KR100491700B1 - Method for immobilization of antithrombotic proteins on polytetrafluoroethylene surface by plasma treatment - Google Patents

Abstract

The present invention relates to a method of immobilizing an antithrombotic protein on a PTFE surface using a plasma process, specifically, generating a carboxyl group on a PTFE surface using a plasma; The surface-activated PTFE is immersed in a solution containing an enzyme polymerization catalyst and anti-thrombotic protein to fix the anti-thrombotic protein comprising a covalent bond through a carboxyl group, the method according to the present invention is a chemical solvent By fixing the antithrombogenic protein in PTFE without the use of the antithrombotic protein, the antithrombotic protein can be effectively applied to various blood contact medical supplies such as vascular catheters. have.

Description

Method for immobilization of antithrombotic proteins on polytetrafluoroethylene surface by plasma treatment}

The present invention relates to a method of immobilizing an antithrombotic protein on a PTFE surface using a plasma process, specifically, generating a carboxyl group on a Teflon (PTFE) surface using a plasma; It relates to a method of immobilizing the anti-thrombotic protein comprising the step of immersing the surface-activated Teflon in a solution containing an enzyme polymerization catalyst and anti-thrombotic protein covalently bonded through a carboxyl group.

Plasma is a gas that is separated into electrons with negative charges and ions with positive charges at high temperature, and refers to a gas having a high degree of charge separation but having a negative number of positive and positive charges as a whole. Plasma currently used has a very low total energy level that does not significantly increase the temperature of the chamber. However, the plasma is very reactive at the level of atoms or ions. have.

As the plasma can be generated at a high temperature or a low temperature, research is being conducted according to each temperature. Applications at high temperatures include plasma vapor deposition, plasma vapor physical deposition, and the like, which cause reactions at high temperatures of 500 ° C. or higher. Efforts have been made to improve the surface properties in a desired direction while using the properties of the existing substrate by changing the physical, mechanical, electrical, and chemical properties of the metal or ceramic material through the above method.

In addition, as a target material that uses a plasma state without heating at room temperature among plasmas generated at low temperatures, research is being conducted to improve the chemical properties of the surface of polymer materials that are concerned about deterioration of material properties due to thermal denaturation. In particular, in the case of medical use, an attempt to apply plasma surface energy change to improvement of antithrombogenicity (Liping T, Yuliang W, Richard BT.Fibrinogen adsorption and host tissue response to plasma functionalized surfaces. J. Biomed. Mater. Res., 1998; 42: 156) and attempted to increase antithrombogenicity through immobilization of bioactive proteins (Yasuhide N, Takehisa M. Novel Surface Fixation Technology of Hydrogen Based on Photochemical Method: Heparin-Immobilized Hydrogelated Surface.J. Polym. Sci., : Part A: Polym. Chem., 1993; 31: 977-82).

In terms of improving antithrombogenicity, materials may be synthesized in new ways (Engbers GH, Feijen J. Current techniques to improve the blood compatibility of biomaterial surfaces.Int . J. Artif. Org ., 1991; 14: 199- 2) using a method of chemically modifying the surface of the material (Takashi S, Takehisa M. Synthesis of polyazido-derivatized substances and photochemical surface modification to immobilize functional groups. J. Biomed. Mater. Res ., 1996; 32: 157-64), chemically modifying the surface of the material and then restraining blood coagulation, such as heparin or urokinase, or immobilizing drugs or enzymes that dissolve fibrin, a major component of blood clots already occurring (Kim SW, Ebert CD). , Mcrea JC, Briggs C, Byun SM, HP Kim.Biological activity of antithrombotic agents immobilized on polymer surfaces.Ann NY Acad. Sci., 1983; 416: 513-24). The methods listed above showed better antithrombogenicity than conventional unmodified materials. In particular, attempts to achieve semi-permanent performance by implanting cells on the surface in contact with blood by improving cell portability and adhesion characteristics as a way to maintain long-term antithrombogenicity (Laura APW, Klaus S, Antony RG, Peter RS, Kathryn RN.Performance of small diameter synthetic vascular prostheses with confluent autologous endothelial cell linings. J. Biomed. Mater. Res., 1996; 30: 221-9).

Meanwhile, polytetrafluoroethylene (hereinafter referred to as 'PTFE') among synthetic polymers used as medical biomaterials has chemically inert and hydrophobic surface properties. These properties make them stable in the body for a long time without stimulating immune function or thrombosis mechanism in vivo. However, these intrinsic properties decrease with the time of transplantation in the body due to the adsorption of proteins.

Therefore, the present inventors tried to fix the antithrombotic protein to the surface of PTFE in order to use PTFE as a biomaterial having excellent surface properties for a long time, and after modifying the surface of the PTFE using a plasma reaction, The present invention was completed by finding a method of immobilization by covalent bonding, and finding that the material obtained by the above-described method was excellent in antithrombogenic reaction as a result of the blood clot dissolution test.

Although the stimulation of thrombosis in the body is low, the bioactive protein on the surface of PTFE is added for the purpose of actively maintaining physiologically active enzymes on the surface. We have developed a method to immobilize.

Accordingly, it is an object of the present invention to provide a method for immobilizing antithrombogenic proteins on the surface of PTFE in order to enhance the ability to inhibit thrombus formation.

Specifically, an object of the present invention is to generate a plasma to generate carboxyl groups on the PTFE surface; The surface-activated PTFE is immersed in a solution containing an enzyme polymerization catalyst and anti-thrombotic protein, and the immobilization method of the anti-thrombotic protein comprising the step of covalently fixing through a carboxyl group.

It is still another object of the present invention to provide PTFE in which an antithrombogenic protein prepared by the above method is immobilized.

In addition, another object of the present invention is to provide a use of the anti-thrombotic protein immobilized PTFE in a variety of blood contact medical supplies such as vascular catheters.

In order to achieve the above object, the present invention provides a method for immobilizing an antithrombotic protein on a surface of polytetrafluoroethylene (hereinafter referred to as PTFE) using a plasma reaction, PTFE obtained thereby, and a use thereof.

Hereinafter, the present invention will be described in more detail (see FIG. 1 ).

In the present invention, to improve the ability to inhibit thrombus formation

Introducing an inert gas to generate a plasma to introduce a carboxyl group to the PTFE surface (step a); And

The surface-activated PTFE is immersed in a solution containing an enzyme polymerization catalyst and an antithrombotic protein (step b) to covalently bind the antithrombotic protein and PTFE through a carboxyl group.

In step a), the PTFE surface is plasma treated to introduce carboxyl groups at a constant concentration.

The plasma is then generated in the presence of a carrier gas using a conventional plasma generator in the art. The carrier gas may include, but is not limited to, inert gases such as argon, carbon dioxide, oxygen, moisture, hydrogen peroxide, acrylic acid, ammonia, and the like. In the embodiment of the present invention, the plasma was generated using argon gas.

At this time, the plasma is generated by a conventional RF plasma generator, DC discharge and microwave.

The concentration of the carboxyl groups introduced is variable depending on the intensity and time of the plasma introduced and the flow rate of the carrier gas. That is, step a) may generate a functional group capable of covalently binding a protein without using a chemical solvent on the PTFE surface by a gaseous plasma reaction. The functional groups generated on the PTFE surface were subjected to toluidine blue staining. The dyeing method is a method of measuring the concentration of the reagent by measuring the absorbance by combining the carboxyl group and the dyeing reagent of the functional groups generated on the surface of the target PTFE specimen and then separating the dyeing reagent bound to the surface. The carboxyl functional groups generated on the surface of PTFE are proportional to the concentration of the dyeing reagent appearing in the absorbance, so it is possible to determine the relative surface functional concentrations according to the plasma treatment conditions.

According to a preferred embodiment, when the plasma intensity is 5 to 150 W, the time is 10 seconds to 10 minutes, and the flow rate of the carrier gas is performed at 5 to 100 cc / min, an appropriate concentration of carboxyl group can be obtained. The above numerical values may be appropriately changed by those skilled in the art depending on the plasma apparatus to be applied.

According to another preferred embodiment, in order to find the conditions under which the maximum carboxyl functional groups are formed on the surface, the relative concentration by the absorbance measurement using the toludiene solution, which is a coloring material, is changed while changing the power and generation time of the plasma generator. As a result of the measurement (see FIGS . 2A and 2B ), the plasma duration was about 300 seconds under the condition that the plasma output was 50w, showing the highest carboxyl group concentration.

However, when the plasma treatment is performed for 300 seconds, the plasma energy is transferred to the polymer and overheated, so that a relatively short time is advantageous at low plasma output, and thus, it is most advantageous to have a plasma duration of 60 seconds at 10 W plasma output.

PTFE of the present invention is not limited in form, and may include any form as long as plasma can be introduced, and includes, for example, sheets, thin films, pellets, and the like.

In step b), the anti-thrombotic protein is covalently bound to the surface-modified PTFE in the carboxyl group.

Specifically, the surface-activated PTFE is carried out by immersion in a solution containing a buffer solution, enzyme polymerization catalyst and antithrombotic protein at 20 ~ 37 ℃ for 10 to 24 hours.

The enzyme polymerization catalyst is 1-ethyl-3- (3- (dimethylamino propyl) carbodiimide (1-ethyl-3- (3- (dimethylamino propyl) carbodiimide, hereinafter referred to as 'EDC') in order to promote a covalent bond reaction. And 0.01 to 0.2 M, preferably 0.03 to 0.06 M.

The buffer solution is used as is commonly used, containing potassium phosphate (KH ₂ PO ₄ ), acetic acid, etc., 0.01 to 0.10 M, preferably 0.1 to 0.5 M and the final pH is 3.5 to 5.5, preferably Is 4.0 to 5.0, most preferably pH 4.4 to 4.7.

Antithrombotic proteins used in the present invention include those commonly recognized in the art and are not limited in the present invention, for example, Lumbrokinase, heparin, hirudin, albumin And lipoproteins. Among the antithrombotic proteins, lumpbrokinase has a thrombolytic ability to dissolve newly formed blood clots, and heparin, hirudin, and liposome q-baek, etc. have the characteristics of reducing the production of blood clots by inhibiting or delaying the progression of blood clots. have.

The antithrombotic protein content is based on 1 mL of the reaction solution per unit surface area of PTFE, and the concentration of the protein in the solution is used in the range of 0.01-2 mg / mL, preferably 0.1-0.3 mg / mL. At this time, if the concentration is out of the above range, the surface improving effect is reduced when the concentration is too low. If the concentration is too high, the requirement of the antithrombotic protein is increased, thereby increasing the cost.

According to a preferred embodiment of the present invention, the anti-thrombotic protein was covalently bonded to PTFE, which was surface-modified with carboxyl groups, by using a room brokinase.

According to another preferred embodiment of the present invention, the clear zone using the fibrin plate in order to determine the thrombolytic ability of the PTFE covalently bonded to the lumpbrokinase, clear zone is clearly generated as shown in FIG . As a result, it was found that rumbrokinase having thrombolytic ability was effectively fixed to the PTFE surface.

After the covalent reaction of step b), a conventional washing process, for example, ultrasonic cleaning is performed to remove the unreacted material attached to the PTFE surface.

Therefore, by using the plasma process of the present invention to generate a functional group on the surface of the PTFE and through the enzyme fixation method using a catalyst it is possible to increase the blood compatibility by lowering the extent of the thrombus generated when the PTFE surface in contact with the blood. In other words, technically engineered plasma processes and biochemical enzyme fixation methods are combined to induce an increase in the antithrombogenicity of medical materials.

As a result, the substance is preferably applicable to medical supplies in contact with blood as the substance has excellent thrombolytic ability. The medical article includes all articles that can be made of PTFE and includes, for example, an artificial blood vessel, a blood vessel catheter, a cannular, a blood oxygenator, and the like.

According to a preferred embodiment of the present invention, a small diameter artificial blood vessel made of PTFE was subjected to a fibrin plate test after plasma treatment and immobilization of lumpbrokinase with an antithrombotic protein according to the present invention, as shown in FIGS . 4A and 4B . As shown, the fibrin region in which black bands were dissolved around the white Teflon sheet showed antithrombogenic effect. As a result, it is possible to prevent unwanted reactions in the living body due to the remaining catalyst and chemicals when the medical article using PTFE immobilized with the antithrombotic protein of the present invention comes into contact with blood in the living body.

The present invention will be described in more detail by the following examples.

However, the following examples are only examples of the present invention and the present invention is not limited thereto.

Example 1 Preparation of PTFE Covalently Bonded with Antithrombotic Protein

Step a) Preparation of PTFE Modified with Carboxyl Group

PTFE was carried out as follows to form a carboxyl group on the surface using a plasma process.

PTFE (TEFLON, manufactured by Shinhwa Bestron Co., Ltd.) sheet (thickness: 0.15 mm, 150 mm x 300 mm width) was cut to a size of width x length 1 x 1 cm, and ultrasonic cleaning was performed for 20 minutes while soaking in alcohol. Subsequently, vacuum drying was performed for 24 hours in a vacuum oven at 45 ° C.

In order to perform the plasma process, the Teflon sample was placed in a chamber of a plasma apparatus (automotive (Korea)) and the vacuum degree was reached to a pressure of 0.05 mmHg using a vacuum pump, and then argon gas was fed at a rate of 20 CC per minute. Flowed into. At this time, the valve of the vacuum pump side was kept open. When the pressure in the vacuum chamber reached a certain level, the plasma generator was operated and the plasma matcher was adjusted to effectively generate the plasma. By such a plasma matcher, the incident wave and the reflected wave generated in the plasma generator may be adjusted to be a sine wave.

At this time, the plasma generation time was changed to 10 watts, 30 watts, and 50 watts to determine the concentration of carboxyl groups formed on the PTFE surface, and the plasma generation time was 10 seconds, 30 seconds, 1 minute, 2 minutes, 3 The test specimen was prepared by proceeding to 5 minutes.

The surface-modified PTFE obtained according to each condition was confirmed by the toludiene blue staining method and the spectrophotometer (spectrophotometer) to determine the presence of the dye, the presence of a carboxyl group on the surface.

Step b): Preparation of PTFE Covalently Bonded with Antithrombotic Protein

In order to immobilize the surface-modified PTFE covalently bonded to the anti-thrombotic protein obtained in the above step was carried out as follows.

A buffer solution containing 0.05 M KH ₂ PO ₄ and 0.03 M 1-ethyl-3- (3- (dimethylamino propyl) carbodiimide (EDC) was prepared, followed by 0.3 mg of lumpbrokinase as an antithrombotic protein. The reaction solution was prepared by adding a concentration of / mL, and the PTFE sample obtained in step a) was added to the solution, and the reaction was continued at room temperature for 16 hours.

After the fixation reaction of the lumpbrokinase was completed, the solution was lightly washed twice with phosphate buffer to remove unreacted substances attached to the surface, and ultrasonic cleaning was performed for 5 minutes.

Experimental Example 1 Measurement of Concentration of Surface Modified Carboxyl Group

In order to determine the concentration of the carboxyl groups present on the surface of the PTFE sheet obtained in step a) of Example 1 with the plasma power and time.

After inducing a reaction by using a colorant reacting with a carboxyl group, the molecules of the reacted colorant were separated into a solution state through a separation reaction and the absorbance was measured to relatively measure the concentration of the carboxyl group present on the surface.

A 5 x 10 ^-5 M toludiene blue solution was prepared using a 10 ^-4 M NaOH solution as a colorant, 3 mL of the solution was added, and a piece of plasma treated PTFE sheet was added thereto and reacted at 25 ° C for 3 hours. . Subsequently, unreacted toludiene blue was removed using 10 ^-4 M NaOH solution (pH 10.0).

The PTFE sheet was immersed in 3 mL of 50% (v / v) acetic acid solution to dissolve toludiene blue bound to the surface, and the absorbance was measured at 631 nm using a spectrophotometer. The toll Louis molecular extinction coefficient of the diene Blue ^{39576.7M -1 cm - 1 (λmax =} 631 nm) is measured by using the three conditions, each sheet of PTFE.

These measurement results are as shown in Table 1 below and FIGS. 2A to 2B .

Carboxyl group concentration measurement (x 10 ^-7 M / cm ² ) Power time 10 W 30 W 50 W 100 W 10 sec 5.84 3.29 4.69 2.79 30 sec 7.20 4.35 5.98 3.79 60 sec 8.09 3.46 3.65 3.55 120 sec 9.48 2.62 4.85 2.19 180 seconds 8.39 4.62 5.65 3.98 300 sec 8.55 3.99 16.6 5.23

According to Table 1 , it can be seen that a high surface functional group concentration appeared at a plasma intensity of 10W. In addition, the plasma duration was 60 seconds at 10W and 300 seconds at 50W showed the highest surface functional group concentration. Figure 2a is a graph showing the concentration of the carboxyl functional group according to the plasma treatment time, it can be seen that the concentration of the carboxyl group has a relatively high value at 120 seconds or more conditions.

In addition, Figure 2b is a graph showing the concentration of the carboxyl group according to the change in the treatment intensity of the plasma, it can be seen that the surface functional group concentration is relatively evenly high at 10W plasma treatment intensity.

Comparing the data of FIG . 2A and FIG. 2B , it can be seen that the processing time of about 60 seconds at the 10 W plasma output is most appropriate in terms of productivity.

Experimental Example 2 Measurement of Thrombus Solubility

In order to determine the thrombolytic ability of the anti-thrombotic protein-covalently bonded PTFE sheet according to the present invention, fibrin plates were used as follows.

Fibrin is a basic structure of the thrombus that is generated in the final stage after the fibrinogen in the blood is triggered by an external stimulus or an internal mechanism and the thrombus reaction proceeds. Removing fibrin almost completely eliminates the problem of blood clots.

Fibrin plates were prepared by pouring a boric acid buffer solution in which fibrinogen was dissolved at a concentration of 0.8% (w / v) in a Petri dish, adding 5 IU of thrombin, and shaking well to ensure uniform mixing. Thrombin converts fibrinogen to fibrin and into a gel that constitutes a thrombus. Fibrin plates prepared by reacting in this way were used for the test for thrombolytic ability after 2 hours.

The occurrence of a clear zone was observed by placing the PTFE sheet obtained in Example 1 (conditions: 10W x 60 seconds treatment) on a fibrin plate, and the results are shown in FIG. 3 . Pure PTFE was used for comparison.

Figure 3 is a clear zone created by the fibrin plate test, the white portion (d) is an undissolved fibrin plate, black in the center of the square around the white Teflon sheet (c) Denotes a clear zone in which fibrin is dissolved by lumpbrokinase immobilized on a PTFE sheet. That is, in the PTFE (a) according to the present invention, a clear zone (c) was formed to dissolve the surrounding fibrin to become a liquid in a transparent state . In FIG. 3 , the background is black to make the difference with fibrin clear. It is a picture taken. In contrast, pure PTFE (b) showed no change.

These results indicate that the antithrombogenic protein is immobilized on the PTFE surface by the present invention, and confirm that the PTFE exhibits an antithrombogenic effect.

Experimental Example 3 Measurement of Thrombus Solubility in Small Bore Artificial Blood Vessels

The fibrin plate was used as follows to determine the thrombolytic ability by directly applying to a small diameter artificial blood vessel using the immobilization method according to the present invention.

Small diameter artificial blood vessels (Vascular graft, Gore-Tex, internal diameter 6mm) were purchased and immobilized after the plasma treatment (plasma power 10W, duration 60 seconds) according to the present invention. Subsequently, the fibroin plate test was performed in the same manner as in Experimental Example 2 by cutting the artificial blood vessels in which the lubrokinase immobilized, and the anti-thrombotic effect was shown in FIGS . 4A and 4B .

Figure 4a is a fibrin plate test of the PTFE (a) treated by the present invention cut in cross section, it can be seen that the black clear zone (b) showing that the fibrin is dissolved.

In addition, Fig. 4b is a fibrin plate of PTFE (a ') treated by the present invention cut in the longitudinal section, as shown in Fig. 4a clear zone (b') that can be seen that dissolves the fibrin around Could confirm.

Thus, the immobilization method of the present invention can be incorporated into any form of PTFE—medical products in contact with blood—and can increase antithrombotic effects.

As described above, by introducing the anti-thrombotic protein into the surface of PTFE through plasma treatment according to the present invention, it is possible to effectively delay or inhibit the generation of blood clots when it comes into contact with blood, and thus to various blood contact medical supplies such as vascular catheters. Applicable

1 is a schematic of an immobilization method according to the present invention.

2A and 2B are graphs showing absorbance measured with an absorbance meter according to voltage ( FIG. 2A ) and time ( FIG. 2B ) of carboxyl functional groups formed on the surface of polytetrafluoroethylene (PTFE) treated with argon gas plasma . to be:

3 is a photograph showing the fibrin plate test:

a: PTFE specimen fixed with lumpbrokinase prepared according to the present invention;

b: pure PTFE specimen; c: clear zone;

d: fibrin solution

4A and 4B are photographs showing a clear zone by plasma treatment of Teflon-made small diameter artificial blood vessel (ePTFE) according to the present invention and fixation of lumpbrokinase followed by fibrin plate test:

a, a ': small caliber artificial blood vessel specimen; b, b ': clearzone;

c, c ': fibrin solution

Claims (10)

Introducing a carboxyl group into polytetrafluoroethylene (PTFE) by injecting a plasma generating gas selected from the group consisting of carbon dioxide, moisture, hydrogen peroxide and acrylic acid to generate a plasma; A method of immobilizing the anti-thrombotic protein on the surface of PTFE by immersing the obtained PTFE in a buffer containing an enzyme polymerization catalyst and an anti-thrombotic protein and covalently binding the carboxyl group introduced into the PTFE and the amine group of the anti-thrombotic protein. The method of claim 1, wherein the plasma is generated by a conventional RF plasma generator, DC discharge, microwave, or the like. delete The method according to any one of claims 1 to 3, wherein the flow rate of the gas for plasma is 10 to 100 CC / min. The method of claim 1, wherein the antithrombotic protein is selected from the group consisting of Lumbrokinase, heparin, hirudin, albumin, and lipoprotein. The method according to any one of claims 1 to 5, wherein the concentration of the antithrombotic protein is 0.01 to 2 mg / mL or more in the reaction solution. The method of claim 1, wherein the immersion is carried out at 20 to 37 ℃ for 10 to 24 hours. The method of claim 1, wherein the buffer solution is characterized in that the pH range of 3.5 to 5.5. PTFE covalently bonded with the antithrombogenic protein obtained by the immobilization method of claim 1. The PTFE covalently bound to the antithrombotic protein of claim 1 is a variety of blood contact medical care, including artificial blood vessels, blood vessel catheter, cannular, blood oxygenator goods.