JP2014018477A - Heparin coating method for stent surface - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a heparin coating method for a stent surface.SOLUTION: The present invention relates to a heparin coating method for stent surfaces, capable of sustaining a coagulation prevention effect. The heparin coating method according to the present invention includes applying heparin to a metal-material stent surface using dopamine, and improving heparin adhesion stability by chemical conjugation between dopamine and heparin. The heparin coating method can sustain the coagulation prevention effect compared with that of the existing heparin coating stent.

Description

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

  Atherogenic arterial stenosis and thrombosis are linked to each other and are one of the most dangerous diseases that kill many lives each year. Is specified by. Stenosis refers to the narrowing or constriction of blood vessels due to the deposition of fat, cholesterol or other substances in the blood vessels of the human body. In severe cases, restenosis can cause the blood vessel to become completely clogged. Thrombosis is when a thrombus occurs or exists in a blood vessel or in the cavity of the heart. Thrombus is caused by coagulation of blood components, mainly platelets and fibrin, when cell components are adsorbed, and thrombosis, like restenosis, induces vascular stenosis at the site where it occurs. .

  One method for treating blood vessels constricted or clogged by restenosis is percutaneous transluminal coronary angioplasty (PTCA), also called coronary angioplasty. This is a method in which a balloon catheter is inserted into a constricted portion of a blood vessel and then inflated by the size of the blood vessel, thereby smoothing the flow of clogged blood flow. However, about one-third of patients who have undergone PTCA treatment must undergo angioplasty again due to the occurrence of restenosis in which the dilated blood vessels narrow again within 6 months after the operation.

  In order to prevent such restenosis of the blood vessel, instead of performing angioplasty on the restenosis portion, a stent as shown in FIG. Alternatively, it is possible to perform a method of inserting the stent together during the treatment. A stent is a tube made of a metal or plastic material, and there are a mesh and a tube. Most stents currently in use are made of metal and are automatically inflated or inflated by a balloon. Whether or not a stent can be inserted depends on the characteristics of restenosis such as the size of the blood vessel and the position of restenosis. The role of the stent is to reinforce the blood vessels that have been widened by the recently performed angioplasty, or to prevent the blood vessels from returning to their original state if no angioplasty is performed. it can. Stents are usually implanted using a catheter. In this method, the stent is first folded to be small and mounted on a balloon catheter. Next, after allowing the catheter to reach the lesion site or the recently tensioned site through the patient's blood vessel, the stent is expanded and secured to the site.

  By such a method, the stent becomes permanently present in the blood vessel, and by opening the blood vessel, the blood flow is smoothed, and the symptoms (mainly chest pain) caused by the cardiovascular disorder can be removed.

  Usually, the blockage of the human body passage such as arterial blockage has been treated with arteriotomy and a drug that dissolves a specific blocking substance, but in recent years, vascular stenosis of the coronary artery using a balloon catheter. The treatment to remove is universal.

  However, when operating with a stent made of reticulated stainless steel or titanium as described above and squeezing the blockage in the blood vessel, the blood in the blood vessel is coagulated on the stent surface, so that the blood vessel is restenulated There was a problem that there were many.

  Therefore, recently, a method is known in which heparin, which is known to have anticoagulability, is coated on the stent surface. As a conventional method for coating heparin on the stent surface, Patent Document 1 discloses that after the surface of the stent that has undergone the surface modulation step is made to have amine groups in diaminocyclohexane (DACH) plasma, A method of coating heparin on the stent surface by washing with deionized water to remove weakly bound heparin after 5 to 80 minutes of immersion in a heparin solution at 50 ° C. (hereinafter referred to as “DACH”). Method)).

  However, in the case of a stent to which heparin is attached by the above-mentioned DACH method, blood coagulation can be prevented and the restenosis rate after treatment can be significantly reduced. However, since the binding between heparin and the stent surface is weak, It has a disadvantage that it is easily eluted and its activity gradually decreases with time.

Korean Patent Application Publication No. 2000-0059680

  The present invention has been made in view of the above-described problems, and the present inventors have coated heparin on the surface of a metallic stent using dopamine, thereby chemically bonding dopamine and heparin. It was confirmed that the adhesion stability of heparin was improved and the effect of preventing blood coagulation could be sustained, and the present invention was achieved.

  An object of the present invention is to provide a coating solution containing heparin and dopamine that can maintain the effect of preventing blood coagulation.

  Another object of the present invention is to provide a method for producing a heparin-coated stent and a heparin-coated stent produced by the method, wherein the coating solution is sprayed onto a metallic stent surface or the stent is immersed in the coating solution. It is to provide.

  According to the present invention for achieving the above-described object, a coating solution containing heparin and dopamine capable of maintaining the effect of preventing blood coagulation is provided.

  According to the present invention, a heparin-coated stent manufactured by spraying a coating solution containing the heparin and dopamine onto the surface of a metallic stent, or immersing the stent in the solution and coating the surface of the stent with heparin, and its A manufacturing method is provided.

  Hereinafter, the present invention will be described in detail.

  The “stent” in the present invention is a metal or plastic tube inserted into a blood vessel or conduit and used to smooth the flow of fluid while keeping the lumen open. It is a medical device for preventing closure due to externally applied pressure or stenosis.

  According to the present invention, a heparin-coated stent is produced by coating heparin on a stent surface by spraying a coating solution of heparin and dopamine onto the surface of a metallic stent or immersing the stent in the solution. A method is provided.

  The coating solution according to the present invention can be prepared by including heparin and dopamine that can maintain the effect of preventing blood coagulation, and further includes a crosslinking activator, a crosslinking activity auxiliary agent, or a mixture thereof. it can.

  In the present invention, the crosslinking activator is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (1-ethyl-3- (3-dimethylaminopropyl) carbohydride hydrochloride, EDC), 1-ethyl-3- (3- (Trimethylammonio) propyl) carbodiimide (1-ethyl-3- (3-trimethylamino) propyl) carbodiimide, ETC), or 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide (1-cyclohexyl-3) -(2-morpholinoethyl) carbodiimide, CMC), preferably 1-ethyl-3- ( - dimethylaminopropyl) carbodiimide hydrochloride (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, EDC) can be used, but is not limited thereto.

  The crosslinking activity auxiliary agent is N-hydroxysuccinimide (N-hydroxysuccinimide, NHS), 1-hydroxybenzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2, 3-benzotriazine (3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine, HOOBt), 1-hydroxy-7-azabenzotriazole (1-hydroxy-7-azabenazotriol, HOAt) , Or any one selected from N-hydroxysulfosuccinimide (Sulfo-NHS), preferably N— Mud carboxymethyl succinimide (N-hydroxysuccinimide, NHS) can be used, but is not limited thereto.

  The coating solution according to the present invention is a coating solution obtained by reacting a mixed solution of heparin and dopamine with addition of EDC (1-Ethyl-3- [3-dimethylaminopropyl] carbohydrate) and NHS (N-hydroxysulfocimide). Features.

  In the present invention, the coating solution produced by reacting the above dopamine, EDC (1-Ethyl-3- [3-dimethylaminopropyl] carbohydrate hydride), NHS (N-hydroxysulfuccinimide), heparin, NH There is an advantage that the effect of preventing blood coagulation can be sustained by improving the stent attachment stability of heparin by chemical bonding of -COOH group and, as a result, controlling the dissolution rate of heparin.

  According to the present invention, a coating solution obtained by adding EDC (1-Ethyl-3- [3-dimethylaminopropyl] carbohydrate) and NHS (N-hydroxysulfocimide) to a mixed solution of heparin and dopamine was reacted, A method for producing a heparin-coated stent is provided, wherein the coating surface is coated with heparin by spraying a coating solution onto a metallic stent surface or immersing the stent in the solution.

  According to the present invention, EDC (1-Ethyl-3- [3-dimethylaminopropyl] carbohydrate) and NHS (N-hydroxysuccinimide) are added to a solution in which heparin and dopamine are mixed at a molar ratio of 1: 1 to 3. There is provided a method for producing a heparin-coated stent, comprising: producing a reacted coating solution; and immersing a metallic stent in the coating solution.

  According to the present invention, a solution in which heparin and dopamine are mixed in a molar ratio of 1: 1.2 to 1.5 is mixed with EDC (1-Ethyl-3- [3-dimethylaminopropyl] carbohydrate hydride) and NHS (N-hydroxysulfocimide). There is provided a method for producing a heparin-coated stent, comprising: a step of producing a coating solution reacted with the addition of a metal solution; and a step of immersing a metallic stent in the coating solution.

  The coating solution according to the present invention is prepared by mixing heparin and dopamine in a molar ratio of 1: 1 to 3, more preferably heparin and dopamine in a molar ratio of 1: 1.2 to 1.5. It is produced by mixing in a number ratio.

  The mixing ratio of heparin and dopamine has an important meaning in the effect of suppressing hemolysis and thrombus formation. As a result of investigating the hemolysis phenomenon when mixing less than 1 mol or more than 3 mol of dopamine with respect to 1 mol of heparin (mol), it becomes impossible to control the amount of hemolysis as time passes. There was a problem that safe stenting could not be performed.

  In addition, as a result of investigating the effect of inhibiting thrombus formation, stable anticoagulant activity cannot be maintained over time, and as a result, the restenosis rate of the treated stent is increased and the blood vessels are restenulated. There was a problem that.

  In the present invention, the metal stent is a biocompatible metal selected from the group consisting of stainless steel, cobalt-chromium alloy, tantalum, nitinol, and gold, and the stent has a tube structure, a spiral structure, or a net-like structure. What has a structure can be used.

  In the present invention, the coating process of the heparin is a process in which the coating solution is added to the surface of the metallic stent and the stent surface is coated with heparin, and a method of immersing the stent in a synthetic solution (dipping coating method), or The stent surface can be coated by a method of spraying a synthetic solution (air spray coating method, spray-coating method) or the like.

  The method may further include drying the stent after the coating solution is coated on the stent. In the process of drying the stent, the solvent is evaporated and removed, and the heparin polymer remains on the surface to form a thin film.

  According to the present invention, a heparin-coated stent manufactured by the above manufacturing method is provided.

  The heparin-coated stent according to the present invention is inserted and fixed by percutaneous balloon angioplasty into a blood vessel having a cardiovascular disease related to a coronary artery using a treatment method suitable for the kind of the stent. A stent fixed in a blood vessel can prevent restenosis in the blood vessel and maintain a dilated state of the blood vessel for a long period of time.

  The heparin-coated stent according to the present invention is a metallic stent using a coating material obtained by reacting dopamine with EDC (1-Ethyl-3- [3-dimethylaminopropyl] carbohydrate) and NHS (N-hydroxysulfocimide). By coating heparin on the metal, it is possible to improve the heparin adhesion stability of the metal stent, thereby controlling the elution rate of heparin, and thus maintaining the effect of preventing blood coagulation.

  The heparin-coated stent according to the present invention has heparin adhesion stability improved by chemical bonding of dopamine and heparin by coating heparin on the surface of a metallic stent using dopamine, and blood compared with existing heparin-coated stents. It has the advantage that the effect of preventing coagulation can be sustained.

1 is a diagram illustrating a normal stent. 4 is a graph illustrating the amount of heparin remaining on the stent surface over time of the heparin-coated stent according to the present invention. 3 is a graph illustrating the hemolysis rate over time of a heparin-coated stent according to the present invention. 4 is a graph illustrating the degree of activation of an anticoagulant over time of a heparin-coated stent according to the present invention. 3 is a photograph illustrating the state of platelet adhesion over time of a heparin-coated stent according to the present invention.

  Hereinafter, the present invention will be described in more detail using 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 spirit and scope of the present invention.

  At this time, unless otherwise defined, technical terms and scientific terms used have a meaning generally understood by those having ordinary knowledge in the technical field to which the present invention belongs.

  In addition, redundant description of the same technical configuration and operation as in the past is omitted.

[Example] Heparin coating According to the present invention, heparin was coated on the stent surface by the following process using dopamine.

  200 mg of heparin and 36 mg of dopamine were mixed in a buffer solution (MES, pH 5.5) and reacted at a temperature of 37 ° C. for 11 hours. The reaction process was performed by adding 405 mg of EDC (1-Ethy1-3- (3-dimethylaminopropyl) carbohydrate) and 121 mg of NHS (N-hydroxysulfuccinimide) for the activation of the —COOH group of heparin. After removing the impurities (EDC, NHS) through dialysis (3 days) and lyophilization (3 days), this was dissolved in pH 8.5, 50 mM TBS (Tris Buffered Saline) to prepare a coating solution. Thereafter, the stent surface was coated with heparin by immersing a cobalt-chromium alloy stent in the solution for 12 hours.

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

[Test Example 1] Test of heparin coating stability of heparin-coated stent In the heparin-coated stent manufactured by the manufacturing method of the above example, in order to confirm the heparin coating stability when heparin was attached to the stent surface, The amount of heparin remaining on the stent surface over time was confirmed using the toluidine blue method.

  The surface of the heparin-coated stent was treated with 0.005% toluidine blue dissolved in PBS (phosphate Buffer Saline) buffer. After reacting while gently shaking at room temperature, 200 μl of the reaction solution was collected every time period, and the value was confirmed at an absorbance of 540 nm using a spectrometer.

  FIG. 2 is a graph illustrating the amount of heparin remaining on the stent surface over time for a heparin-coated stent. As can also be seen from FIG. 2, the stability of the heparin coating on the stent surface decreased slightly over time for the stents coated with heparin using the dopamine of the examples according to the present invention, but the amount is It was very small. On the other hand, in the case of the comparative example stent coated with heparin using the existing DACH method, heparin decreases with time, and the remaining amount is about 50% of the initial adhesion amount on the 28th day, It was confirmed that a large amount was reduced.

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

Test Example 2 Hemolysis Experiment of Heparin-Coated Stent A hemolysis experiment was performed to confirm the interaction between the heparin-coated stent and the blood in the body. When a metal implant, such as a stent, is implanted in the body, hemolysis increases. Accordingly, a stent with heparin attached using the dopamine of the above example (Hepanime), a stent with a comparative example to which heparin is attached by the existing DACH method, and a stent not coated with heparin as a control group (Bare Metal Stnet) BMS) was used to compare the degree of hemolysis for each.

  After collecting fresh blood from healthy pigs, 10% Na-citrate (3.2%) of blood volume was added to prevent clotting. For the experiment, the experiment was divided into the following three groups.

Experimental group: 10 ml NaCl
(−) Control group: 20 ml NaCl
(+) Control group: 10 ml NaCl + 10 ml distilled water

  NaCl was used for hemolysis of erythrocytes by osmotic pressure. After reacting at room temperature for 30 minutes, 0.2 ml of blood prepared in advance was processed into each group, and further reacted for 60 minutes. Then, after centrifuging at 750 rpm for 5 minutes, 0.2 ml of the supernatant was taken, and the numerical value was confirmed at an absorbance of 540 nm using a spectrometer.

  The hemolysis rate was defined as the following formula 1.

[Formula 1]
R = (A−C1) / (C2−C1) × 100
(R: hemolysis rate, A: absorbance of experimental group, C1: absorbance of (−) control group, C2: absorbance of (+) control group)

  FIG. 3 is a graph illustrating the rate of hemolysis over time of a heparin-coated stent according to the present invention. As can also be seen from FIG. 3, it was confirmed that the hemolytic action was significantly reduced in the heparin-coated stents (Examples and Comparative Examples) compared to the untreated stents that were not coated with heparin (control group). It was. In particular, in the case of the stent coated with heparin using the dopamine of the above example, there was almost no change in hemolysis rate, but in the case of the comparative example stent coated with heparin by the existing DACH method, as time passed. It was confirmed that the amount of hemolysis gradually increased and showed a significant difference from the stent coated with heparin using dopamine on the 28th day.

  Therefore, from the above results, in the case of a stent coated with heparin using dopamine according to the present invention, heparin stability is superior due to the chemical bond between dopamine and heparin compared to a stent coated with heparin by the existing DACH method. I think that the.

[Test Example 3] Inactivation experiment of thrombin of heparin-coated stent The blood coagulation process is started while prothrombin is converted into thrombin. A substance called antithrombin 3 suppresses this process. Heparin is also known to increase the activity of antithrombin 3. Thus, it was determined how much heparin-coated stents inactivate thrombin during the blood clotting 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 mixed in Tris-buffer [50 mM Tris (pH 8.4), 1 g / 1 PEO6000, 1 g / 1 BSA, 150 mM NaCl]. And then processed into heparin coated stents. Then 150 μl thrombin (1.2 U / ml) was added. The reaction was carried out at room temperature for 10 minutes, and 50 μl of aceto acid (40%) was added to stop the reaction. 200 μl of the reaction solution was collected, and the numerical value was confirmed at an absorbance of 405 nm using a spectrometer.

  FIG. 4 is a graph illustrating the degree of activation of an anticoagulant over time of a heparin-coated stent according to the present invention. As can be seen from FIG. 4, the stent coated with heparin using the dopamine of the above example shows a stable activity of the anticoagulant regardless of the passage of time. It was confirmed that the activity of the comparative stent coated with is gradually decreased.

[Test Example 4] Platelet adhesion experiment of heparin-coated stent The initial phenomenon in which a thrombus occurs is the collection of platelets. If platelets are prevented from adhering to the stent surface, it will greatly contribute to prevention of thrombus. Therefore, the appearance of platelet adhesion by heparin when treated with platelet-rich plasma on the surface of stents coated with heparin (Examples and Comparative Examples) as compared to untreated stents without heparin (control group). Was observed.

The experiment was conducted by the following method. After collecting fresh blood from healthy pigs, 10% Na-citrate (3.2%) of blood volume was added to prevent clotting. The collected blood was centrifuged at 150 rpm for 15 minutes in order to prepare platelet rich plasma (PRP). Only the supernatant was collected and further centrifuged at 500 rpm for 20 minutes to obtain a precipitate. 3 × 10 ⁷ platelets were treated with each platelet to which heparin was attached, and then reacted at room temperature for 3 hours. In order to confirm the adhesion state using a scanning electron microscope, the test piece was fixed in 2.5% glutaraldehyde for 2 hours, and then dehydrated sequentially with 40, 60, 80, and 90% ethanol.

  FIG. 5 is a photograph illustrating the state of platelet adhesion over time of a heparin-coated stent according to the present invention. As can be seen from FIG. 5, a large number of platelets are observed in the untreated control group stent to which heparin is not attached. On the other hand, in the stent coated with heparin using the dopamine of the above example and the stent coated with heparin by the existing DACH method, a significantly smaller number of platelets was observed compared to the untreated stent.

  From the above results, the heparin-coated stent using dopamine according to the present invention should observe a significantly smaller number of platelet adhesions than the untreated stent and the stent coated with heparin by the existing DACH method. I was able to.

Claims (7)

  A method for producing a heparin-coated stent, wherein heparin is coated on a stent surface by spraying a coating solution of heparin and dopamine onto the surface of a metallic stent or immersing the stent in the solution.   The manufacturing method includes a step of manufacturing a coating solution in which EDC (1-Ethyl-3- [3-dimethylaminopropyl] carbohydrate) and NHS (N-hydroxysulfocimide) are added to a mixed solution of heparin and dopamine and reacted. 2. The heparin coating according to claim 1, further comprising coating the heparin on the stent surface by spraying the coating solution onto a metallic stent surface or immersing the stent in the solution. A method for manufacturing a stent.   The method for producing a heparin-coated stent according to claim 1 or 2, wherein the coating solution is produced by mixing heparin and dopamine in a molar ratio of 1: 1 to 3.   The method for producing a heparin-coated stent according to claim 3, wherein the coating solution is produced by mixing heparin and dopamine in a molar ratio of 1: 1.2 to 1.5.   The heparin-coated stent according to claim 1 or 2, wherein the metallic stent is a biocompatible metal selected from the group consisting of stainless steel, cobalt-chromium alloy, tantalum, nitinol and gold. Production method.   The method for manufacturing a heparin-coated stent according to claim 5, wherein the stent has a tube structure, a spiral structure, or a network structure.   A heparin-coated stent manufactured by the manufacturing method according to any one of claims 1 to 6.

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