparative examples are shown in Table 1.

[0171] Table 1

[0172]

[0173] In Table 1, mass ratio 1 refers to the mass ratio of dopamine to polyamino compound; and mass ratio 2 refers to the mass ratio of heparin and hyaluronic acid.

[0174] Examples 1-5 all exhibit high hydrophilicity, low blood cell and inflammatory cell adhesion properties, and lower levels of inflammatory factor release, indicating that the coating of the present application has good anticoagulant and anti-inflammatory properties. The unmodified polyurethane material in Comparative Example 1 has poor surface biocompatibility; the single heparin coating in Comparative Example 2 has good anticoagulant effect but insufficient anti-inflammatory effect; and the single hyaluronic acid coating in Comparative Example 3 has acceptable anti-inflammatory effect but poor anticoagulant performance. As can be seen from the results of Examples 1-5 and Comparative Examples 1-3 in Table 1, heparin and hyaluronic acid have a synergistic effect, together imparting excellent blood compatibility and anti-inflammatory function to the material surface, and have good clinical application prospects.

[0175] As can be seen from the results of Example 1 and Comparative Examples 4-6, by using the method of co-deposition of dopamine and polyamino compounds to modify the surface of the medical material, the primary amino group density and stability of the surface of the medical material can be significantly enhanced, then by covalently fixing heparin and hyaluronic acid on the surface of the amino-modified medical material through amidation reaction, the stability of the coating is effectively improved, and the biocompatibility and service life of the material surface are significantly improved.

[0176] The technical features of the above-described examples can be combined in any manner. To make the description brief, not all possible combinations of the technical features in the above-described examples are described, however, as long as the combinations of the technical features do not contradict each other, they should be considered within the scope of the present disclosure.

[0177] The above-described examples only express several embodiments of the present application, and the description is relatively specific and detailed, but it should not be construed as limiting the scope of the patent application. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are all within the scope of the present application. Therefore, the scope of the patent protection of the present application should be subject to the appended claims.

Claims

1. A method for the production of a coating for the surface of a medical material, characterized in that, The method comprises the following steps: co-depositing dopamine and a polyamino compound on the surface of a medical material to form an adhesion layer with amino groups on the surface of the medical material; mixing heparin and hyaluronic acid and activating carboxyl groups of the heparin and the hyaluronic acid; contacting the adhesion layer with amino groups with the heparin and the hyaluronic acid after the carboxyl groups are activated to enable covalent grafting reaction between the amino groups and the activated carboxyl groups of the heparin and the hyaluronic acid, thereby obtaining a coating for the surface of a medical material.

2. The method for producing a coating for a medical material surface according to claim 1, wherein The mass ratio of the dopamine and the polyamino compound is (1-2):1; and / or The polyamino compound comprises one or more of polylysine, polyethyleneimine and lysozyme.

3. The method for producing a coating for a medical material surface according to claim 1, wherein The step of co-depositing dopamine and a polyamino compound on the surface of a medical material to perform amino group treatment on the surface of the medical material comprises: dissolving the dopamine and the polyamino compound in a weak alkaline buffer solution with a pH value of 8-9 to obtain a first mixed solution, and immersing the medical material in the first mixed solution to enable co-deposition of the dopamine and the polyamino compound on the surface of the medical material; Optionally, the sum of the mass proportions of the dopamine and the polyamino compound in the first mixed solution is 1%-5%. Optionally, the co-deposition time is 2 hours-24 hours.

4. The method for producing a coating for a medical material surface according to claim 1, wherein The mass ratio of the heparin and the hyaluronic acid is (1-16):4; and / or The step of mixing heparin and hyaluronic acid and activating carboxyl groups of the heparin and the hyaluronic acid comprises: dissolving the heparin and the hyaluronic acid in a weak acid buffer solution with a pH value of 5-6 to obtain a second mixed solution, and then adding a carboxyl activator and a catalyst to the second mixed solution to activate the carboxyl groups of the heparin and the hyaluronic acid; Optionally, the sum of the mass proportions of the heparin and the hyaluronic acid in the second mixed solution is 0.5%-3%. Optionally, the carboxyl activator comprises one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide and N,N'-diisopropylcarbodiimide. Optionally, the molar concentration of the carboxyl activator in the second mixed solution after the carboxyl activator and the catalyst are added is 2mM-20mM. Optionally, the catalyst comprises one or more of 1-hydroxy-2,5-pyrrolidinedione, 1-hydroxy-7-azabenzotriazole and 1,1'-carbonyldiimidazole. Optionally, the molar concentration of the catalyst in the second mixed solution after the carboxyl activator and the catalyst are added is 2mM-20mM. Optionally, the activation temperature is 20°C-25°C, and the activation time is 30 minutes-120 minutes.

5. The method for producing a coating for a medical material surface according to any one of claims 1 to 4, characterized in that, The covalent grafting reaction temperature is 35°C-40°C, and the covalent grafting reaction time is 8 hours-12 hours.

6. A coating for the surface of a medical material, characterized by, The coating comprises an adhesion layer containing a polymer of dopamine and a polyamino compound, and heparin and hyaluronic acid fixed on the surface of the adhesion layer, wherein the heparin and the hyaluronic acid are fixed on the surface of the adhesion layer by an amide bond.

7. The coating for the surface of a medical material according to claim 6, wherein The mass ratio of the heparin to the hyaluronic acid is (1-16):4; and / or, There is a protrusion on the surface of the coating, and the height of the protrusion is 20nm-30nm; and / or, The surface roughness of the coating is 12nm-80nm; and / or, The water contact angle of the surface of the coating is 30°-45°.

8. Use of the coating prepared by the preparation method of any one of claims 1 to 5 or the coating of any one of claims 6 to 7 in the preparation of a medical material having dual functions of anticoagulation and anti-inflammation.

9. A medical material, characterized by The surface of the medical material has the coating prepared by the preparation method of any one of claims 1 to 5 or the coating of any one of claims 6 to 7.

10. The medical material according to claim 9, wherein The medical material comprises one or more of blood-contacting medical devices and implants; Optionally, the medical material comprises one or more of a vascular stent, a heart valve, an artificial blood vessel, an indwelling catheter, a hemodialysis membrane, and an extracorporeal membrane oxygenator. Optionally, the medical material comprises one or more of a vascular stent, a heart valve, an artificial blood vessel, an indwelling catheter, a hemodialysis membrane, and an extracorporeal membrane oxygenator.

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