BVLD-REDV bifunctional polypeptide modified nickel-titanium alloy and preparation method thereof

Abstract

The invention discloses a BVLD-REDV bifunctional polypeptide modified nickel-titanium alloy and a preparation method of the BVLD-REDV bifunctional polypeptide modified nickel-titanium alloy. According to the preparation method, two functional polypeptides, namely BVLD and REDV, are used for carrying out functional modification on the surface of the nickel-titanium alloy, and an anticoagulant polypeptide BVLD and an endothelial cell specific polypeptide REDV are covalently grafted to the surface of the nickel-titanium alloy, so that the compatibility between a stent prepared from the nickel-titanium alloy and a blood vessel environment in a living body is improved. The design of the double-functional surface of the intravascular stent prepared from the nickel-titanium alloy modified by the double-functional polypeptide is expected to have the advantages in the aspects of improving biocompatibility, reducing adverse events such as restenosis in the stent, preventing thrombosis and the like. According to the dual-functional intravascular stent, the two polypeptides with different functions are covalently fixed on the surface of the nickel-titanium alloy substrate, so that the problem that drugs are exposed to the whole body of a patient and the drug tolerance problem possibly exists is effectively avoided, and the risk of adverse reaction generated when the stent is implanted into the human body is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical fields of cell biology and nickel-titanium alloy, and particularly relates to a nickel-titanium alloy modified with BVLD-REDV bifunctional polypeptide and a preparation method thereof. Background Art

[0002] Medical devices play an indispensable role in modern medicine, providing key means for patient diagnosis, treatment, and support. However, with the widespread use of implantable medical devices, especially materials involving blood contact, such as vascular stents and artificial heart valves, thrombosis is prone to occur on their surfaces, leading to serious complications such as embolism and thrombotic diseases. At the same time, the biocompatibility of these material surfaces is also directly related to the effects of cell attachment, growth, and tissue healing.

[0003] In this context, the concept of endothelialization has become a key element in material design. Endothelialization refers to simulating the state of the vascular endothelial cell layer on the material surface, enabling the material to interact better with the surrounding biological tissues and reducing the risk of thrombosis. In this process, the attachment and growth of endothelial cells play a crucial role.

[0004] Nickel-titanium alloy is widely used in medical device manufacturing due to its excellent mechanical properties and biocompatibility. However, there are still a series of problems on its surface, including easy induction of thrombosis and insufficient cell adhesion. Therefore, it is necessary to develop a new type of coating to endow the nickel-titanium alloy surface with dual functions of anticoagulation and endothelial cell adhesion, thereby improving its biocompatibility in vivo. Nowadays, the vascular stents used clinically have developed from bare metal stents to drug-eluting stents, but both of these two types of stents have certain problems during their service in the human body.

[0005] Bare metal stent: Currently, some vascular stents choose to use metal materials as the base materials, such as stainless steel, cobalt-chromium alloy, and nickel-titanium alloy, to maintain vascular patency through the supporting force; its disadvantages are that the bare metal stent may cause allergic reactions after being implanted into the human body, leading to chronic inflammation; the long-term implantation of these simple bare metal stents in the human body may cause early thrombosis and the endothelialization process may lead to in-stent restenosis in the later stage of implantation, limiting its long-term service.

[0006] Drug-eluting stent: Drugs are loaded on the surface of the vascular stent, due to the risk of intimal hyperplasia caused by the unnatural proliferation of smooth muscle cells; its disadvantages are that the drug coating may cause drug tolerance and allergic reactions in patients, and it is difficult to precisely control the drug release rate. During the drug release process, it enters the patient's blood circulation system, resulting in systemic drug exposure and thus causing adverse reactions; due to the functional singularity of the drug-eluting stent, it may also face the risk of thrombosis during long-term service in the human body. At the same time, due to the non-specificity of the loaded drug, while inhibiting hyperplasia, it also inhibits the adhesion and proliferation of endothelial cells required for endothelialization, resulting in insufficient endothelialization of the stent and thus increasing the risk of in-stent restenosis. Summary of the Invention

[0007] In order to overcome the deficiencies and drawbacks of the prior art, the primary object of the present invention is to provide a preparation method of a nickel-titanium alloy modified with BVLD-REDV bifunctional polypeptide. This preparation method uses two functional polypeptides, BVLD and REDV, to functionalize and modify the surface of the nickel-titanium alloy. Specifically, by covalently grafting the anticoagulant polypeptide BVLD and the endothelial cell-specific polypeptide REDV onto the surface of the nickel-titanium alloy, it aims to improve the compatibility of the stent prepared using this nickel-titanium alloy with the vascular environment in the organism.

[0008] The second object of the present invention is to provide a nickel-titanium alloy modified with BVLD-REDV bifunctional polypeptide prepared by the above preparation method.

[0009] The third object of the present invention is to provide a nickel-titanium alloy vascular stent modified with BVLD-REDV bifunctional polypeptide. The nickel-titanium alloy vascular stent modified with BVLD-REDV bifunctional polypeptide of the present invention solves problems such as thrombosis and insufficient endothelialization that may occur during the long-term use of traditional stents. By covalently grafting the endothelial cell-specific polypeptide REDV and the anticoagulant polypeptide molecule BVLD onto the surface of the stent, the technical solution is related to cell biology problems such as the interaction between the technology and cells and cell adhesion.

[0010] The primary object of the present invention can be achieved by adopting the following technical methods:

[0011] A preparation method of a nickel-titanium alloy modified with BVLD-REDV bifunctional polypeptide, comprising the following steps,

[0012] (1) Preparation of hydroxylated nickel-titanium alloy: The nickel-titanium alloy is ultrasonically cleaned with water and absolute ethanol respectively, dried with nitrogen, and hydroxylated by immersing the nickel-titanium alloy in an aqueous NaOH solution to introduce hydroxyl groups on the surface of the nickel-titanium alloy, thus obtaining the hydroxylated nickel-titanium alloy;

[0013] (2) Silanized nickel-titanium alloy: Immerse the hydroxylated nickel-titanium alloy obtained in step (1) completely into an ethynylsilane ethanol solution and incubate it in the dark at room temperature. After the incubation is completed, dehydrate and fix the hydroxylated nickel-titanium alloy in an oven. After the fixation is completed, wash the nickel-titanium alloy with ethanol and dry it with nitrogen to obtain the silanized nickel-titanium alloy;

[0014] (3) Preparation of REDV polypeptide-modified nickel-titanium alloy: Immerse the silanized nickel-titanium alloy obtained in step (2) into a REDV polypeptide solution with an azide group, react at room temperature, wash with deionized water, and dry with nitrogen to obtain the REDV polypeptide-modified nickel-titanium alloy;

[0015] (4) Preparation of BVLD polypeptide-modified nickel-titanium alloy: Immerse the silanized nickel-titanium alloy obtained in step (2) into a BVLD polypeptide solution with an azide group, react at room temperature, wash with deionized water, and dry with nitrogen to obtain the BVLD polypeptide-modified nickel-titanium alloy;

[0016] (5) Preparation of BVLD-REDV bifunctional polypeptide-modified nickel-titanium alloy: Add the BVLD polypeptide solution with an azide group to the REDV polypeptide-modified nickel-titanium alloy obtained in step (3), react at room temperature, wash with deionized water, and dry with nitrogen to obtain the BVLD-REDV bifunctional polypeptide-modified nickel-titanium alloy;

[0017] Or, add the REDV polypeptide solution with an azide group to the BVLD polypeptide-modified nickel-titanium alloy obtained in step (4), react at room temperature, wash with deionized water, and dry with nitrogen to obtain the BVLD-REDV bifunctional polypeptide-modified nickel-titanium alloy;

[0018] The molar mass ratio of the BVLD polypeptide solution with an azide group to the REDV polypeptide solution with an azide group is 1:1.

[0019] Preferably, in step (1), the ultrasonic treatment time is 10 - 30 min, the concentration of the NaOH aqueous solution is 5M - 10M, and the temperature of the hydroxylation treatment is 50 - 100 °C.

[0020] Preferably, in step (1), the ultrasonic treatment time is 20 min, the concentration of the NaOH aqueous solution is 5M, and the temperature of the hydroxylation treatment is 60 °C.

[0021] Preferably, in step (2), the mass concentration of the ethynylsilane ethanol solution is 1% - 10%, the dark incubation time is 12 - 48 h, the oven temperature is 70 - 150 °C, the dehydration and fixation time is 0.5 - 4 h, and the mass fraction of the ethanol is 95%.

[0022] Preferably, in step (2), the mass concentration of the ethynylsilane ethanol solution is 5%, the light-shielded incubation time is 24 h, the oven temperature is 100 °C, the dehydration and fixation time is 1.5 h, and the mass fraction of the ethanol is 95%.

[0023] Preferably, in steps (3), (4) and (5), the room temperature reaction time is 2 - 8 h, and it is washed three times with deionized water.

[0024] Preferably, the concentration of the REDV polypeptide solution with an azide group is 150 μM, and the concentration of the BVLD polypeptide solution with an azide group is 150 μM.

[0025] The second object of the present invention can be achieved by the following technical method:

[0026] A nickel-titanium alloy modified with BVLD-REDV bifunctional polypeptide is prepared by the above preparation method.

[0027] The third object of the present invention can be achieved by the following technical method:

[0028] A vascular stent made of a nickel-titanium alloy modified with BVLD-REDV bifunctional polypeptide is prepared from a nickel-titanium alloy modified with BVLD-REDV bifunctional polypeptide.

[0029] The preparation method of the nickel-titanium alloy modified with BVLD-REDV bifunctional polypeptide of the present invention utilizes two functional polypeptides, BVLD and REDV. REDV helps to promote the adhesion and proliferation of endothelial cells, thereby realizing endothelialization. And BVLD, as a derivative of hirudin, is a direct thrombin inhibitor, which is responsible for providing anticoagulant properties on the surface of the vascular stent to prevent thrombus formation. By co-modifying these two polypeptides on the surface of the vascular stent, the biological functions of in-situ endothelialization and anti-thrombosis of the vascular stent are realized simultaneously, and the synergistic effect of endothelialization and anticoagulation is achieved, providing a more comprehensive solution for the long-term in-vivo application of the vascular stent. Compared with the stent with a single-functional drug coating, the design of the bifunctional surface is more expected to have advantages in improving biocompatibility, reducing the occurrence of adverse events such as in-stent restenosis and preventing thrombus formation. Compared with the drug-eluting stent, the bifunctional vascular stent covalently fixes two polypeptides with different functions on the surface of the nickel-titanium alloy substrate, effectively avoiding the problems of systemic drug exposure of patients and possible drug tolerance, thereby reducing the risk of adverse reactions when the stent is implanted into the human body.

[0030] The nickel-titanium alloy modified with BVLD-REDV bifunctional polypeptide of the present invention utilizes two functional polypeptides, BVLD and REDV. Compared with the prior art, the present invention has the following advantages and beneficial effects.

[0031] Endothelialization promotion: The introduction of REDV polypeptide helps to enhance the specific adhesion of endothelial cells on the stent, promotes the formation of endothelialization, reduces the unnatural migration and proliferation of smooth muscle cells on the stent surface, has good biocompatibility, and reduces the incidence of adverse events such as in-stent restenosis.

[0032] Enhanced anticoagulant effect: The introduction of the thrombin inhibitory peptide BVLD significantly reduces the adhesion of platelets on the stent surface, effectively inhibits the risk of thrombus formation, and reduces the risks such as bleeding caused by anticoagulant drugs during anticoagulation compared with traditional bare metal stents and drug-eluting stents.

[0033] Improved biocompatibility: The bifunctional polypeptide has good biosafety. The modification of the polypeptide is beneficial to improving the biocompatibility of the stent surface, promoting cell adhesion and proliferation, and helping to reduce the inflammatory reaction caused by foreign body reaction after the stent is implanted into the human body;

[0034] Reduced systemic drug burden: Compared with drug-eluting stents, the present invention does not rely on drug release, avoids problems such as drug tolerance and allergic reactions that patients may face, reduces the systemic drug burden of patients, and improves the safety of treatment.

[0035] Due to the above comprehensive advantages, the NiTi alloy stent modified with BVLD-REDV bifunctional polypeptide described in the present invention performs excellently in anti-thrombosis formation and accelerating in-situ endothelialization of vascular stents, providing an innovative, efficient and feasible solution for the design and application of the surface functionalization of vascular stents. Brief Description of the Drawings

[0036] Figure 1 It is the average fluorescence intensity on the surface of the NiTi alloy stent material with different reaction times of REDV in Example 1;

[0037] Figure 2 It is the average fluorescence intensity on the surface of the NiTi alloy stent material with different reaction times of BVLD in Example 1;

[0038] Figure 3 It is the content of lactate dehydrogenase of platelets adhered to the surface of BVLD in Example 2;

[0039] Figure 4 It is the APTT of the NiTi alloy stent material modified with BVLD polypeptide in Example 2;

[0040] Figure 5 It is the PT of the NiTi alloy stent material modified with BVLD polypeptide in Example 2;

[0041] Figure 6 It is the TT of the NiTi alloy stent material modified with BVLD polypeptide in Example 2;

[0042] Figure 7 PTT of the NiTi alloy stent material modified with BVLD polypeptide in Example 2;

[0043] Figure 8 Thrombin inactivation performance of the NiTi alloy stent material modified with BVLD polypeptide in Example 2;

[0044] Figure 9 Fibrinogen adhesion on the surface of the NiTi alloy stent material modified with BVLD polypeptide in Example 2;

[0045] Figure 10 Adhesion of smooth muscle cells on the surface of the NiTi alloy stent material modified with BVLD polypeptide in Example 2;

[0046] Figure 11 Adhesion of endothelial cells on the surface of the NiTi alloy stent material modified with BVLD polypeptide in Example 2;

[0047] Figure 12 Adhesion of smooth muscle cells on the surface of the NiTi alloy stent material modified with REDV polypeptide in Example 3;

[0048] Figure 13 Adhesion of endothelial cells on the surface of the NiTi alloy stent material modified with REDV polypeptide in Example 3;

[0049] Figure 14 Lactate dehydrogenase content of platelets adhered to the surface of BVLD-REDV in Example 4;

[0050] Figure 15 APTT of the NiTi alloy stent material modified with BVLD-REDV bifunctional polypeptide in Example 4;

[0051] Figure 16 PT of the NiTi alloy stent material modified with BVLD-REDV bifunctional polypeptide in Example 4;

[0052] Figure 17 TT of the NiTi alloy stent material modified with BVLD-REDV bifunctional polypeptide in Example 4;

[0053] Figure 18 PTT of the NiTi alloy stent material modified with BVLD-REDV bifunctional polypeptide in Example 4;

[0054] Figure 19 Thrombin inactivation performance of the NiTi alloy stent material modified with BVLD-REDV bifunctional polypeptide in Example 4;

[0055] Figure 20For fibrinogen adhesion on the surface of the NiTi alloy stent material modified with the BVLD-REDV bifunctional polypeptide in Example 4;

[0056] Figure 21 For the adhesion of smooth muscle cells on the surface of the NiTi alloy stent material modified with the BVLD-REDV bifunctional polypeptide in Example 4;

[0057] Figure 22 For the adhesion of endothelial cells on the surface of the NiTi alloy stent material modified with the BVLD-REDV bifunctional polypeptide in Example 4;

[0058] Figure 23 For the characterization of the anticoagulant-promoting endothelial bifunctional performance on the surface of the NiTi alloy stent material modified with the BVLD-REDV bifunctional polypeptide described in Example 4. Detailed implementation manners

[0059] The present invention will be further described in detail below in conjunction with specific embodiments, but the implementation manners of the present invention are not limited thereto. The materials used in the embodiments of the present invention can all be obtained by commercial purchase.

[0060] Example 1

[0061] Method for determining the polypeptide grafting density

[0062] The preparation of the REDV NiTi alloy stent material modified with the fluorescent polypeptide in this example includes the following steps:

[0063] (1) Preparation of hydroxylated NiTi alloy: The NiTi alloy stent material was ultrasonically cleaned with water and absolute ethanol for 10 min respectively, and after being dried with nitrogen, the NiTi alloy stent material was hydroxylated with a 5M NaOH aqueous solution to introduce hydroxyl groups on the surface of the NiTi alloy stent material, and the hydroxylated NiTi alloy was prepared;

[0064] (2) Preparation of silanized NiTi alloy stent material: The hydroxylated NiTi alloy stent material in step (1) was completely immersed in a 5% ethynyl silane ethanol solution and incubated in the dark at room temperature for 24 h. After the incubation was completed, the above NiTi alloy stent material was placed in an oven at 100 °C for dehydration fixation for 1 h. After the fixation was completed, the NiTi alloy stent material was washed three times with 95% ethanol and dried with nitrogen to obtain the silanized NiTi alloy stent material;

[0065] (3) Preparation of the REDV NiTi alloy stent material modified with the fluorescent polypeptide: Add N with a fluorescent group FITC to the silanized NiTi alloy stent material obtained in step (2) 3-REDV-FITC polypeptide solution with a working solution concentration of 150 μM, a working solution volume of 500 μL, and a reaction duration of 1 h - 5 h. After the reaction, the nickel-titanium alloy stent material modified with fluorescent polypeptide was washed with deionized water to remove the unreacted fluorescent polypeptide, and then dried with nitrogen to obtain the REDV nickel-titanium alloy stent material modified with fluorescent polypeptide;

[0066] (4) Add 1 μL of deionized water film to the REDV nickel-titanium alloy stent material modified with fluorescent polypeptide obtained in step (3), and place it under a fluorescence microscope for observation, photographing, and recording the average fluorescence intensity of each regional cell. The average fluorescence intensity of the surface of the nickel-titanium alloy stent material modified with fluorescent polypeptide REDV at different reaction times was statistically analyzed, and the statistical results were plotted as a bar chart, as Figure 1 shown. The results show that 5 h is the reaction time when the grafting of REDV on the surface of the nickel-titanium alloy reaches saturation.

[0067] The preparation steps of the nickel-titanium alloy stent material modified with fluorescent polypeptide BVLD are the same as the above steps of the nickel-titanium alloy stent material modified with fluorescent polypeptide REDV, except that the N 3 -REDV-FITC fluorescent polypeptide solution in step (3) is replaced with the N 3 -BVLD-FITC polypeptide solution to obtain the nickel-titanium alloy stent material modified with fluorescent polypeptide BVLD. The average fluorescence intensity of the regional cells of the nickel-titanium alloy stent material modified with fluorescent polypeptide BVLD was statistically analyzed, and the statistical results were plotted as a bar chart, as Figure 2 shown. The results show that 5 h is the reaction time when the grafting of BVLD on the surface of the nickel-titanium alloy reaches saturation.

[0068] Example 2

[0069] A preparation method of a nickel-titanium alloy sheet modified with BVLD polypeptide, comprising the following steps:

[0070] (1) Preparation of hydroxylated nickel-titanium alloy sheet: The nickel-titanium alloy was hydroxylated with 5 M NaOH aqueous solution at 60 °C to obtain a hydroxylated nickel-titanium alloy sheet;

[0071] (2) Preparation of silanized nickel-titanium alloy sheet: The hydroxylated nickel-titanium alloy sheet obtained in step (1) was completely immersed in a 5% ethynylsilane ethanol solution and incubated in the dark at room temperature for 24 h. After incubation, the stent was placed in an oven at 100 °C for dehydration fixation for 2 h. After fixation, the nickel-titanium alloy sheet was washed three times with 95% ethanol and dried with nitrogen to obtain a silanized nickel-titanium alloy sheet;

[0072] (3) Preparation of BVLD polypeptide solution: Deionized water was added to 5 mg of solid BVLD polypeptide powder and dissolved at room temperature to prepare a 150 μM BVLD polypeptide solution;

[0073] (4) Preparation of BVLD polypeptide-modified nitinol sheet: 400 μL of the BVLD polypeptide solution in step (3) was added to the silanized nitinol sheet, and the reaction was carried out at room temperature for 5 h. After the reaction between the polypeptide solution and the nitinol sheet was completed, the scaffold was washed three times with deionized water and dried with nitrogen to obtain a BVLD polypeptide-modified nitinol sheet.

[0074] Platelet-rich plasma (PRP) was added to the BVLD polypeptide-modified nitinol sheet and incubated in a shaker at 37 °C and 85 rpm for 1 h. After incubation, the surface was washed 3 times with PBS (pH = 7.4) to wash away the unadhered platelets; after washing, the lactate dehydrogenase (LDH) cytotoxicity kit was used to lyse the platelets adhered to the nitinol surface by adding 300 μL of lysis solution to each well and pipetting. After pipetting, the lysis solution in the sample was transferred to a 1.5 mL sterile centrifuge tube and centrifuged at 400 g for 5 min. After centrifugation, the supernatant was poured off, LDH release working solution was added to the centrifuge tube, and it was incubated in an environment at 37 °C for 1 h. After incubation, it was centrifuged at 400 g for 5 min using a centrifuge. After centrifugation, 120 μL of the supernatant was taken from each tube and added to a 96-well plate, and 60 μL of LDH detection working solution was added to each well, mixed, and incubated in the dark at 25 °C for 240 min; after incubation. The absorbance of each sample at 600 nm was detected using an enzyme-linked immunosorbent assay (ELISA) reader, and the results were statistically analyzed and plotted as a bar graph; as Figure 3 shown, BVLD modification on the surface of nitinol stent materials can effectively reduce platelet adhesion on the surface;

[0075] 200 μL of platelet-poor plasma (PPP) was added to the BVLD polypeptide-modified nitinol sheet and incubated in a constant temperature shaker at 37 °C for 30 min. After incubation, the incubated platelet-poor plasma was collected in a 1.5 mL centrifuge tube, and APTT, PTT, TT, and PT detection reagents were added using a fully automatic coagulation tester to detect the effect of BVLD-modified nitinol on prothrombin time, and the results were plotted as a bar graph; as Figures 4 - 7 shown, the BVLD-modified nitinol stent material can effectively prolong the prothrombin time of blood and prevent blood from coagulating rapidly after contacting the material;

[0076] 20 μL of 5 U human thrombin was added dropwise to the surface of the nickel-titanium alloy sheet modified with BVLD polypeptide and co-incubated in a 37 °C cell incubator for 30 min; after incubation, 100 μL of 1% BSA (PBS) was added to each sample to reduce specific adsorption, and the samples were left standing at 25 °C for 10 min. Then, 50 μL of the mixed liquid was transferred to a clean 96-well plate, followed by the addition of 50 μL of 2 mg / mL thrombin chromogenic substrate S2238, and the reaction was carried out in the dark at room temperature for 240 min; after the reaction, the absorbance at 405 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the results were plotted as a bar graph; the results are as Figure 8 shown. Thrombin is used as a hemostatic drug clinically. Excessive thrombin activity in blood vessels may lead to the formation of intravascular thrombus, while the thrombin activity on the surface of the nickel-titanium alloy sheet modified with BVLD decreases, indicating that the presence of BVLD can block the thrombin action pathway in the coagulation cascade and effectively reduce the coagulation situation after vascular stent implantation;

[0077] 500 μL of 1 mg / mL human fibrinogen solution was added to the nickel-titanium alloy sheet modified with BVLD polypeptide and incubated in a constant temperature shaker at 37 °C and 80 rpm for 12 h; after incubation, the human fibrinogen in the sample was aspirated with a pipette pump and the sample was washed with PBS to remove the residual human fibrinogen; after washing, the protein concentration was measured using a BCA kit. 300 μL of BCA working solution was added to the surface of each sample and incubated in the dark in a constant temperature shaker at 37 °C and 80 rpm for 2 h; after incubation, 100 μL of the incubated BCA working solution was transferred to a clean 96-well plate, and the absorbance of each sample at 562 nm was measured using an ELISA reader, and the measured results were plotted as a bar graph;

[0078] Human umbilical vein endothelial cells (HUVEC) and human umbilical artery smooth muscle cells (HUASMC) were cultured on the nickel-titanium alloy sheet modified with BVLD polypeptide for 24 h and 72 h. 500 mL of cell suspension containing 6×10 3 cells of HUVECs or HUASMCs was added to each well. After culturing for 24 h and 72 h, the nickel-titanium alloy sheet was transferred to a new sterile 48-well plate, 300 μL of medium containing cell viability detection working solution (CCK-8) was added, and the plate was placed in a 37 °C cell incubator and incubated in the dark for 2 h. After incubation, 100 μL of the cell viability detection reagent was taken from each well and transferred to a clean 96-well plate, and then the absorbance (abbreviation: OD value) at 450 nm was measured using an ELISA reader. The better the cell viability, the higher the OD value at 450 nm. The results were statistically analyzed and plotted as a bar graph to evaluate the effect of the uncoated group and the BVLD-modified surface on cell viability. The results are as Figures 10 - 11As shown, BVLD has no effect on smooth muscle cell adhesion and can promote endothelial cell adhesion to a certain extent.

[0079] Example 3

[0080] A method for preparing a REDV polypeptide-modified nitinol sheet, comprising the following steps:

[0081] (1) Preparation of hydroxylated nitinol: The nitinol is hydroxylated with 5M NaOH aqueous solution at 60 °C to obtain a hydroxylated nitinol sheet;

[0082] (2) The hydroxylated nitinol sheet obtained in step (1) is completely immersed in a 5% alkynylsilane ethanol solution and incubated in the dark at room temperature for 24 h. After incubation, the scaffold is placed in an oven at 100 °C for dehydration fixation for 2 h. After fixation, the nitinol sheet is washed three times with 95% ethanol and dried with nitrogen to obtain a silanized nitinol;

[0083] (3) Deionized water is added to 5 mg of REDV polypeptide solid powder for dissolution to prepare a 150 μM REDV polypeptide solution;

[0084] (4) 400 μL of the above REDV polypeptide solution is added to the silanized nitinol and reacted at room temperature for 5 h. After the reaction of the polypeptide solution with the nitinol sheet is completed, the scaffold is washed three times with deionized water and dried with nitrogen to obtain a REDV polypeptide-modified nitinol sheet.

[0085] Human umbilical vein endothelial cells (HUVEC) and human umbilical artery smooth muscle cells (HUASMC) are cultured on the REDV bifunctional polypeptide-modified nitinol sheet for 24 h and 72 h. 500 mL of a cell suspension containing 6×10 3 HUVECs or HUASMCs cells is added to each well. After culturing for 24 h and 72 h, the nitinol sheet is transferred to a new sterile 48-well plate, 300 μL of a medium containing a cell viability detection working solution (CCK-8) is added, and it is placed in a cell culture incubator at 37 °C for dark incubation for 2 h. After incubation, 100 μL of the cell viability detection reagent is taken from each well and put into a clean 96-well plate, and then the absorbance at 450 nm (abbreviation: OD value) is measured using an enzyme-linked immunosorbent assay reader. The better the cell viability, the higher the OD value at 450 nm. The results are statistically analyzed and plotted as a bar graph to evaluate the effect of the uncoated group and the REDV-modified surface on cell viability. The results are as Figures 12 - 13 shown. As an endothelial cell-specific adhesion peptide, REDV can effectively promote the adhesion and proliferation of endothelial cells on the surface, and at the same time significantly inhibit the adhesion and proliferation of smooth muscle cells on the surface.

[0086] Example 4

[0087] A preparation method of a nickel-titanium alloy sheet modified with BVLD-REDV bifunctional polypeptide, comprising the following steps:

[0088] (1) Preparation of hydroxylated nickel-titanium alloy sheet: The nickel-titanium alloy is hydroxylated with 5M NaOH aqueous solution at 60 °C to obtain a hydroxylated nickel-titanium alloy;

[0089] (2) Preparation of silanized nickel-titanium alloy sheet: The above hydroxylated nickel-titanium alloy sheet is completely immersed in a 5% alkynylsilane ethanol solution and incubated in the dark at room temperature for 24 h. After incubation, the scaffold is placed in an oven at 100 °C for dehydration fixation for 2 h. After fixation, the nickel-titanium alloy sheet is washed three times with 95% ethanol and dried with nitrogen to obtain a silanized nickel-titanium alloy sheet;

[0090] (3) Preparation of REDV polypeptide solution: Deionized water is added to 5 mg of REDV polypeptide solid powder for dissolution to prepare a 150 μM REDV polypeptide solution;

[0091] (4) Preparation of BVLD polypeptide solution: Deionized water is added to 5 mg of BVLD polypeptide solid powder and dissolved at room temperature to prepare a 150 μM BVLD polypeptide solution;

[0092] (5) Preparation of nickel-titanium alloy sheet modified with BVLD polypeptide: 400 μL of the above BVLD polypeptide solution is added to the silanized nickel-titanium alloy sheet and reacted at room temperature for 5 h. When the reaction between the BVLD polypeptide solution and the silanized nickel-titanium alloy sheet is completed, the scaffold is washed three times with deionized water and dried with nitrogen to obtain a nickel-titanium alloy sheet modified with BVLD polypeptide;

[0093] (6) Preparation of nickel-titanium alloy sheet modified with BVLD-REDV bifunctional polypeptide: 400 μL of the above REDV polypeptide solution is added to the nickel-titanium alloy sheet modified with BVLD polypeptide and reacted at room temperature for 3 h. When the reaction between the REDV polypeptide solution and the nickel-titanium alloy sheet modified with BVLD polypeptide is completed, the scaffold is washed three times with deionized water and dried with nitrogen to obtain a nickel-titanium alloy sheet modified with BVLD-REDV bifunctional polypeptide.

[0094] Add platelet-rich plasma (PRP) to the nickel-titanium alloy sheet modified with BVLD-REDV bifunctional polypeptide, incubate it in a shaker at 37 °C and 85 rpm for 1 h. After the incubation, wash the surface 3 times with PBS (pH = 7.4) to remove the unadhered platelets. After the washing is completed, use a lactate dehydrogenase (LDH) cytotoxicity kit to lyse the platelets adhered to the nickel-titanium alloy surface by adding 300 μL of lysis buffer to each well, and pipette. After pipetting, transfer the lysis buffer in the sample to a 1.5 mL sterile centrifuge tube and centrifuge at 400 g for 5 min. After the centrifugation is completed, pour off the supernatant, add LDH release working solution to the centrifuge tube, and incubate it in an environment at 37 °C for 1 h. After the incubation is completed, centrifuge at 400 g for 5 min with a centrifuge. After the centrifugation is completed, take 120 μL of the supernatant from each tube, add it to a 96-well plate, and add 60 μL of LDH detection working solution to each well, mix well, and incubate in the dark at 25 °C for 240 min. After the incubation is completed. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance of each sample at 600 nm, and statistically analyze the results and plot them as a bar chart; the results are as Figure 14 shown, the surface modified with the dual peptide can still effectively reduce platelet adhesion.

[0095] Add 200 μL of platelet-poor plasma (PPP) to the nickel-titanium alloy sheet modified with BVLD-REDV bifunctional polypeptide, incubate it in a constant-temperature shaker at 37 °C for 30 min. After the incubation is completed, collect the incubated platelet-poor plasma into a 1.5 mL centrifuge tube, use an automatic coagulation tester and add PT, APTT, TT, and PTT detection reagents to detect the effect of the nickel-titanium alloy modified with BVLD-REDV bifunctional polypeptide on prothrombin time, and plot the results as a bar chart; the results are as Figures 15 - 18 shown, the surface modified with BVLD-REDV dual peptide can effectively prolong the prothrombin time of plasma and prevent blood from coagulating rapidly after contacting the vascular stent.

[0096] Drop 20 μL of 5 U human thrombin onto the surface of the nickel-titanium alloy sheet modified with BVLD-REDV bifunctional polypeptide, and co-incubate it in a 37 °C cell culture incubator for 30 min; after the incubation is completed, add 100 μL of 1% BSA (PBS) to each sample to reduce specific adsorption, and let it stand at 25 °C for 10 min. Then, pipette 50 μL of the mixed liquid and transfer it to a clean 96-well plate. Subsequently, add 50 μL of 2 mg / mL thrombin chromogenic substrate S2238, and react in the dark at room temperature for 240 min; after the reaction is completed, use an ELISA reader to measure the absorbance at 405 nm, and plot the results as a bar chart; the results are as Figure 19As shown, thrombin is a key link in the human blood coagulation cascade. The surface modified with dipeptide can reduce thrombin activity, block the process in the thrombin-related blood coagulation cascade pathway, and prevent blood coagulation due to contact with the vascular stent.

[0097] Add 500 μL of 1 mg / mL human fibrinogen solution to the nickel-titanium alloy sheet modified with BVLD-REDV bifunctional polypeptide, and incubate it in a constant temperature shaker at 37 °C and 80 rpm for 12 h; after incubation, use a pipette pump to suck out the human fibrinogen in the sample and wash the sample with PBS to remove the residual human fibrinogen; after washing, use a BCA kit to measure the protein concentration, add 300 μL of BCA working solution to the surface of each sample, and incubate it in the dark in a constant temperature shaker at 37 °C and 80 rpm for 2 h; after incubation, pipette 100 μL of the incubated BCA working solution and transfer it to a clean 96-well plate, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance of each sample at 562 nm, and plot the measured results as a bar chart; as Figure 20 shown, one of the reasons for thrombus formation is the gradual formation of fibrinogen due to fibrin aggregation, while the surface modified with dipeptide can effectively reduce the formation of fibrinogen, thereby inhibiting the formation of thrombus on the surface of the vascular stent;

[0098] Culture human umbilical vein endothelial cells (HUVEC) and human umbilical artery smooth muscle cells (HUASMC) on the nickel-titanium alloy sheet modified with BVLD-REDV bifunctional polypeptide for 24 h and 72 h. Add 500 mL of cell suspension containing 6×10 3 HUVECs or HUASMCs cells to each well. After culturing for 24 h and 72 h, transfer the nickel-titanium alloy sheet to a new sterile 48-well plate, add 300 μL of medium containing cell viability detection working solution (CCK-8), and place it in a cell culture incubator at 37 °C for dark incubation for 2 h. After incubation, take 100 μL of the cell viability detection reagent from each well and transfer it to a clean 96-well plate, and then use an ELISA reader to measure the absorbance (abbreviation: OD value) at 450 nm. The better the cell viability, the higher the OD value at 450 nm. Statistically analyze the results and plot the results as a bar chart to evaluate the cell viability in the uncoated group and the coatings with different ratios of REDV and BVLD polypeptide molecules. The results are as Figures 21 - 22 shown, due to the presence of REDV, it can further promote the adhesion of endothelial cells and effectively inhibit the adhesion and proliferation of smooth muscle cells, which helps to promote the endothelialization of the vascular stent and plays an important role in the reconstruction of vascular endothelial function.

[0099] As can be seen from Examples 1 to 4, after the nickel-titanium alloy is subjected to surface hydroxylation treatment with 5M sodium hydroxide at 60 °C, and then alkynyl groups are introduced on the surface using a 5% silane solution, BVLD and REDV polypeptide solutions with a concentration of 150 μM are grafted respectively. The grafting density reaches saturation at a grafting time of 5 h. When constructing an anticoagulant BVLD polypeptide nickel-titanium alloy surface and a pro-endothelial REDV polypeptide nickel-titanium alloy surface respectively, the two surfaces can respectively show anticoagulant and pro-endothelial effects. When the two polypeptides are simultaneously modified on the nickel-titanium alloy surface, the nickel-titanium alloy surface can simultaneously show anticoagulant and pro-endothelial effects, and its anticoagulant effect and pro-endothelial performance are better than those of the surface modified with a single polypeptide, which can better inhibit the formation of thrombus on the nickel-titanium alloy surface and accelerate the process of surface endothelialization.

[0100] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for preparing a nickel-titanium alloy modified with a BVLD-REDV bifunctional polypeptide, characterized in that: The following steps are included: (1) Preparation of hydroxylated nickel-titanium alloy: ultrasonically cleaning the nickel-titanium alloy with water and anhydrous ethanol, drying with nitrogen, and immersing the nickel-titanium alloy in a NaOH aqueous solution for hydroxylation, thereby introducing hydroxyl groups on the surface of the nickel-titanium alloy to prepare a hydroxylated nickel-titanium alloy; (2) Silanization of nickel-titanium alloy: completely immersing the hydroxylated nickel-titanium alloy in step (1) in an ethanol solution of alkynylsilane and incubating in the dark at room temperature. After the incubation is completed, the hydroxylated nickel-titanium alloy is dehydrated and fixed in an oven. After the fixation is completed, the nickel-titanium alloy is cleaned with ethanol and blown dry with nitrogen to obtain a silanized nickel-titanium alloy; (3) Preparation of REDV polypeptide-modified nickel-titanium alloy: immersing the silanized nickel-titanium alloy in step (2) with the REDV polypeptide solution carrying an azide group, reacting at room temperature, washing with deionized water, and drying with nitrogen to obtain a REDV polypeptide-modified nickel-titanium alloy; (4) Preparation of BVLD polypeptide-modified nickel-titanium alloy: immersing the silanized nickel-titanium alloy in step (2) with the BVLD polypeptide solution carrying an azide group, reacting at room temperature, washing with deionized water, and drying with nitrogen to obtain a BVLD polypeptide-modified nickel-titanium alloy; (5) Preparation of BVLD-REDV bifunctional polypeptide modified nickel-titanium alloy: adding the BVLD polypeptide solution with an azide group to the REDV polypeptide modified nickel-titanium alloy in step (3), reacting at room temperature, washing with deionized water, and drying with nitrogen to obtain the BVLD-REDV bifunctional polypeptide modified nickel-titanium alloy; Alternatively, the azide group REDV polypeptide solution is added to the nickel-titanium alloy modified with the BVLD polypeptide in step (4), reacted at room temperature, washed with deionized water, and dried with nitrogen to obtain a nickel-titanium alloy modified with the BVLD-REDV bifunctional polypeptide; The molar mass ratio of the BVLD polypeptide solution with an azide group to the REDV polypeptide solution with an azide group is 1:

1.

2. The method for preparing the nickel-titanium alloy modified with the BVLD-REDV bifunctional polypeptide according to claim 1, characterized in that: In step (1), the ultrasonic treatment time is 10-30 min, the concentration of the NaOH aqueous solution is 5M-10M, and the temperature of the hydroxylation treatment is 50-100°C.

3. The method for preparing the nickel-titanium alloy modified with the BVLD-REDV bifunctional polypeptide according to claim 1, characterized in that: In step (1), the ultrasonic treatment time is 20 min, the concentration of the NaOH aqueous solution is 5 M, and the temperature of the hydroxylation treatment is 60°C.

4. The method for preparing the nickel-titanium alloy modified with the BVLD-REDV bifunctional polypeptide according to claim 1, characterized in that: The mass concentration of the alkynylsilane ethanol solution in step (2) is 1%-10%, the light-proof incubation time is 12-48h, the oven temperature is 70-150°C, the dehydration fixation time is 0.5-4h, and the mass fraction of ethanol is 95%.

5. The method for preparing the nickel-titanium alloy modified with the BVLD-REDV bifunctional polypeptide according to claim 1, characterized in that: In step (2), the mass concentration of the alkynylsilane ethanol solution is 5%, the light-proof incubation time is 24 hours, the oven temperature is 100° C., the dehydration fixation time is 1.5 hours, and the mass fraction of ethanol is 95%.

6. The method for preparing the nickel-titanium alloy modified with the BVLD-REDV bifunctional polypeptide according to claim 1, characterized in that: The room temperature reaction time in step (3), step (4) and step (5) is 2-8 hours, and the mixture is washed with deionized water three times.

7. The method for preparing the nickel-titanium alloy modified with the BVLD-REDV bifunctional polypeptide according to claim 1, characterized in that: The concentration of the REDV polypeptide solution with an azide group is 150 μM, and the concentration of the BVLD polypeptide solution with an azide group is 150 μM.

8. A nickel-titanium alloy modified with a BVLD-REDV bifunctional polypeptide, characterized in that: The compound is prepared by the preparation method according to any one of claims 1 to 7.

9. A BVLD-REDV bifunctional polypeptide-modified nickel-titanium alloy vascular stent, characterized in that: It is prepared from the nickel-titanium alloy modified with the BVLD-REDV bifunctional polypeptide according to claim 8.

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