KR101770827B1 - Stent for selectively capturing epc and process for producing the same - Google Patents

Abstract

The present invention relates to a stent capable of selectively capturing vascular endothelial progenitor cells and a method for producing the same. More particularly, the present invention relates to a stent in which a silane compound layer, a functional group layer, a hydrophilic polymer layer and a bioligand layer are sequentially bonded on a stent framework, and a method for producing the same.

Description

TECHNICAL FIELD [0001] The present invention relates to a stent capable of selectively capturing vascular endothelial progenitor cells and a method for manufacturing the same.

The present invention relates to a stent capable of selectively capturing vascular endothelial progenitor cells and a method for producing the same. More particularly, the present invention relates to a stent in which a silane compound layer, a functional group layer, a hydrophilic polymer layer and a bioligand layer are sequentially bonded on a stent framework, and a method for producing the same.

Recently, coronary artery disease, such as angina pectoris and myocardial infarction, has been increasing, leading to adult death. Coronary artery disease is a disease that occurs in blood vessels surrounding the heart and causes blood supply disorders to the heart muscle. The most common cause of such coronary artery disease is arteriosclerosis. An increase in the plaque formed by the combination of cholesterol, fat, and other components of the blood in the coronary arteries will cause coronary stenosis, resulting in a decrease in blood supply to the heart muscle, resulting in insufficient nutrients and oxygen . This can lead to chest pain (angina pectoris) or myocardial infarction, and even death in severe cases.

The use of bygraft surgery, balloon angioplasty, and stenting for the treatment of coronary artery disease have recently been reported. In recent years, It is becoming popular.

A stent is a cylindrical tube-shaped precision medical device that is used to normalize blood flow by inserting it into a narrowed or clogged conjunctiva when the blood vessel is clogged by blood clots, malignant or benign disease occurs and the flow is obstructed. The catheter is placed on the balloon at the end of the catheter and moves to the lesion. The balloon is inflated in the lesion, thereby expanding the narrowed blood vessel while expanding the stent to its original diameter. The incidence of restenosis is lower than that of balloon dilatation, and it is now the center of coronary intervention.

However, even after stenting, restenosis, which causes narrowing of the blood vessels at about 20%, occurs, and the restenosis rate is higher when the stent restenosis is performed. The drug-eluting stent, introduced in the early 2000s, has consistently delivered topically effective drugs for restenosis, dramatically reducing intra-stent restenosis. Currently, Cypher, Taxus, Endeavor, etc. are under FDA approval, and most of the lesions have almost no resurrection due to restenosis. Thus, drug-eluting stents have dramatically reduced restenosis, a long-awaited restenosis of coronary intervention, but the inhibition of excessive neointimal hyperplasia has long been associated with the risk of stent thrombosis . According to Iakovou, stent thrombosis occurred in 1.3% of all patients with drug-eluting stents and 45% of patients with stent thrombosis (Iakovou I, et al., Incidence, predictors, and outcome of thrombosis after successful implantation of drug-eluting stents, JAMA 2005; 293: 2126-2130).

Therefore, thrombosis of drug - eluting stents, especially late stent thrombosis occurring after 1 year, is now the most important issue in the field of coronary intervention. To solve this problem, a stent has recently been developed that captures endothelial progenitor cells (EPCs) and rapidly induces endothelialization of endothelial cells in the treated area. However, there is a problem in that the restenosis rate is raised again. (Aoki J, et al., Endothelial progenitor cell capture by stents coated with antibody against CD34, JACC 2005; 45: 1574-1579). In the case of a drug-coated stent, the biocompatibility of the surface may not be very important due to the non-selective effect of the drug. However, in the case of coating a bio-ligand other than a drug, nonspecific adsorption of the protein depending on the biocompatibility of the surface, It is the most important factor that affects performance. This is because the cell selectivity of the antibody that captures vascular endothelial progenitor cells is low, and the biocompatibility of the surface of the stent itself is poor and non-specific adsorption of the protein occurs.

In conclusion, the drug-eluting stent, which solved the problem of restenosis which was the biggest problem in the history of coronary intervention, caused a new problem of stent thrombosis despite various applications. The stent for solving the problem by capturing the vascular endothelial cells is also having a problem of restenosis because the surface properties and the cell selectivity of the biorigand are not excellent. Several stent platforms have been studied, but stents with superior biocompatibility have not yet been developed.

Since the environment of the living body is very complicated and maintains a high order, when the biomaterial is inserted and brought into contact with the living tissue, the biological order is broken at the site, and various biochemical reactions and deformation are forced to occur. Therefore, biocompatibility is one of the most important biomaterials. In order to proceed the biocompatibility of biomaterials without rejection, the non-specific adsorption of proteins, tissue treatment / regeneration the reaction on the biomaterial surface such as tissue healing / regeneration should be able to be effectively controlled by the surface modification technique. Because the stent is no exception, a high degree of surface treatment technology is essential in order to suppress the biotransduction reaction to the stent and properly implement the designed function. The biocompatibility of the stent is determined according to the surface treatment technique, and the loading amount, the diffusion rate, and the in vivo efficacy of the active ingredient such as drug and antibody can be controlled.

In the case of surface-treated stents currently on the market, organic polymers are simply physically adsorbed onto the surface of the stent metal for drug loading. However, the heart is a very dynamic organ and the blood flow of the cardiovascular system is so fast that the physically adsorbed organic polymer can desorb and enter the blood vessels, causing inflammation and thrombogenic reactions. Accordingly, in order to solve the problems of the conventional stent, it is very urgently required to develop a surface treatment technique which is coated with a desired substance more stably but less in a biodejection reaction.

A fundamental object of the present invention is to provide a stent in which a silane compound layer, a functional group layer, a hydrophilic polymer layer, and a bio-ligand layer are sequentially bonded on a stent framework.

Still another object of the present invention is to provide a method for the production of a stent, comprising: (i) introducing a functional group onto a stent skeleton surface into which a hydroxyl group has been introduced through a silanation reaction; (ii) grafting the hydrophilic polymer thin film by coupling the hydrophilic polymer to the functional group; (iii) activating the end of the grafted hydrophilic polymer; And (iv) immobilizing a bioligand capable of capturing the endothelial cell precursor cells on the activated end of the hydrophilic polymer.

The above-described basic object of the present invention can be achieved by providing a stent in which a silane compound layer, a functional group layer, a hydrophilic polymer layer and a bio-ligand layer are sequentially bonded on a stent framework.

In the stent of the present invention, the stent framework may be selected from stainless steel, a nickel-chromium alloy, a nickel-titanium alloy, a cobalt-chromium alloy, tantalum, titanium, aluminum, zirconium, chromium or nickel.

In the stent of the present invention, the functional group may be selected from an amine group, an epoxide group or a carboxyl group.

In the stent of the present invention, the hydrophilic polymer is a polymer containing a hydroxyl group or an ethylene oxide group in the chain, and examples thereof include polyvinyl alcohol, polylysine, polyacrylic acid, polyacrylamide, polyurethane, Nitril-co-acrylic acid), synthetic polymers such as polyethylene glycol or polyethyleneimine, and natural polymers such as chitosan, dextran or cellulose.

In the stent of the present invention, the bioligand may be selected from an antibody, an aptamer, or a peptide that binds to vascular endothelial cadherin.

Still another object of the present invention described above is to provide a method for manufacturing a stent, comprising: (i) introducing a functional group onto a hydroxyl group-introduced stent skeleton surface through a silanation reaction; (ii) grafting the hydrophilic polymer thin film by coupling the hydrophilic polymer to the functional group; (iii) activating the end of the grafted hydrophilic polymer; And (iv) immobilizing a bioligand capable of capturing the endothelial cell precursor cells on the activated end of the hydrophilic polymer.

In the method of the present invention, the stent framework may be selected from stainless steel, a nickel-chromium alloy, a nickel-titanium alloy, a cobalt-chromium alloy, tantalum, titanium, aluminum, zirconium, chromium or nickel.

In the process of the present invention, said silanization reaction is carried out using a compound of formula (I)

X- (CH ₂ ) _n -Si-Y _m ????? (I)

In the above formula (I), X is a functional group capable of coupling with a polymer or an organic compound, and may be an amine group, a halide group, an epoxy group, an aldehyde group, or an acetal group; Y is a functional group which can be liberated by a coupling reaction with a solid surface, which is a halide group, a methoxy group or an ethoxy group; n is an integer from 1 to 25; m is an integer of 1 to 3;

The surface of the stent modified with the new functional group can be directly coupled with the preformed polymer by using the functional group, so that the polymer thin film can be formed. Biopolymers and various synthetic polymers known to be biocompatible have a nucleophile capable of carrying out most chemical reactions. Therefore, it is important to introduce a functional group having high reactivity with the nucleophile through the silanization reaction. Functional groups such as an epoxy group and a carboxyl group are suitable for such a reaction.

In the method of the present invention, the hydrophilic polymer is a polymer containing a hydroxyl group or an ethylene oxide group in the chain. For example, the hydrophilic polymer may be a polyvinyl alcohol, a polylactic acid, a polyacrylic acid, a polyacrylamide, Synthetic polymers such as poly (acrylonitrile-co-acrylic acid), polyethylene glycol or polyethyleneimine, and natural polymers such as chitosan, dextran or cellulose.

It has been reported that the hydrophilicity and fluidity of the above-mentioned polymer inhibits nonspecific adsorption of proteins on its surface. In addition, these polymers have functional groups such as hydroxyl groups and amine groups, and they can immobilize other bioligands such as antibodies after grafting.

To selectively capture only vascular endothelial progenitor cells, the bioligands must be stably immobilized on the biocompatible stent surface. Functional groups introduced in a large amount due to the polymer thin film can be coupled with functional groups of the bioligand through various organic chemical synthesis methods. For example, if a large amount of carboxyl groups is introduced on the surface of the stent, a stable amide bond with the amine group of the antibody can be formed through amide bond formation.

In the method of the present invention, the bioligand may be selected from an antibody, an aptamer, or a peptide that binds to vascular endothelial cadherin. The bioligands are known to be capable of selectively binding to cells.

The method of the present invention is a combination of silanization, polymer grafting, and bioligand immobilization. When a bio-compatible polymer and a bioligand capable of capturing vascular endothelial progenitor cells are used together, the stent restenosis and thrombosis are very effectively inhibited .

The present invention can be used for a stent biomaterial, and there is no particular limitation on its use. For example, it can be a cardiovascular treatment, a stroke treatment, a urethral treatment, or the like.

In addition, the method of the present invention may include other techniques, such as material cleaning, electropolishing, sterilization, etc., which are typically applied to biomaterials as needed.

When the stent according to the present invention is used, the degree of restenosis after the procedure is remarkably reduced, but the vascular endothelialization is rapidly promoted. In addition, according to the present invention, hydrophilic polymers are grafted onto a surface of a general metal stent, and the bioligands are sequentially immobilized thereon, thereby suppressing the adsorption of components such as inflammatory cells or proteins to a maximum extent and selectively capturing only vascular endothelial progenitor cells It is possible to provide a stent surface having excellent biocompatibility.

Figure 1 is one embodiment of the stent of the present invention.
FIG. 2 is a fluorescence microscope photograph showing how EPC and THP-1 change the adsorption behavior of a conventional metal stent and the surface of a stent surface-treated according to the present invention. Green fluorescence represents EPC, and red fluorescence represents THP-1.
3 is a high-resolution electron micrograph showing how EPC is captured on the surface of a surface-treated stent according to the present invention.
FIG. 4 is a graph showing the results of clinical trials of an animal in which the stent of the present invention is applied to rabbits, wherein a and b show how the vascular endothelialization at the stenting site is different from that of the conventional metallic stent and three days after the surface- It is a photograph showing whether there is a difference.
FIG. 5 is a photograph showing how the restenosis progresses at the stenting site 4 weeks after the conventional metal stent and the stent surface-treated according to the present invention are implanted in the rabbit.

Hereinafter, the present invention will be described in more detail with reference to the following examples and drawings. It is to be understood, however, that the description of the embodiments and drawings is intended to illustrate specific embodiments of the invention and is not intended to be exhaustive or to limit the scope of the invention to the precise forms disclosed.

Example  One. Stent  Surface Silanization  Reaction: epoxy-modified Stent  Surface preparation

After standing for about 1 hour in a piranha solution (H ₂ SO ₄ / H ₂ O ₂ (4/1)) with a stent (BMS; 316L stainless steel stent, 3 mm diameter, 15 m length, Humed, Korea) , Ultrasonically washed with distilled water three times for 5 minutes, and washed twice with acetone for 5 minutes. After drying for 1 hour at 30 ° C in a vacuum, a stent surface having a hydroxy group on its surface was prepared. Then, 10% (v / v) 3-glycidoxypropyltrimethoxysilane (GPTS ) / Toluene solution at 30 DEG C to 60 DEG C for 48 hours, washed with toluene three times for 5 minutes, and then ultrasonically washed with dichloromethane twice for 5 minutes. Dried in an oven at 30 캜 in vacuum for 1 hour, and heated at 70 캜 for 3 hours in an argon gas atmosphere to induce stable crosslinking of the silane compound through dehydration reaction.

Example  2. Polymers Grafting : With polyethylene glycol Reformed Stent  Surface preparation

DIEA (3 equivalents) was added to a 10 mM O, O'-bis (2-aminopropyl) polyethylene glycol (PEG, MW = 1,500) / NMP solution to prepare a polymer solution. Solution and surface-treated at 50 ° C for 24 hours. After the surface treatment, ultrasonic washing was performed twice using NMP for 5 minutes and then twice for 5 minutes using dichloromethane. Finally, a PEG grafted stent (PEG / GPTS / stent surface) was prepared by drying in a vacuum oven at 30 ° C for 1 hour.

Example  3. Bio ligand  Immobilization: Antibody immobilized Stent  Surface preparation

First, the amine group of grafted PEG end on the surface of the stent was succinylated with succinic anhydride to replace the carboxyl group. Then, the stent was added to 20 mM DIC (diisopropylcarbodiimide) and HOBt (1-hydroxybenzotriazole) / NMP solution and reacted at 30 ° C for 1 hour to chemically activate the surface of the above-mentioned stent. After the activation reaction, ultrasonic washing was performed twice using dichloromethane for 3 minutes and 5 minutes using NMP for 5 minutes. The activated surface can be rapidly immobilized on the surface by forming a peptide bond with a protein such as an antibody having an amine group. Then, the stent was added to a phosphate buffer (pH = 7.4) containing an antibody capable of selectively capturing EPC, and the stent was reacted at 37 ° C for 24 hours to allow the antibody to stably And immobilized on the surface of the stent (see Figures 2 and 3)

The characteristics of the thus prepared stent surface are shown in Tables 1 and 2. The water surface contact angle and photoelectron spectrum analysis show that the surface of the stent has been modified as intended.

Measurement of water contact angle of surface treated stent surface Stent surface Angle (°) Untreated stent surface 75 Example 1 GTPS / stent surface 60 Example 2 PEG / stent surface 45 Example 3 Antibody / stent surface 48

Auger-electron photoelectron spectrum (APS) measurement of surface-treated stent surface Stent surface C (1s) / Cr (2p) N (1s) / C (1s) C (1s) / O (1s) Untreated stent surface 7.3 Trace 0.057 Example 1 GTPS / stent surface 92.8 Trace 0.6 Example 2 PEG / stent surface 128.8 0.03 1.37 Example 3 Antibody / stent surface 123.8 0.05 1.26

In order to confirm whether the surface-treated stent is biocompatible, and which selectively captures only vascular endothelial progenitor cells to accelerate vascular endothelialization, the surface-treated stent is immersed in an endothelial precursor cell (EPC) solution and THP- 1 cells, respectively, and the degree of adsorption of the cells was confirmed and analyzed by fluorescence microscope and FE-SEM (Field Emission-Scanning Electron Microscope). The reason why THP-1 cells were selected as a comparative example of vascular endothelial progenitor cells is that THP-1 is a representative cell rich in human blood.

A commercially available stainless steel stent and the stent prepared in Example 3 were placed in phosphate buffer (pH = 7.4) of each cell, incubated at 37 ° C for 24 hours, and then treated with phosphate buffered saline and distilled water And washed for 10 minutes. After drying in a vacuum oven at 30 ° C for 1 hour, the surface of the cells adsorbed was observed through a fluorescence microscope. In the case of the untreated general metal stent, EPC and THP-1 were adsorbed in a certain amount without cell selectivity. In the case of the surface-treated stent of Example 3, only a large amount of EPC was selectively captured without adsorption of THP-1. In the case of stents in which EPCs were captured, high-resolution analysis using electron microscopy showed typical cell proliferation patterns. EPC alone can be selectively captured on the surface of the stent to rapidly induce vascular endothelial cell proliferation on the surface of the stent without nonspecific biodegradation. Photographs measured with a fluorescence microscope are shown in FIG. 2, and photographs measured with a high-resolution electron microscope are shown in FIG.

In addition, animal clinical studies were conducted to compare the biological reactions of the conventional metal stent with the surface-treated stent of Example 3 using the rabbit, and as a result, vascular endothelial cells proliferated rapidly in the stent site at 3 days . The results are shown in FIG. 4. This rapid vascular endothelial cellization can inhibit the vivo rejection reaction and essentially inhibit thrombus formation. In addition, after 4 weeks, the cross section of the blood vessel of the rabbit was cut and analyzed. As a result, the conventional metal stent developed restenosis in which the neointimal hyperplasia significantly progressed within 4 weeks and the vessel narrowed again , Whereas in the case of the surface-treated stent of Example 3, it was observed that the expanded blood vessels were maintained by inhibiting the neointimal hyperplasia due to excellent biocompatibility (FIG. 5). That is, it has been confirmed that the stent restenosis and the thrombogenic problem, which are the object of the present invention, can be effectively solved simultaneously by the stent surface treatment technique of the present invention.

Claims (11)

A silane compound layer, a functional group layer, a hydrophilic polymer layer, and a bio-ligand layer are sequentially bonded on the stent framework,
The functional group is formed through a silanation reaction of a compound represented by the following formula (I)
Wherein the hydrophilic polymer layer and the bioligand layer are formed by immobilizing a carboxyl group contained in the hydrophilic polymer layer as an activated end by an amide functional group and an amine functional group contained in the bioligand,
X- (CH ₂ ) _n -Si-Y _m ????? (I)
In the above formula (I), X is an amine group, a halide group, an epoxy group, an aldehyde group, an acetal group or a carboxyl group; Y is a halide group, a methoxy group or an ethoxy group; n is an integer from 1 to 25; m is an integer of 1 to 3; The method of claim 1, wherein the stent framework is a metal selected from the group consisting of stainless steel, a nickel-chromium alloy, a nickel-titanium alloy, a cobalt-chromium alloy, tantalum, titanium, aluminum, zirconium, chromium, Wherein the stent has a structure in which a silane compound layer and a functional group layer are sequentially bonded on a surface of a stent skeleton into which a hydroxy group has been introduced through a reaction. The stent according to claim 1, wherein the functional group is selected from the group consisting of an amine group, an epoxide group and a carboxyl group. The method of claim 1, wherein the hydrophilic polymer is selected from the group consisting of polyvinyl alcohol, polylysine, polyacrylic acid, polyacrylamide, polyurethane, poly (acrylonitrile-co-acrylic acid), polyethylene glycol, polyethyleneimine, chitosan, ≪ / RTI > and cellulose and having an amine group at the end thereof,
Wherein the carboxyl group contained in the hydrophilic polymer layer as an activated end activates a terminal amine group of the hydrophilic polymer. The bioligand of claim 1, wherein an amine group of the bioligand is coupled to an activated carboxyl group of the hydrophilic polymer layer, wherein the bioligand is an antibody binding to vascular endothelial cadherin, < / RTI > aptamers and peptides. (i) introducing a functional group onto the hydroxy group-introduced stent framework surface through a silanization reaction;
(ii) grafting the hydrophilic polymer thin film by coupling the hydrophilic polymer to the functional group;
(iii) activating the end of the grafted hydrophilic polymer with a carboxyl group; And
(iv) immobilizing the activated carboxyl terminal of the hydrophilic polymer by coupling with an amine functional group contained in a bioligand capable of capturing a vascular endothelial cell precursor cell,
Wherein the silanization reaction uses a compound represented by the following formula (I): < EMI ID =
X- (CH ₂ ) _n -Si-Y _m ????? (I)
In the above formula (I), X is an amine group, a halide group, an epoxy group, an aldehyde group, an acetal group or a carboxyl group; Y is a halide group, a methoxy group or an ethoxy group; n is an integer from 1 to 25; m is an integer of 1 to 3; The stent according to claim 6, wherein the stent framework is selected from the group consisting of stainless steel, nickel-chromium alloy, nickel-titanium alloy, cobalt-chromium alloy, tantalum, titanium, aluminum, zirconium, chromium and nickel. Surface treatment method. [7] The method of claim 6, wherein the fixing step (iv) comprises fixing the carboxyl group, which is an active terminal of the hydrophilic polymer, and the amine group contained in the bioligand with an amide bond,
Wherein the carboxyl terminal group that is activated at the hydrophilic polymer layer activates the terminal amine group of the hydrophilic polymer. The stent surface treatment method according to claim 6, wherein the hydrophilic polymer is a polymer having a hydroxyl group or an ethylene oxide group in a chain. 7. The composition of claim 6, wherein the hydrophilic polymer is selected from the group consisting of polyvinyl alcohol, polylysine, polyacrylic acid, polyacrylamide, polyurethane, poly (acrylonitrile-co-acrylic acid), polyethylene glycol, polyethyleneimine, chitosan, And cellulose, and has an amine group at the terminal thereof. 7. The method of claim 6, wherein the bioligand is selected from the group consisting of an antibody, an aptamer, and a peptide that bind to vascular endothelial cadherin.

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