KR101223977B1 - Method of manufacturing nano-structured stent inhibiting toxicity - Google Patents

Abstract

The present invention relates to a stent having a nanostructure on the titanium surface and a method for manufacturing the same. More specifically, in the method for manufacturing a stent made of titanium, the present invention relates to a method for producing a stent having a nanostructure consisting of increasing the deposition rate of an electron beam evaporator and a titanium nanostructure stent. The stent of the present invention can prevent vascular restenosis by the stent by inhibiting the toxic response and immune response of the immune cells.

Description

Method of manufacturing titanium nanostructured stents to inhibit toxicity {Method of manufacturing nano-structured stent inhibiting toxicity}

The present invention relates to a stent having a nanostructure on the titanium surface and a method for manufacturing the same. More specifically, in the method for manufacturing a stent made of titanium, the present invention relates to a method for producing a stent having a nanostructure consisting of increasing the deposition rate of an electron beam evaporator and a titanium nanostructure stent.

In developed countries such as the United States, the number one cause of death is cardiovascular and vascular disease (48%), and cancer accounts for about 23%. Cardiovascular disease is not only a major cause of death, but about 25% of people who die from cardiovascular disease die suddenly. According to recent health statistics, Korea also ranked second with vascular disease (21%). In particular, if the mineral in the blood vessels is located on the inner wall of the blood vessels necrosis of the blood vessels due to the pressure of the blood flow or when completely blocked, oxygen supply through the blood is not necrotic blood vessel cells necrosis causing serious vascular disease.

To prevent this problem, a blood vessel stent is inserted. Stents are spring-shaped metal implants that support blood vessels and are inserted into the vessel walls after the procedure to prevent restenosis, and their use has recently expanded. Although the stent has various procedures, it is mainly performed by balloon dilation, which is inserted with a balloon catheter into blood vessels such as cardiovascular, aortic, and cerebrovascular vessels and expands the coronary passage as the balloon is inflated. have.

Existing stents require elasticity and ductility to expand outwards as the balloon expands and expand to the size of the original vessel passage. That is, the existing stent is required to be flexible for insertion into the complex and curved passages during the procedure of inserting a balloon catheter and fixing the target site and then expanding the balloon to expand the constricted site. After this, conditions such as elasticity are required to prevent the structure of the stent from being deformed by the contracting force of blood vessels (cardiovascular, aortic, cerebral arteries, etc.). Was using

The introduction of metal stents prevented acute vascular occlusion and reduced restenosis after abundant plastic surgery. The problem of stenosis of blood vessels in the area has arisen. As one of the efforts to prevent restenosis, the drug is applied to the surface of the stent, and the drug is applied to the blood vessel after the stent is mounted. The drug therapy suppresses cell proliferation, thereby proliferating neointimal cells. It has a function to suppress.

However, in recent years, the generation of blood clots due to the polymer used for drug release has emerged as a big problem, and a biocompatible material is required as a surface material of the stent.

As described above, the stent material is made of stainless steel, tantalum and titanium-nickel alloys, and various forms such as a balloon type and a tube type have been developed and used. However, even after the stent implantation, 20-40% of the failures occur, because there may be a problem with the biocompatibility of the vascular cells of titanium, which is widely used as a vascular material, and cause immune response and toxicity of immune cells. . In addition, the method of coating the drug to suppress the immune action is commonly used, but since the drug is showing an undesired phenomenon in the differentiation of vascular inner wall cells, it is urgently needed to study the new material of the stent to prevent vascular restenosis .

In order to solve the above problems, the present inventors nano-treated the surface of the titanium used as a material of the cardiovascular stent to investigate the immunological toxic response of macrophages, confirming that the toxic reaction is suppressed, to complete the present invention It became.

Therefore, an object of the present invention is to provide a method for producing a stent with a titanium surface nanostructure.

In another aspect, the present invention provides a stent having a titanium nanostructure, which can prevent vascular restenosis by inhibiting the toxic response of macrophages.

The present invention provides a method for manufacturing a stent made of titanium in order to solve the above problems, comprising the steps of: (1) depositing titanium on a sample surface using an E-beam Evaporator; And (2) increasing the deposition rate to 35-45 A ° / sec to form nanostructures on the titanium surface; It provides a method of manufacturing a stent having a nano-structured surface, including.

The deposition rate of step (1) is preferably 1.5 to 2.5A / sec.

The deposition rate of step (2) is preferably 40A ° / sec.

In the deposition of the step (2) is characterized in that the electron beam current density is 165 to 175 mA / cm ² .

The nanostructure preferably has a thickness of 1 to 50 nm, more preferably 1 to 35 nm.

In another aspect, the present invention provides a stent of the titanium material, the titanium surface has a nanostructure.

The nanostructure of the stent preferably has a thickness of 1 to 50 nm, more preferably 1 to 35 nm.

The stent of the present invention is characterized in that it is produced by the contents disclosed in the manufacturing method.

The stent having a surface of the titanium nanostructure according to the present invention can inhibit the toxic response of macrophages, which is one of immune cells, to prevent vascular narrowing due to the insertion of the implant. In addition, since the thin film thickness of the nanostructure is less than 50 nm and transparent, the application value is also high in thin film coating for thin film thickness and cellular video.

1 illustrates a structure of an e-beam evaparator capable of forming a nanostructure.
2 shows a nanostructured surface formed using an electron beam evaporator.
Figure 3 shows that the shape of the nanostructure is in the form of a circular hemisphere height of about 8-10nm.
Figure 4 shows the results of observing the morphology of macrophages under an optical microscope (Con: control, 1: when treated with titanium, 2: when treated with nano-treated titanium).
Figure 5 shows the results observed by staining the actin (actin) with a fluorescent material to determine the cytoskeleton of macrophages (Con: control, 1: when treated with titanium, 2: nano-treated titanium When processed, red: actin, blue: nucleus).
Figure 6 shows the result of measuring the NO of macrophages (Con: control, 1: when treated with titanium, 2: when treated with nano-treated titanium).
Figure 7 shows the results of Western blotting experiments to determine the degree of iNOS protein expression of macrophages.
8 shows the result of confirming the expression level of iNOS of macrophages using a fluorescent material (green).

The present invention provides a method for manufacturing a stent of titanium material, comprising: (1) depositing titanium on a stent surface using an E-beam Evaporator; And (2) increasing the deposition rate to 35-45 A / sec to form nanostructures on the titanium surface; It relates to a method of manufacturing a stent having a nano-structured surface, including.

The stent is a conventional stent that is not coated with a polymer, the material of which is generally stainless steel or cobalt-chromium alloy.

A "stent" is a columnar metal mesh that inserts when the coronary arteries that supply oxygen and nourishment to the myocardium pumping the heart when it is narrowed or blocked, to restore blood flow.

Vacuum deposition of the electron beam applies a very high voltage to collide the hot electrons emitted from the filament with the evaporation source, thereby evaporating the material to be deposited by the heat generated to deposit it on the substrate.

As used herein, the term "deposition" means heating and evaporating a metal or compound in a vacuum to coat the vapor on a surface of an object with a thin film.

The stent manufacturing method of the present invention is a method of manufacturing a stent of a known titanium material, by using an electron beam evaporator (E-beam Evaporator) to increase the deposition rate to 35 to 45A ° / sec thin nanostructure ( The stent having a protrusion structure).

The evaporator preferably uses ultra-vacuum E-Beam equipment (10-8 torr).

The deposition rate of step (1) is preferably 1.5 to 2.5 A / sec, more preferably 2 A / sec.

Preferably, the deposition rate of step (2) is 40 A ° / sec, and the electron beam current density is 165 to 175 mA / cm ^2. Is preferred, and more preferably 170 mA / cm ² to be.

In another aspect, the present invention relates to a stent, characterized in that the titanium surface has a nano structure in the stent.

A feature of the present invention is that in the known titanium stent structure, the surface has a nano structure (projection structure).

The nanostructure of the stent is 1 to 50 nm, preferably 1 to 35 nm thick.

The nanostructure of the present invention has a thin film thickness of 35 nm or less, and is applicable to cell imaging and microvascularization because of its transparency.

The stent of the present invention can be produced by the manufacturing method described above.

Titanium stents having a nanostructure having a thickness of 50 nm, preferably 35 nm or less, of the present invention can inhibit toxic reactions of macrophages, one of immune cells, to prevent vascular narrowing and restenosis due to implantation. have.

When a conventional titanium stent was inserted into a blood vessel, the rate of revascularization of blood vessels was very high because it causes biocompatibility problems of titanium and immune responses and toxicity of immune cells.

However, the titanium stent having a nanostructure having a thickness of 50 nm, preferably 35 nm or less, of the present invention does not induce a macrophage immune response by less stimulating macrophages, one of the immune cells. In addition, the titanium having a nanostructure of the present invention reduces the amount of NO secreted during inflammation during macrophage immune response, and also reduces the expression of iNOS, an enzyme that produces NO.

That is, the stent of the present invention can prevent vascular restenosis when the stent is inserted into a blood vessel by inhibiting toxic reactions and immune responses of immune cells.

Accordingly, the present invention provides a method for preventing vascular restenosis comprising applying a titanium stent having a nanostructure having a thickness of 50 nm, preferably 35 nm or less, to the vascular endothelium.

Hereinafter, the present invention will be described in detail through examples. The following examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples according to the gist of the present invention to those skilled in the art. Will be self-evident.

Example 1 Preparation of Titanium Nanostructures

The nano-titanium structure was prepared by evaporating a material of pure titanium (99.9%: T-2069, Cerac Inc.) in an ultra-vacuum E-Beam device (10-8 torr) by E-beam and depositing it on the sample surface. Pure titanium was deposited to 50 nm thickness on glass coverslips (12-550-15, Fisher-Scientific, NH) to create a flat titanium surface. The deposition rate at this time was 2A ° / sec, and the electron beam current density was 60 to 70mA / cm ² . In order to form the nanostructure, the sample deposition rate was raised to 40 A ° / sec, and the electron beam current density was 170 mA / cm ² . Increasing the sample deposition rate resulted in the formation of specific nanostructures (projections) starting from the 30nm-thick portion. The surface nanostructure was formed to a thickness of 35 nm or less. The electron beam energy of the evaporator used in the experiment was 7.9 keV.

Example 2 Observation of morphology and actin changes of macrophages

6-well plate (6-well plate) to the normal slides (control), titanium slides, nano slides were fixed and sterilized, and then added to the macrophages were incubated for 12 hours to stabilize. The morphology of the cells was observed using AE31 inverted microscopes (Motic, Wetzlar, Germany). As a result, it was observed that macrophages of the titanium slide extended more than the control (control), far less than the macrophages of the titanium slide on the nano slide. It was found that nano-stimulated macrophages less than conventional titanium stimulates macrophages (FIG. 3).

Actin was stained with red to investigate the cytoskeleton of macrophages based on the observation of the morphology of macrophages. More specifically, in a 6-well plate (6-well plate) containing a sample, the cells were incubated for 12 hours, stabilized, washed twice with PBS, and then 3.7% paraformaldehyde (paraformaldehyde) was used. And fixed. After fixation, the cells were washed with PBS and stained with a fluorescent phalloidin conjugate solution (50 ug / ml) for 40 minutes.

After staining, the cells were washed with PBS and stained with DAPI for 5 minutes. After DAPI staining and washing once again with PBS, the slide was detached from the plate and mounted on an microscope slide using antifade and observed using an LSM 5 exciter (Carl Zeiss, Jena, Germany). As a result, as in the morphology observation, it was observed that the actin of the macrophages on the nano slide is less stretched than the actin of the macrophages on the titanium slide (FIG. 4).

Example 3 Measurement of Nitric Oxide and iNOS in Macrophages

NO can act as a local regulator and neurotransmitter in the body. When blood oxygen levels drop, endothelial cells in the blood vessel walls produce NO. In addition, NO has an effect of activating enzymes that relax muscles by acting on surrounding muscle cells. NO is released from macrophages when inflammation is induced during immune response, and the degree of inflammation can be determined by measuring NO of macrophages.

3-1. NO analysis

 After 6 hours of incubation in a 6-well plate containing samples, the cells were incubated for 4 hours, stabilized, the slides were transferred to a new plate to measure only the cells on the slides, and then incubated for 20 hours (total 24 Time incubation). After incubation, the media of the cells were collected and reacted with Griess solution one-to-one to measure the color development at 540 nm.

As a result of measuring the NO of the macrophages, it was confirmed that NO, which was secreted from titanium more than the control, was reduced in the nanoparticles (FIG. 5).

3-2. iNOS Western blotting

Macrophages were placed in 6-well plates containing samples and incubated for 4 hours to stabilize, then slides were transferred to new plates to measure only cells on the slides and incubated for 20 hours (total 24 hours incubation). After incubation and washing with PBS, the macrophages are transferred to the E-tube. Cells were lysed with extract buffer containing protease inhibitor (PBS solution containing 1% triton X-100, 0.5% sodium deoxycholate, 0.1% SDS). .

Protein was quantified using the Bio-Rad protein assay kit using BSA as a standard. After electrophoresis on 8-50 (w / v) polyacrylamide gel containing 0.1% SDS of 10-50 μg of total cell protein, the protein present in the gel was subjected to electroblotting by nitrocellulose. , NC) was transferred to a membrane. In order to block nonspecific binding, the NC membrane was placed in a tris-buffered saline-tween (TBS-tween) solution containing 5% skim milk powder and allowed to react at room temperature for 1 hour.

After washing once for 15 minutes with TBS-tween solution, again washed twice with fresh TBS-tween solution for 5 minutes. The membrane was placed in a TBS-tween solution containing the antibody to the target protein (iNOS), left in refrigeration for 12 hours and washed in the same manner as above.

The filter was placed in a TBS-tween solution containing a secondary antibody labeled with HRP, and allowed to stand at room temperature for 1 hour and then washed once with TBS-tween solution for 5 minutes and 4 times for 5 minutes. Bands were visualized on the film using Enhanced chemiluminescence (ECL).

The enzyme that produces NO in macrophages is iNOS (inducible nitric oxide synthase). When inflammation is induced, iNOS is expressed to produce NO. In other words, iNOS is directly involved in the inflammatory response of macrophages, which are activated when macrophages are activated to release inflammatory cytokine into the blood and produce NO.

Western blotting experiments were performed to determine the expression level of iNOS protein in macrophages. As a result of the NO assay, iNOS, which was expressed more in titanium than in the control, was reduced in the nanoparticles (Fig. 6).

3-3. Intracellular iNOS Observation

After 6 hours of incubation in a 6-well plate containing samples, the cells were incubated for 4 hours, stabilized, the slides were transferred to a new plate to measure only the cells on the slides, and then incubated for 20 hours (total 24 Time incubation). After incubation and washing twice with PBS, cells were fixed with 3.7% paraformaldehyde. After fixation, the cells were washed with PBS, and then reacted with an antibody against iNOS at a concentration of 10 ug / ml for 1 hour. After the reaction, the plate was washed twice with PBS, and then reacted with a green fluorescent substance (green fluorescent) secondary antibody for 1 hour.

After the reaction was completed, washed with PBS, the slide was removed from the plate and mounted on the microscope slide with antifade and observed using an LSM 5 exciter (Carl Zeiss, Jena, Germany).

As a result of confirming the expression level of iNOS of macrophages using green material, it was observed that the fluorescent material (green), which can stain iNOS, was stained in titanium more than the control group, and in the nano, it was fluorescent than titanium. It was observed that the green was greatly reduced (FIG. 7).

Claims (8)

In the method for producing a stent of titanium material,
(1) depositing titanium on the sample surface using an E-beam Evaporator; And
(2) increasing the deposition rate to 35 to 45 A ° / sec to form protruding nanostructures having a thickness of 1 to 50 nm on the titanium surface; A method of manufacturing a stent having a protruding nanostructured surface comprising a.
The method of claim 1, wherein the deposition rate in the step (1) is 1.5 to 2.5A / sec.
The method of claim 1, wherein the deposition rate in the step (2) is 40A / sec.
The method according to claim 1, wherein the electron beam current density in the deposition of step (2) is 165 to 175 mA / cm ² .
delete A stent of titanium material, the stent having a protruding nanostructure having a thickness of 1 to 50 nm on the titanium surface.
delete A stent characterized by having a protruding nanostructure having a thickness of 1 to 50 nm on a titanium surface, which is prepared by the method according to any one of claims 1 to 4.

Images