Vascular stent coating based on phase transition lysozyme membrane loaded with copper ions and heparin and method for preparing the same

Abstract

The present application belongs to the technical field of blood contact materials, and relates to a vascular stent coating based on phase transition lysozyme film loading copper ions and heparin. The preparation method of the coating comprises the following steps: S1, loading a phase transition lysozyme coating on the surface of a base material of a vascular stent; S2, immersing the base material obtained in step S1 into a copper-containing solution for not less than 4 hours to obtain a copper-loaded coating; and S3, finally immersing the base material covered with the copper-loaded coating obtained in step S2 into an activated heparin solution to obtain a coating jointly loading copper ions and heparin. The cardiovascular stent coating prepared by the present application and loading copper ions and heparin on the phase transition lysozyme film can have long-term stability and continuously release copper ions for 14 days, has good blood compatibility and cell compatibility, and can be used for constructing a surface modification coating of a multifunctional cardiovascular material with antithrombosis and endothelialization.

Description

Technical Field

[0001] The present invention belongs to the technical field of blood contact materials, and specifically relates to a method for preparing a vascular stent coating based on a phase-shift lysozyme membrane loaded with copper ions and heparin, which can be used to construct a surface modified coating of a multifunctional cardiovascular material with anti-thrombotic and endothelial-promoting properties. Background Art

[0002] Cardiovascular stents, as implants, have been extensively studied for the treatment of vascular occlusion caused by atherosclerosis. While cardiovascular stents can re-expand occluded vessels and are highly effective in reestablishing blood flow pathways, they also face several key challenges, such as restenosis, late in-stent thrombosis (LST), and new atherosclerosis. The primary cause of these sequelae is endothelial dysfunction caused by excessive smooth muscle cell (SMC) migration and proliferation or incomplete stent endothelialization. The antithrombotic function of stents has a significant impact on shortening the duration of dual antiplatelet therapy. Therefore, the development of cardiovascular stents that promote and accelerate endothelialization and anti-thrombotic properties is crucial for reducing patient complications and improving prognosis.

[0003] Studies have shown that endothelial-derived nitric oxide (NO) plays an important role in maintaining intravascular physiological homeostasis, resisting atherosclerosis, reducing platelet adhesion and activation through the cyclic guanosine monophosphate (cGMP) pathway, and inhibiting smooth muscle cell growth. However, how to combine NO with heparin to produce cardiovascular stents with both stable anticoagulant and antithrombotic effects still needs to be explored. Coating materials that can promote endothelial cell adhesion and proliferation are the key to achieving early endothelialization. The development of multifunctional surfaces that can reduce thrombosis, promote endothelialization, and inhibit smooth muscle cell proliferation is expected to solve the clinical problems currently posed by vascular stents.

[0004] In addition, heparin is a very important anticoagulant. The anticoagulant mechanism of heparin is believed to be due to the heparin-ATIII complex, which preferentially interacts with antithrombin III (ATIII) and subsequently inhibits key components of the coagulation cascade, such as coagulation factor Xa and thrombin (factor IIa). Heparin coatings have been shown to show favorable results in various medical device-related applications. Stent implantation for percutaneous coronary intervention can cause vascular damage and may cause subacute thrombosis, and heparin-modified cardiovascular stent coatings have been shown to effectively reduce thrombosis. In addition, 300-3000ng / cm 2 The heparin density can promote the proliferation of endothelial cells and inhibit the growth of smooth muscle cells.

[0005] However, the current production strategies of cardiovascular stent coating materials still have some limitations, such as complex processes, unclear biosafety, and difficulty in large-scale application. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a method for preparing a multifunctional cardiovascular stent coating that is simple to operate and requires mild reaction conditions, thereby improving the biocompatibility and stability of the material. The prepared coating exhibits excellent anticoagulant and platelet activation and adhesion resistance, promotes endothelial cell proliferation, and inhibits smooth muscle cell migration and growth.

[0007] Current cardiovascular material coating technologies may have problems such as poor biocompatibility, complex preparation, long reaction time, and unstable coatings. For example, layer-by-layer self-assembly uses two polyelectrolytes to self-assemble layer by layer to construct a coating. Although it can load biomacromolecules well, the coating may change in stability with changes in pH. Polydopamine coatings can adhere to the surface of any material under alkaline conditions, have universality and stability, and can also be modified secondary, but usually require more than 12 hours of reaction to form a film, and the in vivo safety of the reaction by-products is still unknown. Lysozyme is a safe bactericidal active substance with a wide source and FDA certification. Under the action of a reducing agent, lysozyme can form a film at the solid-liquid interface and the gas-liquid interface in a short time. It can be used to construct material surface coatings. The reaction is simple and fast, and it has good biocompatibility. It has been applied to research in the fields of environmental pollution and bone biomaterials, but has not yet been used in research on blood-contact materials. The present invention is completed based on the above-mentioned inventive concept.

[0008] The present invention provides a method for preparing a vascular stent coating, which comprises the following steps in sequence:

[0009] S1, a phase-shift lysozyme coating is loaded on the surface of the base material of the vascular stent;

[0010] S2, immersing the base material obtained in step S1 in a copper-containing solution having a copper ion content of no more than 2.0 mol / L, and immersing the base material in the copper-containing solution for no less than 4 hours to obtain a copper-containing coating;

[0011] S3, finally immersing the base material covered with the copper-loaded coating obtained in step S2 into an activated heparin solution, wherein the concentration of heparin in the heparin solution is 1.0-10.0 mg / mL, and the copper-loaded coating is immersed in the heparin solution for a reaction time of not less than 1 hour to obtain a coating that jointly loads copper ions and heparin.

[0012] Preferably, in step S1, the outer diameter of the base material is smaller than that of the blood vessel and the material is metal or a polymer. For example, in one preferred embodiment of the present invention, the base material is a cobalt-chromium alloy stent; in another preferred embodiment of the present invention, the base material is a polylactic acid stent.

[0013] Preferably, in step S2 of the present invention, base material is immersed in copper-containing solution, for fixing copper ion.The time of soaking needs to spend the night usually, for example, be no less than 5h, 6h, 8h, 9h, 10h, 12h, 15h, 16h, 18h, 20h, 24h, 30h, 32h, or longer. Taking into account effect and preparation cost, the time of soaking can be controlled at 12-36h (hour). In another preference of the present invention, the soaking time in copper-containing solution is 24h.

[0014] Preferably, the copper-containing solution contains soluble copper salts, for example, one or more of the following substances: copper chloride, copper sulfate, copper nitrate, and cuprous chloride.

[0015] Preferably, the concentration of copper ions in the copper-containing solution is 0.05-1.0 mol / L, more preferably 0.1-0.8 mol / L, for example, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, etc.

[0016] Preferably, in step S3, heparin needs to be activated before use, and can be activated using conventional methods, such as activation using the carbodiimide method. In another preferred embodiment of the present invention, the components in the carbodiimide aqueous solution for activating heparin are 4-morpholineethanesulfonic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and N-hydroxysuccinimide, wherein 4-morpholineethanesulfonic acid is 0.05mmol / L and pH is 5.4-5.6; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide concentration is 1-2mg / mL; N-hydroxysuccinimide concentration is 0.24mg / mL-0.48mg / mL.

[0017] Preferably, the concentration of heparin in the heparin solution is 1.5-5.0 mg / mL; for example, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, 4.5 mg / mL. More preferably, the concentration of heparin in the heparin solution is 2.0-4.0 mg / mL.

[0018] The copper-loaded coating is immersed in the heparin solution for a reaction time sufficient to stably coat the activated heparin on the copper coating surface, typically at least 1 hour, for example, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 15 hours, 16 hours, 18 hours, or 20 hours or longer. Preferably, the reaction time is 2-30 hours, and more preferably, 12-24 hours.

[0019] The method for constructing a phase-change lysozyme coating on a substrate surface is the same as the method for forming a thin film on a substrate surface disclosed in the invention patent application publication number CN105153443A. The preparation method of a substrate loaded with a lysozyme phase-change product can comprise the following steps: mixing equal volumes of a 4-hydroxyethylpiperazineethanesulfonic acid buffer solution containing lysozyme and a 4-hydroxyethylpiperazineethanesulfonic acid buffer solution containing tris(2-carboxyethyl)phosphine, adjusting the pH value to 6.0-10.0 with NaOH, wherein the concentration of lysozyme in the resulting mixture is 2-5 mg / mL and the concentration of tris(2-carboxyethyl)phosphine is 20-30 mmol / L; adding the resulting mixture dropwise to the substrate surface, incubating at room temperature in a humid environment for 1-2 hours, then cleaning the substrate surface with ultrapure water and vacuum drying to obtain a substrate loaded with a lysozyme phase-change product.

[0020] The lysozyme described in the present invention is derived from human, chicken, cow, mouse or camel, and is provided by Sigma-Aldrich (Shanghai) Trading Co., Ltd.; the substrate is a polymer, metal, inorganic material, etc., specifically silicon wafer, ITO glass, Cu, Ag, Au, ceramics, cloth, wood, biaxially oriented polypropylene film (BOPP), polyethylene terephthalate plastic (PET), low-density polyethylene (LDPE), cast polypropylene film (CPP), polyimide (PI), polytetrafluoroethylene, etc.

[0021] As disclosed in the invention patent application CN105153443A, a large-sized and stable two-dimensional bioprotein nanofilm prepared using lysozyme can be prepared by the following method: a 5-100 mmol / L tris(2-carboxyethyl)phosphine tris(hydroxymethylaminomethane) buffer solution is adjusted to a pH value of 4.0-6.0 with NaOH, and then the solution is mixed with an equal volume of a 0.1-10 mg / mL lysozyme tris(hydroxymethylaminomethane) buffer solution, and the mixture is allowed to stand at room temperature for 30-50 minutes to form a thin film on the surface of the mixture, namely the bioprotein two-dimensional nanofilm.

[0022] The bioprotein two-dimensional nanofilm is further preferably prepared by the following method: a 40-50 mmol / L tris(2-carboxyethyl)phosphine tris(hydroxymethylaminomethane) buffer solution is adjusted to a pH value of 4.0-6.0 with NaOH, and then the solution is mixed with an equal volume of a 2-5 mg / mL lysozyme tris(hydroxymethylaminomethane) buffer solution, and the mixture is allowed to stand at room temperature for 30-50 minutes to form a thin film on the surface of the mixture, namely the bioprotein two-dimensional nanofilm.

[0023] Alternatively, during the above preparation process, the substrate can be brought into contact with the surface of the mixed solution and allowed to stand at room temperature for 30-50 minutes to form a thin film, namely, a two-dimensional bioprotein nanofilm, on the surface of the substrate.

[0024] The present invention also provides a vascular stent coating, which comprises a phase-shift lysozyme coating, a copper-loaded coating and a heparin coating in sequence.

[0025] Preferably, the vascular stent coating of the present invention can be obtained using the above-mentioned preparation method, and the obtained coating is a phase-shift lysozyme coating, a copper-loaded coating and a heparin coating sequentially coated on the outer surface of the vascular stent.

[0026] Correspondingly, the present invention also provides a vascular stent, wherein the surface of the vascular stent is covered with the above-mentioned vascular stent coating.

[0027] Preferably, a phase-shift lysozyme membrane-loaded copper ion and heparin-based stent coating is prepared that does not swell or rupture for at least 14 days. In a preferred embodiment of the present invention, the prepared phase-shift lysozyme membrane-loaded copper ion and heparin-based stent coating can withstand 30 days or more without swelling or rupture.

[0028] Preferably, the prepared vascular stent coating based on the phase-shift lysozyme membrane loaded with copper ions and heparin continuously releases copper ions when placed in a solution, thereby inhibiting platelet adhesion and prolonging thromboplastin activation time.

[0029] The method for preparing a vascular stent coating based on a phase-shift lysozyme membrane loaded with copper ions and heparin can be used to prepare a vascular stent coating based on a phase-shift lysozyme membrane loaded with copper ions and heparin. A cobalt-chromium alloy or polylactic acid stent smaller than the inner diameter of a blood vessel is immersed in a lysozyme phase-shift product to form a phase-shift lysozyme membrane. The cobalt-chromium alloy or polylactic acid stent coated with the phase-shift lysozyme membrane is then immersed in a copper chloride solution to form a copper-loaded coating. Finally, the cobalt-chromium alloy or polylactic acid stent coated with the copper-loaded coating is immersed in a heparin solution activated by a carbodiimide method to obtain a phase-shift lysozyme coating that co-immobilizes copper ions and heparin.

[0030] Preferably, the cardiovascular stent coating based on the phase-shift lysozyme membrane loaded with copper ions and heparin continuously produces nitric oxide in the presence of a donor, promotes endothelial cell proliferation, and inhibits smooth muscle cell proliferation; the donor contains S-nitroso-N-acetylpenicillamine and reduced glutathione.

[0031] The donor is a substance that simulates NO precursors present in the blood, and corresponding substances can also be used as substitutes. For example, S-nitrosothiols that can stably generate nitric oxide (NO) through a reaction can be used. Equal concentrations of S-nitroso-N-acetylpenicillamine and reduced glutathione can be used. In a preferred embodiment of the present invention, the donors used are 10 μM S-nitroso-N-acetylpenicillamine and 10 μM reduced glutathione.

[0032] The present invention uses a phase-transition lysozyme (PTL) membrane as a carrier to prepare a cardiovascular stent coating loaded with copper ions and heparin. The substrate is immersed in a lysozyme phase transition product to form a phase-transition lysozyme membrane. The substrate is then immersed in a copper chloride solution, and the copper ions are fixed by active groups to prepare a copper-loaded coating. Finally, the copper-loaded coating is immersed in a heparin solution activated by a carbodiimide method. Heparin is further grafted onto the coating by covalent bonding to obtain a phase-transition lysozyme coating that co-fixes copper ions and heparin. The cardiovascular stent coating loaded with copper ions and heparin prepared by the present invention can have long-term stability (at least 30 days), and can continuously release copper ions for 14 days. The grafted heparin exhibits anticoagulant activity and has good blood compatibility and cell compatibility. The application of blood-contact materials has a wide range of application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solution of the present application, the following is a brief introduction to the drawings required for use in the embodiments. Obviously, each drawing described below is for some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0034] Figure 1 This is a high-resolution spectrum of copper elements from X-ray electron spectroscopy testing of the cardiovascular stent coating P-Cu-Hep based on the phase-transition lysozyme membrane loaded with copper ions and heparin and the phase-transition lysozyme coating PTL prepared in Example 1;

[0035] Figure 2 The heparin grafting amount on the P-Cu-Hep coating prepared in Example 1 was monitored in real time using a quartz crystal microbalance;

[0036] Figure 3 1 is a scanning electron micrograph of the initial surface of the PTL coating and the P-Cu-Hep coating prepared in Example 1 and the surface after being immersed in PBS for 30 days;

[0037] Figure 4 is the cumulative concentration of copper ions released by the PTL coating and the P-Cu-Hep coating prepared in Example 1 within 14 days;

[0038] Figure 5 is the cumulative concentration of NO produced by the PTL coating and the P-Cu-Hep coating prepared in Example 1 in the presence of a donor over 14 days;

[0039] Figure 6 is a scanning electron micrograph of platelet adhesion between the P-Cu-Hep coating prepared in Example 1 and the surface of the untreated sample Blank;

[0040] Figure 7 is the activated partial thromboplastin time of the P-Cu-Hep coating prepared in Example 1 and the sample Blank;

[0041] Figure 8 Endothelial cell proliferation activity detection (CCK-8 assay) on the P-Cu-Hep coating prepared in Example 1 and the Blank sample;

[0042] Figure 9 The smooth muscle cell proliferation activity detection (CCK-8 detection method) on the P-Cu-Hep coating prepared in Example 1 and the sample Blank;

[0043] Figure 10 This is a diagram of cells cultured after the PTL coating was treated with 0.5 mg / mL copper concentration in Example 2.

[0044] Figure 11 3 is a scanning electron microscope image of the P-Cu-Hep coating prepared on the polylactic acid stent in Example 2 and the sample Blank. DETAILED DESCRIPTION

[0045] The present invention discloses a method for preparing a cardiovascular stent coating based on a phase-shift lysozyme membrane loaded with copper ions and heparin. The method uses the phase-shift lysozyme membrane as a carrier, utilizes the abundant active groups on the membrane surface to immobilize copper ions on the material surface, and then covalently grafts heparin molecules via the carbodiimide method to prepare a cardiovascular stent coating that co-immobilizes copper ions and heparin. The coating exhibits long-term stability, can continuously release copper ions, and catalyzes NO production in the presence of a donor. It also exhibits good blood and cell compatibility and can be used for surface modification of cardiovascular materials to promote stent endothelialization. The coating preparation process is simple, the reaction conditions are mild, and it can be applied to the surfaces of various materials, suggesting broad application prospects for blood-contact materials.

[0046] The following examples of the present application will clearly and completely describe the technical solution. Obviously, the described examples are only some preferred embodiments of the present application, not all embodiments. Based on the examples in the present application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0047] Example 1

[0048] (1) Preparation of lysozyme phase transition solution: dilute 1M 4-hydroxyethylpiperazineethanesulfonic acid buffer solution to 10mM and adjust the pH to 7.4 with sodium hydroxide; take 20mg of lysozyme and dissolve it in 10mL of 4-hydroxyethylpiperazineethanesulfonic acid buffer (10mM, pH=7.4) to prepare a 2mg / mL lysozyme solution; take 573mg of tris(2-carboxyethyl)phosphine solution and dissolve it in 40mL of 4-hydroxyethylpiperazineethanesulfonic acid buffer solution (10mM, pH=7.2) to prepare a 50mM solution, and adjust the pH to 7.2 with sodium hydroxide; uniformly mix the 2mg / mL lysozyme solution and tris(2-carboxyethyl)phosphine solution in a volume ratio of 1:1 to prepare a fresh lysozyme phase transition solution.

[0049] (2) Preparation of copper-loaded coating: Immerse a cobalt-chromium alloy sheet or a stent of the same material in a lysozyme phase transition solution and incubate at room temperature for 1 hour to form a phase transition lysozyme nanofilm on the substrate surface; then immerse the alloy sheet or stent in a 0.25 mg / mL copper chloride solution and react for 24 hours to obtain a copper-loaded coating. Heparin loading on the copper-loaded coating: Use ultrapure water to prepare a 10 mg / mL 4-morpholineethanesulfonic acid buffer solution and adjust the pH to 5.4 with sodium hydroxide; prepare a carbodiimide aqueous solution, take a certain amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide and dissolve them in 4-morpholineethanesulfonic acid buffer (pH = 5.4) to a final concentration of 2 mg / mL and 0.48 mg / mL, take a certain amount of heparin and add it to the carbodiimide aqueous solution to a final concentration of 4 mg / mL, and activate for 15 minutes; immerse the copper-loaded coating in the activated heparin solution and react for 6 hours.

[0050] The inventors used X-ray electron spectroscopy, electron scanning electron microscopy, quartz crystal microbalance, inductively coupled plasma and NO detection experiments to characterize the obtained samples. The results are shown in Figure 1-5 .Depend on Figure 1 X-ray electron spectroscopy analysis shows that Cu 2p Split into Cu 2p3 / 2 (932.7eV) and Cu 2p1 / 2 The peaks at (952.6 eV) indicate the presence of Cu(II) and Cu(I), respectively, proving that copper ions are successfully fixed on the PTL coating. Therefore, it is believed that the coating is successfully prepared in the present invention.

[0051] Figure 2 The quartz crystal microbalance experiment proved the successful grafting of heparin. Even after washing, the density of heparin did not drop significantly.

[0052] Figure 3Scanning electron microscopy (SEM) images of the coating showed its surface morphology and good stability after 30 days of PBS immersion. After 30 days of PBS immersion, the coating surface was intact and contained aggregates of lysozyme nanospheres, but no swelling or cracking of the coating occurred.

[0053] Figure 4 Inductively coupled plasma testing demonstrated that the coating could release copper ions for at least 14 days.

[0054] Figure 5 The results demonstrate that the coating can continuously catalyze NO production in the presence of a donor. These results demonstrate that the surface phase-transition lysozyme membrane can load copper ions and heparin, exhibit long-term stability, and can mimic endothelial function to continuously catalyze NO production.

[0055] Blood compatibility evaluation

[0056] Figure 6 Platelet adhesion experiments demonstrated that the coating effectively inhibits platelet adhesion in the presence of a donor. The left image shows a blank control. In the absence of the coating, a large number of partially spread, dendritic, and fully spread platelets are observed under the microscope, indicating activated platelets and a high adherence count. The coating on the right shows no radiating platelets. Compared to the blank control on the left, the coating of the present invention effectively inhibits platelet adhesion.

[0057] Figure 7 In the activated partial thromboplastin time experiment, it was demonstrated that the heparin immobilized in the coating was active and could prolong the partial thromboplastin activation time. PPP refers to platelet-poor plasma.

[0058] Cytocompatibility evaluation

[0059] CCK-8 assay of human umbilical vein endothelial cells and human umbilical artery smooth muscle cells cultured on the coating: Samples were grouped into with donor and without donor (10 μM S-nitroso-N-acetylpenicillamine and 10 μM reduced glutathione) and placed in 24-well culture plates at 2×10 4The cells were seeded into well plates at a density of cells / mL and cultured at 37°C and 5% CO2, with donors added every 6 hours. After 24 and 72 hours of cell culture, the samples were removed and washed twice with PBS, and 300 μL of freshly prepared DMEM culture medium (human umbilical vein endothelial cells) and MEM culture medium (human umbilical artery smooth muscle cells) containing 10% CCK-8 were added to each well of the 24-well plate, respectively, and then cultured in a cell culture incubator at 37°C and 5% CO2 for 2.5 hours. After shaking evenly, the culture medium was gently aspirated into a 96-well plate, and the absorbance at 450 nm was measured directly on a microplate reader (SpectraMax Id3). All samples were measured 4 times, and the results were averaged.

[0060] Depend on Figure 8 It can be seen that after culturing for 24 and 72 hours, the heparin- and copper-ion-loaded coating can promote the proliferation of endothelial cells in the absence of donors due to the presence of heparin. After adding donors, the coating further promotes the growth of endothelial cells.

[0061] Figure 9 It was shown that in the absence of donors, after 24 and 72 hours of culture, the heparin in the heparin-loaded and copper ion coating inhibited the growth of smooth muscle cells. After adding donors, the NO catalyzed by the coating synergistically enhanced the inhibitory effect of heparin on smooth muscle cell growth.

[0062] Further tests show that the coating of the present invention can not only promote the proliferation of endothelial cells and inhibit the proliferation of smooth muscle cells, but also inhibit the migration of smooth muscle cells.

[0063] Example 2

[0064] In this embodiment, the copper chloride concentration of Example 1 was changed to 0.1 mg / mL, and the other steps were the same as in Example 1. The test results show that the coating of the present invention can be prepared by changing the copper chloride concentration to 0.1 mg / mL. Although theoretically, as long as it contains copper or copper ions, it can be used, in actual operation, the present invention has found that the copper salt concentration cannot be too high, such as Figure 10 The results showed that compared with PTL and blank samples, a copper ion concentration of 0.5 mg / mL inhibited endothelial cell growth, and a concentration exceeding 2.0 mg / mL severely inhibited endothelial cell growth.

[0065] Example 3

[0066] (1) Preparation of lysozyme phase transition solution: dilute 1M 4-hydroxyethylpiperazineethanesulfonic acid buffer solution to 10mM and adjust the pH to 7.4 with sodium hydroxide; take 20mg of lysozyme and dissolve it in 10mL of 4-hydroxyethylpiperazineethanesulfonic acid buffer (10mM, pH=7.4) to prepare a 2mg / mL lysozyme solution; take 573mg of tris(2-carboxyethyl)phosphine solution and dissolve it in 40mL of 4-hydroxyethylpiperazineethanesulfonic acid buffer solution (10mM, pH=7.2) to prepare a 50mM solution, and adjust the pH to 6.8 with sodium hydroxide; uniformly mix the 2mg / mL lysozyme solution and tris(2-carboxyethyl)phosphine solution in a volume ratio of 1:1 to prepare a fresh lysozyme phase transition solution.

[0067] (2) Preparation of copper-loaded coating: Immerse the polylactic acid stent in a lysozyme phase transition solution and incubate at room temperature for 1 hour to form a phase transition lysozyme nanofilm on the surface of the material; then immerse the material in a 0.5 mg / mL copper chloride solution and react for 24 hours to obtain a copper-loaded coating. Heparin loading on the copper-loaded coating: Use ultrapure water to prepare a 10 mg / mL 4-morpholineethanesulfonic acid buffer solution and adjust the pH to 5.4 with sodium hydroxide; prepare a carbodiimide aqueous solution, take a certain amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide and dissolve them in a 4-morpholineethanesulfonic acid buffer solution (pH = 5.4) to a final concentration of 1 mg / mL and 0.24 mg / mL, take a certain amount of heparin and add it to the carbodiimide aqueous solution to a final concentration of 2 mg / mL, and activate for 15 minutes; immerse the copper-loaded coating in the activated heparin solution and react for 6 hours. (See Figure 11 ).

[0068] The embodiments described above are merely specific implementations of the present application, but the scope of protection of the present application is not limited thereto. Any changes or substitutions that can be imagined by any person skilled in the art within the technical scope disclosed in the present application without resorting to creative effort should be included within the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims in the present application.

Claims

1. A method for preparing a vascular stent coating, characterized in that: The following steps are included in sequence: S1, loading a phase-shift lysozyme coating on the surface of a substrate material of a vascular stent, wherein the substrate material is a cobalt-chromium alloy stent or a polylactic acid stent; S2, immersing the base material obtained in step S1 in a copper-containing solution having a copper ion content of no more than 2.0 mol / L for no less than 4 hours to obtain a copper-containing coating; S3, finally immersing the base material covered with the copper-loaded coating obtained in step S2 into an activated heparin solution, wherein the heparin concentration in the heparin solution is 1.0-10.0 mg / mL, and the heparin is activated using the carbodiimide method. The copper-loaded coating is immersed in the heparin solution for a reaction time of not less than 1 hour to obtain a coating that is jointly loaded with copper ions and heparin.

2. The method for preparing a vascular stent coating according to claim 1, wherein: In step S1 , the outer diameter of the base material is smaller than that of the blood vessel.

3. The method for preparing a vascular stent coating according to claim 1, wherein: In step S2, the base material is immersed in a copper-containing solution for not less than 12 hours to fix copper ions; The copper-containing solution contains one or more of the following substances: copper chloride, copper sulfate, copper nitrate, cuprous chloride; or, The concentration of copper ions in the copper-containing solution is 0.05-1.0 mol / L.

4. The method for preparing a vascular stent coating according to claim 1, wherein: In step S3, the copper-loaded coating is immersed in a heparin solution for a reaction time of 2-24 h; or, The heparin concentration in the heparin solution is 1.5 - 5.0 mg / mL.

5. The method for preparing a stent coating according to any one of claims 1 to 4, characterized in that: The concentration of copper ions in the copper-containing solution is 0.1 - 0.8 mol / L; Immerse the base material in a copper-containing solution for no less than 24 hours; The heparin concentration in the heparin solution is 2.0 - 4.0 mg / mL; The copper-loaded coating is immersed in the heparin solution for a reaction time of not less than 6 hours.

6. A vascular stent coating prepared according to the method of any one of claims 1 to 5, characterized in that: The outer surface of the blood vessel stent comprises a phase-shift lysozyme coating, a copper-loaded coating and a heparin coating in sequence.

7. A vascular stent, characterized in that: The vascular stent coating according to claim 6 is provided, wherein the outer surface of the vascular stent is coated with a phase transition lysozyme coating, a copper-loaded coating and a heparin coating in sequence.

8. The use of the preparation method according to claim 1, characterized in that: A vascular stent coating based on a phase-shift lysozyme membrane loaded with copper ions and heparin is prepared, which does not swell or rupture for at least 14 days; alternatively, it is placed in a solution to continuously release copper ions, inhibit platelet adhesion, and prolong the thromboplastin activation time.

9. The use according to claim 8, characterized in that: A cobalt-chromium alloy or polylactic acid stent with a size smaller than the inner diameter of a blood vessel is immersed in a lysozyme phase transition product to form a phase transition lysozyme film. The cobalt-chromium alloy or polylactic acid stent coated with the phase transition lysozyme film is then immersed in a copper chloride solution to prepare a copper-loaded coating. Finally, the cobalt-chromium alloy or polylactic acid stent coated with the copper-loaded coating is immersed in a heparin solution activated by a carbodiimide method to obtain a phase transition lysozyme coating that co-fixes copper ions and heparin.

10. The use according to claim 8, characterized in that: A cardiovascular stent coating based on a phase-shift lysozyme membrane loaded with copper ions and heparin continuously produces nitric oxide in the presence of a donor, promoting endothelial cell proliferation and inhibiting smooth muscle cell proliferation; the donor contains S-nitroso-N-acetylpenicillamine and reduced glutathione.

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