CN113244462B

CN113244462B - Drug-coated intravascular stent for preventing restenosis in stent and preparation method thereof

Info

Publication number: CN113244462B
Application number: CN202110549030.8A
Authority: CN
Inventors: 安美文; 郭继强; 张锦; 高宇平; 高颖; 王立; 刘阳; 杨彩仙; 吕营; 岳志杰; 曹烨; 李芳�; 王昭月
Original assignee: Taiyuan University of Technology; Shanxi Bethune Hospital of Shanxi Academy Of Medical Sciences
Current assignee: Taiyuan University of Technology; Shanxi Bethune Hospital of Shanxi Academy Of Medical Sciences
Priority date: 2021-05-20
Filing date: 2021-05-20
Publication date: 2022-05-24
Anticipated expiration: 2041-05-20
Also published as: CN113244462A

Abstract

The invention discloses a composite medicine intravascular stent which comprises a bare metal stent (prepared by nickel-titanium memory alloy materials), wherein a stent body is loaded with recombinant human collagen and folic acid, and HMGB1 fragment peptide and VEGFR-2 antibody are crosslinked on the surface of the bare metal stent. Specifically, a composite spinning solution of recombinant human collagen, folic acid, HMGB1 fragment peptide and VEGFR-2 antibody is loaded on a nickel-titanium memory alloy bare metal stent by using a dynamic liquid electrostatic spinning technology to form a vascular stent loaded with the recombinant human collagen, the folic acid, the cross-linked HMGBI fragment peptide and the VEGFR-2 antibody, so that the capture, homing, proliferation and differentiation of EPCs are promoted, and the rapid endothelialization of blood vessels is realized. The invention can be used for coronary artery stents, cerebrovascular stents, renal artery stents, aortic stents, etc.

Description

Drug-coated intravascular stent for preventing restenosis in stent and preparation method thereof

Technical Field

The invention relates to the technical field of medical vascular stents implanted into human bodies, in particular to a vascular stent which loads recombinant human collagen, folic acid and cross-linked High Mobility Group protein 1 (HMGB 1) fragment peptide and human vascular Endothelial growth factor receptor 2 antibody (VEGFR-2/KDR/CD 309 antibody), aiming at further specifically promoting the capture, homing, proliferation and differentiation of Endothelial Progenitor Cells (EPCs) on the basis of meeting the requirements of a common vascular stent, inhibiting the excessive hyperplasia of intima and simultaneously realizing the rapid endothelialization of blood vessels and increasing the biocompatibility of the surface of the stent, thereby achieving the postoperative effects of reducing the restenosis in the stent and the formation of late stent thrombosis.

Background

Nowadays, with the improvement of living standard and the change of living habits and eating habits of people, the incidence rate of cardiovascular and cerebrovascular diseases is high, and the incidence rate of cardiovascular and cerebrovascular diseases is more and more younger. Interventional therapy is an effective method for treating cardiovascular and cerebrovascular thrombotic diseases at present, but in-Stent restenosis (ISR) and Late Stent Thrombosis (LST) seriously affect the long-term curative effect. Stenting is the most important and effective method of treating arterial occlusions. The stent placement procedure is generally: after the catheter-stent-balloon system is placed in a lesion area, the balloon enables the stent to be permanently deformed under the action of pressure; after the saccule and the catheter are taken out, the stent still remains in the blood vessel to maintain an expanded state, thereby achieving continuous blood fluency. The stent implantation is to restore the patency of a stenosed blood vessel by mechanical support of a stent, thereby possibly causing adaptive reaction of the blood vessel. Endothelial Cell (ECs) damage is the initiating factor of vascular responses. The stent damages the inner wall of the blood vessel, leads to endothelial denudation and promotes local thrombosis.

An ideal vascular stent should contain the following properties: the biocompatibility is good; the reticular structure enables the stent to have certain deformation capacity, and the compliance of the blood vessel stent is ensured; after being placed into the blood vessel, the stent can provide certain mechanical strength to support the blood vessel and maintain smooth blood flow; fourthly, the method has the functions of capturing the EPCs, promoting the migration, proliferation and differentiation of the EPCs into ECs and realizing the rapid endothelialization of the blood vessel; fifthly, the composition has the characteristics of extremely low immunological rejection, anticoagulation and certain affinity with endothelial cells; sixthly, the blood homocysteine (Hcy) level can be effectively reduced, the vascular endothelial function is improved, and the elasticity of blood vessels is improved.

At present, the medicament coating metal intravascular stent clinically used on a large scale plays a role of permanent support, but the problems of thrombus and restenosis caused by the implantation of foreign matters, and simultaneously, the medicament coating inhibits the growth of intima and the growth of endothelium. Existing stent coatings often block intimal hyperplasia while at the same time preventing the full functional and structural regeneration of the endothelium, thus there remains the potential for late stage thrombosis due to insufficient regeneration of the endothelial covering. The CD34 antibody is by far the most commonly used EPCs for stent coating to capture bioactive molecules. Although the CD34 antibody has gained wide acceptance in the field of mediating capture of EPCs, there are problems with the use of CD34 antibodies. CD34 is not completely specific for EPCs, but is also expressed on the surface of other cell types, such as hematopoietic stem cells and platelets. Therefore, the CD34 antibody stent has poor effect of promoting endothelialization to resist in-stent restenosis in a short period. Other antibodies useful for capturing EPCs were sought which, in addition to being specifically expressed in EPCs, are also expressed in differentiated ECs, and antibodies more specific for capturing EPCs than the CD34 antibody are of great interest in further reducing in-stent restenosis and late stent thrombosis.

Disclosure of Invention

The invention aims to provide a drug-coated vascular stent for preventing in-stent restenosis and a preparation method thereof, and particularly relates to a vascular stent loaded with folic acid, recombinant human collagen, cross-linked HMGB1 fragment peptide and VEGFR-2 antibody and a preparation method thereof. The VEGFR-2 antibody can enable injured parts of blood vessels to capture EPCs quickly, the HMGB1 can promote migration, proliferation and differentiation of EPCs into ECs, re-endothelialization of injured blood vessels is accelerated, the recombinant human collagen can inhibit deposition of thrombotic components on the injured parts of the blood vessels, and folic acid can enhance elasticity and compliance of the blood vessels.

The invention is realized by adopting the following technical scheme:

a drug-coated vascular stent for preventing in-stent restenosis comprises a bare metal stent, wherein recombinant human collagen, folic acid, HMGB1 fragment peptide and VEGFR-2 antibody are cross-loaded on the bare metal stent.

Further preferably, the bare metal stent is made of a nickel titanium memory alloy material.

A preparation method of a drug-coated vascular stent for preventing in-stent restenosis comprises the following steps:

(1) preparing a reticular nickel-titanium memory alloy bare metal stent: weaving the nickel-titanium alloy into metal wires, and bending the metal wires into a nickel-titanium alloy net-shaped framework;

(2) and (3) pretreatment of the stent: using propanol analysis pure solution or medical ethanol solvent to the obtained reticular nickel-titanium memory alloy bare metal stent, cleaning the stent body by using ultrasonic waves, removing impurities on the surface of the stent body, cleaning the stent body by using distilled water and ultrasonic waves, and drying the cleaned stent body;

(3) and preparing a composite spinning solution: dissolving the recombinant human collagen, folic acid, HMGB1 polypeptide and VEGFR-2 antibody in a solvent, and uniformly mixing to obtain a composite spinning solution;

(4) continuously electrospinning the composite spinning solution by adopting a dynamic liquid electrostatic spinning method to generate composite nanofibers with orientation, loose fiber structures and large pore diameters, and winding the obtained composite nanofibers on a bare metal stent to form a drug-coated intravascular stent;

(5) and freeze-drying and crosslinking the obtained drug-coated intravascular stent to obtain the drug-coated intravascular stent.

In the step (1), the nickel-titanium alloy mesh framework is formed by bending a nickel-titanium alloy wire into a plurality of wave-shaped bending rings which are connected in sequence, or a nickel-titanium alloy wire is bent into a single ring, and then the nickel-titanium alloy wire is used for connecting a plurality of independent wave-shaped bending rings into the pipe-shaped nickel-titanium alloy framework. Or the nickel-titanium alloy reticular skeleton is formed by engraving a nickel-titanium alloy pipe by laser, and has wavy patterns after expansion.

In the step (2), the concentration of a propanol analytically pure solution is 99.5 percent, and the concentration of a medical ethanol solvent is 75 percent; the frequency of ultrasonic cleaning the bracket body for two times is 28-100 khz, and the cleaning time is 5-15 min; the drying temperature is 30-40 ℃, and the drying time is 30-60 min.

In the step (3), the substrate solvent is a polylactic acid solution, and the polylactic acid is dissolved in one of organic solvents such as 1, 4-dioxane, dichloromethane and dimethyl sulfoxide according to the concentration of 0.01-0.1 g/ml in an anaerobic operation box and is uniformly stirred to obtain the polylactic acid solution. The concentration of the recombinant human collagen is 0.1-5 mg/ml, the weight percentage of folic acid is 0.1-10% (by weight of solvent), the weight percentage of HMGB1 polypeptide is 0.1-10% (by weight of solvent), and the concentration of VEGFR-2 antibody is 10-100 mg/ml.

In the step (4), the technological parameters of the dynamic liquid electrostatic spinning are as follows: spinning voltage is 8-20 kV, a rotating metal rod is used for receiving fibers, the diameter of the metal rod is 2-4 mm, the receiving distance is 8-20 cm, the rotation rate of a stainless steel rod is 200-1000 rmp, the spinning speed is 0.1-5 ml/h, and the temperature environment is 60 +/-5 ℃.

In the step (5), the temperature of freeze drying is-20 to-60 ℃, and the time is 1 to 5 hours; 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (1- [3- (Dimethylamino) propyl ] -3-Ethylcarbodiimide, EDC), N-Hydroxysuccinimide (NHS) and Phosphate Buffer Saline (PBS) solution are used as cross-linking agents, 20mg of EDC and 50mg of NHS are added into 1ml of PBS solution, the mixture coated on the stent is modified and cross-linked on a naked metal stent at 25 ℃ for 25 min.

The invention relates to a vascular stent for preventing in-stent restenosis, in particular to a vascular stent loaded with recombinant human collagen, folic acid, cross-linked HMGB1 fragment peptide and VEGFR-2 antibody. The VEGFR-2 antibody can enable injured parts of blood vessels to capture EPCs quickly, the HMGB1 can promote migration, proliferation and differentiation of EPCs into ECs, re-endothelialization of injured blood vessels is accelerated, the recombinant human collagen can inhibit deposition of thrombotic components on the injured parts of the blood vessels, and folic acid can enhance elasticity and compliance of the blood vessels.

The mechanism of loading recombinant human collagen and folic acid on the vascular stent and crosslinking HMGB1 fragment peptide and VEGFR-2 antibody is as follows:

from the anatomical structure of blood vessels, arterial blood vessels generally consist of three layers of membranes, the innermost layer is the intima, the part in direct contact with blood consists of a large number of endothelial cells, and the endothelial cells are supported by a basement membrane; the middle layer is a tunica media and consists of a large number of smooth muscle cells and extracellular matrix; the outermost layer is the adventitia, which is composed of loose connective tissue and fibroblasts. Among them, ECs play an extremely important role in the internal structure. ECs provide an antithrombotic surface by separating blood components from the subendothelial matrix proteins and maintaining the balance between the coagulation and fibrinolytic systems through the synthesis and release of active molecules. Injury of the intima exposes the underlying endothelium, which contains collagen and tissue factor, to the blood circulation, causing activation of platelets and the coagulation system. Therefore, the formation of an intact endothelial cell coating on the intimal surface of the blood vessel, and the formation of a natural barrier between the subintimal tissue and the blood stream, are important conditions for ensuring the normal function of the blood vessel and maintaining the integrity of the blood vessel. The injury and slow repair of vascular endothelial cells after injury are common pathophysiological basis of vascular injury diseases such as restenosis and coronary heart disease after vascular interventional therapy. Vascular repair after injury is very complex and is a process of multiple mechanisms involved and interacting. Poor repair of damaged vessels can lead to excessive thickening of the vessel intima and narrowing of the lumen. Severe reduction in blood perfusion can cause diseases such as myocardial infarction, cerebral infarction, etc.

Currently, promoting In situ endothelialization of Stent surfaces In order to prevent In Stent Restenosis (ISR) and Late Stent Thrombosis (LST) from occurring has become a research hotspot. Generally, when stents are implanted in vivo to achieve in situ endothelialization, there are two major sources of ECs, adhesion, proliferation and aggregation of EPCs and ECs at the surface of the stent. The undamaged endothelial cells can migrate to the damaged part within 1-2 cm from the implantation point of the stent. The biological active molecules are fixed on the surface of the material, so that the adhesion and proliferation of cells on the surface of the material can be promoted, and the endothelialization of the damaged part of the blood vessel is finally realized. EPCs are adult stem cells that have the ability to self-renew and differentiate into mature endothelial cells. ECs are derived from the differentiation and proliferation of EPCs. Under the stimulation of physiological or pathological factors, EPCs can mobilize from bone marrow to peripheral blood, chemotaxis to damaged parts of endothelium, and then differentiate and proliferate into ECs to replace damaged ECs to achieve normal functions. EPCs overcome the defect of pure endothelial cell proliferation, and make the rapid endothelialization of the surface of the stent possible. Rapid endothelialization after stent placement is an effective method to reduce intrastent thrombosis and intrastent restenosis. The vascular stent capable of promoting migration, adhesion and proliferation of EPCs is constructed, so that not only can the homing of EPCs on the surface of the material be promoted, EPCs in circulating blood be captured and deposited on the surface of the material, and the differentiation of EPCs towards endothelial cells is induced, but also the surface material of the stent can promote the proliferation of endothelial cells. The combined use of the two methods can prevent stent restenosis and thrombosis.

In situ endothelialization not only achieves capture and differentiation promotion of EPCs, but also maintains the ability of ECs for a long period of time. Although a number of approaches have been explored, future research may combine these strategies to mimic the ECs production process, combining captured biomolecules, growth stimulating factors and appropriate matrix materials. EPCs play a role in endovascular repair, differentiate into mature endothelial cells by secreting various cytokines, and promote the proliferation and migration of previously remaining endothelial cells and endothelial progenitor cells, thereby repairing damaged blood vessels. And their function depends on the synergistic effects of the local microenvironment and other populations on vascular damage. The cell membranes of the EPCs express CD34, CD133 and VEGFR-2 membrane proteins, the coronary artery stent can be improved by using an antibody with specificity to an EPCs surface receptor, the antibody of the EPCs surface receptor is fixed on the surface of the stent, the EPCs are rapidly captured after the stent is placed, and in-situ endothelialization of a vascular injury part is realized. The CD34 antibody is by far the most commonly used EPCs for stent coating to capture bioactive molecules. Although the CD34 antibody has gained wide acceptance in the field of mediating EPCs capture, there are several problems with the use of CD34 antibodies. CD34 is not completely specific for EPCs, but is also expressed on the surface of other cell types, such as hematopoietic stem cells and platelets. Therefore, the CD34 antibody stent has poor effect of promoting endothelialization to resist in-stent restenosis in a short period. VEGFR-2 antibody is another antibody used to capture EPCs, and VEGFR-2 is also expressed in differentiated ECs in addition to its specific expression in EPCs, indicating that VEGFR-2 antibody is more specific for capturing EPCs than CD34 antibody.

ECs injury is the initiating factor of the vascular response. The stent damages the inner wall of the blood vessel, leading to endothelial denudation and promoting local thrombosis. The extent of thrombosis is closely related to the depth of stent entry into the vessel. Inflammation is a process of vascular self-repair, with monocytes and polymorphonuclear leukocytes adhering to the elastic intima. Smooth muscle cells migrate to the intima and proliferate, secrete a large amount of protein and collagen with the transition from the high-differentiation-contractile type to the low-differentiation-synthesis type, deposit on the media and adventitia of the blood vessel, and undergo fibrosis.

HMGB1 is a non-histone DNA binding protein that promotes the assembly of nuclear protein complexes in the nucleus, and as an inflammatory factor, triggers various pathological processes including inflammatory cytokine release, cell migration and angiogenesis by mediating receptors such as TLR2, TLR4 and RAGE. It is not only shown to be an Advanced mediator of infection, but is also thought to regulate angiogenesis in pathological states, mediating activation of ECs by binding to receptors expressed on EC membranes, the Receptor for Advanced Glycation End product Receptors (RAGE). The target HMGB1 polypeptide has certain therapeutic potential in angiogenesis-related diseases. The HMGB1 fragment peptide is composed of a part of amino acids of HMGBI protein, and has the function of stimulating cell migration. Preferably, the peptide is the smallest peptide fragment in the fragment with the cell migration stimulating activity, namely the HMGB1 fragment peptide at least comprising the 17 th-25 th amino acid sequence of HMGB1 protein. The fragment peptide composed of a part of the amino acids of the HMGBI protein is not particularly limited as long as it has a cell migration stimulating activity, and examples of the fragment peptide include, but are not limited to, a fragment peptide including at least HMGB1 fragment peptide (1 to 44) and having as its upper limit any HMGBI-derived fragment peptide excluding the entire HMGB1 protein. The HMGB1 fragment peptide and the VEGFR-2 antibody are cross-linked on the vascular stent, so that the stent can be promoted to rapidly capture EPCs, the differentiation and proliferation of the EPCs are promoted, and the rapid endothelialization of blood vessels is realized.

The stent implantation is to restore the patency of a stenotic lesion blood vessel by mechanical support of a stent, thereby possibly causing adaptive reaction of the blood vessel. The stent implantation is a mechanical process, and factors influencing the local mechanical environment of the blood vessel include: balloon inflation pressure, mechanical properties of the vessel wall member, and the like. Stent hyperexpansion refers to the state of a stent immediately after PCI surgery where the stent is wholly or partially beyond its respective reference vessel boundary. The diameter of the stent is usually enlarged to be larger than the diameter of the lumen of a reference blood vessel so as to ensure that the stent and the vessel wall are attached to each other well, the positioning is accurate, and the stent has larger lumen acquisition and storage capacity and ideal blood flow recovery degree. The larger the stent diameter is, the more serious the injury to the vessel wall and plaque in the expansion process is, the higher the restenosis rate is easily caused, and even serious malignant complications such as vessel rupture and the like are caused. At present, through further optimization or surface modification of the drug-loaded coating, the recombinant human collagen has the characteristics of extremely low immune rejection, anticoagulation and certain affinity with endothelial cells, and promotes re-endothelialization of the damaged part of the blood vessel.

High Homocysteine (HHcy) causes the function of the inner skin of an arterial vessel to be weakened, so that elastic fibers of the arterial vessel are degraded, the elasticity of the blood vessel is reduced, the elasticity and the compliance of the arterial vessel are reduced, the High Homocysteine participates in the formation and the development of hypertension, the blood vessel atherosclerosis and the hypertension have obvious synergistic effect on cardiovascular and cerebrovascular events. Supplementing water-soluble vitamin folic acid, can effectively reduce homocysteine level in blood, improve vascular endothelial function, and improve elasticity of blood vessel. Hyperhomocysteinemia most elastic layers are disorganized, elastic fibers are broken or are in a flaky shape, smooth muscle cells are hypertrophic and rearranged, the ratio of elastin is obviously reduced, and the compliance of the artery wall is reduced. Folic acid, an important coenzyme for the methionine cycle, plays an important role as a methyl group donor in the synthesis of methionine by Hcy. When the folic acid content in the body is higher, the Hcy can be promoted to generate methionine through a re-methylation way, so that the Hcy level is reduced.

The scheme of the invention has the following advantages:

(1) the invention prepares the vascular stent loaded with recombinant human collagen, folic acid, HMGB1 polypeptide and VEGFR-2 antibody for the first time. On the basis of meeting the requirements of a common vascular stent, the method further specifically promotes the capture, homing, proliferation and differentiation of EPCs, inhibits the excessive hyperplasia of intima, simultaneously realizes the rapid endothelialization of blood vessels, and has good biocompatibility and compliance.

(2) The concentration of the polylactic acid solution in the composite spinning solution is 0.01-0.1 g/ml; the concentration of the recombinant human collagen is 0.1-5 mg/ml, the weight percentage of folic acid is 0.1-10%, the weight percentage of HMGB1 polypeptide is 0.1-10%, and the concentration of VEGFR-2 antibody is 10-100 mg/ml.

(3) The method adopts a dynamic liquid electrostatic spinning method to realize the loading of the functional medicine on the surface of the bare metal stent, is firmer compared with a soaking method and a spraying method, can not only protect the biological activity of the medicine or the factors to the maximum extent, but also can ensure that the medicine or the factors can be controllably and continuously released through diffusion action, a degradation mechanism and the like.

The invention has reasonable design, and the nanofiber obtained by loading the four medicaments on a bare metal stent by loading recombinant human collagen, folic acid, HMGB1 polypeptide and VEGFR-2 antibody and adopting a dynamic liquid electrostatic spinning method can obtain the vascular stent with short-term anticoagulation performance and long-term endothelialization promotion.

Detailed Description

The following provides a detailed description of specific embodiments of the present invention.

The composite medicine coating intravascular stent comprises a bare metal stent (prepared from a nickel-titanium memory alloy material), wherein a stent body is loaded with recombinant human collagen and folic acid, and HMGB1 fragment peptide and VEGFR-2 antibody are crosslinked on the surface of the bare metal stent. Dissolving recombinant human collagen, HMGB1 polypeptide, VEGFR-2 antibody and folic acid in a solvent to obtain a composite spinning solution; the composite spinning solution of the recombinant human collagen, the folic acid, the HMGB1 fragment peptide and the VEGFR-2 antibody is loaded on the nickel-titanium memory alloy bare metal stent by using a dynamic liquid electrostatic spinning technology, the composite spinning solution is continuously electrospun to generate a composite nano layer with certain orientation, a loose fiber structure and a large pore diameter, so that the vascular stent loaded with the recombinant human collagen, the folic acid, the cross-linked HMGBI fragment peptide and the VEGFR-2 antibody is formed, the capture, homing, proliferation and differentiation of EPCs are promoted, and the rapid endothelialization of blood vessels is realized. The invention can be used for coronary artery stents, cerebrovascular stents, renal artery stents, aortic stents, etc.

The electrostatic spinning technology is a common method for preparing a bracket in tissue engineering research, can continuously prepare nano-scale to micron-scale fibers, and can simulate the size and the structure of natural extracellular matrix, thereby being beneficial to the adhesion and the growth of endothelial cells and preventing the proliferation and the adhesion of other cells. Meanwhile, the raw materials of the electrostatic spinning method are widely selected, and synthetic polymer materials, natural polymer materials and blended compounds can be prepared by the electrostatic spinning method. In recent years, based on the requirements of tissue engineering scaffolds on fiber structure and function, electrostatic spinning methods are gradually developed and applied. The coaxial electrostatic spinning method is mainly used for preparing fibers loaded with functional drugs or factors, the functional drugs can be loaded on the surface of the stent by the method, the biological activity of the drugs or the factors can be protected to the maximum degree, and the drugs or the factors can be controllably and continuously released through diffusion, degradation mechanisms and the like. According to the invention, the vascular stent with short-term anticoagulation performance and long-term endothelialization promotion can be obtained by loading recombinant human collagen, HMGB1 polypeptide, VEGFR-2 antibody and folic acid and adopting a dynamic liquid electrostatic spinning method to obtain nanofibers from the four medicines. The fiber material prepared by the electrostatic spinning technology has a structure with large aperture and high porosity so as to maintain the transmission of nutrients and ensure the three-dimensional migration and growth of cells.

Example 1

A specific preparation method of a drug-coated intravascular stent for preventing in-stent restenosis comprises the following steps:

(1) preparing a reticular nickel-titanium memory alloy bare metal stent: the nickel-titanium alloy is woven into metal wires, and the metal wires are bent into a nickel-titanium alloy net-shaped framework. Specifically, a nickel-titanium alloy wire is bent into a plurality of wavy bent rings which are connected in sequence to form a pipeline-shaped nickel-titanium alloy framework.

(2) And (3) pretreatment of the stent: using a propanol analytically pure solution or a medical ethanol solvent to the obtained reticular nickel-titanium memory alloy bare metal stent, wherein the concentration of the propanol analytically pure solution is 99.5%, and the concentration of the medical ethanol solvent is 75%; cleaning the bracket body by using ultrasonic waves to remove impurities on the surface of the bracket body, and cleaning the bracket body by using the ultrasonic waves through distilled water, wherein the frequency of the bracket body is 50kHz through twice ultrasonic cleaning, and the cleaning time is 10 min; and drying the cleaned stent body at the drying temperature of 30-40 ℃ for 50-60 min.

(3) And preparing a composite spinning solution: dissolving the recombinant human collagen, folic acid, HMGB1 polypeptide and VEGFR-2 antibody in a substrate solvent, and mixing uniformly to obtain the composite spinning solution.

The substrate solvent is polylactic acid solution, and the polylactic acid is dissolved in 1, 4-dioxane according to the concentration of 0.08g/ml in an anaerobic operation box and is uniformly stirred to obtain the polylactic acid solution.

The concentration of the recombinant human collagen is 0.5mg/ml, the weight percentage of folic acid is 10%, the weight percentage of HMGB1 polypeptide is 5%, and the concentration of VEGFR-2 antibody is 10 mg/ml.

(4) And continuously electrospinning the composite spinning solution by adopting a dynamic liquid electrostatic spinning method to generate composite nanofibers with orientation, loose fiber structures and large pore diameters, and winding the obtained composite nanofibers on a bare metal stent to form the drug-coated intravascular stent.

The technological parameters of the dynamic liquid electrostatic spinning are as follows: spinning voltage is 12-15 kV, a rotating metal rod is used for receiving fibers, the diameter of the metal rod is 2-4 mm, the receiving distance is 10-20 cm, the rotation rate of a stainless steel rod is 800-1000 rmp, the spinning speed is 3-5 ml/h, and the temperature environment is 60 +/-5 ℃.

(5) And freeze-drying and crosslinking the obtained drug-coated intravascular stent to obtain the drug-coated intravascular stent. Wherein the temperature of freeze drying is-50 to-60 ℃, and the time is 1 h; EDC, NHS and PBS solution are used as cross-linking agents, 20mg of EDC and 50mg of NHS are added into 1ml of PBS solution, the mixture coated on the stent is modified, and the modified mixture is cross-linked on a bare metal stent at the temperature of 25 ℃ for 25 min.

Example 2

(1) preparing a reticular nickel-titanium memory alloy bare metal stent: the nickel-titanium alloy is woven into metal wires, and the metal wires are bent into a nickel-titanium alloy net-shaped framework. Specifically, a nickel-titanium alloy wire is bent into a single ring, and then a plurality of independent wave-shaped bent rings are connected into a pipeline-shaped nickel-titanium alloy framework through the nickel-titanium alloy wire.

(2) And (3) pretreatment of the stent: using a propanol analytically pure solution or a medical ethanol solvent to the obtained reticular nickel-titanium memory alloy bare metal stent, wherein the concentration of the propanol analytically pure solution is 99.5%, and the concentration of the medical ethanol solvent is 75%; cleaning the bracket body by using ultrasonic waves, removing impurities on the surface of the bracket body, cleaning the bracket body by using distilled water by using ultrasonic waves, wherein the frequency of the bracket body is 100kHz through ultrasonic cleaning twice, and the cleaning time is 5-10 min; and drying the cleaned stent body at the drying temperature of 30-40 ℃ for 30-50 min.

The substrate solvent is polylactic acid solution, and the polylactic acid is dissolved in dimethyl sulfoxide according to the concentration of 0.05g/ml in an anaerobic operation box and is uniformly stirred to obtain the polylactic acid solution.

The concentration of the recombinant human collagen is 5mg/ml, the weight percentage of folic acid is 5%, the weight percentage of HMGB1 polypeptide is 5%, and the concentration of VEGFR-2 antibody is 10 mg/ml.

The technological parameters of the dynamic liquid electrostatic spinning are as follows: spinning voltage is 10-15 kV, a rotating metal rod is used for receiving fibers, the diameter of the metal rod is 2-4 mm, the receiving distance is 8-12 cm, the rotation rate of a stainless steel rod is 800-1000 rmp, the spinning speed is 0.1-1 ml/h, and the temperature environment is 60 +/-5 ℃.

(5) And freeze-drying and crosslinking the obtained drug-coated intravascular stent to obtain the drug-coated intravascular stent. Wherein the temperature of freeze drying is-40 to-60 ℃, and the time is 3 hours; EDC, NHS and PBS solution are used as cross-linking agents, 20mg of EDC and 50mg of NHS are added into 1ml of PBS solution, the mixture coated on the stent is modified, and the modified mixture is cross-linked on a bare metal stent at the temperature of 25 ℃ for 25 min.

Example 3

(1) preparing a reticular nickel-titanium memory alloy bare metal stent: the nickel-titanium alloy is woven into metal wires, and the metal wires are bent into a nickel-titanium alloy net-shaped framework. Specifically, the nickel-titanium alloy reticular skeleton is formed by engraving a nickel-titanium alloy pipe by laser, and has wavy patterns after expansion.

(2) And (3) pretreatment of the stent: using a propanol analytically pure solution or a medical ethanol solvent to the obtained reticular nickel-titanium memory alloy bare metal stent, wherein the concentration of the propanol analytically pure solution is 99.5%, and the concentration of the medical ethanol solvent is 75%; cleaning the bracket body by using ultrasonic waves, removing impurities on the surface of the bracket body, cleaning the bracket body by using distilled water by using ultrasonic waves, wherein the frequency of the bracket body is 80kHz by using ultrasonic waves for two times, and the cleaning time is 15 min; and drying the cleaned stent body at the drying temperature of 30-40 ℃ for 40-60 min.

(3) And preparing a composite spinning solution: dissolving the recombinant human collagen, folic acid, HMGB1 polypeptide and VEGFR-2 antibody in a solvent, and mixing uniformly to obtain the composite spinning solution.

The substrate solvent is polylactic acid solution, and the polylactic acid is dissolved in dichloromethane according to the concentration of 0.05g/ml in an anaerobic operation box and is uniformly stirred to obtain the polylactic acid solution.

The concentration of the recombinant human collagen is 0.5mg/ml, the weight percentage of folic acid is 10%, the weight percentage of HMGB1 polypeptide is 5%, and the concentration of VEGFR-2 antibody is 100 mg/ml.

The technological parameters of the dynamic liquid electrostatic spinning are as follows: spinning voltage is 15-20 kV, a rotating metal rod is used for receiving fibers, the diameter of the metal rod is 2-4 mm, the receiving distance is 10-20 cm, the rotation rate of a stainless steel rod is 200-500 rmp, the spinning speed is 1-5 ml/h, and the temperature environment is 60 +/-5 ℃.

(5) And freeze-drying and crosslinking the obtained drug-coated intravascular stent to obtain the drug-coated intravascular stent. Wherein the temperature of freeze drying is-20 to-50 ℃, and the time is 5 hours; EDC, NHS and PBS solution are used as cross-linking agents, 20mg of EDC and 50mg of NHS are added into 1ml of PBS solution, the mixture coated on the stent is modified, and the modified mixture is cross-linked on a bare metal stent at the temperature of 25 ℃ for 25 min.

The size of the nanofiber prepared by the embodiment of the invention is beneficial to the adhesion and growth of endothelial cells; the recombinant human collagen has the effects of anticoagulation and thrombosis prevention, folic acid can improve the elasticity of blood vessels, and the long-term endothelialization promotion can be realized by loading HMGB1 polypeptide and VEGFR-2 antibody; the loose and porous structure is beneficial to the axial parallel arrangement and three-dimensional growth of endothelial cells, and prevents the adhesion and proliferation of other cells; fragment peptides (1-44) of HMGB1 protein are prepared, and the polypeptide has the activity of inducing cell migration.

Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the detailed description is made with reference to the embodiments of the present invention, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which shall be covered by the claims of the present invention.

Claims

1. A drug-coated vascular stent for preventing in-stent restenosis comprises a bare metal stent, and is characterized in that: the preparation method is characterized by comprising the following steps of preparing the artificial collagen by adopting a dynamic liquid electrostatic spinning method, wherein recombinant human collagen, folic acid, HMGB1 fragment peptide and VEGFR-2 antibody are cross-loaded on the bare metal bracket; the concentration of the recombinant human collagen in the composite spinning solution is 0.1-5 mg/ml, the weight percentage of folic acid is 0.1-10%, the weight percentage of HMGB1 polypeptide is 0.1-10%, and the concentration of VEGFR-2 antibody is 10-100 mg/ml.

2. A drug-coated vascular stent to prevent in-stent restenosis as in claim 1, wherein: the bare metal stent is prepared from a nickel-titanium memory alloy material.

3. A preparation method of a drug-coated intravascular stent for preventing restenosis in the stent is characterized by comprising the following steps: the method comprises the following steps:

(3) and preparing a composite spinning solution: dissolving the recombinant human collagen, folic acid, HMGB1 polypeptide and VEGFR-2 antibody in a solvent, and uniformly mixing to obtain a composite spinning solution; the concentration of the recombinant human collagen is 0.1-5 mg/ml, the weight percentage of folic acid is 0.1-10%, the weight percentage of HMGB1 polypeptide is 0.1-10%, and the concentration of VEGFR-2 antibody is 10-100 mg/ml;

4. A method of preparing a drug-coated vascular stent to prevent in-stent restenosis as claimed in claim 3, wherein: in the step (1), the nickel-titanium alloy reticular framework is formed by bending a nickel-titanium alloy wire into a plurality of wave-shaped bending rings which are connected in sequence, or a nickel-titanium alloy wire is bent into a single ring, and then the nickel-titanium alloy wire is used for connecting a plurality of independent wave-shaped bending rings into a pipeline-shaped nickel-titanium alloy framework;

or the nickel-titanium alloy reticular skeleton is formed by engraving a nickel-titanium alloy pipe by laser, and has wavy patterns after expansion.

5. A method for preparing a drug-coated vascular stent to prevent in-stent restenosis as claimed in claim 3 or 4, wherein: in the step (2), the concentration of a propanol analytically pure solution is 99.5 percent, and the concentration of a medical ethanol solvent is 75 percent; the frequency of ultrasonic cleaning the bracket body for two times is 28-100 khz, and the cleaning time is 5-15 min; the drying temperature is 30-40 ℃, and the drying time is 30-60 min.

6. The method for preparing a drug-coated vascular stent for preventing in-stent restenosis as claimed in claim 5, wherein: in the step (3), the solvent is polylactic acid solution, and the polylactic acid is dissolved in one of 1, 4-dioxane, dichloromethane and dimethyl sulfoxide organic solvents according to the concentration of 0.01-0.1 g/ml in an anaerobic operation box and is uniformly stirred to obtain the polylactic acid solution.

7. The method for preparing a drug-coated vascular stent for preventing in-stent restenosis as claimed in claim 6, wherein: in the step (4), the technological parameters of the dynamic liquid electrostatic spinning are as follows: spinning voltage is 8-20 kV, a rotating metal rod is used for receiving fibers, the diameter of the metal rod is 2-4 mm, the receiving distance is 8-20 cm, the rotation rate of a stainless steel rod is 200-1000 rmp, the spinning speed is 0.1-5 ml/h, and the temperature environment is 60 +/-5 ℃.

8. The method for preparing a drug-coated vascular stent for preventing in-stent restenosis as claimed in claim 7, wherein: in the step (5), the temperature of freeze drying is-20 to-60 ℃, and the time is 1 to 5 hours; 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, N-hydroxysuccinimide and PBS (phosphate buffer solution) are used as cross-linking agents, 20mg of EDC and 50mg of NHS are added into 1ml of PBS, the mixture coated on the stent is modified, and the modified mixture is cross-linked on a bare metal stent at the temperature of 25 ℃ for 25 min.

9. The drug-coated vascular stent for preventing in-stent restenosis prepared by the preparation method of claim 3 is used for coronary stents, cerebrovascular stents, renal artery stents or aortic stents.