KR101178793B1 - Method for coating heparin on surface of stent - Google Patents

Abstract

PURPOSE: A method for coating heparin on the surface of a stent is provided to maintain a blood clot coagulation effect by improving heparin attachment stability. CONSTITUTION: A method for coating heparin on the surface of a stent is as follows. A coating solution is prepared by adding EDC(1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and NHS(N-hydroxysulfosuccinimide) to a mixture solution of heparin and dopamine. A metal stent is dipped in the coating solution. The metal stent is made of a material selected from a group consisting of stainless steel, Co-Cr alloy, tantalum, and gold.

Description

Method for coating heparin on surface of stent}

The present invention relates to a method for coating heparin on the surface of the stent, and more particularly, to a method for coating heparin on the surface of the stent, which can maintain the effect of preventing blood clotting.

Atogenic artierial narrowing and thrombosis are interrelated diseases, one of the most dangerous diseases that kill people every year, and has been identified by many health organizations around the world, including the United States. Stenosis refers to the narrowing or shrinking of blood vessels by the deposition of fat, cholesterol or other substances in the blood vessels of the human body. In severe cases, restenosis often results in clogged blood vessels. Thrombosis is when a blood clot develops or exists inside a blood vessel or in a cavity of a heart. A thrombus is caused by the coagulation of blood elements, mainly platelets or fibrin, when cell components are adsorbed. Thrombosis, like restenosis, also causes vascular stenosis in areas that occur.

One method of treating blood vessels constricted or blocked by restenosis is percutaneous transluminal coronary angioplasty (CPTA), also called coronary angioplasty. This is done by inserting a balloon catheter into the constricted part of the blood vessel and then expanding it to the size of the blood vessel to facilitate the flow of blocked blood flow. However, about one-third of patients undergoing PTCA developed a restenosis that narrowed the enlarged blood vessels within six months of the procedure, requiring angioplasty.

Instead of performing angioplasty on the restenosis in order to prevent the restenosis of such vessels, there is a method of inserting a stent as shown in FIG. 1 in a simple manner or inserting a stent together during the procedure. A stent is a tube made of metal or plastic, which is made of mesh and not of mesh. Most stents in use today are made of metal and are automatically inflated or inflated by balloons. Stent insertion is determined by the nature of the restenosis, such as the size of the vessel or the location of restenosis. The role of the stent may be to reinforce the vessels widened by the recent angioplasty, or to prevent the elastic recoil from returning to the original state if the angioplasty is not performed. The stent is usually implanted using a catheter. This method involves first folding the stent down and placing it on a balloon catheter so that the catheter is reached through the patient's blood vessels to the site of the lesion or recently expanded, and then the stent is swollen and held in place.

In this way, the stent stays permanently in the blood vessels and opens up the blood vessels, thus facilitating blood flow and eliminating the symptoms of cardiovascular disorders (mostly pain in the chest).

In general, blockage in the human body, such as arterial blockage, has been treated with arteriotomy and drugs that dissolve specific blockers, but recently, a procedure is used to remove vascular stenosis of the coronary artery using a balloon catheter. It is becoming commonplace.

However, when the blood vessels are squeezed to the stent surface when the blockage in the blood vessels is compressed by using a stent made of mesh stainless steel or titanium as described above, the blood vessels are restrained. It was.

Therefore, recently, a method of coating a stent surface with heparin, which is known to have anticoagulant ability, is known. In the conventional method of coating the surface of the stent with heparin, Korean Patent Publication No. 2000-0059680 discloses that the surface of the stent undergoing a surface modulation process has an amine group in diaminocyclohexane (DACH) plasma. After dipping in heparin solution at 25-50 ° C. for 5 to 80 minutes, washing with deionized water to remove weakly bound heparin to coat heparin on the surface of the stent (hereinafter referred to as 'DACH method'). ) Is disclosed.

However, in the case of the stent with heparin attached by the above DACH method, the coagulation of heparin significantly prevents the restenosis rate after the procedure, but the binding of heparin to the stent surface is weak and the heparin is rapidly eluted. The disadvantage is that the activity gradually decreases.

The present invention was conceived by recognizing the above points, an object of the present invention is to coat the heparin on the surface of the metal stent using dopamine to improve the stability of heparin adhesion by chemical bonding of dopamine and heparin prevents the clotting effect It was confirmed that can be sustained, the present invention was completed.

It is an object of the present invention to provide a coating solution containing heparin and dopamine that can sustain the effect of preventing blood clotting.

Still another object of the present invention is to provide a method for preparing a heparin-coated stent, or a heparin-coated stent prepared by the method, by spraying the coating solution onto a metal stent surface or immersing the stent in the solution.

In order to achieve the above object, the present invention provides a coating solution containing hepamine and dopamine that can maintain the effect of preventing blood clotting.

The present invention provides a method for producing a heparin coated stent, and a heparin coated stent prepared by the method for spraying the coating solution on the surface of the metal stent or immersing the stent in the solution.

The method for producing a heparin coated stent according to the present invention is characterized by using a coating solution containing heparin and dopamine.

The coating solution according to the present invention is prepared by mixing heparin and dopamine in a molar ratio of 1: 1 to 3.

The method for producing a heparin coated stent according to the present invention is characterized in that the coating solution prepared by the above method is sprayed on the surface of the metal stent, or by dipping the stent in the solution to coat the heparin on the surface of the stent.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for producing a heparin coated stent, characterized in that the coating solution of heparin and dopamine is sprayed on the surface of the metal stent or immersing the stent in the solution to coat the heparin on the surface of the stent.

The coating solution is prepared by mixing heparin and dopamine in a molar ratio of 1: 1 to 3, and more preferably prepared by mixing heparin and dopamine in a molar ratio of 1: 1.2 to 1.5.

In the present invention, the metal stent is a biocompatible metal selected from the group consisting of stainless steel, cobalt-chromium alloy, tantalum, nitimol and gold, the structure of the stent is a tube structure, spiral structure and / or network structure It can be used to have.

In the present invention, the coating process of heparin is a process of coating the heparin on the surface of the stent by applying the coating solution to the metal stent surface, immersing the stent in the composite solution, or spraying the composite solution on the surface of the stent It can be coated by the method.

The present invention provides a heparin coated stent prepared by the above method.

The heparin coated stent according to the present invention improves the stability of heparin adhesion by chemical bonding of dopamine and heparin by coating heparin on the surface of the metal stent using dopamine, so that the heparin-coated stent can maintain the effect of preventing blood clotting as compared to the conventional heparin coated stent. Has an advantage.

1 is a view showing a general stent,
2 is a graph showing the amount of heparin remaining on the surface of the stent over time of the heparin coating stent according to the present invention,
3 is a graph showing the hemolysis rate over time of the heparin coated stent according to the present invention,
Figure 4 is a graph showing the degree of activation of anticoagulant (Anticoagluant) over time of the heparin coating stent according to the present invention,
Figure 5 is a photograph showing the platelet adhesion state over time of the heparin coating stent according to the present invention.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited by the following examples, and it is apparent to those skilled in the art that various changes or modifications can be made within the idea and scope of the present invention.

Here, unless otherwise defined in the technical terms and scientific terms used, those having ordinary skill in the art to which the present invention belongs have a generally understood meaning.

Repeated descriptions of the same technical constitution and operation as those of the conventional art will be omitted.

EXAMPLES Heparin Coating

According to the present invention, heparin was coated on the stent surface using dopamine by the following process.

200 mg of heparin and 36 mg of dopamine were mixed in a buffer solution (MES, pH 5.5) and reacted at 37 ° C. for 11 hours. The reaction was performed by adding 405 mg of EDC (1-Ethy1-33 (3-dimethylaminopropy1) carbodiimide hydrochloride) and 121 mg of NHS (N-hydroxysulfosuccinimide) to activate the -COOH group of heparin. After removal of dialysis (3 days) and lyophilization (3 days) for removal, the solution was dissolved in 50 mM TBS (Tris Buffered Saline) at pH 8.5 to prepare a coating solution, and then stent of cobalt-chromium alloy in the solution. Was soaked for 12 hours to coat heparin on the surface of the stent.

On the other hand, as a comparative example for comparison, heparin was coated on the stent surface of the cobalt-chromium alloy using the DACH method disclosed in Korean Patent Laid-Open No. 2000-0059680.

Test Example 1 Heparin Coating Stability Test of Heparin Coating Stent

In the heparin coated stent prepared by the manufacturing method of the above embodiment, the amount of heparin remaining on the surface of the stent was checked by using a toluidine blue method to confirm the stability of heparin coating when heparin was attached to the surface of the stent.

0.005% of toluidine blue dissolved in PBS (hosphate buffered saline) buffer was treated on the surface of the heparin coated stent. After the reaction was slowly shaken at room temperature, 200 μl of the reaction solution was collected at each time period, and the value was confirmed at an absorbance of 540 nm using a spectrometer.

FIG. 2 is a graph showing the amount of heparin remaining on the surface of the stent over time of the heparin coated stent. As can be seen in Figure 2, the heparin coating stability on the stent surface was slightly decreased over time in the case of the stent coated with heparin using the dopamine of the embodiment according to the present invention, but the amount was insignificant. On the other hand, in the case of the stent of the comparative example coated with heparin using the conventional DACH method, heparin decreased with time, and on the 28th, it was confirmed that the remaining amount was reduced to about 50% of the initial adhesion amount.

From the above results, it was confirmed that the heparin-coated stent using dopamine according to the present invention has improved adhesion stability of heparin by chemical bonding of dopamine and heparin.

[ Test Example  2] heparin coating Stent  Hemolysis

Hemolysis experiments were performed to determine the interaction between heparin-coated stents and the blood in the body. If a metal graft such as a stent is placed in the body, hemolysis is increased. Therefore, heparin-attached stents (Hepanime) using the dopamine of the above embodiment, heparin-attached stents using the conventional DACH method, and heparin-coated stents (Bare Metal Stnet; BMS) as controls, respectively. The degree of hemolysis was compared.

Fresh blood was collected from healthy pigs and 10% Na-citrate (3.2%) was added to prevent coagulation. For the experiment, the experiment was divided into the following three groups.

Experimental group: 10 ml NACl

(-) Control: 20 ml NACl

(+) Control: 10 ml NACl + 10 ml distilled water

NaCl was used for red blood cell hemolysis by osmotic pressure. After reacting for 30 minutes at room temperature, 0.2 ml of blood prepared in advance was treated in each group and further reacted for 60 minutes. After centrifugation at 750 rpm for 5 minutes, the supernatant of 0.2 ml was taken and its value was confirmed at 540 nm by absorbance using a spectrometer.

Hemolysis rate was defined as in Equation 1 below.

Figure 3 is a graph showing the hemolysis rate over time of the heparin coating stent according to the present invention. As can be seen in Figure 3, it was confirmed that hemolytic action is significantly less in the heparin-coated stents (Examples and Comparative Examples) compared to the untreated stent (control) without heparin-coated. In particular, heparin-coated stents showed little change in hemolysis rate using the dopamine of the above example, but in the case of the stent of the comparative example coated with heparin by the conventional DACH method, hemolysis was gradually increased over time. Was confirmed to show a significant difference with the heparin-coated stent using dopamine.

Therefore, the heparin stability of the heparin coated stent using dopamine according to the present invention is considered to be superior to the heparin stability by chemical bonding of dopamine and heparin by the conventional DACH method. .

[ Test Example  3] heparin coating Stent Thrombin  Inactivation experiment

The coagulation process begins with the conversion of prothrombin to thrombin. Antithrombin III inhibits this process. Heparin is known to increase the activity of antithrombin 3. Therefore, it was confirmed how heparin-coated stent inactivates thrombin during the coagulation process. This experiment was performed using a substrate specialized for thrombin.

0.2 mg / ml thrombin substrate S-2238 and 70 mU / ml antithrombin 3 were added to Tris-buffer [50 mM Tris (pH 8.4), 1 g / 1 PEO6000, 1 g / 1 BSA, 150 mM NaCl]. After mixing, heparin-coated stents were treated. Then 150 μl thrombin (1.2 U / mL) was added. The reaction was stopped at room temperature for 10 minutes, and 50 µl of acetic acid (40%) was added to stop the reaction. 200 µl of the reaction solution was taken and its value was confirmed at an absorbance of 405 nm using a spectrometer.

Figure 4 is a graph showing the degree of activation of anticoagulant (Anticoagluant) over time of the heparin coating stent according to the present invention. As can be seen in Figure 4, the heparin-coated stent using the dopamine of the embodiment shows the stabilized activity of the anticoagulant irrespective of time, whereas the activity of the heparin-coated stent of the comparative example by the conventional DACH method This could be seen to decrease gradually.

[ Test Example  4] heparin coating Stent Antiplatelet  Adhesion experiment

The initial phenomenon of thrombus formation is the accumulation of platelets. If platelets are inhibited from adhering to the stent surface, it will be a great help in preventing thrombus. Therefore, heparin platelet adhesion was observed when heparin-coated stents (Examples and Comparative Examples) were treated with platelet-rich plasma, compared to untreated stents (control).

The experiment was conducted in the following manner. Fresh blood was collected from healthy pigs and 10% Na-citrate (3.2%) was added to prevent coagulation. The blood collected to prepare platelet rich plasma (PRP) was centrifuged at 150 rpm for 15 minutes. The precipitate was collected by further centrifugation at 500 rpm for 20 minutes to see only the supernatant. 3 × 10 platelets were treated with heparin-coated platelets and reacted at room temperature for 3 hours. In order to confirm the adhesion pattern using a scanning electron microscope, the specimen was fixed for 2 hours with 2.5% glutaaldehyde and sequentially dehydrated with 40, 60, 80, and 90% ethanol.

Figure 5 is a photograph showing the platelet adhesion state over time of the heparin coating stent according to the present invention. As can be seen from FIG. 5, a large number of platelets could be observed in the untreated control stent without heparin. In contrast, in the stent coated with heparin using the dopamine of the example and the stent coated with heparin by the conventional DACH method, a significantly smaller number of platelets was observed.

From the above results, the heparin-coated stent using the dopamine according to the present invention, including the untreated stent, heparin stability was observed significantly less platelet adhesion than the case of the stent coated heparin by the conventional DACH method Could.

Claims (6)

delete delete Preparing a coating solution in which heparin and dopamine were mixed at a molar ratio of 1: 1.2 to 1.5 by adding EDC (1-Ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride) and NHS (N-hydroxysulfosuccinimide) Wow;
Immersing the metal stent in the coating solution;
Including, heparin-coated stent manufacturing method. The method of claim 3, wherein
The metal stent is a method for manufacturing a heparin coated stent, characterized in that the biocompatible metal selected from the group consisting of stainless steel, cobalt-chromium alloy, tantalum, nitimol and gold. delete delete

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