CN113244462B - A kind of drug-coated vascular stent for preventing restenosis in stent and preparation method thereof - Google Patents

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

A kind of drug-coated vascular stent for preventing restenosis in stent and preparation method thereof

technical field

The invention relates to the technical field of medical blood vessel stents implanted in the human body, in particular to the construction of a human blood vessel loaded with recombinant human collagen and folic acid, cross-linked high mobility group box 1 (High Mobility Group Box 1, HMGB1) fragment peptide and human blood vessel The vascular stent of endothelial growth factor receptor 2 antibody (VEGFR-2/KDR/CD309 antibody) is designed to further specifically promote the capture and homing of endothelial progenitor cells (EPCs) on the basis of meeting the general requirements of vascular stents , proliferation, differentiation, inhibit intimal hyperplasia while achieving rapid endothelialization of blood vessels and increasing the biocompatibility of the stent surface, so as to achieve the postoperative effect of reducing in-stent restenosis and late stent thrombosis.

Background technique

Nowadays, with the improvement of people's living standards and changes in living and eating habits, the incidence of cardiovascular and cerebrovascular diseases remains high, and the incidence of cardiovascular and cerebrovascular diseases is getting younger and younger. Interventional therapy is currently an effective method for the treatment of cardiovascular and cerebrovascular thrombotic diseases, but in-stent restenosis (ISR) and late stent thrombosis (Late Stent Thrombosis, LST) seriously affect its long-term efficacy. Stent placement is the most important and effective method for the treatment of arterial occlusion. The stent placement process is generally as follows: after the catheter-stent-balloon system is placed in the lesion area, the balloon permanently deforms the stent under the action of pressure; after the balloon and catheter are removed, the stent remains in the blood vessel to maintain an expanded state, thereby achieving Sustained blood flow. Stent implantation restores patency of stenotic diseased blood vessels through the mechanical support of stents, which may cause an adaptive response of blood vessels. Endothelial Cells (ECs) injury is the initiating factor of vascular response. The stent damages the inner wall of the blood vessel, leading to endothelial denudation and promoting local thrombosis.

An ideal vascular stent should contain the following properties: ① good biocompatibility; ② mesh structure, which enables the stent itself to have a certain deformability to ensure the compliance of the vascular stent; ③ can provide a certain mechanical strength after being placed in the blood vessel It supports blood vessels and maintains blood flow; ④ has the function of capturing EPCs and promoting the migration, proliferation and differentiation of EPCs into ECs, and achieving rapid endothelialization of blood vessels; The characteristics of the affinity; ⑥ can effectively reduce the level of homocysteine (Hcy) in the blood, improve vascular endothelial function, and improve the elasticity of blood vessels.

At present, the large-scale clinical use of drug-coated metal vascular stents plays a role of permanent support, but the implantation of foreign bodies causes thrombosis and restenosis problems. At the same time, the drug coating inhibits intimal growth and also inhibits endothelial growth. Existing stent coatings often hinder intimal hyperplasia while hindering the overall functional and structural regeneration of the endothelium, so there is still the potential for late thrombosis due to insufficient endothelial covering regeneration. CD34 antibody is by far the most commonly used EPCs for stent coating to capture bioactive molecules. Although CD34 antibodies have been widely recognized in the field of mediating the capture of EPCs, there are some problems with the use of CD34 antibodies. CD34 is not entirely specific to 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 a poor effect on promoting endothelialization in the short term and resisting in-stent restenosis. Looking for other antibodies that can be used to capture EPCs, this antibody is expressed in differentiated ECs in addition to specific expression in EPCs, capture EPCs more specific than CD34 antibody for further reduction of in-stent restenosis and late stenting Thrombosis is of great significance.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a drug-coated vascular stent for preventing restenosis in the stent and a preparation method thereof, especially loading folic acid and recombinant human collagen, cross-linked HMGB1 fragment peptide and VEGFR-2 antibody on the vascular stent and its preparation method. Preparation. VEGFR-2 antibody can rapidly capture EPCs at the injured site of blood vessels, HMGB1 can promote the migration, proliferation and differentiation of EPCs into ECs, and accelerate the re-endothelialization of injured blood vessels. Recombinant human collagen can inhibit the deposition of thrombotic components at the site of vascular injury. , Folic acid enhances the elasticity and compliance of blood vessels.

The present invention adopts following technical scheme to realize:

A drug-coated vascular stent for preventing in-stent restenosis includes a bare metal stent, on which the bare metal stent is cross-linked and loaded with recombinant human collagen, folic acid, HMGB1 fragment peptide and VEGFR-2 antibody.

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

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

(1) Preparation of mesh nickel-titanium memory alloy bare metal stent: weaving nickel-titanium alloy into metal wire, and bending the metal wire to make a nickel-titanium alloy mesh frame;

(2) Pretreatment of stents: The obtained reticulated nickel-titanium memory alloy bare metal stents are used analytically pure solution of propanol or medical ethanol solvent, and ultrasonic waves are used to clean the stent body to remove impurities on the surface of the stent body, and then use distilled water for use. Ultrasonic cleaning of the stent body, drying the cleaned stent body;

(3) Preparation of composite spinning solution: Dissolve recombinant human collagen, folic acid, HMGB1 polypeptide, and VEGFR-2 antibody in a solvent and mix well to obtain a composite spinning solution;

(4) Using the dynamic liquid electrospinning method, continuous electrospinning of the composite spinning solution generates composite nanofibers with orientation, loose fiber structure and large pore size, and the obtained composite nanofibers are wrapped around a bare metal stent to form a drug-coated blood vessel. bracket;

(5) The drug-coated vascular stent obtained above is freeze-dried and cross-linked to obtain the drug-coated vascular stent.

In step (1), the nickel-titanium alloy mesh skeleton is bent into a plurality of successively connected wave-shaped bending rings by using a nickel-titanium alloy wire, or a single nickel-titanium alloy wire is bent into a single ring, and then Then, a plurality of independent zigzag bending rings are connected to form a pipe-shaped nickel-titanium alloy skeleton with a nickel-titanium alloy wire. Alternatively, the nickel-titanium alloy mesh skeleton is laser-engraved from a nickel-titanium alloy tube, and has a wavy pattern after expansion.

In step (2), the concentration of the analytically pure solution of propanol is 99.5%, and the concentration of the medical ethanol solvent is 75%; the frequency of the two ultrasonic cleaning of the stent body is 28~100khz, and the cleaning time is 5~15min; the drying temperature is 30~40°C , the drying time is 30~60min.

In step (3), the base solvent is a polylactic acid solution, and in an anaerobic operation box, polylactic acid is dissolved in 1,4-dioxane, dichloromethane, and dimethyl at a concentration of 0.01-0.1 g/ml. One of organic solvents such as sulfoxide, stir evenly to obtain a polylactic acid solution. The concentration of recombinant human collagen is 0.1~5mg/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), the concentration of VEGFR-2 antibody 10~100mg/ml.

In step (4), the process parameters of dynamic liquid electrospinning are: the spinning voltage is 8~20kV, the fiber is received by a rotating metal rod, the diameter of the metal rod is 2~4mm, the receiving distance is 8~20cm, and the stainless steel rod is rotated The speed is 200~1000rmp, the spinning speed is 0.1~5ml/h, and the temperature environment is 60±5℃.

In step (5), the temperature of freeze-drying is -20~-60°C, and the time is 1~5h; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (1-[ 3-(Dimethylamino)propyl]-3-Ethylcarbodiimide, EDC), N-Hydroxysuccinimide (NHS), Phosphate BufferSaline (PBS) solution as cross-linking agent, each 1ml of PBS 20 mg of EDC and 50 mg of NHS were added to the solution to modify the mixture coated on the stent and cross-link it on the bare metal stent at a temperature of 25° C. and a time of 25 min.

The vascular stent for preventing in-stent restenosis according to the present invention is particularly loaded with recombinant human collagen and folic acid, cross-linked HMGB1 fragment peptide and VEGFR-2 antibody on the vascular stent. VEGFR-2 antibody can rapidly capture EPCs at the injured site of blood vessels, HMGB1 can promote the migration, proliferation and differentiation of EPCs into ECs, and accelerate the re-endothelialization of injured blood vessels. Recombinant human collagen can inhibit the deposition of thrombotic components at the site of vascular injury. , Folic acid enhances the elasticity and compliance of blood vessels.

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

From the perspective of the anatomical structure of blood vessels, arterial vessels are generally composed of three layers of membranes, the innermost layer is the intima, the part in direct contact with blood is composed of a large number of endothelial cells, and the endothelial cells are supported by the basement membrane; the middle layer is the media, which is composed 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 subendothelial matrix proteins and maintaining the balance between coagulation and fibrinolytic systems by synthesizing and releasing active molecules. Damage to the intima exposes the subendothelial layer containing collagen and tissue factor to the blood circulation, causing activation of platelets and the coagulation system. Therefore, it is an important condition to ensure the normal function of blood vessels and maintain the integrity of blood vessels to form a complete coverage of endothelial cells on the intima surface of blood vessels and form a natural barrier between the subintimal tissue and blood flow. Vascular endothelial cell injury and slow repair after injury are the 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 involving and interacting with multiple mechanisms. Poor repair of damaged blood vessels can lead to excessive thickening of the intima and narrowing of the lumen. Serious reduction of blood perfusion can cause myocardial infarction, cerebral infarction and other diseases.

At present, in order to prevent the occurrence of in-stent restenosis (ISR) and late stent thrombosis (Late Stent Thrombosis, LST), promoting the in situ endothelialization of the stent surface has become a research hotspot. Generally, when scaffolds are implanted in vivo to achieve in situ endothelialization, there are two main sources of ECs, EPCs and adhesion, proliferation and aggregation of ECs on the scaffold surface. Uninjured endothelial cells can migrate to the damaged site within 1-2 cm from the stent implantation site. The immobilization of bioactive molecules on the surface of the material can also promote the adhesion and proliferation of cells on the surface of the material, and ultimately achieve the endothelialization of damaged blood vessels. EPCs are adult stem cells with 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 be mobilized from bone marrow to peripheral blood, chemotactic to the site of endothelial injury, and then differentiate and proliferate into ECs to replace damaged ECs to achieve normal function. EPCs overcome the defect of pure endothelial cell proliferation and enable rapid endothelialization of the scaffold surface. Rapid endothelialization after stent placement is an effective method to reduce stent thrombosis and in-stent restenosis. The construction of a vascular scaffold that can promote the migration, adhesion and proliferation of EPCs can not only promote the homing of EPCs on the surface of the material, capture the EPCs in the circulating blood, and deposit them on the surface of the material to induce their differentiation into endothelial cells, but also on the surface of the scaffold. The material can promote the proliferation of endothelial cells. Combining the two approaches can prevent stent restenosis and thrombosis.

In situ endothelialization not only achieves the capture and promotion of differentiation of EPCs, but also maintains the capacity of ECs for a long time. While many approaches have been explored, future studies may combine these strategies to mimic the ECs production process by combining captured biomolecules, growth stimulators, and suitable matrix materials. EPCs play a role in endovascular repair by secreting a variety of cytokines, differentiating into mature endothelial cells, and promoting the proliferation and migration of original residual endothelial cells and endothelial progenitor cells, thereby repairing damaged blood vessels. And their function relies on the synergistic effect of the local microenvironment and other populations on vascular injury. The membrane proteins of CD34, CD133 and VEGFR-2 are expressed on the cell membrane of EPCs. Coronary stents can be improved by using antibodies specific to the receptors on the surface of EPCs. The antibodies of the receptors on the surface of EPCs can be immobilized on the surface of the stent. Rapid capture of EPCs to achieve in situ endothelialization at the site of vascular injury. CD34 antibody is by far the most commonly used EPCs for stent coating to capture bioactive molecules. Although CD34 antibodies have been widely recognized in the field of mediating the capture of EPCs, there are some problems with the use of CD34 antibodies. CD34 is not entirely specific to 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 a poor effect on promoting endothelialization in the short term and resisting in-stent restenosis. VEGFR-2 antibody is another antibody used to capture EPCs. In addition to its specific expression in EPCs, VEGFR-2 is also expressed in differentiated ECs, which indicates that VEGFR-2 antibody captures EPCs more specifically than CD34 antibody .

ECs injury is the initiating factor of vascular response. The stent damages the inner wall of the blood vessel, leading to endothelial denudation and promoting local thrombosis. The degree of thrombosis is closely related to the depth of the stent into the vessel. Inflammation is a process by which blood vessels repair themselves, and monocytes and polynuclear leukocytes adhere to the elastic intima. Smooth muscle cells migrate to the intima and proliferate. With the transition from a highly differentiated contractile type to a poorly differentiated synthetic type, they secrete a large amount of protein and collagen, which are deposited in the media and adventitia of blood vessels, resulting in fibrosis.

HMGB1 is a non-histone DNA-binding protein that promotes the assembly of nuclear protein complexes in the nucleus and acts as an inflammatory factor that triggers various pathological processes, including inflammatory cytokines, by mediating receptors such as TLR2, TLR4 and RAGE release, cell migration and angiogenesis. Not only has it been shown to be an advanced mediator of infection, but it is also thought to regulate angiogenesis in pathological conditions by interacting with a receptor expressed on the EC membrane, the receptor for advanced glycation end products (RAGE). ) combined to mediate the activation of ECs. Targeting 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 effect of stimulating cell migration. Preferably, the smallest peptide fragment among the fragments having cell migration stimulating activity, that is, the HMGB1 fragment peptide comprising at least the 17th to 25th amino acid sequence of the HMGB1 protein. Fragment peptides composed of a part of amino acids of HMGBI protein have cell migration stimulating activity, and are not particularly limited. Examples of such fragment peptides include at least HMGB1 fragment peptides (1-44) and excluding the full length of HMGB1 protein. Any HMGBI-derived fragment peptides in the above are the upper limit peptides, but are not limited to these. Cross-linking HMGB1 fragment peptide and VEGFR-2 antibody on vascular stent can promote the rapid capture of EPCs by the stent, promote the differentiation and proliferation of EPCs, and achieve rapid endothelialization of blood vessels.

Stent implantation restores patency of stenotic diseased blood vessels through the mechanical support of stents, which may cause an adaptive response of blood vessels. Stent implantation is a mechanical process, and the factors affecting the local mechanical environment of blood vessels include balloon inflation pressure and mechanical properties of blood vessel wall components. Stent overexpansion refers to the stent state in which the overall or local stent exceeds its corresponding reference vessel boundary immediately after PCI. Usually, the diameter of the stent is enlarged to a size larger than the lumen diameter of the reference vessel to ensure that the stent adheres well to the vessel wall, is positioned accurately, has a large lumen collection and preservation ability, and an ideal blood flow recovery degree. The larger the diameter of the stent, the more serious the damage to the vessel wall and plaque during the expansion process, which is likely to lead to an increase in the rate of restenosis, and even lead to serious malignant complications such as vessel rupture. Now, through further optimization or surface modification of the drug-loaded coating, recombinant human collagen is characterized by extremely low immune rejection, anticoagulant, and a certain affinity with endothelial cells, which promotes vascular damage. Re-endothelialization.

High homocysteine (High Homocysteine, HHcy) leads to the weakening of arterial endothelial function, the degradation of arterial elastic fibers, resulting in decreased blood vessel elasticity, which in turn causes arterial elasticity and compliance to decrease. Atherosclerosis also has a significant synergistic effect with hypertension in causing cardiovascular and cerebrovascular events. Supplementing the water-soluble vitamin folic acid can effectively reduce the level of homocysteine in the blood, improve vascular endothelial function, and improve the elasticity of blood vessels. In hyperhomocysteinemia, most of the elastic layers are disordered, elastic fibers are broken or fragmented, smooth muscle cells are hypertrophied and rearranged, the elastin ratio is significantly reduced, and the compliance of the arterial wall is reduced. As an important coenzyme in the methionine cycle, folic acid plays an important role as a methyl donor in the process of Hcy synthesis of methionine. When the content of folic acid in the body is high, it can promote the remethylation of Hcy to generate methionine, thereby reducing the level of Hcy.

The scheme of the present invention has the following advantages:

(1) The present invention is the first to prepare a vascular stent loaded with recombinant human collagen, folic acid, HMGB1 polypeptide, and VEGFR-2 antibody. On the basis of meeting the requirements of general vascular stents, it further specifically promotes the capture, homing, proliferation and differentiation of EPCs, inhibits excessive intimal hyperplasia and achieves rapid endothelialization of blood vessels, and has good biocompatibility and compliance.

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

(3) The method of dynamic liquid electrospinning is used to realize the loading of functional drugs on the surface of bare metal stents. Compared with the soaking method and the spraying method, this method can not only protect the biological activity of drugs or factors to the greatest extent. , the drug or factor can also be released in a controllable and sustained manner through diffusion and degradation mechanisms.

The present invention has a reasonable design. By loading recombinant human collagen, folic acid, HMGB1 polypeptide and VEGFR-2 antibody, and using dynamic liquid electrospinning method, the nanofibers obtained from the above four drugs are loaded on a bare metal stent, thereby obtaining The vascular stent with short-term anticoagulation performance and long-term endothelialization promotion can be used not only for coronary stents, but also for cerebrovascular stents, renal artery stents and aortic stents, and has good practical application value.

Detailed ways

Specific embodiments of the present invention will be described in detail below.

The composite drug-coated vascular stent described in the present invention comprises a bare metal stent (made of nickel-titanium memory alloy material), and the stent body is loaded with recombinant human collagen, folic acid, HMGB1 fragment peptide and VEGFR-2 antibody Cross-linked to the surface of bare metal stents. Specifically, the recombinant human collagen, HMGB1 polypeptide, VEGFR-2 antibody and folic acid are dissolved in a solvent to obtain a composite spinning solution; the recombinant human collagen, folic acid, HMGB1 fragment peptide and VEGFR are separated by dynamic liquid electrospinning technology. The composite spinning solution of -2 antibody is loaded on a nickel-titanium memory alloy bare metal scaffold, and the composite spinning solution is continuously electrospun to form a composite nanolayer with certain orientation, loose fiber structure and large pore size, forming a loaded recombinant human source. The vascular scaffold of collagen and folic acid, cross-linked HMGBI fragment peptide and VEGFR-2 antibody promotes EPCs capture, homing, proliferation, differentiation, and achieves rapid endothelialization of blood vessels. The present invention can be used not only for coronary stents, but also for cerebrovascular stents, renal artery stents, aortic stents, and the like.

Electrospinning technology is a common method for preparing scaffolds in tissue engineering research. It can continuously prepare nano-scale to micro-scale fibers, which mimic the size and structure of natural extracellular matrix, which is conducive to the adhesion and growth of endothelial cells and prevents the growth of other cells. Proliferation and adhesion. At the same time, the electrospinning method has a wide selection of raw materials, and synthetic polymer materials, natural polymer materials and blended composites can be prepared by electrospinning method. In recent years, based on the requirements of tissue engineering scaffolds for fiber structure and function, electrospinning methods have been gradually developed and applied. The coaxial electrospinning method is mainly used to prepare fibers loaded with functional drugs or factors. By this method, functional drugs can be loaded on the surface of the stent, which can not only protect the biological activity of the drugs or factors to the greatest extent, but also pass Diffusion, degradation mechanisms, etc. enable controlled and sustained release of drugs or factors. In the present invention, by loading recombinant human collagen, HMGB1 polypeptide, VEGFR-2 antibody and folic acid, and using dynamic liquid electrospinning, nanofibers obtained from the above four drugs can be obtained with short-term anticoagulation performance and long-term endothelial promotion. vascular stents. Fiber materials prepared by electrospinning technology have large pore size and high porosity structure to maintain the transport of nutrients and ensure the three-dimensional migration and growth of cells.

Example 1

A specific preparation method of a drug-coated vascular stent for preventing in-stent restenosis is as follows:

(1) Preparation of a reticulated nickel-titanium memory alloy bare metal stent: the nickel-titanium alloy is woven into a metal wire, and the metal wire is bent to form a nickel-titanium alloy mesh frame. Specifically, a nickel-titanium alloy wire is bent into a plurality of successively connected wave-shaped bending rings and connected to form a pipe-shaped nickel-titanium alloy skeleton.

(2) Pretreatment of stents: use the obtained reticulated nickel-titanium memory alloy bare metal stents with a propanol analytically pure solution or a medical ethanol solvent, the concentration of the analytically pure propanol solution is 99.5%, and the concentration of the medical ethanol solvent is 75% ; Then use ultrasonic to clean the stent body to remove impurities on the surface of the stent body, and then use ultrasonic waves to clean the stent body through distilled water. The frequency of the two ultrasonic cleaning of the stent body is 50 kHz, and the cleaning time is 10 min; the cleaned stent body is dried at the drying temperature. The temperature is 30~40℃, and the drying time is 50~60min.

(3) Preparation of composite spinning solution: Dissolve recombinant human collagen, folic acid, HMGB1 polypeptide and VEGFR-2 antibody in a base solvent and mix well to obtain a composite spinning solution.

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

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

(4) Using the dynamic liquid electrospinning method, continuous electrospinning of the composite spinning solution generates composite nanofibers with orientation, loose fiber structure and large pore size, and the obtained composite nanofibers are wrapped around a bare metal stent to form a drug-coated blood vessel. bracket.

The process parameters of dynamic liquid electrospinning are: the spinning voltage is 12~15kV, the fiber is received by a rotating metal rod, the diameter of the metal rod is 2~4mm, the receiving distance is 10~20cm, the rotation rate of the stainless steel rod is 800~1000rmp, The spinning speed is 3~5ml/h, and the temperature environment is 60±5℃.

(5) The drug-coated vascular stent obtained above is freeze-dried and cross-linked to obtain the drug-coated vascular stent. Among them, the freeze-drying temperature was -50~-60 °C, and the time was 1 h; EDC, NHS, and PBS solutions were used as cross-linking agents, and 20 mg of EDC and 50 mg of NHS were added to each 1 ml of PBS solution. Modification was carried out, and it was cross-linked on a bare metal scaffold at a temperature of 25 °C and a time of 25 min.

Example 2

A specific preparation method of a drug-coated vascular stent for preventing in-stent restenosis is as follows:

(1) Preparation of a reticulated nickel-titanium memory alloy bare metal stent: the nickel-titanium alloy is woven into a metal wire, and the metal wire is bent to form a nickel-titanium alloy mesh frame. Specifically, a nickel-titanium alloy wire is used to bend into a single ring, and then a plurality of independent zigzag-shaped bending rings are connected with the nickel-titanium alloy wire to form a pipe-shaped nickel-titanium alloy skeleton.

(2) Pretreatment of stents: use the obtained reticulated nickel-titanium memory alloy bare metal stents with a propanol analytically pure solution or a medical ethanol solvent, the concentration of the analytically pure propanol solution is 99.5%, and the concentration of the medical ethanol solvent is 75% ; Then use ultrasonic to clean the stent body to remove impurities on the surface of the stent body, and then use ultrasonic waves to clean the stent body through distilled water. The frequency of the two ultrasonic cleaning of the stent body is 100kHz, and the cleaning time is 5~10min; the cleaned stent body is dried. The drying temperature is 30~40℃, and the drying time is 30~50min.

(3) Preparation of composite spinning solution: Dissolve recombinant human collagen, folic acid, HMGB1 polypeptide and VEGFR-2 antibody in a base solvent and mix well to obtain a composite spinning solution.

The base solvent is a polylactic acid solution, and in an anaerobic operation box, polylactic acid is dissolved in dimethyl sulfoxide at a concentration of 0.05 g/ml, and stirred evenly to obtain a polylactic acid solution.

The concentration of recombinant human collagen is 5 mg/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.

(4) Using the dynamic liquid electrospinning method, continuous electrospinning of the composite spinning solution generates composite nanofibers with orientation, loose fiber structure and large pore size, and the obtained composite nanofibers are wrapped around a bare metal stent to form a drug-coated blood vessel. bracket.

The process parameters of dynamic liquid electrospinning are: the spinning voltage is 10~15kV, the fiber is received by a rotating metal rod, the diameter of the metal rod is 2~4mm, the receiving distance is 8~12cm, the rotation rate of the stainless steel rod is 800~1000rmp, The spinning speed is 0.1~1ml/h, and the temperature environment is 60±5℃.

(5) The drug-coated vascular stent obtained above is freeze-dried and cross-linked to obtain the drug-coated vascular stent. Among them, the temperature of freeze-drying is -40~-60 °C, and the time is 3h; EDC, NHS, PBS solution is used as cross-linking agent, and 20 mg of EDC and 50 mg of NHS are added to each 1 ml of PBS solution. Modification was carried out, and it was cross-linked on a bare metal scaffold at a temperature of 25 °C and a time of 25 min.

Example 3

A specific preparation method of a drug-coated vascular stent for preventing in-stent restenosis is as follows:

(1) Preparation of a reticulated nickel-titanium memory alloy bare metal stent: the nickel-titanium alloy is woven into a metal wire, and the metal wire is bent to form a nickel-titanium alloy mesh frame. Specifically, the nickel-titanium alloy mesh skeleton is made of a nickel-titanium alloy tube by laser engraving, and has a wavy pattern after expansion.

(2) Pretreatment of stents: use the obtained reticulated nickel-titanium memory alloy bare metal stents with a propanol analytically pure solution or a medical ethanol solvent, the concentration of the analytically pure propanol solution is 99.5%, and the concentration of the medical ethanol solvent is 75% ; Then use ultrasonic to clean the stent body to remove impurities on the surface of the stent body, and then use ultrasonic waves to clean the stent body through distilled water. The frequency of the two ultrasonic cleaning of the stent body is 80kHz, and the cleaning time is 15min; the cleaned stent body is dried at a drying temperature. The temperature is 30~40℃, and the drying time is 40~60min.

(3) Preparation of composite spinning solution: Dissolve recombinant human collagen, folic acid, HMGB1 polypeptide and VEGFR-2 antibody in a solvent and mix well to obtain a composite spinning solution.

The base solvent is a polylactic acid solution. In an anaerobic operation box, polylactic acid is dissolved in dichloromethane at a concentration of 0.05 g/ml, and stirred evenly to obtain a polylactic acid solution.

The concentration of recombinant human collagen is 0.5 mg/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.

(4) Using the dynamic liquid electrospinning method, continuous electrospinning of the composite spinning solution generates composite nanofibers with orientation, loose fiber structure and large pore size, and the obtained composite nanofibers are wrapped around a bare metal stent to form a drug-coated blood vessel. bracket.

The process parameters of dynamic liquid electrospinning are: the spinning voltage is 15~20kV, the fiber is received by a rotating metal rod, the diameter of the metal rod is 2~4mm, the receiving distance is 10~20cm, the rotation rate of the stainless steel rod is 200~500rmp, The spinning speed is 1~5ml/h, and the temperature environment is 60±5℃.

(5) The drug-coated vascular stent obtained above is freeze-dried and cross-linked to obtain the drug-coated vascular stent. Among them, the temperature of freeze-drying was -20~-50°C, and the time was 5h; EDC, NHS, PBS solution was used as cross-linking agent, and 20 mg of EDC and 50 mg of NHS were added to each 1 ml of PBS solution. Modification was carried out, and it was cross-linked on a bare metal scaffold at a temperature of 25 °C and a time of 25 min.

The size of the nanofibers prepared in the examples of the present invention is beneficial to the adhesion and growth of endothelial cells; the recombinant human collagen has the effect of anti-coagulation and preventing thrombosis, folic acid can improve the elasticity of blood vessels, and at the same time load HMGB1 polypeptide and VEGFR-2 antibody It can achieve long-term promoting endothelialization; its loose and porous structure is conducive to the parallel arrangement and three-dimensional growth of endothelial cells around the axis, preventing the adhesion and proliferation of other cells; the fragment peptide (1-44) of the HMGB1 protein is prepared, which is a Has the activity of inducing cell migration.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the embodiments of the present invention have been described in detail, those of ordinary skill in the art should understand that the technical solutions of the present invention should be modified. Or equivalent replacements do not depart from the spirit and scope of the technical solutions of the present invention, and all should be included in the protection scope of the claims of the present invention.

Claims (9)

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:

(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; 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) 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.

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.