Cardiovascular stent coating capable of rapidly reconstructing endothelium and releasing nitric oxide for long time, preparation method and stent

Abstract

The invention discloses a cardiovascular stent coating capable of rapidly reconstructing endothelium and releasing nitric oxide for a long time, a preparation method and a stent. The cardiovascular stent coating comprises a dopamine / hexamethylenediamine-copper coating and multiple functional substances combined with the dopamine / hexamethylenediamine-copper coating, and the multiple functional substances comprise an NO precursor, endothelial polysaccharide with the anticoagulation characteristic and growth factors capable of specifically adhering EC. The cardiovascular stent coating provided by the invention is grafted with various functional substances such as endothelial polysaccharide, growth factors capable of specifically adhering EC, NO precursors and the like, so that the defects of low NO yield, poor anticoagulation effect, incapability of quickly promoting re-endothelialization in an early stage and the like of a traditional coating are favorably improved. According to the invention, the cardiovascular stent is endowed with various functions of adhering EC, increasing anticoagulation, providing enough NO under the condition of existence or absence of an endogenous NO donor and the like, so that rapid reendothelialization is promoted, thrombosis and restenosis are prevented, and the long-term curative effect and safety of the cardiovascular stent are finally improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cardiovascular stents, and particularly relates to a rapid endothelial reconstruction and long-acting nitric oxide release anti-embolic multifunctional cardiovascular stent coating, a preparation method thereof and a cardiovascular stent. BACKGROUND

[0002] Cardiovascular disease is one of the main causes of disability and premature death worldwide, and more than 18 million people die from it every year. Cardiovascular disease is caused by vascular obstruction caused by the deposition, growth and calcification of plaques. At present, cardiovascular stent implantation is a common way to treat vascular obstruction, although it has a certain effect, but the stent surgery will cause damage to the vascular endothelial structure, thereby causing multi-level and full-time vascular microenvironment disorder, which limits its long-term clinical efficacy. Specifically, in the first stage, platelets are activated, and platelets and leukocytes rapidly adhere and aggregate at the implantation site, leading to thrombosis and vascular occlusion. In the second stage, macrophages adhere and polarize, release inflammatory factors, and inflammatory reactions are activated, delaying endothelial repair. In the third stage, due to the inevitable damage to endothelial cells (EC) caused by the implantation process, EC migration inhibition and delayed re-endothelialization are caused; at the same time, the damage to the endothelial structure will reduce the activity of endothelial nitric oxide synthase (eNOS), thereby affecting the production of NO (a gas molecule secreted by EC), so that the endothelial function is reduced. In the fourth stage, under the inflammatory environment, smooth muscle cells (SMC) will migrate to the lesion site for wound repair, but the long-term over-proliferation of SMC will cause neointimal hyperplasia, leading to vascular lumen restenosis. These vascular microenvironment imbalances eventually lead to the failure of cardiovascular stents and vascular reocclusion.

[0003] In healthy physiological conditions, vascular microenvironment homeostasis is maintained by healthy endothelium, in which endothelial glycocalyx (e.g., heparin (Hep)) and EC-secreted therapeutic gas NO, collectively maintain an anti-inflammatory and anti-coagulant environment. NO can promote EC growth, reduce macrophage pro-inflammatory polarization, and further inhibit platelet adhesion and activation (anti-thrombosis) and SMC migration and proliferation (anti-vascular stenosis) by activating the NO-cyclic guanosine monophosphate (cGMP) pathway. However, in inflammatory environments, NO is consumed and inactivated by reactive oxygen species (ROS), leading to reduced bioavailability and NO levels. Glycocalyx, particularly Hep, is an important component of the endothelial surface that not only suppresses ROS production but also promotes the release of NO from the NO precursor L-arginine (Arg) by enhancing the activity of eNOS in endothelial cells. Incorporating Hep and NO donors into stents can reduce inflammation and thrombosis, but the rapid recruitment of ECs to promote early re-endothelialization remains challenging, as an incomplete endothelium provides opportunities for platelet recruitment and SMC invasion. Vascular endothelial growth factor (VEGF) is a key mediator of cell-matrix interactions that can promote EC adhesion and spreading during angiogenesis. In addition, VEGF binding to EC receptors, including α3β1 and αvβ3 integrins, can activate many important intracellular signaling molecules and mediate EC migration and proliferation. Therefore, the combination of VEGF with glycocalyx modification and NO-generating stent coatings has great potential in reducing inflammatory responses and promoting EC adhesion to achieve re-endothelialization, inhibiting platelet adhesion and SMC proliferation, and ultimately inhibiting thrombosis and restenosis.

[0004] A series of strategies to improve vascular endothelium have thus emerged. For example, a nanoscale dopamine-copper (DA-Cu) coating prepared by complexing copper (Cu) and dopamine (DA) has strong catalytic NO production capacity. Since Cu has similar activity to glutathione peroxidase (GPx), this coating can continuously decompose endogenous NO donors S-nitrosothiols (RSNOs) into NO, thereby maintaining vascular NO supply. However, this strategy overlooks some key requirements. First, endogenous donors are degraded in the lesion environment, leading to a lack of endogenous donors and thus insufficient NO production. Second, an unrelenting inflammatory response accelerates NO depletion and renders it physiologically inactive, and persistent inflammation is detrimental to endothelial repair and increases the risk of vascular restenosis. Finally, the inability of the coating to rapidly capture ECs will delay re-endothelialization of the stent.

[0005] Therefore, designing a multifunctional (sufficient long-term NO production, inflammation elimination, vascular endothelial structure remodeling, and rapid EC recruitment for re-endothelialization) vascular stent on demand is crucial for preventing thrombosis and restenosis after stent implantation. SUMMARY

[0006] In view of the above deficiencies of the prior art, the purpose of the present application is to provide a kind of anti-thrombus multifunctional cardiovascular stent coating of endothelium and long-acting release nitric oxide for quick reconstruction, and its preparation method and cardiovascular stent, to solve the problems of low NO yield, poor anticoagulation effect, and inability to promote early reendothelialization quickly of existing coating.

[0007] The technical scheme of the present application is as follows:

[0008] In the first aspect of the present application, a kind of cardiovascular stent coating of endothelium and long-acting release nitric oxide for quick reconstruction is provided, wherein the cardiovascular stent coating includes dopamine / hexanediamine-copper coating, a plurality of functional substances combined with the dopamine / hexanediamine-copper coating, and the plurality of functional substances include NO precursor, endothelial glycosaminoglycan with anticoagulant properties and growth factor that can specifically adhere EC.

[0009] Optionally, the NO precursor is arginine.

[0010] Optionally, the endothelial glycosaminoglycan with anticoagulant properties is heparin.

[0011] Optionally, the growth factor that can specifically adhere EC is vascular endothelial growth factor.

[0012] In the second aspect of the present application, a kind of preparation method of the cardiovascular stent coating of endothelium and long-acting release nitric oxide for quick reconstruction described in the present application is provided, wherein it includes the following steps:

[0013] providing dopamine / hexanediamine-copper coating;

[0014] grafting a plurality of functional substances on the surface of the dopamine / hexanediamine-copper coating to obtain the cardiovascular stent coating; wherein the plurality of functional substances include NO precursor, endothelial glycosaminoglycan with anticoagulant properties and growth factor that can specifically adhere EC.

[0015] Optionally, it specifically includes the following steps:

[0016] providing dopamine / hexanediamine-copper coating;

[0017] immersing the dopamine / hexanediamine-copper coating in arginine solution to obtain DA / HD-Cu-Arg coating;

[0018] immersing the DA / HD-Cu-Arg coating in heparin solution to obtain DA / HD-Cu-Arg-Hep coating;

[0019] immersing the DA / HD-Cu-Arg-Hep coating in vascular endothelial growth factor solution to obtain DA / HD-Cu-Arg-Hep-VEGF cardiovascular stent coating.

[0020] Optionally, the method for preparing the dopamine / hexanediamine-copper coating comprises the steps of:

[0021] adding dopamine and hexanediamine into a Tris-HCl solution to obtain a first mixed solution, and then adding a copper salt into the first mixed solution to obtain a second mixed solution;

[0022] immersing the bare stent into the second mixed solution at room temperature for a predetermined time to obtain the dopamine / hexanediamine-copper coating on the surface of the stent.

[0023] In a third aspect, the present application provides a cardiovascular stent, wherein the cardiovascular stent is coated with the cardiovascular stent coating for rapid re-endothelization and long-term release of nitric oxide according to the present application.

[0024] Beneficial effects: The present application provides a nanoscale dopamine / hexanediamine-copper (DA / HD-Cu) coating grafted with multiple functional substances, which can rapidly re-endothelialize and maintain long-term production of nitric oxide (NO). The coating is prepared by an organic solvent-free immersion technique, and the coating is modified on the surface of the stent by using the DA catechol structure of the mussel. In addition, multiple functional substances with carboxyl or amino structures are grafted onto the DA / HD-Cu coating through amino carboxyl condensation and Schiff base Michael addition reaction, thereby obtaining a multifunctional cardiovascular stent coating that can generate NO for a long time, inhibit inflammation, promote early endothelialization, prevent thrombosis and restenosis. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 A schematic diagram of the multifunctional coating provided by the embodiments of the present application.

[0026] Figure 2 Electron paramagnetic resonance spectroscopy results of a bare stainless steel (316L SS) sample, DA, and DA / HD-Cu coating provided by the embodiments of the present application.

[0027] Figure 3 Matrix-assisted laser desorption time-of-flight mass spectrometry results of the DA / HD-Cu coating provided by the embodiments of the present application.

[0028] Figure 4 X-ray photoelectron spectroscopy wide scan measurement spectroscopy results of the DA / HD-Cu-VEGF coating and the DA / HD-Cu-Arg-Hep-VEGF coating provided by the embodiments of the present application.

[0029] Figure 5 Attenuated total reflection Fourier transform infrared spectroscopy results of a bare 316L SS sample, DA / HD-Cu-VEGF coating, and DA / HD-Cu-Arg-Hep-VEGF coating provided by the embodiments of the present application.

[0030] Figure 6 Grafting amount quantitative result chart of Arg, Hep and VEGF in multifunctional DA / HD-Cu-Arg-Hep-VEGF coating provided by the embodiment of the present application.

[0031] Figure 7 Scanning electron microscope result chart of DA / HD-Cu-Arg-Hep-VEGF coating stent before and after expansion provided by the embodiment of the present application.

[0032] Figure 8 Long-term NO generation quantitative result chart in the presence of NO donor provided by the embodiment of the present application.

[0033] Figure 9 Long-term NO generation quantitative result chart in human umbilical vein endothelial cells (HUVEC) in the absence of NO donor provided by the embodiment of the present application.

[0034] Figure 10 ROS scavenging result chart provided by the embodiment of the present application.

[0035] Figure 11 Macrophage polarization result chart provided by the embodiment of the present application.

[0036] Figure 12 Scanning electron microscope result chart of platelet adhesion experiment on bare stent and DA / HD-Cu, DA / HD-Cu-Arg, DA / HD-Cu-Hep, DA / HD-Cu-VEGF, DA / HD-Cu-Arg-Hep and DA / HD-Cu-Arg-Hep-VEGF coating stent provided by the embodiment of the present application.

[0037] Figure 13 Cell growth behavior result chart observed after HUVEC was planted on bare stent and DA / HD-Cu, DA / HD-Cu-Arg, DA / HD-Cu-Hep, DA / HD-Cu-VEGF, DA / HD-Cu-Arg-Hep and DA / HD-Cu-Arg-Hep-VEGF coating stent for 3 days provided by the embodiment of the present application.

[0038] Figure 14 Cell growth behavior result chart observed after human umbilical artery smooth muscle cells (HUASMC) was planted on bare stent and DA / HD-Cu, DA / HD-Cu-Arg, DA / HD-Cu-Hep, DA / HD-Cu-VEGF, DA / HD-Cu-Arg-Hep and DA / HD-Cu-Arg-Hep-VEGF coating stent for 3 days provided by the embodiment of the present application.

[0039] Figure 15 A schematic diagram of a rabbit extracorporeal circulation experimental device provided for an embodiment of the present application.

[0040] Figure 16 A cross-sectional result diagram of a bare stent and a stent coated with DA / HD-Cu, DA / HD-Cu-Arg, DA / HD-Cu-Hep, DA / HD-Cu-VEGF, DA / HD-Cu-Arg-Hep and DA / HD-Cu-Arg-Hep-VEGF after 2 hours of in-vitro blood circulation experiment provided for an embodiment of the present application.

[0041] Figure 17 A scanning electron microscope result diagram of a bare stent and a stent coated with DA / HD-Cu-Arg-Hep-VEGF after 1 week of in-vivo implantation provided for an embodiment of the present application, and a laser confocal microscope result diagram for evaluating re-endothelialization of the stent by CD31, phalloidin and 4,6-diamidino-2-phenylindole (DAPI) staining.

[0042] Figure 18 A histomorphology analysis result diagram of a bare stent and a stent coated with DA / HD-Cu-Arg-Hep and DA / HD-Cu-Arg-Hep-VEGF after 3 months of in-vivo implantation provided for an embodiment of the present application.

[0043] Figure 19 A neointima area and stenosis analysis result diagram of a bare stent and a stent coated with DA / HD-Cu-Arg-Hep and DA / HD-Cu-Arg-Hep-VEGF after 3 months of in-vivo implantation provided for an embodiment of the present application. DETAILED DESCRIPTION

[0044] The present application provides a rapid re-endothelialization and long-acting nitric oxide release anti-thrombosis multifunctional cardiovascular stent coating, a preparation method thereof and a cardiovascular stent. To make the purpose, technical solutions and effects of the present application clearer and more explicit, the present application is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present application and not to limit the present application.

[0045] Existing strategies for improving vascular endothelium ignore some key requirements. First, endogenous donors are degraded in the lesion environment, resulting in insufficient endogenous donors, and thus it is difficult to produce sufficient NO. Second, the inflammation reaction cannot be eliminated, which accelerates the loss of NO and makes it lose physiological activity, and the persistent inflammation is not conducive to endothelial repair and increases the risk of vascular restenosis. Finally, the coating cannot quickly capture ECs, which will delay the re-endothelialization of the stent.

[0046] Therefore, it is crucial to design a multifunctional cardiovascular stent (long-term production of sufficient NO, elimination of inflammation, remodeling of vascular endothelial structure, rapid recruitment of EC to achieve re-endothelialization) to prevent thrombosis and restenosis after stent implantation.

[0047] Specifically, the cardiovascular stent coating capable of long-term release of NO grafted with multiple functional substances provided by the embodiments of the present application comprises a dopamine / hexamethylene diamine-copper (DA / HD-Cu) coating, and multiple functional substances combined with the dopamine / hexamethylene diamine-copper coating, wherein the multiple functional substances comprise an NO precursor, an endothelial polysaccharide with anticoagulant properties, and a growth factor capable of specifically adhering to EC.

[0048] In view of the low local concentration and short half-life of endogenous NO donors in a pathological microenvironment, an NO precursor can be provided to compensate for the insufficient level of RSNOs in an actual disease environment. For example, arginine (Arg) as an NO precursor can generate NO under the action of eNOS, avoiding the reduction of NO generation caused by insufficient endogenous RSNOs, and ensuring sufficient supply of NO. In order to further achieve remodeling of the structure of the vascular endothelial surface, an endothelial polysaccharide with anticoagulant properties (for example, Hep) can be modified on the stent coating to simulate the endothelial surface, and the excellent anticoagulant properties thereof can maintain vascular homeostasis, increase the activity of eNOS, promote Arg to generate NO, and provide necessary conditions for long-term stable generation of NO. In order to achieve anti-inflammation, the introduction of DA, Arg, and Hep, which are antioxidant components, can eliminate ROS in the inflammatory environment, and VEGF and NO can promote the polarization of macrophages to the anti-inflammatory M2 type. In addition, for rapid EC recruitment, a growth factor capable of specifically adhering to EC, such as vascular endothelial growth factor (VEGF), is introduced into the coating system to promote early EC adhesion, so that the endothelial layer can be formed on the stent surface to achieve re-endothelialization.

[0049] Therefore, the coating provided by the embodiments of the present application is grafted with multiple functional substances such as an endothelial polysaccharide, a growth factor capable of specifically adhering to EC, and an NO precursor, which helps to improve the defects of the conventional coating, such as low NO yield, poor anticoagulant effect, and inability to rapidly promote re-endothelialization in the early stage.

[0050] In a preferred embodiment, the NO precursor is arginine, the endothelial polysaccharide with anticoagulant properties is Hep, and the growth factor capable of specifically adhering to EC is vascular endothelial growth factor.

[0051] The coating provided by the embodiments of the present application is grafted with an endothelial polysaccharide Hep, a growth factor VEGF capable of specifically adhering to EC (i.e., recruiting EC), and an NO precursor Arg, which can better improve the defects of the conventional coating, such as low NO yield, poor anticoagulant effect, and inability to rapidly promote re-endothelialization in the early stage.

[0052] In summary, the embodiment of the present application provides a multifunctional cardiovascular stent coating capable of long-term release of NO, which reconstructs the healthy vascular endothelial microenvironment by combining different functional substances (such as Arg, Hep and VEGF) with the DA / HD-Cu network. The coating preparation technology adopted is a solvent-free impregnation technology, in which DA is firmly adhered to the surface of the stent by mimicking the catechol structure of mussels, DA and Cu form coordination compounds by complexation, and HD provides sufficient amino groups for surface grafting. Through amino carboxyl condensation and Schiff base Michael addition reaction, a variety of functional substances with carboxyl or amino structure are grafted onto the DA / HD-Cu coating, thereby obtaining a multifunctional vascular stent coating capable of long-term generation of NO, inhibition of inflammation, promotion of early endothelialization, prevention of thrombosis and restenosis. After implantation in vivo, the coating quickly eliminates ROS in the inflammatory microenvironment through the antioxidant properties of DA, Hep and Arg, ensuring the physiological role of the generated NO. Since Cu has a GPx-like activity, the coating can catalyze the in situ generation of NO by decomposing endogenous RSNOs. In order to avoid the reduction of NO production caused by insufficient endogenous NO donors, the NO precursor Arg can be grafted onto DA through Schiff base Michael addition reaction, and Arg can release NO under the action of eNOS. This sufficient supply of NO overcomes the potential defects of endogenous RSNOs. Subsequently, the generated NO guides the polarization of macrophages into anti-inflammatory M2 type, providing a healthy environment for vascular repair. In order to enhance the anticoagulant effect, the endothelial polysaccharide Hep can be condensed with the amino groups on the surface of the coating. Hep not only simulates the endothelial structure, but also enhances the blood compatibility and eNOS activity, promotes the generation of NO by Arg, and forms a self-sustaining NO generating coating. In addition, in order to promote rapid re-endothelialization after stent implantation, VEGF can be covalently immobilized on the coating. The introduction of VEGF can quickly adhere EC to achieve early endothelial layer formation after implantation, which is beneficial to inhibit the migration of SMC and platelet adhesion. As can be seen, the coating can realize the on-demand grafting of multiple substances, and has great design flexibility.

[0053] The embodiment of the present application considers the different needs of the timing reaction after stenting and the reconstruction of endothelial structure, and endows the stent with the functions of adhering EC, increasing anticoagulation, and providing sufficient NO in the presence or absence of endogenous NO donors, thereby promoting rapid re-endothelialization, preventing thrombosis and restenosis, and ultimately improving the long-term efficacy and safety of cardiovascular stents.

[0054] The embodiment of the present application provides a preparation method of the rapid re-endothelialization and long-acting release of nitric oxide cardiovascular stent coating, wherein the preparation method comprises the following steps:

[0055] Providing a dopamine / hexanediamine-copper coating;

[0056] grafting a plurality of functional substances on the surface of the dopamine / hexanediamine-copper coating to obtain the cardiovascular stent coating; wherein the plurality of functional substances comprises a NO precursor, an endothelial polysaccharide with anticoagulation properties, and a growth factor capable of specifically adhering to ECs.

[0057] In one embodiment, the method specifically comprises the following steps:

[0058] providing a dopamine / hexanediamine-copper coating (denoted as DA / HD-Cu);

[0059] immersing the dopamine / hexanediamine-copper coating in an arginine solution to obtain a DA / HD-Cu-Arg coating;

[0060] immersing the DA / HD-Cu-Arg coating in a heparin solution to obtain a DA / HD-Cu-Arg-Hep coating;

[0061] immersing the DA / HD-Cu-Arg-Hep coating in a vascular endothelial growth factor solution to obtain a DA / HD-Cu-Arg-Hep-VEGF cardiovascular stent coating.

[0062] In one embodiment, the method for preparing the dopamine / hexanediamine-copper coating comprises the following steps:

[0063] adding dopamine and hexanediamine into a Tris-HCl solution to obtain a first mixed solution, and then adding a copper salt into the first mixed solution to obtain a second mixed solution;

[0064] immersing the bare stent into the second mixed solution at room temperature for a predetermined time (e.g., 24 hours) to obtain a dopamine / hexanediamine-copper coating on the surface of the stent.

[0065] The present embodiment provides a cardiovascular stent, wherein the cardiovascular stent is combined with the cardiovascular stent coating for rapid re-endothelization and long-term release of nitric oxide according to the present embodiment.

[0066] The present embodiment provides a cardiovascular stent capable of long-term release of nitric oxide by grafting a plurality of functional substances, which plays a role through long-term release of nitric oxide and the grafted functional substances, enhances anti-inflammatory and anticoagulation effects, rapidly promotes early re-endothelization, and reduces the incidence of vascular embolism or restenosis after stent intervention.

[0067] The present application will be further described in detail through specific embodiments.

[0068] Embodiment 1

[0069] The present embodiment provides a multifunctional molecule grafted coating, and the preparation method thereof comprises the following steps:

[0070] As Figure 1 The chemical structure of the coating is shown in the figure, and the carboxyl-containing substances such as Arg, Hep, VEGF, etc. are connected to the stent. A specific embodiment is described as follows.

[0071] Step one, preparation of DA / HD-Cu coating

[0072] A mixed solution of 1 mg / mL DA and 2.44 mg / mL HD was prepared in a 10 mM Tris-HCl solution with pH = 8.5, and then CuCl2·2H2O was added to obtain a 5 μg / mL CuCl2 solution. The 316L SS stent was immersed in the above solution at room temperature for 24 hours to obtain the DA / HD-Cu coating.

[0073] Step two, preparation of DA-Cu-Arg coating

[0074] The DA / HD-Cu coating sample was immersed in a 1 mg / mL Arg solution (dissolved in PBS, pH = 7.4) for 24 hours to obtain the DA / HD-Cu-Arg coating.

[0075] Step three, preparation of DA / HD-Cu-Arg-Hep coating

[0076] A 0.5 mg / mL N-hydroxysuccinimide (NHS) and 1 mg / mL 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide (EDC) solution was prepared in a 10 mg / mL 2-(N-morpholino) ethanesulfonic acid (MES) solution (pH = 5.5), and Hep (1 mg / mL) was added and stirred for 15-30 minutes to pre-activate Hep. The DA / HD-Cu-Arg coating sample prepared in step two was immersed in the above pre-activated Hep solution for 24 hours and washed with ultrapure water to obtain the DA / HD-Cu-Arg-Hep coating.

[0077] Step four, preparation of DA / HD-Cu-Arg-Hep-VEGF coating

[0078] The DA / HD-Cu-Arg-Hep coating sample was immersed in a 0.5 μg / mL VEGF solution (dissolved in PBS, pH = 7.4) for 12 hours to obtain the DA / HD-Cu-Arg-Hep-VEGF coating.

[0079] The coating prepared in Example 1 was tested as follows.

[0080] Test Example 1

[0081] First, the coordination reaction between DA and Cu in the DA / HD-Cu coating was characterized by electron paramagnetic resonance spectrum, as shown inFigure 2 As shown, there is almost no Cu signal in DA sample, but a Cu-related broad signal existing in DA / HD-Cu at 3440 mT was found, indicating the successful coordination. Then the chemical structure of DA / HD-Cu chelate was analyzed by mass spectrum to study the polymerization mechanism of DA / HD-Cu, as shown in Fig. 2. Figure 3 As shown, the peaks existing at 212, 335, 610 m / z further confirmed the chelation between Cu and DA derivatives. Then the surface elemental composition and distribution of the coating were analyzed by X-ray photoelectron spectroscopy. As shown in Fig. 3, Figure 4 As shown, there is a strong Cu signal in all coatings, and DA / HD-Cu-Arg-Hep-VEGF also shows the S signal belonging to Hep, indicating the increase of chemical components in the coating. In order to study the chemical structure of the coating, the chemical structure of the coating was determined by infrared spectroscopy, as shown in Fig. 4. Figure 5 As shown, each coating has the same -COOH, -NH, -OH and -C=C peaks, confirming the formation of DA network; in the DA / HD-Cu-Arg-Hep-VEGF sample, the -C=N bond of Arg at 1600 cm -1 and the -S=O bond of Hep at 1000 cm -1 were also detected, indicating that Arg and Hep were successfully modified on the coating. Subsequently, the grafting amount of Arg, Hep and VEGF modified on the coating was quantified by QCM-D, as shown in Fig. 5. Figure 6 As shown, about 346±11 ng / cm 2 of Arg, 609±7 ng / cm 2 of Hep and 232±4 ng / cm 2 of VEGF were successfully connected to the sample surface, further confirming the successful grafting of multiple functional molecules. Finally, the surface morphology of the sample was observed by SEM, as shown in Fig. 6. Figure 7 As shown, after expansion, the DA / HD-Cu-Arg-Hep-VEGF coating did not appear to be delaminated or cracked, showing good mechanical properties.

[0082] Test Example 2

[0083] This test example is an evaluation of long-term NO production. In the presence of NO donor, DA / HD-Cu and DA / HD-Cu-Arg-Hep-VEGF stents were immersed in a PBS solution (pH = 7.4) containing 10 μM glutathione (GSH) and 10 μM S-Nitroso-N-acetylpenicillamine (SNAP) (NO donor), and replaced every 12 hours. The content of NO was detected by total NO detection kit at 1, 5, 10, 15 and 30 days, respectively, to study the NO production ability under the catalysis of Cu. Figure 8The results show that DA / HD-Cu-Arg-Hep-VEGF coating can produce NO in the presence of NO donor, which indicates that the modification of Arg, Hep and VEGF does not affect the catalytic ability of Cu. The coating still maintains NO production after 30 days of immersion, which indicates that it can release NO for a long time.

[0084] In addition, in the absence of NO donor, in order to study the NO production ability of NO precursor Arg in the coating, the scaffolds immersed in PBS were taken out at 0, 7, 14 and 28 days, and then 5×10 4 cells / cm 2 of HUVECs were inoculated on the surface modified scaffolds in the culture medium without NO donor, and the produced NO was determined by total NO detection kit. The quantitative data are shown in Figure 9 , which indicates that DA / HD-Cu-Arg-Hep-VEGF coating can achieve long-term NO release at the cell level.

[0085] Test Example 3

[0086] This test example is a study on the growth behavior of macrophages on the developed coating. Macrophages RAW264.7 were inoculated on each sample at a density of 1×10 4 cells / cm 2 , and lipopolysaccharide (LPS, 1 μg / mL) and NO donor were added for incubation for 3 days. For ROS scavenging experiment, the cells on the sample were collected and analyzed by flow cytometry using reactive oxygen species kit. For macrophage polarization immunofluorescence staining, all samples were washed and fixed, and CD86 and CD206 antibodies were used to label M1 and M2 type macrophages respectively, and the cells were observed using fluorescence microscope.

[0087] As shown in Figure 10 , the bare scaffold showed strong ROS signal, while the coating samples showed obvious ROS scavenging effect, among which the DA / HD-Cu-Arg-Hep-VEGF coating group could scavenge about 65.5% of ROS. In Figure 11 , compared with the high number of pro-inflammatory M1 type macrophages in the bare scaffold, the M1 type macrophages in the coating group gradually decreased, and the anti-inflammatory M2 type macrophages increased, among which the DA / HD-Cu-Arg-Hep-VEGF coating group had the least M1 type macrophages and the most M2 type macrophages, showing strong anti-inflammatory effect.

[0088] Test Example 4

[0089] This test example is an evaluation of the platelet adhesion ability of the stents. Fresh blood of healthy SD rats (male, 350-450 g) was used. The whole blood was centrifuged at 1500 rpm for 15 minutes to obtain high concentration platelet plasma (PRP). The PRP was added to the sample surface and co-incubated with the NO donor at 37°C for 2 hours. After washing all samples with PBS, they were stored in 2.5% glutaraldehyde solution at 4°C overnight, and then subjected to stepwise dehydration and alcoholization with ethanol (30, 50, 70, 90 and 100%). The platelet adhesion of each sample was observed using SEM.

[0090] As shown in Figure 12 , the bare stent did not show inhibition of platelet adhesion, while the coated samples showed significant inhibition of platelet adhesion, with only a small amount of platelets on the DA / HD-Cu-Arg-Hep and DA / HD-Cu-Arg-Hep-VEGF coated groups.

[0091] Test Example 5

[0092] This test example is a study of the growth behavior of HUVECs and HUASMCs on the developed coatings. 5 x 10 4 cells / cm 2 of HUVECs or HUASMCs were seeded onto the blank and coated samples (DA, DA / HD-Cu, DA / HD-Cu-Arg, DA / HD-Cu-Hep, DA / HD-Cu-Arg-Hep and DA / HD-Cu-Arg-Hep-VEGF coatings) of 316L SS substrates, respectively, and the cells were cultured in the presence of the NO donor. After 72 hours, the samples were fixed with 4% paraformaldehyde and then stained with FTIC-phalloidin staining, and the cell growth was observed using a fluorescence microscope.

[0093] As shown in Figure 13 , the growth of HUVECs, the stent surface of the DA / HD-Cu-Arg-Hep and DA / HD-Cu-Arg-Hep-VEGF groups was completely covered with cells after 72 hours of culture, compared to the bare stent surface. Quantitative data showed that the amount of HUVECs grown in the DA / HD-Cu-Arg-Hep-VEGF group was 2.98 times that of the bare stent group. As shown in Figure 14 , the growth of HUASMCs was significantly inhibited after 72 hours of culture in the presence of the NO donor. These experiments show that the present embodiment can promote the growth of ECs and inhibit the proliferation of SMCs, which is beneficial for promoting re-endothelialization and preventing restenosis.

[0094] Test Example 6

[0095] This test example is an evaluation of the in vitro anti-thrombosis efficacy of the stent coating. An in vitro rabbit venous shunt model was established as shown in Figure 15 , in which adult New Zealand white rabbits (3.5-4.0 kg) were anesthetized, the left carotid artery and right external carotid artery were exposed, and a PVC tube was used to connect the left carotid artery and right external carotid artery of the rabbit to allow blood flow. The blank sample and DA, DA / HD-Cu, DA / HD-Cu-Arg, DA / HD-Cu-Hep, DA / HD-Cu-Arg-Hep, and DA / HD-Cu-Arg-Hep-VEGF coated samples were placed in the middle of the tube. After 2 hours of extracorporeal circulation, the circuit was terminated, the samples were collected, and evaluation was performed.

[0096] As shown in Figure 16 , the bare sample without coating was completely blocked by blood clots, while the other samples with coating had reduced blood clots on the wall, among which the DA / HD-Cu-Arg-Hep and DA / HD-Cu-Arg-Hep-VEGF sample groups had only a small amount of thrombus on the wall.

[0097] Test Example 7

[0098] This test example is an evaluation of the in vivo re-endothelialization and anti-vascular stenosis efficacy of the stent coating. Adult New Zealand white rabbits (3.5-4.0 kg) were anesthetized with 1% sodium pentobarbital, the rabbit's femoral artery was exposed, and the blank sample (Primtech TM , commercial 316L SS bare stent) and DA / HD-Cu-Arg-Hep-VEGF coated sample were implanted into the opposite femoral artery. Sampling was performed at 1 and 3 months, respectively.

[0099] To verify the endothelial coverage of the sample surface, CD31 immunofluorescence staining was performed on the sample at 1 week, in which red fluorescence indicates cytoskeleton, green fluorescence indicates endothelial cells, and blue fluorescence indicates cell nucleus, as shown in Figure 17 , SEM results and laser confocal microscopy results after 1 week of implantation. The results show that the DA / HD-Cu-Arg-Hep-VEGF coated stent is completely covered by endothelium, while the endothelial coverage of the control group bare stent is limited, confirming the re-endothelialization effect of the DA / HD-Cu-Arg-Hep-VEGF coated stent.

[0100] At 3 months of implantation, Van Gieson staining was used for histomorphological analysis to verify the anti-restenosis effect of the sample. The results are shown in Figure 18 , in which the Primtech TM stent showed severe neointimal hyperplasia, while the average neointimal area and stenosis percentage of the DA / HD-Cu-Arg-Hep-VEGF coated stent were Figure 19) are obviously reduced, and the surface of the embodiment can effectively inhibit restenosis.

[0101] To sum up, the application provides a kind of anti-thrombosis multifunctional cardiovascular stent coating for quickly reconstructing endothelium and long-acting releasing nitric oxide and a preparation method and cardiovascular stent thereof.The stent coating has multifunctional designability, and various functional molecules can be grafted according to different needs, for example, Arg can be introduced to realize sufficient NO generation with or without endogenous NO donor when NO donor is insufficient;Hep can be introduced to remodel vascular endothelial structure and improve eNOS expression to promote NO generation when inflammation occurs and anticoagulation effect is poor;VEGF can be introduced to promote rapid EC adhesion to promote rapid reconstruction of endothelial layer when endothelialization is slow.These designs can prevent thrombosis and restenosis, improve long-term efficacy and safety of the cardiovascular stent.In general, the stent supports functional substances with amino and carboxyl groups to be modified to the coating surface to realize flexible functional design.

[0102] It should be understood that the application is not limited to the above examples, and those skilled in the art can make improvements or changes according to the above description, and all these improvements and changes shall belong to the protection scope of the appended claims of the application.

Claims

1. A cardiovascular stent coating for rapid endothelial reconstruction and long-lasting nitric oxide release, characterized in that, The cardiovascular stent coating includes a dopamine / hexamethylenediamine-copper coating and a variety of functional substances bound to the dopamine / hexamethylenediamine-copper coating. The various functional substances include NO precursors, endothelial polysaccharides with anticoagulant properties, and growth factors that can specifically adhere to ECs.

2. The cardiovascular stent coating for rapid endothelial reconstruction and long-acting nitric oxide release according to claim 1, characterized in that, The NO precursor is arginine.

3. The cardiovascular stent coating for rapid endothelial reconstruction and long-acting nitric oxide release according to claim 1, characterized in that, The endothelial polysaccharide with anticoagulant properties is heparin.

4. The cardiovascular stent coating for rapid endothelial reconstruction and long-acting nitric oxide release according to claim 1, characterized in that, The growth factor that specifically adheres to ECs is vascular endothelial growth factor.

5. A method for preparing a cardiovascular stent coating with rapid endothelial reconstruction and long-acting nitric oxide release as described in any one of claims 1-4, characterized in that, Including the following steps: Offers dopamine / hexamethylenediamine-copper coating; Multiple functional substances are grafted onto the surface of the dopamine / hexamethylenediamine-copper coating to obtain the cardiovascular stent coating; wherein, the multiple functional substances include NO precursor, endothelial polysaccharide with anticoagulant properties, and growth factors that can specifically adhere to ECs.

6. The method for preparing a cardiovascular stent coating with rapid endothelial reconstruction and long-acting nitric oxide release according to claim 5, characterized in that, The specific steps include: Offers dopamine / hexamethylenediamine-copper coating; The dopamine / hexamethylenediamine-copper coating was immersed in an arginine solution to obtain a DA / HD-Cu-Arg coating. The DA / HD-Cu-Arg coating was immersed in a heparin solution to obtain the DA / HD-Cu-Arg-Hep coating; The DA / HD-Cu-Arg-Hep coating was immersed in a vascular endothelial growth factor solution to obtain the DA / HD-Cu-Arg-Hep-VEGF cardiovascular stent coating.

7. The method for preparing a cardiovascular stent coating for rapid endothelial reconstruction and long-acting nitric oxide release according to claim 5 or 6, characterized in that, The method for preparing the dopamine / hexamethylenediamine-copper coating includes the following steps: Dopamine and hexamethylenediamine were added to Tris-HCl solution to obtain a first mixed solution. Copper salt was then added to the first mixed solution to obtain a second mixed solution. The bare scaffold is immersed in the second mixed solution at room temperature for a predetermined time to obtain a dopamine / hexamethylenediamine-copper coating on the surface of the scaffold.

8. A cardiovascular stent, characterized in that, The cardiovascular stent surface is coated with a cardiovascular stent coating that rapidly reconstructs the endothelium and releases nitric oxide as described in any one of claims 1-4.

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