Bivalirudin-protein coating as well as preparation method and application thereof

Abstract

The invention discloses a bivalirudin-protein coating as well as a preparation method and application thereof, and belongs to the technical field of biological materials. The bivalirudin-protein coating comprises a bivalirudin-protein layer arranged on the surface of a base material, the bivalirudin-protein layer is formed by carrying out structural rearrangement on a bivalirudin-protein co-assembly coating with a micro-nano pore structure in the presence of an anion solution. The bivalirudin-protein coating is good in stability and high in density, and can realize long-acting and controllable slow release compared with the bivalirudin, so that a modified base material is endowed with an excellent long-acting anticoagulant effect, and the bivalirudin-protein coating can be used for preparing blood contact devices and the like. The preparation method comprises the following steps: mixing a base material with a mixed solution containing an oxidizing agent, bivalirudin and protein, so that bivalirudin and protein are subjected to a co-assembly reaction, and a co-assembly coating with a micro-nano pore structure is obtained; the co-assembled coating is mixed with an anion solution, so that the co-assembled coating is subjected to structural rearrangement to realize self-packaging.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a bivalirudin-protein coating and a preparation method and application thereof. Background Art

[0002] With the widespread clinical use of blood-contacting devices (such as vascular stents, catheters, and artificial hearts), the development of safe, biocompatible coatings with long-lasting anticoagulant properties has become a key research topic in the biomaterials field. To prevent blood from forming thrombi on the device surface, anticoagulant coatings are widely used in these devices. However, existing anticoagulant coating technologies still face challenges in terms of sustained-release efficiency and stability.

[0003] For example, the most common traditional anticoagulant coatings use heparin or low-molecular-weight heparin (LMWH) as an anticoagulant. While heparin coatings effectively inhibit coagulation factors in the blood and prevent thrombosis, they are susceptible to blood flow and mechanical friction, leading to coating shedding or failure. Furthermore, coating shedding or blood flow can lead to rapid drug release, making the anticoagulant effect less sustained and reducing its effectiveness. Furthermore, long-term, high-dose heparin use can cause side effects such as bleeding and thrombocytopenia.

[0004] Compared to traditional heparin anticoagulants, bivalirudin, as a direct thrombin inhibitor, can reversibly bind to the catalytic active site of thrombin, effectively inhibiting thrombin activity and thereby blocking the coagulation cascade. This offers a higher level of anticoagulant safety and is particularly suitable for patients at high risk of bleeding. Due to its excellent anticoagulant properties and low bleeding side effects, numerous studies in recent years have focused on immobilizing bivalirudin on the surfaces of blood-contact materials. By constructing bivalirudin-functionalized coatings, continuous regulation of the local blood environment can be achieved, effectively preventing blood coagulation and improving the hemocompatibility of medical devices. However, due to the small size of bivalirudin molecules and the suboptimal microstructural design of current bivalirudin coatings, stable and sustained drug release is difficult to achieve. This makes it difficult to maintain the anticoagulant effect of bivalirudin during long-term blood contact. Furthermore, existing bivalirudin sustained-release coatings also suffer from poor stability and are prone to shedding.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide a bivalirudin-protein coating and a preparation method and application thereof, so as to solve or improve the above technical problems.

[0007] The present invention can be achieved like this: In a first aspect, the present invention provides a bivalirudin-protein coating, which comprises a bivalirudin-protein layer disposed on a surface of a substrate; The bivalirudin-protein layer is formed by structural rearrangement of a bivalirudin-protein co-assembled coating with a micro-nanoporous structure in the presence of an anionic solution.

[0008] In an alternative embodiment, the bivalirudin-protein co-assembled coating contains micro-nano protein-bivalirudin particles.

[0009] In an optional embodiment, the particle size of the micro-nano protein-bivalirudin particles is 900 nm to 1100 nm.

[0010] In an optional embodiment, the micro-nano protein-bivalirudin particles are formed by a co-assembly reaction of bivalirudin and protein under the induction of oxygen free radicals.

[0011] In an optional embodiment, the number of layers of the bivalirudin-protein co-assembly coating is 1 to 10 layers.

[0012] In a second aspect, the present invention provides a method for preparing a bivalirudin-protein coating according to any of the aforementioned embodiments, comprising the following steps: mixing a substrate with a mixed solution containing an oxidant, bivalirudin and a protein, so that the bivalirudin and the protein undergo a co-assembly reaction under the induction of oxygen free radicals to obtain a bivalirudin-protein co-assembly coating having a micro-nanoporous structure; mixing the bivalirudin-protein co-assembly coating with an anionic solution to cause the bivalirudin-protein co-assembly coating to undergo structural rearrangement to obtain a bivalirudin-protein layer.

[0013] In an optional embodiment, when the number of layers of the bivalirudin-protein co-assembly coating is n, n≥2, after obtaining the m-th layer of the bivalirudin-protein co-assembly coating, the obtained m-th layer of the bivalirudin-protein co-assembly coating is cleaned, and the next layer of the bivalirudin-protein co-assembly coating is prepared under the same co-assembly reaction conditions until the n-th layer of the bivalirudin-protein co-assembly coating is obtained; wherein the value of m is 1 to n-1.

[0014] In an optional embodiment, the temperature of the co-assembly reaction is 30°C to 45°C.

[0015] In an optional embodiment, the temperature of the co-assembly reaction is 35°C to 40°C.

[0016] In an optional embodiment, the co-assembly reaction time is not less than 2 hours.

[0017] In an optional embodiment, the co-assembly reaction time is 2 hours to 72 hours.

[0018] In an optional embodiment, the co-assembly reaction time is 6 h to 12 h.

[0019] In an optional embodiment, the temperature of the co-assembly reaction is 35° C. to 40° C., and the time is 6 h to 12 h.

[0020] In an optional embodiment, the pH value of the co-assembly reaction is 3-7.

[0021] In an optional embodiment, the pH value of the mixed solution is 3-7.

[0022] In an optional embodiment, the pH value of the mixed solution is 4-6.

[0023] In an optional embodiment, the concentration of the oxidant in the mixed solution is 0.01 mg / mL to 100 mg / mL.

[0024] In an optional embodiment, the concentration of the oxidant in the mixed solution is 0.05 mg / mL to 20 mg / mL.

[0025] In an optional embodiment, the concentration of the oxidant in the mixed solution is 0.5 mg / mL to 4 mg / mL.

[0026] In an optional embodiment, the concentration of the protein in the mixed solution is 0.01 mg / mL to 100 mg / mL.

[0027] In an optional embodiment, the concentration of the protein in the mixed solution is 0.5 mg / mL to 5 mg / mL.

[0028] In an optional embodiment, the concentration of the protein in the mixed solution is 1 mg / mL to 2 mg / mL.

[0029] In an optional embodiment, the concentration of bivalirudin in the mixed solution is 0.01 mg / mL to 100 mg / mL.

[0030] In an optional embodiment, the concentration of bivalirudin in the mixed solution is 0.05 mg / mL to 5 mg / mL.

[0031] In an optional embodiment, the concentration of bivalirudin in the mixed solution is 0.1 mg / mL to 1 mg / mL.

[0032] In an optional embodiment, the mixed solution is obtained by mixing an oxidant solution, a protein solution and a bivalirudin solution, wherein the volume ratio of the oxidant solution, the protein solution and the bivalirudin solution is 1:1:0.01 to 1:1:1; the molar ratio of protein to bivalirudin in the final mixed solution is 0.1:1 to 20:1, and the concentration of the oxidant in the mixed solution is 0.01 mg / mL~100 mg / mL.

[0033] In an optional embodiment, the mixed solution is obtained by mixing an oxidant solution, a protein solution and a bivalirudin solution, wherein the volume ratio of the oxidant solution, the protein solution and the bivalirudin solution is 1:1:0.01 to 1:1:1; the total concentration of the protein and bivalirudin in the reaction system is 0.01 mmol / L~5 mmol / L, and the concentration of the oxidant in the mixed solution is 0.01 mg / mL~100 mg / mL.

[0034] In an optional embodiment, the isoelectric point of the protein is 3-7.

[0035] In an alternative embodiment, the protein comprises at least one of serum albumin, casein, fibroin, arginine kinase, lactoferrin, and whey protein.

[0036] In an optional embodiment, the protein is serum albumin with an isoelectric point of 3-7.

[0037] In an alternative embodiment, the oxidant comprises at least one of an organic oxidant and an inorganic oxidant.

[0038] In an alternative embodiment, the organic oxidant comprises at least one of dichlorine peroxide and peracetic acid.

[0039] In an alternative embodiment, the inorganic oxidizing agent comprises at least one of persulfate, chlorate, perchlorate, perchlorate, dichromate, periodate, permanganate, nitrate, and an inorganic peroxide.

[0040] In an alternative embodiment, the inorganic peroxide includes at least one of Na2O2, K2O2, MgO2, CaO2, BaO2, and H2O2.

[0041] In an optional embodiment, distilled water with a pH value of 3-7 and a temperature of 10° C.-45° C. is used for cleaning.

[0042] In an optional embodiment, distilled water with a pH value of 4-6 and a temperature of 25° C.-37° C. is used for cleaning.

[0043] In an alternative embodiment, the bivalirudin-protein co-assembled coating is dried before being mixed with the anionic solution.

[0044] In an optional embodiment, the drying temperature is 10°C to 45°C.

[0045] In an optional embodiment, the mixing time of the bivalirudin-protein co-assembled coating and the anion solution is not less than 1 min.

[0046] In an optional embodiment, the mixing time of the bivalirudin-protein co-assembled coating and the anion solution is 5 min to 60 min.

[0047] In an optional embodiment, the temperature of the anion solution is 4°C to 45°C.

[0048] In an optional embodiment, the temperature of the anion solution is 4°C to 37°C.

[0049] In an optional embodiment, the concentration of the anion solution is not less than 0.1 mmol / L.

[0050] In an optional embodiment, the concentration of the anion solution is 0.1 mmol / L to 10 mmol / L.

[0051] In an optional embodiment, the concentration of the anion solution is 5 mmol / L to 10 mmol / L.

[0052] In an optional embodiment, the anion solution includes at least one of a phosphoric acid solution, a phosphate solution, a hydrogen phosphate solution, a dihydrogen phosphate solution, a sulfurous acid solution, a sulfite solution, a sulfite solution, a sulfonic acid solution, and a sulfonate solution.

[0053] In an optional embodiment, the anion solution includes at least one of a phosphate solution, a hydrogen phosphate solution, and a dihydrogen phosphate solution.

[0054] In an optional embodiment, the substrate includes at least one of a metal material, an inorganic material, a polymer material, a natural biological material, and an artificially synthesized polypeptide hydrogel material.

[0055] In an optional embodiment, the metal material includes at least one of cobalt-based alloys, titanium and its alloys, nickel-titanium alloys, platinum and its alloys, magnesium and its alloys, iron and its alloys, and zinc and its alloys.

[0056] In an optional embodiment, the inorganic material includes at least one of titanium oxide and its nanotubes, carbon materials, silicon, silicon dioxide, calcium phosphate, silicon nitride, silicon carbide, aluminosilicate, calcium aluminum system, bioglass, titanium nitride and biomedical micro-nanoparticles.

[0057] In an optional embodiment, the polymer material includes at least one of polyester, polyethylene, polyvinyl chloride, polytetrafluoroethylene, polyurethane, polystyrene, polyvinyl alcohol, polypropylene, polyoxymethylene, polycarbonate, carbon copolymer, polyglycolic acid, polymethyl methacrylate, polyvinyl acetate, polylactic acid, glycolide-lactide copolymer, polytrimethylene carbonate, polycaprolactone, polyhydroxyalkanoate, polybutylene succinate, polyamide, polydioxanone, epoxy resin, silicone rubber, silicone gel, polyacrylic acid and its derivatives, polyethylene glycol and its derivatives.

[0058] In an optional embodiment, the biomedical micro-nanoparticles include at least one of ferroferric oxide nanoparticles, silicon dioxide nanoparticles, titanium oxide nanoparticles, and zinc oxide nanoparticles.

[0059] In an alternative embodiment, the natural biomaterial comprises at least one of decellularized tissues and organs of animal origin, gelatin, collagen, fibrin, silk protein, keratin, and polysaccharide.

[0060] In alternative embodiments, the animal-derived decellularized tissues and organs include at least one of blood vessels, valves, hearts, bones, lungs, ligaments, bladders, mucosa, and corneas.

[0061] In an optional embodiment, the polysaccharide includes at least one of sodium hyaluronate, sodium alginate, agarose, plastic starch material, cellulose, hemicellulose, lignin, chitin and derivatives thereof.

[0062] In an optional embodiment, the artificially synthesized polypeptide hydrogel material includes at least one of L-lysine and poly-L-glutamic acid.

[0063] In a third aspect, the present invention further provides an application of the bivalirudin-protein coating according to any one of the aforementioned embodiments, for example, using the coating to prepare a blood-contact device.

[0064] In a fourth aspect, the present invention further provides a blood-contacting device comprising the bivalirudin-protein coating according to any one of the aforementioned embodiments.

[0065] In an optional embodiment, the blood-contacting device includes at least one of a vascular stent, a catheter, and an artificial heart.

[0066] The beneficial effects of the present invention include: The bivalirudin-protein coating provided by the present invention comprises a bivalirudin-protein layer arranged on the surface of a substrate; the bivalirudin-protein layer is formed by structural rearrangement of a bivalirudin-protein co-assembled coating with a micro-nanoporous structure in the presence of an anionic solution.

[0067] The above-mentioned structural rearrangement mainly refers to the collapse of the hydrophobic nucleation and / or protein conformation in the bivalirudin-protein co-assembled coating under the induced extrusion of the hydrophilic anionic solution. During the collapse process, based on the trend of thermodynamic driving towards minimum interfacial energy, the bivalirudin-protein layer gradually becomes dense and flat, thereby having an encapsulation effect on the micro-nanopore structure.

[0068] The bivalirudin-protein coating has good stability and high density, and can achieve long-term, controllable sustained release of bivalirudin, thereby giving the modified substrate an excellent long-term anticoagulant effect. It can be used to prepare blood-contact devices, etc., and has great application prospects and value. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0070] Figure 1 This is a graph showing the results of the platelet adhesion test in Experimental Example 1; Figure 2 This is the result of the fluorescence staining test in Experimental Example 2; Figure 3 This is a scanning electron microscope image of a stainless steel sample having a bivalirudin-protein co-assembled coating prepared in Comparative Example 2 in Experimental Example 3; Figure 4 This is a scanning electron microscope image of the stainless steel sample having the bivalirudin-protein co-assembled coating prepared in Example 1 of Experimental Example 3; Figure 5 This is a graph showing the particle size results corresponding to different reaction times in Example 1 and Comparative Example 3 in Test Example 4; Figure 6 The graph is a thrombin time test result diagram of samples immersed at different time points in Experimental Example 5; Figure 7 The thrombin time test results of the immersion solution at different time points in Experimental Example 5; Figure 8 Graph showing the thrombin time test results for each sample in Test Example 6; Figure 9 3D profilometer morphology results of the samples provided in Example 1 and Comparative Example 4 in Experimental Example 6; Figure 10 3D profilometer morphology results of the samples prepared in Example 1 of Experimental Example 7 and samples 7-1 to 7-4; Figure 11 3D profilometer morphology results of the samples prepared in Example 1 of Experimental Example 8 and samples 8-1 to 8-7; Figure 12 3D profilometer morphology results of the samples prepared in Example 1 of Experimental Example 9 and samples 9-1 to 9-7; Figure 13 3D profilometer morphology results of the sample prepared in Example 1 of Experimental Example 10 and samples 10-1 to 10-7; Figure 14The thrombin time test results of samples immersed at different time points in Experimental Example 11 are shown; Figure 15 The thrombin time test results of samples immersed at different time points in Experimental Example 12 are shown in FIG. Figure 16 1 is the scanning electron microscope result of different samples in ① of Experimental Example 13; Figure 17 The scanning electron microscope results of different samples in ② of Experimental Example 13; Figure 18 The scanning electron microscope results of different samples in ③ of Experimental Example 13; Figure 19 These are the scanning electron microscope results of different samples in ④ of Experimental Example 13. DETAILED DESCRIPTION

[0071] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0072] The bivalirudin-protein coating provided by the present invention and its preparation method and application are described in detail below.

[0073] The present invention provides a bivalirudin-protein coating, which comprises a bivalirudin-protein layer arranged on the surface of a substrate; the bivalirudin-protein layer is formed by structural rearrangement of a bivalirudin-protein co-assembled coating with a micro-nanoporous structure in the presence of an anionic solution.

[0074] Compared with the bivalirudin-protein co-assembled coating, the bivalirudin-protein layer has a significantly higher density. The above-mentioned structural rearrangement mainly refers to the collapse of the hydrophobic nucleation and / or protein conformation in the bivalirudin-protein co-assembled coating. During the collapse process, based on the trend of thermodynamic driving towards minimum interfacial energy, the bivalirudin-protein layer gradually becomes denser and smoother, thereby having an encapsulation effect on the micro-nano pore structure.

[0075] The aforementioned bivalirudin-protein co-assembled coating is formed by stacking protein-bivalirudin micro-nano particles. In some optional embodiments, the particle size of the micro-nano protein-bivalirudin particles can range from 900 nm to 1100 nm. Compared to particles formed solely from an oxidant and protein, these micro-nano protein-bivalirudin particles can rapidly assemble and aggregate into larger particles within a short period of time (4 hours). Once formed, the particles rapidly stack on the substrate surface due to gravity (and then form a coating based on hydrophobic interactions and hydrogen bonds), thereby facilitating the formation of micro-nanopore structures between the particles. Particles formed solely from an oxidant and protein, on the other hand, require a longer time to assemble and aggregate, with smaller particles primarily deposited on the surface and stacking and fusing.

[0076] The above-mentioned micro-nano protein-bivalirudin particles are formed by the co-assembly reaction of bivalirudin and protein under the induction of oxygen free radicals. Specifically, under the induction of oxygen free radicals, the disulfide bonds that maintain the stability of the molecular structure in the protein molecules are broken, and oxidation forms a stable intermediate sulfide. In this process, the protein structure is stretched, and its side chains expose a large number of hydrophobic groups. In an aqueous solution system, the hydrophobic groups interact with each other and are more likely to assemble together, forming hydrophobic nucleation sites to more easily avoid water. That is, the hydrophobic groups exposed by the protein side chains are close to the hydrophobic groups of bivalirudin and combine to form hydrophobic nucleation sites, thereby forming micro-nano protein-bivalirudin particles and depositing them on the surface of the substrate, thereby obtaining a bivalirudin-protein co-assembly coating. Through the above-mentioned co-assembly process, bivalirudin is inserted into the reaction between the oxidant and the protein, thereby increasing the degree of intermolecular aggregation and the aggregation efficiency, and the assembled particles of the protein rapidly become larger, thereby forming a micro-nano pore structure in the process of rapid deposition and stacking on the surface of the substrate under the action of gravity, and at the same time enhancing the thermal stability of the coating.

[0077] In some optional embodiments, the number of layers of the bivalirudin-protein co-assembly coating can be 1 to 10 layers, such as 1 layer, 2 layers, 3 layers, 4 layers, 5 layers, 6 layers, 7 layers, 8 layers, 9 layers or 10 layers. If the number of layers of the bivalirudin-protein co-assembly coating exceeds 10 layers, the resulting coating is too thick and has poor mechanical properties, and may peel off or crack from the surface of the implantable device during service. In some exemplary embodiments, the number of layers of the bivalirudin-protein co-assembly coating can be 1 to 3 layers, such as 1 layer, 2 layers or 3 layers. Setting the number of layers of the bivalirudin-protein co-assembly coating to 1 to 3 layers can, on the one hand, ensure good mechanical properties of the coating, and on the other hand, maintain long-term sustained release of bivalirudin, thereby imparting long-term anticoagulant and antithrombotic properties to the device surface.

[0078] Accordingly, the present invention also provides a method for preparing the above-mentioned bivalirudin-protein coating, which comprises the following steps: S1: mixing a substrate with a mixed solution containing an oxidant, bivalirudin, and a protein, so that the bivalirudin and the protein undergo a co-assembly reaction under the induction of oxygen free radicals, thereby obtaining a bivalirudin-protein co-assembly coating having a micro-nanoporous structure; S2: mixing the bivalirudin-protein co-assembled coating with an anion solution, so that the anions are adsorbed on the bivalirudin-protein co-assembled coating to cause structural rearrangement, thereby obtaining a bivalirudin-protein layer.

[0079] When the number of layers of the bivalirudin-protein co-assembly coating is n, n≥2, after obtaining the m-th layer (m ranges from 1 to n-1) of the bivalirudin-protein co-assembly coating, the obtained m-th layer of the bivalirudin-protein co-assembly coating is cleaned, and the next layer of the bivalirudin-protein co-assembly coating is prepared under the same co-assembly reaction conditions until the n-th layer of the bivalirudin-protein co-assembly coating is obtained.

[0080] It should be noted that the components and proportions of the mixed solutions used to prepare each layer of the bivalirudin-protein co-assembly coating are the same.

[0081] For example, when the number of layers of the bivalirudin-protein co-assembly coating is 2, the preparation of the bivalirudin-protein co-assembly coating includes: mixing a substrate with a first mixed solution containing an oxidant, bivalirudin and a protein, so that the bivalirudin in the first mixed solution and the protein undergo a co-assembly reaction under the induction of the oxidant to obtain a first layer of bivalirudin-protein co-assembly coating; washing the substrate with the first layer of bivalirudin-protein co-assembly coating, and then mixing it with a second mixed solution containing an oxidant, bivalirudin and the protein, so that the bivalirudin in the second mixed solution and the protein undergo a co-assembly reaction under the induction of the oxidant, thereby obtaining a second layer of bivalirudin-protein co-assembly coating on the surface of the first layer of bivalirudin-protein co-assembly coating.

[0082] For example, when the number of layers of the bivalirudin-protein co-assembly coating is greater than 2, the preparation of the bivalirudin-protein co-assembly coating includes: mixing a substrate with a first mixed solution containing an oxidant, bivalirudin and protein, so that the bivalirudin in the first mixed solution and the protein undergo a co-assembly reaction under the induction of the oxidant to obtain a first layer of bivalirudin-protein co-assembly coating; washing the substrate with the first layer of bivalirudin-protein co-assembly coating, and then mixing it with a second mixed solution containing an oxidant, bivalirudin and protein, so that the bivalirudin in the second mixed solution and the protein undergo a co-assembly reaction under the induction of the oxidant, thereby obtaining a second layer of bivalirudin-protein co-assembly coating on the surface of the first layer of bivalirudin-protein co-assembly coating; preparing other layers of bivalirudin-protein co-assembly coating in the above manner until the nth layer of bivalirudin-protein co-assembly coating is obtained, wherein n is the preset number of layers of the bivalirudin-protein co-assembly coating.

[0083] In some optional embodiments, the co-assembly reaction temperature can be 30°C to 45°C, such as 30°C, 32°C, 35°C, 38°C, 40°C, 42°C, or 45°C, or other values or ranges within the range of 30°C to 45°C. If the co-assembly reaction temperature is lower than 30°C, the coating may be deposited too thinly; if the co-assembly reaction temperature is higher than 45°C, the coating may be deposited too thickly, which may affect the activity of the protein and bivalirudin. In some preferred embodiments, the co-assembly reaction temperature can be 35°C to 40°C. Performing the co-assembly reaction within this temperature range is beneficial for obtaining a coating with optimal thickness and efficacy.

[0084] In some optional embodiments, the co-assembly reaction time is not less than 2 hours, such as 2 hours to 72 hours, such as 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 hours, 50 hours, 52 hours, 54 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours or 72 hours, etc., or other values within the range of 2 hours to 72 hours. If the co-assembly reaction time is less than 2 hours, the coating may be deposited too thinly; if the co-assembly reaction time is longer than 72 hours, the coating may be deposited too thickly, which may affect the activity of the protein and bivalirudin. In some preferred embodiments, the co-assembly reaction time can be 6h~12h, such as 6h, 6.5h, 7h, 7.5h, 8h, 8.5h, 9h, 9.5h, 10h, 10.5h, 11h, 11.5h or 12h, etc., to obtain a coating with better thickness and efficacy.

[0085] In some preferred embodiments, the temperature of the co-assembly reaction can be set to 35°C~40°C, and the time can be set to 6h~12h. When the co-assembly reaction is carried out under these conditions, the coating thickness can be controlled within an appropriate range without affecting the activity of the protein and bivalirudin.

[0086] In some optional embodiments, the pH value of the co-assembly reaction can be 3 to 7, such as 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7, or other values or ranges within the range of 3 to 7. If the pH value of the co-assembly reaction is lower than 3 or higher than 7, the activity of the protein or bivalirudin will be affected, making it difficult to form a coating. By specifically controlling the pH value of the co-assembly reaction within the range of 3 to 7, the binding of groups during the co-assembly process is more favorable, avoiding electrostatic repulsion.

[0087] In some optional embodiments, the pH value of the mixed solution may be 3 to 7, such as 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5 or 7, or other values or ranges within the range of 3 to 7. Similarly, if the pH value of the mixed solution is less than 3, it is not conducive to maintaining the biological activity of bivalirudin; if the pH value of the mixed solution is greater than 7, it is difficult to form a stable coating, which is not conducive to the long-term sustained release of bivalirudin from the coating. In some preferred embodiments, the pH value of the mixed solution may be 4 to 6. Controlling the pH of the mixed solution to 4 to 6 can protect the biological activity of bivalirudin to the greatest extent during the reaction process, while facilitating the formation of protein polypeptide micro-nanoparticles, thereby constructing a protein polypeptide-based micro-nanostructure coating with good stability and good mechanical properties.

[0088] In some optional embodiments, the concentration of the oxidant in the mixed solution can be 0.01 mg / mL to 100 mg / mL, such as 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 8 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL or 100 mg / mL, or other values within the range of 0.01 mg / mL to 100 mg / mL.

[0089] If the concentration of the oxidant in the mixed solution is less than 0.01 mg / mL, the degree of protein denaturation will be too low, making it difficult to form a protein coating; if the concentration of the oxidant in the mixed solution is greater than 100 mg / mL, the protein and bivalirudin will be excessively denatured and lose their biological functions.

[0090] In some preferred embodiments, the concentration of the oxidant in the mixed solution can be 0.05 mg / mL to 20 mg / mL, such as 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL or 20 mg / mL, or other values within the range of 0.05 mg / mL to 20 mg / mL.

[0091] In some more preferred embodiments, the concentration of the oxidant in the mixed solution can be 0.5 mg / mL to 4 mg / mL, such as 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL or 4 mg / mL, or other values within the range of 0.5 mg / mL to 4 mg / mL.

[0092] By controlling the concentration of the oxidant in the mixed solution within the above-mentioned preferred or better range, a more suitable reaction environment can be obtained, which can not only destroy some disulfide bonds of the protein and extend the protein molecules to a certain extent, which is conducive to the formation of nanoparticles by hydrophilic and hydrophobic interactions and hydrogen bonds after the insertion of bivalirudin, but also protect the biological functional activity of bivalirudin to the greatest extent.

[0093] In some optional embodiments, the concentration of the protein in the mixed solution can be 0.01 mg / mL to 100 mg / mL, such as 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 8 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL or 100 mg / mL, or other values within the range of 0.01 mg / mL to 100 mg / mL.

[0094] If the concentration of the protein in the mixed solution is less than 0.01 mg / mL, the concentration is too low and is not conducive to the formation of the coating; if the concentration of the protein in the mixed solution is greater than 100 mg / mL, the concentration is too high and is not conducive to the oxidant's role in destroying the disulfide bonds of the protein, thereby affecting the assembly and formation of the coating.

[0095] In some preferred embodiments, the concentration of the protein in the mixed solution can be 0.5 mg / mL to 5 mg / mL, such as 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL or 5 mg / mL, or other values within the range of 0.5 mg / mL to 5 mg / mL.

[0096] In some more preferred embodiments, the concentration of the protein in the mixed solution can be 1 mg / mL to 2 mg / mL.

[0097] By controlling the concentration of the protein in the mixed solution within the above-mentioned preferred or better range, a more stable, uniform, and long-lasting bivalirudin sustained-release coating can be obtained.

[0098] In some optional embodiments, the concentration of bivalirudin in the mixed solution can be 0.01 mg / mL to 100 mg / mL, such as 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 8 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL or 100 mg / mL, or other values within the range of 0.01 mg / mL to 100 mg / mL.

[0099] If the concentration of bivalirudin in the mixed solution is less than 0.01 mg / mL, it is not conducive to maintaining the effective loading amount of bivalirudin in the coating to meet the requirements of long-term anti-thrombotic effect; if the concentration of bivalirudin in the mixed solution is greater than 100 mg / mL, it is not conducive to the formation of the coating, and will accelerate the assembly and aggregation process, resulting in the formation of huge protein-bivalirudin particles in the solution, which will not be able to stack and assemble on the coating surface to form a stable coating.

[0100] In some preferred embodiments, the concentration of bivalirudin in the mixed solution can be 0.05 mg / mL to 5 mg / mL, such as 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL or 5 mg / mL, etc., or other values within the range of 0.05 mg / mL to 5 mg / mL.

[0101] In some more preferred embodiments, the concentration of bivalirudin in the mixed solution may be 0.1 mg / mL to 1 mg / mL.

[0102] It should be noted that the present invention does not impose any particular limitation on the preparation method of the mixed solution, as long as the concentration ranges of the oxidant, protein and bivalirudin in the mixed solution are met.

[0103] In some optional embodiments, the mixed solution can be obtained by mixing an oxidant solution, a protein solution and a bivalirudin solution, wherein the volume ratio of the oxidant solution, the protein solution and the bivalirudin solution is 1:1:0.01 to 1:1:1, such as 1:1:0.01, 1:1:0.02, 1:1:0.03, 1:1:0.04, 1:1:0.05, 1:1:0.06, 1:1:0.07, 1:1:0.08, 1:1:0.09 or 1:1:1, etc., or other values within the range of 1:1:0.01 to 1:1:1. In the final mixed solution, the molar ratio of protein to bivalirudin can be 0.1:1 to 20:1, such as 0.1:1, 0.2:1, 0.5:1, 0.8:1, 1:1, 2:1, 5:1, 8:1, 10:1, 12:1, 15:1, 18:1, or 20:1, or other values within the range of 0.1:1 to 20:1. The concentration of the oxidant in the mixed solution is 0.01 mg / mL to 100 mg / mL.

[0104] In other optional embodiments, the mixed solution is obtained by mixing an oxidant solution, a protein solution and a bivalirudin solution, wherein the volume ratio of the oxidant solution, the protein solution and the bivalirudin solution is 1:1:0.01 to 1:1:1, such as 1:1:0.01, 1:1:0.02, 1:1:0.03, 1:1:0.04, 1:1:0.05, 1:1:0.06, 1:1:0.07, 1:1:0.08, 1:1:0.09 or 1:1:1, etc., and can also be other values within the range of 1:1:0.01 to 1:1:1. The total concentration of the protein and bivalirudin in the reaction system can be 0.01 mmol / L to 5 mmol / L, such as 0.01 mmol / L, 0.02 mmol / L, 0.05 mmol / L, 0.08 mmol / L, 0.1 mmol / L, 0.2 mmol / L, 0.5 mmol / L, 0.8 mmol / L, 1 mmol / L, 1.5 mmol / L, 2 mmol / L, 2.5 mmol / L, 3 mmol / L, 3.5 mmol / L, 4 mmol / L, 4.5 mmol / L, or 5 mmol / L, or other values within the range of 0.01 mmol / L to 5 mmol / L. The concentration of the oxidant in the mixed solution is 0.01 mg / mL to 100 mg / mL.

[0105] In some optional embodiments, the oxidant used in the present invention may include at least one of an organic oxidant and an inorganic oxidant. The organic oxidant may, by way of example but not limitation, include at least one of dichlorine peroxide and peracetic acid. The inorganic oxidant may, by way of example but not limitation, include at least one of persulfate, chlorate, perchlorate, perchlorate, dichromate, periodate, permanganate, nitrate, and an inorganic peroxide. The inorganic peroxide may, by way of example but not limitation, include at least one of Na2O2, K2O2, MgO2, CaO2, BaO2, and H2O2.

[0106] In some optional embodiments, the isoelectric point of the protein can be 3-7, such as 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5 or 7, or other values or ranges within the range of 3-7.

[0107] It should be noted that by specifically controlling the isoelectric point of the protein in the range of 3 to 7, it is more conducive to the combination of groups during the co-assembly process, avoiding electrostatic repulsion and being more conducive to assembly.

[0108] In some optional embodiments, the protein may illustratively but not limitatively include at least one of serum albumin, casein, fibroin, arginine kinase, lactoferrin, and whey protein.

[0109] In some preferred embodiments, the protein is serum albumin, which has an isoelectric point of 3-7. Albumin acts as a transport protein in the body, effectively protecting drugs from immune system recognition and degradation, providing a degree of "immune escape" and potentially prolonging the drug's shelf life. Furthermore, the albumin coating exhibits excellent passive anti-adhesion properties, which, combined with the active anticoagulant function of bivalirudin, further enhances the long-lasting anticoagulant effect of the bivalirudin-protein coating. In the formulation combining bivalirudin with serum albumin, which has an isoelectric point of 3-7, the pH of the solvent in the mixed solution is preferably controlled between 5 and 7, and more preferably at 6.

[0110] In the present invention, when the number of layers of the bivalirudin-protein co-assembly coating is n (n ≥ 2), the cleaning process involved in the preparation of the bivalirudin-protein co-assembly coating is primarily to remove loosely adhered substances on the surface. In some optional embodiments, distilled water with a pH of 3 to 7 and a temperature of 10°C to 45°C can be used for cleaning. The pH value of the distilled water can be 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7, or other values within the range of 3 to 7. The temperature of the distilled water can be 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, or 45°C, or other values within the range of 10°C to 45°C. In some preferred embodiments, distilled water with a pH of 4 to 6 and a temperature of 25°C to 37°C can be used for cleaning.

[0111] In the present invention, after the final bivalirudin-protein co-assembly coating is obtained, the bivalirudin-protein co-assembly coating is dried and then mixed with the anion solution.

[0112] The drying process described above eliminates stress within the bivalirudin-protein co-assembly coating, further facilitating the collapse of hydrophobic nucleation and / or protein conformation within the bivalirudin-protein co-assembly coating. Excessive stress within the bivalirudin-protein co-assembly coating can make it difficult or impossible for the hydrophobic nucleation and / or protein conformation within the bivalirudin-protein co-assembly coating to collapse, leading to an inability to effectively encapsulate the micro-nanoporous structure and a difficulty in obtaining a dense, smooth bivalirudin-protein coating.

[0113] Illustratively, the drying temperature may be 10°C to 45°C, such as 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C or 45°C, or other values within the range of 10°C to 45°C.

[0114] If the drying temperature is lower than 10°C, it is not conducive to the evaporation of moisture inside the coating, and it also fails to eliminate the residual stress of the coating; if the drying temperature is higher than 45°C, it is not conducive to protecting the biological activity of bivalirudin.

[0115] In some optional embodiments, the mixing time of the bivalirudin-protein co-assembled coating and the anionic solution is no less than 1 minute. If the mixing time is less than 1 minute, the anions do not have enough time to induce complete reorganization of the micro-nanoporous coating, and a partial pore structure is retained. In some preferred embodiments, the mixing time of the bivalirudin-protein co-assembled coating and the anionic solution can be 5 minutes to 60 minutes, such as 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes, or other values within the range of 5 minutes to 60 minutes.

[0116] In some optional embodiments, the temperature of the anion solution can be 4° C. to 45° C., such as 4° C., 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., or 45° C., or other values within the range of 4° C. to 45° C. In some preferred embodiments, the temperature of the anion solution can be 4° C. to 37° C.

[0117] If the temperature of the anion solution is lower than 4°C, it is not conducive to coating encapsulation and affects the long-acting sustained-release ability of bivalirudin; if the temperature of the anion solution is higher than 45°C, it is not conducive to maintaining the functional activity of bivalirudin.

[0118] In some optional embodiments, the concentration of the anion solution is not less than 0.1mmol / L, such as 0.1mmol / L to 10mmol / L, such as 0.1mmol / L, 0.5mmol / L, 1mmol / L, 1.5mmol / L, 2mmol / L, 2.5mmol / L, 3mmol / L, 3.5mmol / L, 4mmol / L, 4.5mmol / L, 5mmol / L, 5.5mmol / L, 6mmol / L, 6.5mmol / L, 7mmol / L, 7.5mmol / L, 8mmol / L, 8.5mmol / L, 9mmol / L, 9.5mmol / L or 10mmol / L, etc., and can also be other values within the range of 0.1mmol / L to 10mmol / L. In some preferred embodiments, the concentration of the anion solution can be 5mmol / L to 10mmol / L.

[0119] If the concentration of the anionic solution is lower than 0.1 mmol / L, the coating will not completely rearrange and self-encapsulate, and some pores will remain, potentially leading to excessive initial burst release of the drug. Higher anionic solution concentrations increase the encapsulation rate and shorten the encapsulation time. For example, at a 10 mmol / L anionic solution concentration, the coating can achieve complete encapsulation in approximately 1 minute.

[0120] In some optional embodiments, the anion solution may illustratively but not limitatively include at least one of a phosphoric acid solution, a phosphate solution, a hydrogen phosphate solution, a dihydrogen phosphate solution, a sulfurous acid solution, a sulfite solution, a sulfonic acid solution, and a sulfonate solution. In some preferred embodiments, the anion solution may include at least one of a phosphate solution, a hydrogen phosphate solution, and a dihydrogen phosphate solution.

[0121] It should be noted that the present invention preferably uses above-mentioned anionic solution, and it all has stronger hydrophilicity, and has stronger hydrophilicity compared to protein, thus more easily combines with water and generates hydrogen bond than protein.More specifically, bivalirudin-protein co-assembly coating has the hydrophilic group positioned at the outside and the hydrophobic nucleation positioned at the inside, in the process that bivalirudin-protein co-assembly coating mixes with anionic solution, anionic solution has stronger hydrophilicity than bivalirudin-protein co-assembly coating, therefore, anion can be compared with bivalirudin-protein co-assembly coating under solution system and rushes to combine the water molecule in solution system, is equivalent to further squeezing hydrophobic nucleation, thus makes hydrophobic nucleation collapse.In collapse process, based on the effect of water and the trend driven by thermodynamics to minimum interfacial energy, hydrophobic nucleation can change to the state of minimum interfacial energy, thus drives bivalirudin-protein layer to gradually tend to dense, smooth.

[0122] After the bivalirudin-protein co-assembled coating undergoes structural rearrangement after reacting with the anionic solution, it is further dried. For example, the drying process can be carried out in a 37° C. constant temperature drying oven for 12 hours.

[0123] In some optional embodiments, the substrate may illustratively but not limitatively include at least one of metal materials, inorganic materials, polymer materials, natural biological materials, and artificially synthesized polypeptide hydrogel materials.

[0124] The metal material may illustratively but not limitatively include at least one of cobalt-based alloys, titanium and its alloys, nickel-titanium alloys, platinum and its alloys, magnesium and its alloys, iron and its alloys, and zinc and its alloys.

[0125] The inorganic material may illustratively but not limitatively include at least one of titanium oxide and its nanotubes, carbon materials, silicon, silicon dioxide, calcium phosphate, silicon nitride, silicon carbide, aluminosilicate, calcium aluminum system, bioglass, titanium nitride and biomedical micro-nanoparticles.

[0126] The biomedical micro-nanoparticles may illustratively but not limitatively include at least one of ferroferric oxide nanoparticles, silicon dioxide nanoparticles, titanium oxide nanoparticles, and zinc oxide nanoparticles.

[0127] The polymer material may illustratively but not limitatively include at least one of polyester, polyethylene, polyvinyl chloride, polytetrafluoroethylene, polyurethane, polystyrene, polyvinyl alcohol, polypropylene, polyoxymethylene, polycarbonate, carbon copolymer, polyglycolic acid, polymethyl methacrylate, polyvinyl acetate, polylactic acid, glycolide-lactide copolymer, polytrimethylene carbonate, polycaprolactone, polyhydroxyalkanoate, polybutylene succinate, polyamide, polydioxanone, epoxy resin, silicone rubber, silicone gel, polyacrylic acid and its derivatives, polyethylene glycol and its derivatives.

[0128] The natural biological material may illustratively but not limitatively include at least one of decellularized tissues and organs of animal origin, gelatin, collagen, fibrin, silk protein, keratin, and polysaccharide.

[0129] Examples of animal-derived decellularized tissues and organs include, but are not limited to, at least one of blood vessels, valves, hearts, bones, lungs, ligaments, bladders, mucosa, and corneas. Examples of polysaccharides include, but are not limited to, sodium hyaluronate, sodium alginate, agarose, plastic starch materials, cellulose, hemicellulose, lignin, chitin, and their derivatives. Examples of synthetic polypeptide hydrogel materials include, but are not limited to, at least one of L-lysine and poly-L-glutamic acid.

[0130] In addition, the present invention also provides an application of the above-mentioned bivalirudin-protein coating, for example, using it to prepare blood-contact devices.

[0131] Correspondingly, the present invention also provides a blood-contact device comprising the above-mentioned bivalirudin-protein coating.

[0132] In some optional embodiments, the blood-contact device may illustratively but not limitatively include at least one of a vascular stent, a catheter, and an artificial heart.

[0133] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0134] Example 1 This embodiment provides a bivalirudin-protein coating, the preparation process of which is as follows: S1: The substrate is mixed with a mixed solution containing an oxidant, bivalirudin and a protein, so that the bivalirudin and the protein undergo a co-assembly reaction under the induction of oxygen free radicals, thereby obtaining a bivalirudin-protein co-assembly coating (one layer) having a micro-nanoporous structure.

[0135] Specifically, ultrapure water, adjusted to pH 6 with HCl, was used as the solvent to dissolve serum albumin (SA, isoelectric point 4.7), bivalirudin (BVLD, isoelectric point 3.99), and sodium persulfate (SPS) powders, respectively, to prepare a 1 mg / mL serum albumin solution, a 1 mg / mL bivalirudin solution, and a 4 mg / mL sodium persulfate solution. The SA, BVLD, and SPS solutions were then mixed in a 5:1:5 volume ratio to form a mixed solution (pH 6, with approximately 1.82 mg / mL sodium persulfate, 0.45 mg / mL serum albumin, and 0.09 mg / mL bivalirudin). The solution was then added to a clean stainless steel surface and incubated at 37°C for a total of 12 hours. After taking out, it was washed with ultrapure water (pH 6, temperature 25°C) to remove the loosely adhered substances on the surface, and then dried at 37°C for 12 hours to obtain a bivalirudin-protein co-assembled coating.

[0136] S2: mixing the bivalirudin-protein co-assembled coating with an anion solution, so that the anions are adsorbed on the bivalirudin-protein co-assembled coating to cause structural rearrangement, thereby obtaining a bivalirudin-protein layer.

[0137] The bivalirudin-protein co-assembled coating prepared in S1 was immersed in a phosphate solution at a temperature of 25° C. and a concentration of 10 mmol / L for 30 minutes, and then dried in a constant temperature drying oven at 37° C. for 12 hours to obtain a bivalirudin-protein coating.

[0138] This embodiment adjusts the conditions such as the oxidant concentration, the pH value of the co-assembly reaction, and the reaction time to maximize the retention of the anticoagulant activity of bivalirudin while partially destroying the disulfide bonds within the albumin molecules, so that the albumin molecules and the added bivalirudin molecules are re-coassembled to form a bivalirudin-protein co-assembly coating; then, by reacting the bivalirudin-protein co-assembly coating with an anionic solution, a dense bivalirudin-protein coating is obtained, thereby achieving long-term sustained release of bivalirudin and imparting excellent anticoagulant function to the surface of the stainless steel substrate.

[0139] Example 2 This embodiment provides a bivalirudin-protein coating, which differs from Example 1 in that: The mixed solution is prepared by mixing a 1 mg / mL whey protein solution (isoelectric point of whey protein is 4.6), a 1 mg / mL bivalirudin solution, and a 1 mg / mL sodium persulfate solution in a volume ratio of 5:1:5. The pH value of the mixed solution is 6, and the concentrations of sodium persulfate, whey protein, and bivalirudin in the mixed solution are approximately 0.45 mg / mL, 0.45 mg / mL, and 0.09 mg / mL, respectively.

[0140] The total assembly reaction time was 8 h.

[0141] The rest are the same as in Example 1.

[0142] This embodiment maximizes the retention of bivalirudin's anticoagulant activity while partially destroying the disulfide bonds within the whey protein molecules by adjusting conditions such as the oxidant concentration, the pH value of the co-assembly reaction, and the reaction time, so that the whey protein molecules and the added bivalirudin molecules can be re-coassembled to form a bivalirudin-protein co-assembly coating; then, by reacting the bivalirudin-protein co-assembly coating with an anionic solution, a dense bivalirudin-protein coating is obtained, achieving long-term sustained release of bivalirudin, and imparting excellent anticoagulant and osteogenic functions to the surface of the stainless steel substrate.

[0143] Example 3 This embodiment provides a bivalirudin-protein coating, which differs from Example 1 in that: The mixed solution is prepared by mixing a 1 mg / mL pepsin solution (pepsin has an isoelectric point of 3), a 1 mg / mL bivalirudin solution, and a 2 mg / mL sodium persulfate solution in a volume ratio of 5:1:5. The pH of the mixed solution is 4, and the concentrations of sodium persulfate, pepsin, and bivalirudin in the mixed solution are approximately 0.9 mg / mL, 0.45 mg / mL, and 0.09 mg / mL, respectively.

[0144] The rest are the same as in Example 1.

[0145] This embodiment maximizes the retention of bivalirudin's anticoagulant activity while partially destroying the disulfide bonds within the pepsin molecules by adjusting conditions such as the oxidant concentration, the pH value of the co-assembly reaction, and the reaction time, so that the pepsin molecules and the added bivalirudin molecules can be re-coassembled to form a bivalirudin-protein co-assembly coating; then, by reacting the bivalirudin-protein co-assembly coating with an anionic solution, a dense bivalirudin-protein coating is obtained, thereby achieving long-term sustained release of bivalirudin and imparting excellent anticoagulant function to the surface of the stainless steel substrate.

[0146] Comparative Example 1 The only difference between this comparison and Example 1 is that no oxidant was added during the preparation process.

[0147] Comparative Example 2 The only difference between this comparative example and Example 1 is that there is no S2 step.

[0148] Comparative Example 3 The only difference between this comparative example and Example 1 is that no bivalirudin was added during the preparation process.

[0149] Comparative Example 4 The only difference between this comparative example and Example 1 is that the temperature of the co-assembly reaction is room temperature (25° C.).

[0150] Comparative Example 5 The only difference between this comparative example and Example 1 is that the temperature of the co-assembly reaction is 50°C.

[0151] Test Example 1 The anti-platelet adhesion test was performed on the bivalirudin-protein co-assembled coating obtained in step S1 of Examples 1 to 3. The test method and conditions were as follows: 100 μL of platelet-rich plasma was added to the surface of the bivalirudin-protein co-assembled coating obtained in step S1 of Examples 1 to 3, and the coating was incubated at 37°C for 30 minutes. The surface was washed with deionized water, and the platelet adhesion was observed under a scanning electron microscope after dehydration and drying. The results are shown in FIG. Figure 1 shown.

[0152] Depend on Figure 1 It can be seen that the bivalirudin-protein co-assembled coatings prepared in step S1 in Examples 1 to 3 have good anti-platelet adhesion ability, and the bivalirudin-protein co-assembled coatings all have a porous structure.

[0153] Test Example 2 Fluorescence staining tests were performed on the stainless steel samples with bivalirudin-protein coatings prepared in Example 1 and Comparative Example 1. The specific method and conditions were as follows: 100 μL of pre-prepared FITC-NHS activated ester was added to the surface of the bivalirudin-protein co-assembled coating obtained in step S1 of Examples 1 to 4, and the mixture was incubated overnight in a refrigerator at 4°C. The surface was washed with deionized water, dried, and the binding of the fluorescent dye to the protein coating was observed under an inverted fluorescence microscope. The results are shown in FIG. Figure 2 shown.

[0154] Depend on Figure 2 It can be seen that the sample in Example 1 exhibited fluorescence, while the sample in Comparative Example 1 did not. This is because the N-hydroxysuccinimide (NHS) molecules only chemically grafted onto the primary amino groups of the bivalirudin-protein coating and barely reacted with the substrate. Comparative Example 1 did not use an oxidizing agent during the preparation process, which failed to induce co-assembly between bivalirudin and the protein, preventing the formation of a successful bivalirudin-protein coating. Consequently, the sample in Comparative Example 1 failed to exhibit fluorescence in the fluorescence staining test. This further demonstrates that a bivalirudin-protein coating can only be prepared in the presence of an oxidizing agent.

[0155] Test Example 3 The protein coatings obtained in Comparative Example 2 and Example 1 were observed by scanning electron microscopy, and the results were as follows: Figure 3 and Figure 4 shown.

[0156] in, Figure 3 (a) is a surface scanning electron micrograph of a stainless steel sample having a bivalirudin-protein co-assembled coating prepared in Comparative Example 2; (b) is a cross-sectional scanning electron micrograph of the sample; (c) is a partial enlarged view of (a); and (d) is a partial enlarged view of (b).

[0157] Figure 4 (a) is a surface scanning electron micrograph of a stainless steel sample having a bivalirudin-protein co-assembled coating prepared in Example 1; (b) is a cross-sectional scanning electron micrograph of the sample; (c) is a partial enlarged view of (a); and (d) is a partial enlarged view of (b).

[0158] Depend on Figure 3 and Figure 4 By comparison, it can be seen that the bivalirudin-protein co-assembled coating has an obvious micro-nanopore structure before being mixed and reacted with the anionic solution; after the bivalirudin-protein co-assembled coating is mixed and reacted with the anionic solution, the obtained bivalirudin-protein coating is dense and flat, indicating that the micro-nanopore structure in the bivalirudin-protein co-assembled coating is encapsulated.

[0159] Test Example 4 The particle size test was performed on the preparation process of Example 1 and Comparative Example 3. The specific method and conditions are as follows: set the time nodes to 0h, 0.5h, 1h, 2h, 4h and 6h, take out the reaction solution at the above time points, and use nanoparticle size and Zeta potential instrument to detect the particle size of the particles in the reaction solution. The results are as follows: Figure 5 shown.

[0160] Depend on Figure 5 It can be seen that: Example 1 ( Figure 5 (a)) compared with comparative example 3 ( Figure 5 In (b), micronized particles are formed faster and the film is formed earlier, so the coating has more pores inside.

[0161] Test Example 5 This experiment investigates the effect of anionic solution concentration on the performance of bivalirudin-protein coating.

[0162] ①. TT test was performed on the stainless steel samples with bivalirudin-protein coating prepared in Example 1 and Comparative Example 2. The specific method and conditions are as follows: A portion of the stainless steel samples with bivalirudin-protein coating prepared in Example 1 and Comparative Example 2 was taken out at different soaking time points, 100 μL of platelet-poor plasma (PPP) was added to the sample surface, and incubated in a 37°C incubator for 30 minutes. 100 microliters of PPP was taken out and mixed with an equal amount of thrombin reagent. The time for the mixed reaction liquid to coagulate was observed and recorded. The thrombin time (TT) test results of the samples at different soaking time points are as follows: Figure 6 shown.

[0163] Depend on Figure 6 It can be seen that the bivalirudin-protein coating obtained in Example 1 can still have a high anticoagulant activity during the 50-day immersion period.

[0164] ②. The immersion solutions with bivalirudin-protein coating prepared in Example 1 and Comparative Example 2 were subjected to TT test respectively. The specific method and conditions are as follows: A portion of the immersion solution with bivalirudin-protein coating prepared in Example 1 and Comparative Example 2 was taken out at different immersion time points, 100 μL of platelet-poor plasma (PPP) was added to the sample surface, and incubated in a 37°C incubator for 30 minutes. 100 μL of PPP was taken out and mixed with 100 microliters of thrombin reagent, and the time for the mixed reaction solution to coagulate was observed and recorded. The thrombin time (TT) test results of the immersion solution at different immersion time points are as follows: Figure 7 shown.

[0165] Depend on Figure 7It can be seen that the bivalirudin-protein coating obtained in Example 1 can continuously release bivalirudin with biological activity during the 50-day immersion period, and the coating can achieve long-term sustained release of bivalirudin.

[0166] Test Example 6 This study investigates the effect of co-assembly reaction temperature on the performance of bivalirudin-protein coatings.

[0167] ①, TT test was performed on the stainless steel samples with bivalirudin-protein coating prepared in Example 1, Comparative Example 4 and Comparative Example 5. The specific method and conditions are shown in Test Example 5. The thrombin time (TT) test results of each sample are as follows: Figure 8 shown.

[0168] Depend on Figure 8 It can be seen that: if the temperature of the co-assembly reaction is too low or too high, it will have an adverse effect on the coagulation effect of the product. The reason may be that if the temperature of the co-assembly reaction is too low, the coating cannot be formed or the coating formed is small, resulting in poor coagulation effect; if the temperature of the co-assembly reaction is too high, it will affect the activity of bivalirudin, resulting in poor coagulation effect.

[0169] ②, the stainless steel samples with bivalirudin-protein coating prepared in Example 1 and Comparative Example 4 were observed by 3D profilometer morphology, and the results are as follows: Figure 9 shown.

[0170] Depend on Figure 9 It can be seen that the temperature of the co-assembly reaction is too low, and the coating cannot be formed or the formed coating is small.

[0171] Test Example 7 This study investigated the effect of solvent pH on the performance of bivalirudin-protein coatings.

[0172] This experimental example provides samples 7-1 to 7-4. The only difference between samples 7-1 to 7-4 and Example 1 is that the pH value of the solvent in the mixed solution involved in the co-assembly reaction is different. The pH values of the corresponding solvents in samples 7-1 to 7-4 are 4, 5, 7, and 8, respectively. The pH values are obtained by adjusting ultrapure water using HCl and NaOH.

[0173] The stainless steel samples with bivalirudin-protein coating prepared in Example 1 and samples 7-1 to 7-4 were observed by 3D profilometer morphology. The results are as follows: Figure 10 shown.

[0174] Depend on Figure 10It can be seen that in the co-assembly scheme of bivalirudin and albumin, a solvent pH of 6 resulted in the best film-forming coating, followed by pH values of 5 and 7, and then pH values of 4 and 8. Correspondingly, a mixed solution with a pH of 4 to 6 exhibited better results than a mixed solution with a pH of 3 to 4 (not included) and 6 (not included) to 7.

[0175] Test Example 8 This experimental example studies the effect of the co-assembly reaction time on the performance of the bivalirudin-protein coating. Specifically, this experimental example sets samples 8-1 to 8-7. The only difference between samples 8-1 to 8-7 and Example 1 is that the co-assembly reaction time is different. The co-assembly reaction times corresponding to samples 8-1 to 8-7 are 1h, 2h, 6h, 24h, 48h, 72h and 84h, respectively.

[0176] The stainless steel samples with bivalirudin-protein coating prepared in Example 1 and samples 8-1 to 8-7 were observed by 3D profilometer morphology. The results are as follows: Figure 11 shown.

[0177] Depend on Figure 11 It can be seen that the bivalirudin-protein coating obtained by the process used in Example 1 just completely covers the substrate. In other processes, the deposition time is too short and the resulting coating is thin and uneven, and cannot cover the surface of the substrate; while if the deposition time is too long, the formed coating is thicker, affecting the mechanical properties of the coating.

[0178] Test Example 9 This test example studies the effect of the concentration of the oxidant in the mixed solution on the performance of the bivalirudin-protein coating. Specifically, this test example sets samples 9-1 to 9-7. The only difference between samples 9-1 to 9-7 and Example 1 is that the concentration of the oxidant in the mixed solution is different. The concentrations of the oxidant in the mixed solution corresponding to samples 9-1 to 9-7 are 0.005 mg / mL, 0.01 mg / mL, 0.05 mg / mL, 0.5 mg / mL, 20 mg / mL, 100 mg / mL and 120 mg / mL, respectively.

[0179] The stainless steel samples with bivalirudin-protein coating prepared in Example 1 and samples 9-1 to 9-7 were observed by 3D profilometer morphology. The results are as follows: Figure 12 shown.

[0180] Depend on Figure 12 It can be seen that: if the concentration of the oxidant is too low, the coating cannot be formed; if the concentration is too high, the protein conformation will change extremely quickly, forming huge particles, which are unstable when combined with the substrate.

[0181] Test Example 10 This test example studies the effect of the concentration of protein in the mixed solution on the performance of the bivalirudin-protein coating. Specifically, this test example sets samples 10-1 to 10-8. The only difference between samples 10-1 to 10-8 and Example 1 is that the concentration of protein in the mixed solution is different. The concentrations of protein in the mixed solution corresponding to samples 10-1 to 10-8 are 0.005 mg / mL, 0.01 mg / mL, 1 mg / mL, 2 mg / mL, 5 mg / mL, 10 mg / mL, 100 mg / mL and 120 mg / mL, respectively.

[0182] The stainless steel sample with bivalirudin-protein coating prepared in Example 1 and samples 10-1 to 10-8 were observed by 3D profilometer morphology. The results are as follows: Figure 13 shown.

[0183] Depend on Figure 13 It can be seen that if the protein concentration is too low or too high, the coating cannot be formed.

[0184] Test Example 11 This test example studies the effect of the concentration of bivalirudin in the mixed solution on the performance of the bivalirudin-protein coating. Specifically, this test example sets samples 11-1 to 11-8. The only difference between samples 11-1 to 11-8 and Example 1 is that the concentrations of bivalirudin in the mixed solution are different. The concentrations of bivalirudin in the mixed solution corresponding to samples 11-1 to 11-8 are 0.005 mg / mL, 0.01 mg / mL, 0.05 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 100 mg / mL and 120 mg / mL, respectively.

[0185] The performance test method refers to Test Example 5, and the results are as follows Figure 14 shown.

[0186] Depend on Figure 14 It can be seen that when the concentration of bivalirudin added to the reaction is too low, less bivalirudin participates in the formation of the coating, and the thrombin time detected by the coating is relatively short (there is a significant difference compared with Example 1), and the coating cannot achieve the performance of long-term, controlled sustained release of bivalirudin; and when the concentration of bivalirudin is too high, the thrombin time of the coating will not be significantly prolonged, indicating that the bivalirudin-protein coating constructed using this process has a certain limit on the loading amount of bivalirudin. After reaching this limit, the bivalirudin loading amount will not increase with increasing concentration.

[0187] Test Example 12 This test example studies the effect of the isoelectric point of the protein concentration in the mixed solution on the performance of the bivalirudin-protein coating. Specifically, this test example sets samples 12-1 to 12-2. The only difference between samples 12-1 and 12-2 and Example 1 is that proteins with different isoelectric points are used. Sample 12-1 is pepsin with an isoelectric point of <3, and sample 12-2 is lactoferrin with an isoelectric point of >7.

[0188] The performance test method refers to Test Example 5, and the results are as follows Figure 15 shown.

[0189] Depend on Figure 15 It can be seen that the thrombin time (TT) of sample 12-1 and sample 12-2 is significantly reduced compared with Example 1. It is speculated that this may be because the main method of construction of sample 12-1 and sample 12-2 is electrostatic assembly rather than hydrophilic-hydrophobic interaction. After superhydrophilic anion treatment, a large amount of BVLD will be released suddenly, which seriously affects the anti-coagulation performance of the coating.

[0190] Test Example 13 This experimental example studies the effects of the temperature, type, concentration of the anionic solution, and the mixing time of the anionic solution with the bivalirudin-protein co-assembled coating on the performance of the bivalirudin-protein coating. Specifically, this experimental example sets samples 13-1 to 13-13.

[0191] ①. The only difference between Samples 13-1 and 13-2 and Example 1 is that the temperatures of the anion solutions are different. The temperatures of the anion solutions corresponding to Samples 13-1 and 13-2 are 4° C. and 37° C., respectively.

[0192] The morphology of the stainless steel sample with bivalirudin-protein coating prepared in Example 1 and samples 13-1 to 13-2 was observed using a scanning electron microscope. The results are as follows: Figure 16 shown.

[0193] Depend on Figure 16 It can be seen that the temperature of the anion solution affects the rate of coating reconstruction, and the two are positively correlated.

[0194] ②. The only difference between samples 13-3 to 13-5 and Example 1 is that the types of anionic solutions are different. The anionic solution used in sample 13-3 is sodium sulfite solution, the anionic solution used in sample 13-4 is sulfonic acid solution, and the anionic solution used in sample 13-5 is benzoic acid (hydrophobic).

[0195] The morphology of the stainless steel sample with bivalirudin-protein coating prepared in Example 1 and samples 13-3 to 13-5 was observed using a scanning electron microscope. The results are as follows: Figure 17 shown.

[0196] Depend on Figure 17 It can be seen that the more hydrophilic the anion, the easier it is to induce reconstruction of the bivalirudin-protein coating.

[0197] ③. The only difference between samples 13-6 to 13-9 and Example 1 is that the mixing time of the bivalirudin-protein co-assembled coating and the anionic solution is different. The mixing time for sample 13-6 is 1 min, the mixing time for sample 13-7 is 5 min, the mixing time for sample 13-8 is 10 min, and the mixing time for sample 13-9 is 60 min.

[0198] The morphology of the stainless steel sample with bivalirudin-protein coating prepared in Example 1 and samples 13-6 to 13-9 was observed using a scanning electron microscope. The results are as follows: Figure 18 shown.

[0199] Depend on Figure 18 It can be seen that within a certain time range, the time of anion solution treatment affects the self-encapsulation speed of the coating, and the two are positively correlated. When the reaction lasts about 30 minutes, the anion-induced coating reconstruction is completed. There is no significant change in the coating even if the reaction time is increased subsequently.

[0200] ④. The only difference between samples 13-10 to 13-13 and Example 1 is that the concentration of the anion solution for treating the bivalirudin-protein coating is different. The anion concentration corresponding to sample 13-10 is 1 mmol / L, the mixing time corresponding to sample 13-11 is 2 mmol / L, the mixing time corresponding to sample 13-12 is 4 mmol / L, and the mixing time corresponding to sample 13-13 is 5 mmol / L.

[0201] The morphology of the stainless steel sample with bivalirudin-protein coating prepared in Example 1 and samples 13-10 to 13-13 was observed using a scanning electron microscope. The results are as follows: Figure 19 shown.

[0202] Depend on Figure 19 It can be seen that the concentration of the anion solution affects the self-encapsulation speed of the coating, and the two are positively correlated.

[0203] In summary, the bivalirudin-protein coating provided by the present invention has good stability and high density, and can achieve long-term, controllable sustained release of bivalirudin, thereby giving the modified substrate an excellent long-term anticoagulant effect. It can be used to prepare blood-contact devices, etc., and has great application prospects and value.

[0204] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A bivalirudin-protein coating, characterized in that The bivalirudin-protein coating comprises a bivalirudin-protein layer disposed on the surface of a substrate; The bivalirudin-protein layer is formed by structural rearrangement of a bivalirudin-protein co-assembled coating with a micro-nano pore structure in the presence of an anion solution.

2. The bivalirudin-protein coating according to claim 1, characterized in that The bivalirudin-protein co-assembly coating contains micro-nano protein-bivalirudin particles.

3. The bivalirudin-protein coating according to claim 2, characterized in that The particle size of the micro-nano protein-bivalirudin particles is 900nm-1100nm.

4. The bivalirudin-protein coating according to claim 2, characterized in that The micro-nano protein-bivalirudin particles are formed by the co-assembly reaction of bivalirudin and protein under the induction of oxygen free radicals.

5. The bivalirudin-protein coating according to any one of claims 1 to 4, characterized in that The number of layers of the bivalirudin-protein co-assembly coating is 1 to 10 layers.

6. A method for preparing the bivalirudin-protein coating according to any one of claims 1 to 5, characterized in that: The following steps are involved: The substrate is mixed with a mixed solution containing an oxidant, bivalirudin, and a protein, so that the bivalirudin and the protein undergo a co-assembly reaction under the induction of oxygen free radicals, thereby obtaining a bivalirudin-protein co-assembly coating having a micro-nanoporous structure; The bivalirudin-protein co-assembly coating is mixed with an anion solution to cause the bivalirudin-protein co-assembly coating to undergo structural rearrangement, thereby obtaining the bivalirudin-protein layer.

7. The preparation method according to claim 6, characterized in that When the number of layers of the bivalirudin-protein co-assembly coating is n, n≥2, after obtaining the m-th layer of bivalirudin-protein co-assembly coating, the obtained m-th layer of bivalirudin-protein co-assembly coating is cleaned, and the next layer of bivalirudin-protein co-assembly coating is prepared under the same co-assembly reaction conditions until the n-th layer of bivalirudin-protein co-assembly coating is obtained; wherein the value of m is 1 to n-1.

8. The preparation method according to claim 6 or 7, characterized in that The temperature of the co-assembly reaction is 30°C~45°C.

9. The preparation method according to claim 8, characterized in that The temperature of the co-assembly reaction is 35°C~40°C.

10. The preparation method according to claim 6 or 7, characterized in that: The total assembly reaction time is not less than 2 h.

11. The preparation method according to claim 10, characterized in that: The total assembly reaction time is 2h~72h.

12. The preparation method according to claim 11, characterized in that The total assembly reaction time is 6h~12h.

13. The preparation method according to claim 6 or 7, characterized in that: The temperature of the co-assembly reaction is 35°C~40°C, and the time is 6h~12h.

14. The preparation method according to claim 6 or 7, characterized in that: The pH value of the co-assembly reaction is 3~7.

15. The preparation method according to claim 6 or 7, characterized in that: The pH value of the mixed solution is 3-7.

16. The preparation method according to claim 15, characterized in that The pH value of the mixed solution is 4-6.

17. The preparation method according to claim 6 or 7, characterized in that: The concentration of the oxidant in the mixed solution is 0.01 mg / mL to 100 mg / mL.

18. The preparation method according to claim 17, characterized in that: The concentration of the oxidant in the mixed solution is 0.05 mg / mL to 20 mg / mL.

19. The preparation method according to claim 18, characterized in that The concentration of the oxidant in the mixed solution is 0.5 mg / mL to 4 mg / mL.

20. The preparation method according to claim 6 or 7, characterized in that: The concentration of the protein in the mixed solution is 0.01 mg / mL to 100 mg / mL.

21. The preparation method according to claim 20, characterized in that The concentration of the protein in the mixed solution is 0.5 mg / mL to 5 mg / mL.

22. The preparation method according to claim 21, characterized in that The concentration of the protein in the mixed solution is 1 mg / mL to 2 mg / mL.

23. The preparation method according to claim 6 or 7, characterized in that: The concentration of the bivalirudin in the mixed solution is 0.01 mg / mL to 100 mg / mL.

24. The preparation method according to claim 22, characterized in that The concentration of the bivalirudin in the mixed solution is 0.05 mg / mL to 5 mg / mL.

25. The preparation method according to claim 24, characterized in that The concentration of the bivalirudin in the mixed solution is 0.1 mg / mL to 1 mg / mL.

26. The preparation method according to claim 6 or 7, characterized in that: The mixed solution is obtained by mixing an oxidant solution, a protein solution and a bivalirudin solution, wherein the volume ratio of the oxidant solution, the protein solution and the bivalirudin solution is 1:1:0.01 to 1:1:1; in the mixed solution, the molar ratio of protein to bivalirudin in the final mixed solution is controlled between 0.1:1 and 20:1, and the concentration of the oxidant in the mixed solution is 0.01 mg / mL~100 mg / mL.

27. The preparation method according to claim 6 or 7, characterized in that: The mixed solution is obtained by mixing an oxidant solution, a protein solution and a bivalirudin solution, wherein the volume ratio of the oxidant solution, the protein solution and the bivalirudin solution is 1:1:0.01 to 1:1:1; the total concentration of the protein and the bivalirudin in the reaction system is 0.01 mmol / L~5 mmol / L, and the concentration of the oxidant in the mixed solution is 0.01 mg / mL~100 mg / mL.

28. The preparation method according to claim 6 or 7, characterized in that: The isoelectric point of the protein is 3-7.

29. The preparation method according to claim 28, characterized in that The protein includes at least one of serum albumin, casein, fibroin, arginine kinase, lactoferrin and whey protein.

30. The preparation method according to claim 29, characterized in that The protein is serum albumin with an isoelectric point of 3 to 7.

31. The preparation method according to claim 6 or 7, characterized in that: The oxidant includes at least one of an organic oxidant and an inorganic oxidant.

32. The preparation method according to claim 31, characterized in that The organic oxidant includes at least one of dichlorine peroxide and peracetic acid.

33. The preparation method according to claim 32, characterized in that The inorganic oxidant includes at least one of persulfate, chlorate, perchlorate, perchlorate, dichromate, periodate, permanganate, nitrate and inorganic peroxide.

34. The preparation method according to claim 33, characterized in that The inorganic peroxide includes at least one of Na2O2, K2O2, MgO2, CaO2, BaO2 and H2O2.

35. The preparation method according to claim 7, characterized in that Use distilled water with a pH value of 3 to 7 and a temperature of 10°C to 45°C for cleaning.

36. The preparation method according to claim 35, characterized in that Use distilled water with a pH value of 4 to 6 and a temperature of 25°C to 37°C for cleaning.

37. The preparation method according to claim 6, characterized in that The bivalirudin-protein co-assembled coating was dried and then mixed with the anionic solution.

38. The preparation method according to claim 37, characterized in that The drying temperature is 10℃~45℃.

39. The preparation method according to claim 6 or 7, characterized in that The mixing time of the bivalirudin-protein co-assembly coating and the anion solution is not less than 1 minute.

40. The preparation method according to claim 39, characterized in that The mixing time of the bivalirudin-protein co-assembly coating and the anion solution is 5 min to 60 min.

41. The preparation method according to claim 6 or 7, characterized in that The temperature of the anion solution is 4°C to 45°C.

42. The preparation method according to claim 41, characterized in that The temperature of the anion solution is 4°C to 37°C.

43. The preparation method according to claim 6 or 7, characterized in that The concentration of the anion solution is not less than 0.1 mmol / L.

44. The preparation method according to claim 43, characterized in that The concentration of the anion solution is 0.1 mmol / L to 10 mmol / L.

45. The preparation method according to claim 44, characterized in that The concentration of the anion solution is 5 mmol / L to 10 mmol / L.

46. The preparation method according to claim 6 or 7, characterized in that The anion solution includes at least one of a phosphoric acid solution, a phosphate solution, a hydrogen phosphate solution, a dihydrogen phosphate solution, a sulfurous acid solution, a sulfite solution, a sulfite solution, a sulfonic acid solution, and a sulfonate solution.

47. The preparation method according to claim 46, characterized in that The anion solution includes at least one of a phosphate solution, a hydrogen phosphate solution, and a dihydrogen phosphate solution.

48. The preparation method according to claim 6 or 7, characterized in that The substrate comprises at least one of metal materials, inorganic materials, polymer materials, natural biological materials and artificially synthesized polypeptide hydrogel materials.

49. The preparation method according to claim 48, characterized in that The metal material includes at least one of cobalt-based alloys, titanium and its alloys, nickel-titanium alloys, platinum and its alloys, magnesium and its alloys, iron and its alloys, and zinc and its alloys.

50. The preparation method according to claim 48, characterized in that The inorganic material includes at least one of titanium oxide and its nanotubes, carbon materials, silicon, silicon dioxide, calcium phosphate, silicon nitride, silicon carbide, aluminosilicate, calcium aluminum series, bioglass, titanium nitride and biomedical micro-nano particles.

51. The preparation method according to claim 50, characterized in that The biomedical micro-nanoparticles include at least one of ferroferric oxide nanoparticles, silicon dioxide nanoparticles, titanium oxide nanoparticles and zinc oxide nanoparticles.

52. The preparation method according to claim 48, characterized in that The polymer material includes at least one of polyester, polyethylene, polyvinyl chloride, polytetrafluoroethylene, polyurethane, polystyrene, polyvinyl alcohol, polypropylene, polyoxymethylene, polycarbonate, carbon copolymer, polyglycolic acid, polymethyl methacrylate, polyvinyl acetate, polylactic acid, glycolide-lactide copolymer, polytrimethylene carbonate, polycaprolactone, polyhydroxyalkanoate, polybutylene succinate, polyamide, polydioxanone, epoxy resin, silicone rubber, silicone gel, polyacrylic acid and its derivatives, polyethylene glycol and its derivatives.

53. The preparation method according to claim 48, characterized in that The natural biological material includes at least one of decellularized tissues and organs of animal origin, gelatin, collagen, fibrin, silk protein, keratin and polysaccharide.

54. The preparation method according to claim 53, characterized in that The animal-derived decellularized tissues and organs include at least one of blood vessels, valves, hearts, bones, lungs, ligaments, bladders, mucosa, and corneas.

55. The preparation method according to claim 53, characterized in that The polysaccharide includes at least one of sodium hyaluronate, sodium alginate, agarose, plastic starch material, cellulose, hemicellulose, lignin, chitin and derivatives thereof.

56. The preparation method according to claim 48, characterized in that The artificially synthesized polypeptide hydrogel material includes at least one of L-lysine and poly-L-glutamic acid.

57. A use of the bivalirudin-protein coating according to any one of claims 1 to 5, characterized in that: The bivalirudin-protein coating is used for preparing blood-contact devices.

58. A blood contact device, characterized in that: The blood-contact device contains the bivalirudin-protein coating according to any one of claims 1 to 5.

59. The blood contact device according to claim 58, characterized in that: The blood-contact device includes at least one of a vascular stent, a catheter and an artificial heart.

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