Fucoidin-bioactive peptide coating intravascular stent and preparation method thereof

Abstract

The invention discloses a fucoidin-bioactive peptide coated intravascular stent and a preparation method thereof, and belongs to the technical field of medical treatment. The surface of the fucoidin-bioactive peptide coating intravascular stent is coated with a composite coating, and the composite coating is composed of fucoidin and bioactive peptide. The invention discloses a fucoidin-bioactive peptide coated intravascular stent and a preparation method thereof, and the fucoidin-bioactive peptide coated intravascular stent not only solves the problems of poor blood compatibility and slow endothelialization, but also has multiple functions of anticoagulation, anti-proliferation and repair promotion. The intravascular stent coating technology is comprehensively upgraded, and a safer, more effective and more lasting solution is provided for treatment of cardiovascular diseases.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a fucoidan-bioactive peptide coated vascular stent and a preparation method thereof. Background Art

[0002] As an important cardiovascular interventional treatment device, vascular stents are widely used in the treatment of coronary heart disease, myocardial infarction and other diseases. In recent years, with the continuous development of technology, vascular stent coating technology has also made significant progress. At present, the classic vascular stent coating technology is to coat a poly-L-lactic acid (PLLA) coating loaded with rapamycin on a stainless steel stent to prepare a drug-eluting stent (DES). Poly-L-lactic acid, as a carrier material for anti-smooth muscle cell proliferation drugs, can gradually degrade and release rapamycin in the body, thereby inhibiting the excessive proliferation of vascular smooth muscle cells and reducing the incidence of restenosis in the stent.

[0003] However, the existing technology still has some shortcomings. First, poly-L-lactic acid and rapamycin are both hydrophobic organic compounds, which makes the coating highly hydrophobic and easily adsorbs plasma proteins and platelets, thus affecting blood compatibility. Secondly, although rapamycin can effectively inhibit the proliferation of smooth muscle cells, it may also delay the endothelialization of the stent, which is not conducive to vascular repair and blood circulation reconstruction. In addition, the existing coating technology still has limitations in promoting endothelial cell growth and repair, and it is difficult to meet the clinical requirements for high performance of vascular stents. Summary of the invention

[0004] The present invention aims to provide a fucoidan-bioactive peptide coated vascular stent and a preparation method thereof. The fucoidan-bioactive peptide coated vascular stent not only solves the problems of poor blood compatibility and slow endothelialization, but also has multiple functions of anti-coagulation, anti-proliferation and repair promotion, realizing a comprehensive upgrade of vascular stent coating technology, and providing a safer, more effective and lasting solution for the treatment of cardiovascular diseases.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] A fucoidan-bioactive peptide coated vascular stent, the surface of which is coated with a composite coating consisting of fucoidan and bioactive peptides.

[0007] Preferably, the molecular weight of the fucoidan is 10 kDa-100 kDa.

[0008] Preferably, the amino acid sequence of the bioactive peptide is one or more of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6.

[0009] Preferably, the composite coating has a thickness of less than 5 μm.

[0010] Preferably, the material of the vascular stent body is selected from one or both of non-degradable metals and degradable metals.

[0011] Preferably, the non-degradable metal includes one or more of stainless steel, nickel-titanium alloy, and cobalt-chromium alloy.

[0012] Preferably, the degradable metal includes one or more of magnesium alloy, iron alloy and zinc alloy.

[0013] The present invention also provides a method for preparing the fucoidan-bioactive peptide coated vascular stent, comprising the following steps:

[0014] S1, placing the vascular stent body material in a dopamine solution, and subjecting the material to a constant temperature oscillation reaction. After the reaction is completed, the material is washed with a phosphate buffer solution, and dried to obtain a pretreated vascular stent sample;

[0015] S2. Dissolve the fucoidan and bioactive peptides in a phosphate buffer solution at room temperature, and mix them evenly to obtain a fucoidan-bioactive peptide mixed solution;

[0016] S3, placing the pretreated vascular stent sample obtained in step S1 into the mixed solution of fucoidan-bioactive peptide obtained in step S2, and reacting the mixture under constant temperature oscillation to obtain the fucoidan-bioactive peptide coated vascular stent.

[0017] Preferably, in step S1, the constant temperature oscillation reaction temperature is 25-35°C, the constant temperature oscillation reaction time is 12-36h, and the oscillation speed is 80-120rpm.

[0018] Preferably, in step S3, the constant temperature oscillation reaction temperature is 20-40°C, the constant temperature oscillation reaction time is 4-8h, and the oscillation speed is 80-160rpm.

[0019] Compared with the prior art, the present invention has the following advantages and technical effects:

[0020] The present invention constructs a composite coating of fucoidan and bioactive peptides on the surface of the vascular stent, effectively reducing plasma protein adsorption and platelet adhesion, significantly improving the blood compatibility of the vascular stent, and reducing the risk of thrombosis. At the same time, it accelerates the endothelialization process on the surface of the vascular stent, reduces inflammatory reactions, and promotes vascular repair and blood circulation reconstruction.

[0021] In addition, the synergistic effect of fucoidan and bioactive peptides can effectively inhibit the excessive proliferation of vascular smooth muscle cells, reduce the incidence of restenosis in the stent, and improve the long-term patency of the vascular stent. Through dopamine pretreatment technology, the composite coating can firmly adhere to the surface of the vascular stent, ensuring the stability of the coating in the in vivo environment. At the same time, the use of natural biomaterials reduces the risk of tissue immune response and inflammatory response.

[0022] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A statistical graph of the hemolysis rate of the vascular stents provided in Examples 2-4 of the present invention and Comparative Example 1;

[0024] Figure 2 Platelet adhesion SEM images of the vascular stents provided in Examples 2-4 of the present invention and Comparative Example 1;

[0025] Figure 3 The fibrinogen adhesion and denaturation results of the vascular stents provided in Examples 2-4 and Comparative Example 1 of the present invention, wherein: Figure 3 A in the figure is the fibrinogen adhesion statistics chart. Figure 3 B in the figure is the denaturation diagram of fibrinogen;

[0026] Figure 4 The results of endothelial cell fibrinogen adhesion and degenerated endothelial cell adhesion and growth of the vascular stents provided in Examples 2-4 of the present invention and Comparative Example 1;

[0027] Figure 5 A statistical graph of endothelial cell proliferation levels on the surface of the vascular stents provided in Examples 2-4 of the present invention and Comparative Example 1;

[0028] Figure 6 The results of fibrinogen adhesion and degenerated endothelial cell adhesion and growth on the surface of smooth muscle cells of vascular stents provided in Examples 2-4 of the present invention and Comparative Example 1;

[0029] Figure 7 A statistical graph of the proliferation level of smooth muscle cells on the surface of the vascular stent provided in Examples 2-4 of the present invention and Comparative Example 1;

[0030] Figure 8The co-culture results of endothelial cells and smooth muscle cells in the vascular stents provided in Examples 2-4 of the present invention and Comparative Example 1 are shown. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0032] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0033] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in the art and can be purchased through commercial channels.

[0034] Sources of materials used in the present invention: Stainless steel vascular stents were purchased from Shandong Weigao Medical Instrument Co., Ltd.;

[0035] Bioactive peptides were purchased from Shanghai Jier Biochemical (Shanghai) Co., Ltd.

[0036] Solution preparation used in the embodiment:

[0037] Dopamine solution, the solute is dopamine, the concentration is 2 mg / mL, the solvent is a mixed solution of tris(hydroxymethyl)aminomethane and hydrogen chloride, and the pH value is 8.5.

[0038] Phosphate buffer solution, pH 7.4.

[0039] Example 1

[0040] Bioactive peptides were purchased from Shanghai Jier Biochemical (Shanghai) Co., Ltd.

[0041] Table 1 Amino acid sequences of bioactive peptides

[0042] serial number Amino acid sequence Sequence ID 1 AGEDPHGYFLPGQFA SEQ ID NO.1 2 C-AGEDPHGYFLPGQFA SEQ ID NO.2 3 CG-AGEDPHGYFLPGQFA SEQ ID NO.3 4 G-AGEDPHGYFLPGQFA SEQ ID NO.4 5 GG-AGEDPHGYFLPGQFA SEQ ID NO.5 6 CGG-AGEDPHGYFLPGQFA SEQ ID NO.6

[0043] Example 2 This example provides a fucoidan-bioactive peptide coated vascular stent, the surface of the vascular stent is coated with a composite coating, and the composite coating is composed of fucoidan and bioactive peptides.

[0044] The molecular weight of fucoidan is 20kDa.

[0045] The amino acid sequence of the bioactive peptide is the amino acid sequence shown in SEQ ID NO.1 in Example 1.

[0046] This embodiment also provides a method for preparing a fucoidan-bioactive peptide coated vascular stent, comprising the following steps:

[0047] S1. Place the stainless steel vascular stent material in a dopamine solution with a concentration of 2 mg / mL, react in a constant temperature air bath oscillator at 25°C for 24 hours, and the speed of the constant temperature air bath oscillator is 100 rpm. After the reaction is completed, wash with a phosphate buffer solution, and dry in a vacuum drying oven at 50°C for 12 hours to obtain a pretreated vascular stent sample;

[0048] S2. Dissolve 5 mg of fucoidan with a molecular weight of 20 kDa and 10 mg of bioactive peptide in 10 mL of phosphate buffer solution at room temperature, mix them evenly, and obtain a fucoidan-bioactive peptide mixed solution, wherein the concentration of fucoidan in the fucoidan-bioactive peptide mixed solution is 0.5 mg / mL, and the concentration of the bioactive peptide is 1 mg / mL;

[0049] S3. Place the pretreated vascular stent sample obtained in step S1 into the mixed solution of fucoidan-bioactive peptide obtained in step S2, and react in a constant temperature air bath oscillator at 40° C. for 6 hours at a speed of 100 rpm to obtain a fucoidan-bioactive peptide coated vascular stent.

[0050] The preparation method of Example 3 is the same as that of Example 2, except that the concentration of the bioactive peptide in the fucoidan-bioactive peptide mixed solution is 3 mg / mL.

[0051] The preparation method of Example 4 is the same as that of Example 2, except that the concentration of the bioactive peptide in the fucoidan-bioactive peptide mixed solution is 5 mg / mL.

[0052] Comparative Example 1 The vascular stent provided in this comparative example is an untreated stainless steel vascular stent.

[0053] The effects of the vascular stents provided in the above-mentioned Examples 2-4 and Comparative Example 1 were verified by the following experiments, wherein the sample in Comparative Example 1 is represented by A, and Examples 2-4 are represented by B, C, and D, respectively.

[0054] 1. Hemolysis rate: The specific operation of the hemolysis experiment is as follows: Place the front of the A, B, C, and D samples with the sides and bottoms sealed with silicone in a 15mL centrifuge tube containing 10mL of normal saline, and use normal saline and ultrapure water as the negative control group and positive control group, respectively. Place the centrifuge tube in a 37°C constant temperature water bath for 30 minutes, then add 200μL of diluted healthy human blood (blood: normal saline = 4:5 (v / v)) to the centrifuge tube. After incubation at 37°C for 1 hour, transfer the solution in the original centrifuge tube to a new centrifuge tube and centrifuge at 2500rpm for 15 minutes. Subsequently, take out 100μL of supernatant in a 96-well plate, use an enzyme reader to measure its absorbance at a wavelength of 540nm, and calculate the hemolysis rate of samples A, B, C, and D. The results are as follows. Figure 1 .

[0055] Depend on Figure 1 It can be seen that the hemolysis rate of sample A in comparative example 1 is the highest, which is 2.78%, but it is also lower than the requirement of 5%. The hemolysis rates of samples B, C, and D in Examples 2-4 are significantly lower than those of sample A, among which the hemolysis rates of samples B, C, and D are 1.18%, 0.45%, and 0.42%, respectively. That is, the hemolysis rates of the stainless steel vascular stents modified with the fucoidan-bioactive peptide composite coating provided in Examples 2-4 of the present invention are all at a relatively low level, meeting the prerequisite for the clinical use of vascular stents.

[0056] 2. Platelet adhesion: Fresh blood from healthy volunteers was centrifuged at 1500rpm for 15 minutes to obtain platelet-rich plasma (PRP). Then, 80μL of PRP was dripped on the surface of samples A, B, C, and D, and incubated at 37°C for 60 minutes, and then the surface was gently rinsed three times with normal saline. Subsequently, 4% paraformaldehyde was added for fixation for 30 minutes, and then gently rinsed three times with normal saline, and then dehydrated with 25%, 50%, 75%, and 100% ethanol in a gradient manner for 15 minutes. Finally, the sample was dried in an oven at 37°C for 6 hours, taken out and sprayed with gold, and the adhesion of platelets on the surface of samples A, B, C, and D was observed by SEM. The results are as follows Figure 2 .

[0057] Depend on Figure 2 It can be seen that the number of platelets on the surface of sample A is the largest, and the risk of thrombosis is higher. Compared with sample A, no obvious platelet adhesion was observed on the surfaces of samples B, C, and D, which shows that the fucoidan-bioactive peptide composite coating can effectively inhibit the adhesion of platelets on the surface of modified stainless steel samples, thereby significantly reducing the risk of thrombosis induced by samples B, C, and D.

[0058] 3. Fibrinogen adhesion and denaturation: First, seal the bottom and sides of the A, B, C, and D samples to be tested with silica gel, dry and set aside. Centrifuge the fresh blood of healthy volunteers at 3000 rpm for 15 minutes to obtain platelet-poor plasma (PPP). Then add 80 μL PPP to the surface of A, B, C, and D samples, incubate at 37°C for 30 minutes, rinse gently with saline three times, add 5% bovine serum albumin (BSA) blocking solution and incubate for 30 minutes. After rinsing three times with saline, add 50 μL of HRP-coupled goat anti-human fibrinogen adsorption antibody (antibody: PBS = 1:500) to the surface, incubate at 37°C for 60 min, and then gently rinse three times with saline. Then add 100 μL of TMB colorimetric solution to the surface. After reacting in the dark for 10 min, add 50 μL of ELISA sulfuric acid stop solution. After the foam disappears, absorb 120 μL of supernatant into a 96-well plate, and measure the absorbance at a wavelength of 450 nm using an enzyme reader.

[0059] The fibrinogen denaturation test procedure is the same as the fibrinogen adhesion test procedure, except that the HRP-coupled goat anti-human fibrinogen adsorption antibody is replaced with the HRP-coupled goat anti-human fibrinogen denaturation antibody. Figure 3 .

[0060] Depend on Figure 3 It can be seen that among all the samples, sample A showed the highest level of fibrinogen adhesion and denaturation, poor blood compatibility, and a higher risk of thrombosis. Compared with sample A, the adhesion and denaturation levels of fibrinogen in samples B, C, and D were lower. Taking sample A as the control group, the fibrinogen adhesion levels on the surfaces of samples B, C, and D were 23.2%, 26.5%, and 22.6% of sample A, respectively. And the fibrinogen denaturation levels of samples B, C, and D were 23.3%, 25.3%, and 22.6% of sample A, respectively. After modification with the composite coating of fucoidan and bioactive peptides, the blood compatibility of the stainless steel sample was significantly improved, and the blood compatibility was significantly improved, making it suitable for use as a vascular stent material.

[0061] 4. Endothelial cell adhesion and growth experiment: First, use silica gel to seal the bottom and sides of samples A, B, C, and D, and sterilize the bottom and front of samples A, B, C, and D with ultraviolet for 30 minutes before use. Then prepare 1640 complete medium, and the preparation method is as follows: prepare 1640 complete medium according to the ratio of 89% 1640 medium, 10% BI serum, and 1% penicillin-streptomycin mixture. Place the front of the sample in a 24-well non-adhesive plate and place it on the clean bench for another ultraviolet sterilization for ten minutes. Endothelial cells are inoculated on the surface of samples A, B, C, and D at an inoculation density of 8000 cells / mL. Cell recovery and passage must be performed before endothelial cell inoculation. After endothelial cells are inoculated, they are placed in a CO2 constant temperature incubator for 1 day and 3 days, during which 1640 complete medium is replaced every day. After 1 day and 3 days of culture, the spent culture medium is extracted, gently rinsed three times with PBS, and then fixed with 4% paraformaldehyde fixative for 1 hour. The fixative was then removed, and after rinsing gently with PBS three times, BSA blocking solution was added to block for 30 minutes. The blocking solution was then removed, and after rinsing gently with PBS three times, 0.5% Triton-X-100 solution diluted with PBS was added to permeabilize for 5 minutes. The permeation solution was then removed, and after rinsing gently with PBS three times, 100 μL of diluted phalloidin solution (phalloidin: PBS = 1:200) was added to the sample surface, and stained for 50 minutes under light-proof conditions. The phalloidin solution was then removed, and after rinsing gently with PBS three times, 80 μL of diluted DAPI solution (10 μg / mL) was added to the surface of samples A, B, C, and D, and stained for 4 minutes under light-proof conditions. The DAPI solution was then removed, and PBS was used to rinse gently three times. Finally, the stained A, B, C, and D samples were placed under a super-resolution fluorescence confocal microscope to take pictures and observe the fluorescent staining of endothelial cells after 24 hours and 72 hours of culture on samples A, B, C, and D. Among them, the cell nucleus was blue after DAPI staining, and the cytoskeleton was green after phalloidin staining. The results are as follows Figure 4 .

[0062] Depend on Figure 4 It can be seen that sample A is not suitable for endothelial cell growth. The number of cells is the least after 24h and 72h of culture. Sample A has poor cell compatibility. This is because the pure stainless steel surface has poor biological activity, which is not conducive to endothelial cell growth. Sample groups B, C, and D are all suitable for endothelial cell growth. When the culture time is 24h, the endothelial cells are not fully spread, and there are still a few cells on the sample surface that are round. After 72h of culture, except for sample A, endothelial cells adhere and proliferate normally on all samples, and the morphology is fully spread and spindle-shaped. The results of endothelial cell fluorescence staining experiments show that after modification with the composite coating of fucoidan and bioactive peptides, vascular endothelial cells can adhere and grow on the stainless steel surface, and the endothelial cells show a relatively good growth state.

[0063] 5. Endothelial cell activity experiment: The CCK-8 test method was used to evaluate the in vitro cell activity of endothelial cells on the coating surface of samples A, B, C, and D. The specific operation is as follows: The endothelial cells were inoculated on the surface of samples A, B, C, and D at a seeding density of 8000 cells / mL, and then placed in a CO2 constant temperature incubator for 1 day and 3 days, during which the 1640 culture medium was replaced every day. After 1 day and 3 days of culture, the spent culture medium was removed, and the plates were gently rinsed three times with PBS. Then 700 μL of CCK-8 dilution solution (CCK-8: pure 1640 culture medium = 1:9) was added to each well, and the plates were incubated in a CO2 constant temperature incubator for 3 hours in the dark. Then, 100 μL of supernatant was extracted into a 96-well plate using a pipette, and its absorbance at 450 nm was measured using an enzyme reader. The relative cell activity of the endothelial cells was calculated based on the absorbance. The results are as follows. Figure 5 .

[0064] Depend on Figure 5 It can be seen that sample A has the worst cell compatibility and the lowest cell proliferation level. The absorbance values ​​of sample groups B, C, and D are much higher than sample A, indicating that the cell proliferation level of stainless steel samples modified by fucoidan-bioactive peptide composite coating is much higher than sample A, and endothelial cells have good compatibility with the functional composite coating on the surface. Moreover, the cell proliferation level of endothelial cells after 72 hours of culture is much higher than that of 24 hours of culture, which indicates that endothelial cells can adhere and proliferate normally on the surface of samples B, C, and D, which is mainly attributed to the growth-promoting effect of fucoidan and bioactive peptide composite coating on vascular endothelial cells.

[0065] 6. Smooth muscle cell adhesion and growth: First, use silica gel to seal the bottom and sides of samples A, B, C, and D, and sterilize the bottom and front of samples A, B, C, and D with ultraviolet for 30 minutes before use. Then prepare iCell complete medium, and the preparation method is as follows: prepare iCell complete medium according to the ratio of 89% iCell medium, 10% Corning serum, and 1% penicillin-streptomycin mixture. Place the front of samples A, B, C, and D in a 24-well non-adhesive plate and place it on the clean bench for another ultraviolet sterilization for ten minutes. Smooth muscle cells are inoculated on the surface of samples A, B, C, and D at an inoculation density of 6000 cells / mL. Cell recovery and passage must be performed before inoculation of smooth muscle cells. After inoculation, smooth muscle cells are placed in a CO2 constant temperature incubator for 1 day and 3 days, during which iCell complete medium is replaced every day. After 1 day and 3 days of culture, the nuclei of smooth muscle cells are stained with DAPI dilution solution, and the cytoskeleton of smooth muscle cells is stained with phalloidin dilution solution. Finally, the stained samples A, B, C, and D were placed under a super-resolution fluorescence confocal microscope to take pictures and observe the adhesion and growth of smooth muscle cells on the surface of samples A, B, C, and D.

[0066] Depend on Figure 6 It can be seen that smooth muscle cells can adhere and grow on the surface of sample A and composite coating modified samples after 24h and 72h of culture. The smooth muscle cells did not grow well on the surface of sample A, there were fewer cells in the fluorescence image, and the cell diffusion area was small, which was due to the biological inertness of stainless steel. Most of the smooth muscle cells on the surfaces of samples B, C, and D showed a flat spherical morphology after 24h of culture, proving that the cell diffusion was insufficient, which was attributed to the inhibitory effect of fucoidan and bioactive peptides on smooth muscle cell proliferation, adhesion and migration. After 72h of culture, there was no obvious cell migration and proliferation of smooth muscle cells on the surface of stainless steel samples modified by fucoidan and bioactive peptide composite coatings, and the number of smooth muscle cells on the sample surface did not increase significantly, indicating that fucoidan and bioactive peptides can regulate the cell growth of vascular smooth muscle cells on the surface of modified stainless steel samples.

[0067] 7. The CCK-8 test method was used to evaluate the in vitro cell activity of smooth muscle cells on the sample coating surface. The specific operation is as follows: smooth muscle cells were inoculated on the surface of samples A, B, C, and D at a seeding density of 6000 cells / mL, and then placed in a CO2 constant temperature incubator for 1 day and 3 days, during which the iCell complete culture medium was replaced every day. After 1 day and 3 days of culture, the cell activity of smooth muscle cells was evaluated using the CCK-8 kit. The specific operation steps were consistent with the endothelial cell activity experiment. The results are shown in Figure 2. Figure 7 .

[0068] Depend on Figure 7 It can be seen that the cell compatibility of sample A is the worst, and the proliferation level of smooth muscle cells is the lowest, which is related to the poor cell compatibility of sample A. At the same time point (24h and 72h), the trend of smooth muscle cell proliferation is consistent. After 24h of culture, the activity of smooth muscle cells in all groups was at a low level. After 72h of culture, except for the C experimental group, the smooth muscle cell proliferation level of the B experimental group and the D experimental group did not increase significantly, which indicates that the fucoidan-bioactive peptide composite coating is related to the growth regulation of smooth muscle cells on the sample surface.

[0069] 8. Endothelial cell and smooth muscle cell co-culture experiment: The competitive adhesion and growth of endothelial cells and smooth muscle cells on the surfaces of samples A, B, C, and D were evaluated by using the co-culture experiment of endothelial cells and smooth muscle cells. In short, the dyes for endothelial cells and smooth muscle cells were prepared for use, that is, the endothelial cell dye was prepared according to the ratio of red fluorescent tracer probe (Cyto TraceTM RedCFDA) reagent: PBS = 1:1000; the smooth muscle cell dye was prepared according to the ratio of green fluorescent tracer probe (CytoTraceTM Green CFDA) reagent: PBS = 1:1000. The endothelial cells and smooth muscle cells were cultured with iCell complete medium for 2 days, and then the waste liquid was drawn out, and the above-prepared endothelial cell dye and smooth muscle cell dye were added to replace the iCell complete medium and continued to be cultured for 30 minutes. Then the waste liquid was drawn out, and after gently rinsing with PBS, the iCell complete medium was added to culture for 30 minutes to successfully mark and stain the endothelial cells and smooth muscle cells. Endothelial cells and smooth muscle cells were then seeded onto the sample surface at a density of 3000 cells / mL and placed in a CO2 constant temperature incubator for 1 and 3 days, during which iCell complete medium was replaced every day. After 1 and 3 days of culture, the competitive adhesion growth of endothelial cells and smooth muscle cells on the surface of samples A, B, C, and D was observed using a super-resolution fluorescence confocal microscope.

[0070] Depend on Figure 8 It can be seen that after 24 hours of co-culture, there is basically no growth of endothelial cells and smooth muscle cells on the surface of sample A, while endothelial cells and smooth muscle cells can adhere and grow on the surfaces of samples B, C, and D, and the number of vascular smooth muscle cells on the surfaces of samples B, C, and D is significantly less than that of vascular endothelial cells, which indicates that endothelial cells have a competitive growth advantage over smooth muscle cells on samples B, C, and D, which is mainly due to the growth regulation of fucoidan and bioactive peptides on vascular endothelial cells and smooth muscle cells. After modification with the fucoidan-bioactive peptide composite coating, the stainless steel surface can promote the growth of endothelial cells and inhibit the excessive proliferation of smooth muscle cells, which is beneficial to improve the application performance of stainless steel vascular stent materials.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A fucoidan-bioactive peptide coated vascular stent, characterized in that: The surface of the vascular stent is coated with a composite coating, which is composed of fucoidan and bioactive peptides.

2. The fucoidan-bioactive peptide coated vascular stent according to claim 1, characterized in that: The molecular weight of the fucoidan is 10 kDa-100 kDa.

3. The fucoidan-bioactive peptide coated vascular stent according to claim 1, characterized in that: The amino acid sequence of the bioactive peptide is one or more of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.

6.

4. The fucoidan-bioactive peptide coated vascular stent according to claim 1, characterized in that: The material of the vascular stent body is selected from one or both of non-degradable metals and degradable metals.

5. A method for preparing the fucoidan-bioactive peptide coated vascular stent according to any one of claims 1 to 4, characterized in that: The steps include: S1, placing the vascular stent body material in a dopamine solution, and subjecting the material to a constant temperature oscillation reaction. After the reaction is completed, the material is washed with a phosphate buffer solution, and dried to obtain a pretreated vascular stent sample; S2. Dissolve the fucoidan and bioactive peptides in a phosphate buffer solution at room temperature, and mix them evenly to obtain a fucoidan-bioactive peptide mixed solution; S3, placing the pretreated vascular stent sample obtained in step S1 into the mixed solution of fucoidan-bioactive peptide obtained in step S2, and reacting the mixture under constant temperature oscillation to obtain the fucoidan-bioactive peptide coated vascular stent.

6. The preparation method according to claim 5, characterized in that: In step S1, the constant temperature oscillation reaction temperature is 25-35°C, the constant temperature oscillation reaction time is 12-36h, and the oscillation speed is 80-120rpm.

7. The preparation method according to claim 5, characterized in that: In step S3, the constant temperature oscillation reaction temperature is 20-40°C, the constant temperature oscillation reaction time is 4-8h, and the oscillation speed is 80-160rpm.

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