Gene delivery stent using titanium oxide thin film coating and method for fabricating the same

Abstract

The present invention relates to a gene delivery stent using titanium oxide thin film coating and a method for fabricating the gene delivery stent. The gene delivery stent according to the present invention may be loaded with a drug having anti-inflammatory and anti-thrombotic effects and simultaneously deliver a gene capable of inhibiting proliferation of vascular smooth muscle cells. Accordingly, late thrombosis and metal allergy may be reduced, and vascular restenosis in the stent region may be prevented, thereby making it possible to increase treatment effects of the bare metal stent.

Claims

1. A gene delivery stent using titanium oxide thin film coating, the gene delivery stent comprising: a titanium oxide thin film obtained by coating a surface of a metal stent with TiO.sub.2-xN.sub.x (0.001x1) and modifying the coated surface to introduce a hydroxyl group; a drug layer containing a drug having a functional group bound to a hydroxyl group of the titanium oxide thin film to thereby be adhered onto the titanium oxide thin film; and an oligonucleotide layer containing oligonucleotide bound to the drug to thereby be adhered onto the drug layer, wherein the drug is at least one of abciximab, alpha lipoic acid (ALA), and heparin.

2. The gene delivery stent of claim 1, wherein the oligonucleotide is selected from a group consisting of gDNA, cDNA, pDNA, mRNA, tRNA, rRNA, siRNA, miRNA, and antisense-oligonucleotide.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a view showing a process of sequentially coating a titanium oxide thin film and a drug on a surface of a metal stent and coating oligonucleotide thereon again in a gene delivery stent using titanium oxide thin film coating according to an exemplary embodiment of the present invention;

(2) FIG. 2 is a mimetic view of a PECVD apparatus for coating titanium dioxide or nitrogen-doped titanium oxide (TiO.sub.2-xN.sub.x (0.001x1)) used in the gene delivery stent using titanium oxide thin film coating according to the exemplary embodiment of the present invention;

(3) FIG. 3 is a mimetic view of a state in which alpha-lipoic acid (ALA) is adhered to the titanium oxide thin film in the gene delivery stent using titanium oxide thin film coating according to the exemplary embodiment of the present invention;

(4) FIG. 4 is graphs obtained by electron spectroscopy for chemical analysis (ESCA) of a TiO.sub.2 thin film deposited at 5 W and 400 C. for 4 hours in the gene delivery stent using titanium oxide thin film coating according to the exemplary embodiment of the present invention.

(5) FIG. 5 is graphs obtained by ESCA of a nitrogen-doped TiO.sub.2 thin film deposited at 5 W and 400 C. for 4 hours in the gene delivery stent using titanium oxide thin film coating according to the exemplary embodiment of the present invention;

(6) FIG. 6 is a graph showing a contact angle change according to a discharge power applied to surface modification in the gene delivery stent using titanium oxide thin film coating according to the exemplary embodiment of the present invention;

(7) FIG. 7 is a simple mimetic view of a plasmid allowing whether or not the gene is transfected to be known by generating beta-galactosidase as a plasmid coated in the gene delivery stent using titanium oxide thin film coating according to the exemplary embodiment of the present invention;

(8) FIG. 8 is a graph showing an amount of genes coated on each of three drug (abciximab, heparin, ALA) coating layers in the gene delivery stent using titanium oxide thin film coating according to the exemplary embodiment of the present invention;

(9) FIG. 9 is photographs showing that the genes are safely coated on a titanium oxide/Abciximab/plasmid composite layer, a titanium oxide/Heparin/plasmid composite layer, and a titanium oxide/ALA/plasmid composite layer without damage in functions of the gene, respectively, and all of the genes (g-Wiz lacZ plasmid) are normally expressed in cells, in the gene delivery stent using titanium oxide thin film coating according to the exemplary embodiment of the present invention;

(10) FIG. 10 is photographs showing that the genes are transfected into tissues after metal pieces coated with each of the titanium oxide/Abciximab/plasmid composite layer, the titanium oxide/Heparin/plasmid composite layer, and the titanium oxide/ALA/plasmid composite layer are grafted in bodies of rats for an experiment, in the gene delivery stent using titanium oxide thin film coating according to the exemplary embodiment of the present invention;

(11) FIG. 11 is cross-sectional views of the tissues into which the genes are transfected after the metal piece coated with each of the titanium oxide/Abciximab/plasmid composite layer, the titanium oxide/Heparin/plasmid composite layer, and the titanium oxide/ALA/plasmid composite layer are grafted in bodies of rats for the experiment, in the gene delivery stent using titanium oxide thin film coating according to the exemplary embodiment of the present invention; and

(12) FIG. 12 is a photograph showing that when porcine coronary vascular smooth muscle cells are cultured on the metal pieces coated with the titanium oxide/Abciximab/plasmid composite layer, the genes are transfected into the cells.

BEST MODE

(13) (1) Titanium Oxide Thin Film Coating on Surface of Metal

(14) A metal plate having a size of 1 cm1 cm was fabricated in a disk shape using stainless steel was fabricated among materials used for a stent, and titanium oxide thin film coating was performed on a surface of the metal plate.

(15) The metal plate was fixed to a plasma generator as a stent H shown in FIG. 2 in a vacuum chamber connected to a radio frequency (RF) plasma generator generating plasma and a vacuum pump, and a temperature of the plasma vacuum chamber was maintained at 400. Firstly, in order to improve adhesion between a substrate of the metal plate and a thin film, the surface of the metal was subjected to a plasma pre-treatment process by flowing argon and oxygen before thin film coating to wash the surface of the metal. Titanium isopropoxide was put into a bubbler evaporator, mixed with oxygen, which was reaction gas using argon (Ar), which was carrier gas while maintaining a temperature of the bubbler at 50, and then introduced in a reaction chamber, followed by generating plasma to perform a reaction for 4 hours, thereby coating the surface of the metal plate with a titanium dioxide thin film. In this case, a flow rate of argon (Ar), which was the carrier gas, was 100 sccm, and a flow rate of oxygen, which was the reaction gas, was maintained at 20 sccm. Discharge power was variously applied in a range of 5 to 200 W to coat the thin film. In order to fabricate a nitrogen-doped titanium dioxide thin film, the above-mentioned experimental conditions were equally maintained except that argon, oxygen, and nitrogen were supplied at flow rates of 100 sccm, 10 sccm, and 1 sccm, respectively.

(16) The discharge power may be variously applied from 5 to 200 W at the time of coating the titanium dioxide thin film, but it was confirmed that as the larger the discharge power, the higher the surface roughness. Root mean square (Rms) values of results obtained by an atomic force microscope (AFM) of the thin film fabricated for 4 hours while changing discharge power at 5, 10, and 15 W were shown in the following Table 1.

(17) TABLE-US-00001 TABLE 1 Sample NT5 NT10 NT25 T5 T10 T15 Rms 3.571 5.142 7.119 7.760 9.403 13.862 T: titanium dioxide coated thin film, NT: nitrogen-doped titanium dioxide coated thin film, numbers (5, 10, 15): discharge power applied at the time of depositing titanium dioxide thin film

(18) As shown in Table 1, it was confirmed that in the case of the nitrogen-doped thin film, the surface was more uniform than in the case of the titanium dioxide thin film and the lower the discharge power, the more uniform the obtained thin film. It was known that the roughness of the thin film affects blood compatibility, and as the surface roughness is reduced, blood compatibility becomes excellent.

(19) In addition, the thin film fabricated at 5 W had the lowest surface roughness. Therefore, all of the titanium dioxide thin film deposition for surface modification was performed while fixing discharge power at 5 W and maintaining a temperature 400 C. for 4 hours, thereby fabricating a stent coated with titanium dioxide.

(20) In FIG. 4 showing results of ESCA of the titanium dioxide thin film fabricated at 5 W, peaks corresponding to Ti.sup.4+ in TiO.sub.2 were confirmed at 458.8 eV (2p3/2) and 464.7 eV (2p1/2), and an O1s peak corresponding to a TiO bond in TiO.sub.2 was confirmed at 530.4 eV.

(21) In FIG. 5 showing results of ESCA of the nitrogen-doped titanium dioxide thin film, Ti peaks and O1s peaks were confirmed at positions similar to that in the titanium dioxide thin film, and an N1s peak was confirmed at 399 eV at a content ratio of 0.8%, such that it was confirmed that the surface of the titanium dioxide was doped with nitrogen.

(22) (2) Modification of Titanium Oxide Thin Film Coating Layer for Generating Hydroxyl Group

(23) In order to adhere the drug onto the surface of the coated titanium dioxide, a functional group capable of chemically binding to a functional group in drug molecules needs to exist in the surface of the titanium oxide.

(24) Therefore, in the present invention, in order to introduce OH group in the surface of the titanium dioxide layer capable of chemically binding to the drug, the surface of the thin film was modified by non-thermal plasma using deionized water (H.sub.2O). After the metal plate coated with titanium dioxide was fixed to a tubular non-thermal plasma reactor made of Pyrex and the bubbler was filled with tertiary deionized water, water vapor was introduced into the plasma reactor at 10 sccm and discharge power was changed in a range of 10 to 100 W, thereby modifying the titanium dioxide thin film for 10 minutes using a non-thermal plasma process.

(25) In order to introduce the hydroxyl group in the surface of the titanium dioxide thin film, after the surface was modified using H.sub.2O, the surface was washed with deionized water once, and then a contact angle was measured. Results between the discharge power and the contact angle applied for modification were shown in FIG. 6.

(26) As a result, as shown in FIG. 6, entirely, the contact angle was reduced by about 40 (degrees) than the contact angle before modification, it was confirmed that a hydrophilic functional group was introduced in the surface. In addition, it was confirmed that the contact angle was reduced when the applied discharge power was in a range of to 50 W, and as the discharge power became higher than 60 W, there was an increasing tendency in contact angle.

(27) This may be because as the discharge power was increased, the titanium dioxide thin film, which was a target for modification, was slightly etched by the plasma, or a structure of titanium dioxide was changed.

(28) (3) Drug Adhesion

(29) The used drugs were as follows.

(30) (1) -lipoic acid (Thiocticaid(ALA); Bukwang Pharm. Co. Ltd)

(31) (2) Abciximab (ReoPro; Eli Lilly and Company, Indianapolis, Ind.)

(32) (3) Heparin sodium salt (Grade1-A, From Porcine Intestinal Mucosa, Sigma-Aldrich)

(33) (4) 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide methiodide (DCC; 98%, Alfa Aesar)

(34) (5) 4-(Dimethylamino)pyridine (DMAP; 99%, Sigma-Aldrich Co.)

(35) (6) Sodium bicarbonate (99%, Dae Jung Chemicals & Metals Co., Ltd)

(36) In order to adhere ALA onto the surface of the titanium dioxide coating layer, 0.0124 g of ALA, 0.005 g of sodium bicarbonate were sufficiently dissolved by adding 2 mL of deionized water, respectively, to prepare an ALA solution, 0.8915 g of DCC and 0.07 g of DMAP were dissolved in 150 mL of deionized water, respectively. 2 mL of the prepared ALA solution and 1.5 mL of the DMAP+DCC mixed solution were harvested, respectively, and put into a vessel, followed by mixing. Then, the mixed solution was left at 35 for 1 hour to activate the mixed solution, and the surface-modified metal plate fabricated in Surface-modified metal plate was put into the activated mixed solution and stored at 35 C. for 2 hours, thereby adhering the drug to the surface of the titanium dioxide layer.

(37) Heparin was adhered by the same method except that 1.5 mL of the prepared DMAP+DCC mixed solution was put into 0.0065 g of heparin sodium salt.

(38) Abciximab was adhered by the same method except that 1.5 mL of the DMAP-DCC mixed solution was put into 0.25 mL of abciximab.

(39) (4) Gene Adhesion

(40) The drug adhered to the surface of the metal plated coated with titanium dioxide had various kinds of chemical functional groups. For example, there were a carboxyl group, an amine group, a disulfide (SS) bond, and the like. A plasmid was put into an aqueous solution maintained at a pH of 6 to 7, and the stent containing the drug adhered thereto was added thereto and maintained at room temperature for a predetermined time (1 to 5 hours). In this case, various physical interactions were generated between various functional groups in the drug adhered to the surface of the metal plate coated with the titanium dioxide thin film and functional groups existing in the plasmid, such that the plasmid was adhered to the surface of the metal plate in several layers.

(41) In order to confirm whether or not the gene was delivered from the metal plate composed of the titanium dioxide/drug/gene complex fabricated by the above-mentioned method, gWIZ--gal plasmid was purchased from Genlantis Company. When gWIZ-beta gal plasmid is delivered into the cells, the cells generate -Galactosidase, wherein -Galactosidase, which is an enzyme hydrolyzing -galactoside, is used as a reporter gene in eukaryotic transfection experiment. In cells in which the gene is transfected, -Galactosidase cleaves 5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside(X-gal) to generate blue precipitates. Since this blue color may be observed in tissue or cell through a microscope or by the naked eyes, it is possible to determine whether the gene was transfected or not.

(42) FIG. 7 shows a simple mimetic view of the plasmid allowing whether or not the gene is transfected to be known by generating beta-galactosidase as gWIZ-R-gal plasmid.

[Experimental Example 1] Quantification of Adhered Gene

(43) The metal plate fabricated by the methods in Examples (1) to (3) was positioned on a 12-well plate, and a solution containing genes (total plasmid content: 20 ug/200 ul DW) was put onto the metal plate. After the metal plate was left for 8 to 12 hours, the metal plate was immersed again in sterile deionized water (DW) for 30 minutes to remove extra DNA non-specifically adhered thereto. A concentration of the plasmid in washing DW was measured using Nanodrop ND-1000 spectrophotometer (Thermo scientific, USA). After the plate was washed and dried in a sterile bench, subsequent experiments were performed. The amount of DNA adhered to the flask was estimated by arithmetically subtracting a measured amount of the DNA in the washing DW from initial 20 ug of plasmid according to the following Equation 1.
DNA binding amounts (ug)=20ugDNA amount (ug) in washing DW[Equation 1]

(44) FIG. 8 is a graph showing the amount of the gene coated on each of three drug (abciximab, heparin, ALA) coating layers in the gene delivery stent using titanium oxide thin film coating according to the exemplary embodiment of the present invention. In FIG. 8, it may be confirmed that in the case of a TiO.sub.2 single coating group, in the case of an abciximab coating group, in the case of a heparin coating group, and in the case of ALA coating group, amounts of plasmid coated at an area of 1 cm.sup.3 were approximately 2.7 ug, 7.2 ug, 5.6 ug, and 2.5 ug, respectively.

[Experimental Example 2] Intracellular Expression of Gene Eluted from Metal/Titanium Dioxide/Drug/Gene Complex

(45) In order to confirm whether or not functional deformation was generated in the plasmid after the gene was bound to the surface of the metal plate in a form of titanium/drug/gene (plasmid) complex (hereinafter, referred to as a metal flask), after the metal flask was generated, the gene was artificially separated, followed by measuring whether or not the gene had an activity.

(46) In order to elute the gene from the metal flask, the metal flask was put in a 12-well plate, and 100 uL of 0.1TE buffer (pH 8.0) was added thereto again and left at room temperature for 30 minutes. After 30 minutes, 0.1TE buffer was harvested, and an amount of the eluted plasmid was measured using Nanodrop ND-1000 spectrophotometer (Thermo Scientific, USA).

(47) In order to confirm whether or not the eluted plasmid was normally active, the gene was transfected in cells under in vivo conditions, using Lipofectamine 2000, which is a transfection product fabricated by Invitrogen Company. As described below, human embryonic kidney 293 T cells (HEK 293 T cell) were seeded in a 12-well plate at 110.sup.5/well and cultured. After 24 hours, a culture medium was treated with 6 ug of the eluted plasmid using Lipofectamine, and after 4 hours, the culture medium was stirred, followed by continuously culturing for 48 hours. Then, X-gal staining was performed.

(48) After fixing the cell, the cell was stained with an X-gal stain solution at 37 C. for 24 hours. Thereafter, in the case of observing whether the cell was stained or not, the positively stained cell was observed as a blue stained cell in an optical microscope.

(49) As a result, as shown in FIG. 9, it may be confirmed that the genes were safely coated on each of the gene delivery stents using titanium oxide thin film coating according to the exemplary embodiment of the present invention, that is, the titanium dioxide/abciximab/plasmid composite layer, the titanium dioxide/heparin/plasmid composite layer, and the titanium dioxide/ALA/plasmid composite layer without damaging the functions of the gene, and all of the genes (g-Wiz lacZ plasmid) were normally expressed in cells.

(50) In addition, as shown in FIG. 9, it was confirmed that all of the plasmid eluted from abciximab, ALA, and heparin normally allowed the cell to generate -galactosidase. As the result, it may be confirmed that the functions of the gene coated in the present invention were maintained.

[Experimental Example 3] Confirmation of Whether or not Genes are Delivered in Abdominal Wall of Rats for Experiment

(51) The metal flask fabricated in the Examples was grafted in abdominal wall of rats for the experimental. After 7 days of grafting, muscles of the abdominal wall were harvested and the X-gal staining was performed by the same method as that in Experimental Example 2. After staining, the muscle in abdominal wall was cut, and a stained site was confirmed.

(52) FIG. 10 is a photograph showing that the genes were transfected into tissues after the gene delivery stent using titanium oxide thin film coating according to the exemplary embodiment of the present invention, that is, metal pieces coated with each of the titanium dioxide/abciximab/plasmid composite layer, the titanium dioxide/Heparin/plasmid composite layer, and the titanium dioxide/ALA/plasmid composite layer were grafted in bodies of the rats for the experiment.

(53) As shown in FIG. 10, it may be confirmed that the sites at which the tissue in the abdominal wall was stained as a blue color were observed by the naked eyes. The results shows that the -gal gene-coated plate successfully transfected the gene in the abdominal tissue.

(54) In addition, as shown in FIG. 11, it may confirmed that the cells of the tissue in which the gene was transfected by grafting the metal piece coated with each of the titanium dioxide/abciximab/plasmid composite layer, the titanium dioxide/Heparin/plasmid composite layer, and the titanium dioxide/ALA/plasmid composite layer, in the body of the rats for the experiment delivered the gene to a connective tissue (red arrow) on the abdominal wall, and at the same time, the stained sites were also observed in the tissue cells (yellow arrow) under the connective tissue by the microscope.

[Experimental Example 4] Confirmation of Whether or not Genes are Delivered in Vascular Smooth Muscle Cells

(55) In order to confirm whether or not the gene may be transfected in the vascular smooth muscle cell capable of being a main graft site of the stent besides the connective tissue and the muscle tissue from the plate coated with the gene as confirmed in Experimental Example 3, the vascular smooth muscle cells were directly cultured in the surface of the metal plate, followed by confirming whether the gene was transfected in the cell.

(56) The vascular smooth muscle cells were separated from the porcine coronary artery separated in a sterile state. After separating the coronary artery from a heart of the pig, all of the connective tissues in the outside of the blood vessel were removed, and vascular endothelial cells were removed by scratching the inside of the blood vessel using forceps. The blood vessel tissue from which the connective tissue and the endothelial cell were removed was put into a solution containing collagenase, elastase, and tryptase and finely cut using scissors, followed by reacting with each other in a shaking culture equipment at 37 and 60 rpm for 60 minutes to separate the cells. Then, the separated cells were cultured in Dulbecco's Modified Eagles (DME) media. After culturing, the cultured cells were proliferated up to passage 3 to 4, and then the immuno-staining was performed using anti-smooth muscle actin Ab (anti-SMC Ab) to confirm that the proliferated cells were vascular smooth muscle cells. The confirmed cells were used in the subsequent experiments. The metal plate coated with -gal gene was positioned in a 12-well plate, and the vascular smooth muscle cells were cultured thereon at 510.sup.4/well using DME media containing 10% fetal bovine serum (FBS). After 7 days of culture, the metal plate in which the cells were cultured to thereby be adhered thereto was picked out and fixed, and then the staining was performed by the same method as the X-gal staining method in Experimental Example 2.

(57) The stained metal pieces were observed under a microscope. As a result, as shown in FIG. 12 (photograph showing that when porcine coronary vascular smooth muscle cells were cultured on the metal pieces coated with the titanium dioxide/abciximab/plasmid composite layer, the genes were transfected into the cells), it may be confirmed that the cells (arrow) adhered to the surface of the metal was observed as a blue stained cell. The result indicates that the metal/titanium dioxide/drug/gene complex may transfect the gene in the vascular smooth muscle cells.