KR20210157023A - Artificial vessel having fibrinolytic activity and preparation method thereof - Google Patents

Abstract

The present invention relates to artificial blood vessels with thrombolytic activity and a manufacturing method thereof, in which the artificial blood vessels with fucoidan bound thereto can prevent neointimal hyperplasia and thrombokinesis when used in vascular interventional procedures or reconstruction procedures, and can prevent or treat angiostenosis. In particular, thrombi are dissolved by fucoidan, so that patency rates can be improved as compared to existing artificial blood vessels.

Description

Artificial blood vessel having thrombolytic activity and its manufacturing method {Artificial vessel having fibrinolytic activity and preparation method thereof}

The present invention relates to an artificial blood vessel having thrombolytic ability and a method for manufacturing the same.

Cardiovascular disease is the leading cause of death worldwide, with 17.3 million deaths in 2008, and 7.3 million deaths due to coronary heart disease in particular. Bypass surgery is common to allow peripheral bypass coronary artery revascularization, and transplantation with own blood vessel (internal diameter (ID) < 6 mm) is the most widely used. Nevertheless, autologous transplantation has several drawbacks, including significant morbidity and disease associated with it, and lack of usefulness due to previous organ harvesting. Synthetic materials such as polyethylene terephthalate (PET) and expanded polytetrafluoroethylene (ePTFE) have been successfully used as prostheses for replacement of medium-to-large diameter vessels (ID > 6 mm) where conditions of high blood flow and low resistance predominate. used [Bordenave et al, 2008]. However, synthetic implants used under intra-articular bypass and coronary artery bypass (ID < 6 mm) have limitations in that the patency rate cannot be sustained for a long time. The patency period of ePTFE prostheses was reported to be 40-50% when proximal femoral artery bypass was used for 5 years, and 20% when using infrapopliteal artery bypass after 3 years [Bordenave et al, 2008]. It has been reported that the use of PET or ePTFE for small-diameter vessels induces several complications such as aneurysm, intimal hyperplasia, calcification, thrombosis, infection, and lack of growth potential in childhood [Dean et al, 2012, Rathore et al, 2012].

After vascular intervention or reconstruction using artificial blood vessels, neointimal proliferation in blood vessels, especially in the anastomosis, induces and promotes thrombus formation, leading to stenosis. It is the most effective way to increase the patency rate by preventing and treating it. Currently, ePTFE, which is currently most used as an artificial blood vessel, has advantages such as flexibility, spontaneous closure of a wound when a needle is inserted into a dialysis machine, and hemostasis. There is no way to prevent this, and even in the heparin-coupled ePTFE artificial blood vessel, which has been compensated for this problem, the patency rate is improved by about 20% through the inhibition of thrombus formation, but there is a problem in that the vascular occlusion occurs through the formation of the neointimal in the end.

Accordingly, the present inventors completed the present invention by preparing a new artificial blood vessel using fucoidan, which exhibits thrombolytic ability and anti-neointimal properties, as a result of research to develop a new artificial blood vessel.

Japanese Patent Laid-Open No. 1997-327509

An object of the present invention is to provide an artificial blood vessel having thrombolytic ability using fucoidan.

Another object of the present invention is to provide a method for manufacturing the artificial blood vessel.

One aspect of the present invention provides an artificial blood vessel in which fucoidan is covalently fixed to the inner surface of the artificial blood vessel.

As used herein, the term 'artificial blood vessel' refers to an artificial organ that replaces a living blood vessel that cannot be cured by surgery or pharmacological treatment. These artificial blood vessels satisfy the vascular function, can be sterilized so that there are no abnormalities during or after transplantation, do not twist, have adequate permeability and sutureability, and meet the mechanical and physical properties of elasticity and flexibility to withstand continuous contraction and expansion. , it should not be toxic in the human body, it should not cause infection, inflammation and immune response, and blood should not clot inside the blood vessel. Therefore, the artificial blood vessel is preferably a polymer having at least one of blood compatibility, anti-calcification properties, and histocompatibility.

The artificial blood vessel may be a biodegradable polymer or a non-degradable polymer as a biocompatible polymer, or a polymer obtained by polymerizing a biodegradable polymer and a non-degradable polymer.

According to one embodiment of the present invention, the artificial blood vessel is polytetrafluoroethylene (PTFE), expanded polytetrafluoroethylene (ePTFE), polyurethane (polyurethane), Dacron (Dacron) and It may be made of one or more biocompatible polymers selected from the group consisting of collagen. The collagen may be derived from decellularized xenogeneic blood vessels. In this case, it is preferable that the artificial blood vessel be ePTFE that can be used semi-permanently because the physical properties are stably maintained for a long period of time.

On the other hand, as the inner surface of the artificial blood vessel is adsorbed and rearranged by interaction with proteins in the blood, a hydrophobic adsorption phenomenon occurs. The hydrophobic adsorbed protein does not desorb from the blood vessel surface and acts as a substance attracting platelets, white blood cells and other tissue cells, thus causing thrombosis. In order to improve or prevent the hydrophobic adsorption of the artificial blood vessel, it may be to change the inner or inner surface of the artificial blood vessel to be hydrophilic, or to chemically surface-modify the inner surface of the artificial blood vessel by introducing an antithrombotic material to the inner surface of the artificial blood vessel.

According to one embodiment of the present invention, the inner surface of the artificial blood vessel may be surface-modified with fucoidan having a thrombolytic ability, and at this time, in order to improve the binding force between the artificial blood vessel and fucoidan, and at the same time to modify the overall inner surface of the artificial blood vessel. The hydrophilic polymer may also be surface-modified.

According to one embodiment of the present invention, the inner surface of the artificial blood vessel is collagen, poly(1,8-octanediol-co-citric acid) (poly(1,8-octanediol-co-citric acid)), Polyethylene glycol (PEG), poly(acrylic acid), poly(acrylate), poly(acrylamide), poly (vinyl ester)-based (poly(vinylester)), polyoxide-based (polyoxide), polyvinyl alcohol-based (poly(vinyl alcohol)), polystyrene-based (polystyrene) and at least one selected from the group consisting of derivatives or mixtures thereof It may be surface-modified with a hydrophilic polymer.

According to one embodiment of the present invention, the surface concentration of the hydrophilic polymer is 0.0001 to 100 μg/cm ² , 0.0005 to 50 μg/cm ² , 0.001 to 10 μg/cm ² , 0.005 to 5 μg/cm ² , 0.01 to 1 μg/cm ² , or 0.05 to 0.5 μg/cm ² It may be one. At this time, if the surface concentration of the hydrophilic polymer is ^{less than 0.0001 μg/cm 2} , proteins in the blood may be adsorbed and cause thrombus formation, and ^{if it exceeds 100 μg/cm 2} , the hydrophilicity is too high, so the binding of fucoidan is weak can be done

The fucoidan (fucoidan) is a material extracted from brown algae, and is used as a food, dietary supplement, additive, etc. as a functional raw material. This fucoidan helps excretion of cholesterol, lowers blood cholesterol levels, can prevent vascular disease, prevents obesity from developing into adult diseases, and has recently been reported to have blood clotting inhibitory or thrombolytic properties.

According to an embodiment of the present invention, the fucoidan is seaweed ( Undaria pinnatifida ), Fucus evanescens , Saccharina cichorioides , seaweed ( Costaria costata ), rhubarb ( Eisenia bicyclis ), Fucus vesiculosus, etc. It may be purified or processed from the extract extracted from .

According to one embodiment of the present invention, the fucoidan may be at least one selected from the group consisting of fucoidan, recombinant fucoidan, fucoidan derivative, and fucoidan analog having properties similar to fucoidan.

According to one embodiment of the present invention, the surface concentration of the fucoidan is 0.001 to 10 μg/cm ² , 0.005 to 5 μg/cm ² , 0.01 to 1 μg/cm ² , or 0.05 to 0.5 μg/cm ^{2 It} may be one have. At this time, if the surface concentration of fucoidan is ^{less than 0.001 μg/cm 2} , thrombus dissolution may not be smooth and may cause thrombus formation, and ^{if it exceeds 10 μg/cm 2} , hemostasis may be delayed during endothelial damage .

The fucoidan may be provided through a covalent bond with the surface of an artificial blood vessel surface-modified with a hydrophilic polymer. Here, the 'covalent bond' refers to a chemical bond formed by sharing electrons between atoms. The type of covalent bond may be an amine bond, an azide bond, an amide bond, etc. In particular, an amide bond is more preferable due to the ease of formation of a covalent bond or stability after bonding. Such a covalent bond may exhibit a characteristic in which fucoidan is not eluted even when the artificial blood vessel is washed by a solvent dissolving fucoidan.

In addition, another aspect of the present invention provides a method for manufacturing an artificial blood vessel.

According to one embodiment of the present invention, the manufacturing method of the artificial blood vessel comprises the steps of: a) coating a hydrophilic polymer on the inner surface of the artificial blood vessel; And d) the hydrophilic polymer-coated artificial blood vessel may be one comprising the step of imparting fucoidan as a covalent bond.

Step a) is a process of primary surface modification of the inner surface of the artificial blood vessel with a hydrophilic polymer.

The description of the artificial blood vessel and the hydrophilic polymer is the same as described above.

According to one embodiment of the present invention, the artificial blood vessel of step a) is polytetrafluoroethylene (PTFE), expanded polytetrafluoroethylene (ePTFE), polyurethane, dacron (Dacron) and collagen (collagen) may be made of one or more biocompatible polymers selected from the group consisting of.

According to one embodiment of the present invention, the hydrophilic polymer in step a) is collagen, poly(1,8-octanediol-co-citric acid) (poly(1,8-octanediol-co-citric acid)) , polyethylene glycol (PEG), poly (acrylic acid), poly (acrylate), poly (acrylamide), One selected from the group consisting of poly(vinylester), polyoxide, poly(vinyl alcohol), polystyrene, and derivatives or mixtures thereof It may be more than

According to one embodiment of the present invention, the surface concentration of the hydrophilic polymer in step a) may be in the ^{range of 0.0001 to 100 μg/cm 2 .}

Step b) is a process of secondary surface modification by imparting fucoidan to the hydrophilic polymer coated on the artificial blood vessel through a covalent bond.

The description of the fucoidan is the same as described above.

According to one embodiment of the present invention, the fucoidan in step b) may be one or more selected from the group consisting of fucoidan, recombinant fucoidan, fucoidan derivative, and fucoidan analog having properties similar to fucoidan.

According to an embodiment of the present invention, the surface concentration of fucoidan in step b) may be ^{0.001 to 10 μg/cm 2 .}

The artificial blood vessel prepared through the above step may be one in which a hydrophilic polymer is introduced as a spacer into a polymer matrix made of a biocompatible polymer, and then fucoidan is covalently immobilized.

According to one embodiment of the present invention, the artificial blood vessel may have a structure in which a biocompatible polymer, a hydrophilic polymer, and a fucoidan are sequentially stacked.

Fucoidan-coupled artificial blood vessel according to the present invention can prevent neointimal proliferation and thrombus formation when used for vascular intervention or reconstruction, and can prevent or treat vascular clotting. In particular, since the thrombus is dissolved by fucoidan, the patency rate can be improved compared to the existing artificial blood vessel.

1 is a schematic schematic diagram of an artificial blood vessel according to an embodiment of the present invention.
Figure 2 is a picture of the spin shearing equipment used in an embodiment of the present invention.
3 is an FT-IR result confirming the amide bond between fucoidan and POC of a fucoidan-coupled ePTFE artificial blood vessel prepared according to an embodiment of the present invention.
4 is an SEM result of the inner surface of the POC-coated ePTFE artificial blood vessel prepared according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail. However, these descriptions are provided for illustrative purposes only to help the understanding of the present invention, and the scope of the present invention is not limited by these illustrative descriptions.

Example 1. Preparation of fucoidan-coupled ePTFE artificial blood vessels

1-1. POC synthesis

To synthesize poly(1,8-octanediol-co-citric acid) (POC), citric acid and 1,8-octanediol were mixed at a molar concentration of 1:1 at 140°C for 15 minutes. Then, the mixture was mixed by mixing at 120 °C for 1 hour. For purification, the mixture (prepolymer) was put in distilled water to precipitate, distilled water was removed, and then freeze-drying was performed.

1-2. POC coating

In order to coat an ePTFE tube (diameter 6 mm) with POC, a glass rod with a diameter of 5 mm is immersed in 10% POC (using ethanol or 1,4-dioxane solvent), and then the glass rod is spin shearing equipment (see Fig. 2). The ePTFE tube was inserted into the unfixed side of the glass rod. The inside of the ePTFE tube was coated by manually rotating the ePTFE tube counterclockwise while rotating the glass rod clockwise. The coating process was repeated 3 times and then the ePTFE tube was dried in an oven at 80° C. for 48 hours.

1-3. Aminated fucoidan preparation

Fucoidan was prepared by further purifying fucoidan from seaweed spore leaf extract-fucoidan purchased from Haewon Biotech Co., Ltd. by chromatography. 500 mg of the prepared fucoidan was dissolved in 7.5 mL of water, and then mixed with 2.5 mL of 2.6 N epichlorohydrin at 40°C for 2 hours. The mixture was dialyzed in water at 25°C for 2 days, freeze-dried, and dissolved in 3 mL of 30% aqueous ammonia and reacted at 40°C for 90 minutes. Thereafter, it was dialyzed in water at 25° C. for 2 days and freeze-dried.

1-4. Manufacturing of fucoidan-conjugated ePTFE artificial blood vessels

In 1-2 above, the POC-coated ePTFE tube was washed with PBS solution at 37° C. for 3 days, and then dried naturally at room temperature for one day. Then, immersed in 0.1 M MES buffer (buffer) for 1 hour, transferred to 50 mM DH solution (including 0.1 M MES buffer 100 ml, 300 mM EDC and 150 mM NHS) and soaked at room temperature for 5 hours. It was washed with NaCl and distilled water.

In 0.1 M MES buffer mixed with 200 mM EDC and 100 mM NHS, 0.2 mM aminated fucoidan prepared in 1-3 above was mixed. The POC-coated ePTFE tube was immersed in the mixed solution, mixed for one day, and then washed with 2 M NaCl and distilled water to remove the residue and dried. The preparation of fucoidan-conjugated ePTFE artificial blood vessels was completed.

Experimental Example 1. Analysis of physical properties of fucoidan-coupled ePTFE artificial blood vessels

The physical properties of the fucoidan-coupled ePTFE artificial blood vessel prepared in Example 1 were analyzed.

To confirm the amide bond (peptide bond) between fucoidan and POC in fucoidan-conjugated ePTFE artificial blood vessels (Fuco-cojugated ePTFE), Fourier transform infrared spectroscopy (FT-IR) spectroscopy was used to determine fucoidan-free ePTFE artificial blood vessels (POC). ePTFE) and comparative analysis.

As a result, referring to FIG. 3 , in the fucoidan-coupled ePTFE artificial blood vessel prepared in Example 1, it was confirmed that amide-bonded amides I and II between fucoidan and POC were formed.

In addition, in order to confirm the inner surface of the POC-only coated ePTFE artificial blood vessel (POC ePTFE), a scanning electron microscope (SEM) was used to compare and analyze the POC-coated ePTFE artificial blood vessel.

As a result, referring to FIG. 4 , it was confirmed that the surface of the POC-coated ePTFE artificial blood vessel was modified through the POC coating.

So far, the present invention has been looked at with respect to preferred embodiments thereof. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (13)

An artificial blood vessel in which fucoidan is covalently fixed to the inner surface of the artificial blood vessel.
The method according to claim 1,
The artificial blood vessel is selected from the group consisting of polytetrafluoroethylene (PTFE), expanded polytetrafluoroethylene (ePTFE), polyurethane, Dacron and collagen. An artificial blood vessel made of one or more biocompatible polymers.
The method according to claim 1,
The inner surface of the artificial blood vessel is collagen, poly(1,8-octanediol-co-citric acid) (poly(1,8-octanediol-co-citric acid)), polyethylene glycol (PEG) , poly(acrylic acid), poly(acrylate), poly(acrylamide), poly(vinylester) )), polyoxide, poly(vinyl alcohol), polystyrene, and derivatives or mixtures thereof blood vessel.
The method according to claim 1,
The surface concentration of the hydrophilic polymer is 0.0001 to 100 μg/cm ² Artificial blood vessels.
The method according to claim 1,
The fucoidan is at least one selected from the group consisting of fucoidan, recombinant fucoidan, fucoidan derivatives and fucoidan analogues having properties similar to those of fucoidan.
The method according to claim 1,
The surface concentration of the fucoidan is 0.001 to 10 μg/cm ² Artificial blood vessels.
a) coating a hydrophilic polymer on the inner surface of the artificial blood vessel; and
d) imparting fucoidan to the artificial blood vessel coated with the hydrophilic polymer as a covalent bond
A method of manufacturing an artificial blood vessel comprising a.
8. The method of claim 7,
The artificial blood vessel in step a) is made of polytetrafluoroethylene (PTFE), expanded polytetrafluoroethylene (ePTFE), polyurethane, Dacron and collagen. A method consisting of one or more biocompatible polymers selected from the group consisting of.
8. The method of claim 7,
The hydrophilic polymer of step a) is collagen, poly(1,8-octanediol-co-citric acid) (poly(1,8-octanediol-co-citric acid)), polyethylene glycol (PEG) ), poly(acrylic acid), poly(acrylate), poly(acrylamide), poly(vinyl ester) vinylester)), polyoxide-based (polyoxide), polyvinyl alcohol-based (poly(vinyl alcohol)), polystyrene-based (polystyrene), and a method that is at least one selected from the group consisting of derivatives or mixtures thereof.
8. The method of claim 7,
The surface concentration of the hydrophilic polymer in step a) is 0.0001 to 100 μg/cm ^{2 The} method.
8. The method of claim 7,
The fucoidan of step b) is at least one selected from the group consisting of fucoidan, recombinant fucoidan, fucoidan derivative, and fucoidan analog having properties similar to fucoidan.
8. The method of claim 7,
The surface concentration of fucoidan in step b) is 0.001 to 10 μg/cm ^{2 The} method is.
8. The method of claim 7,
The artificial blood vessel is a method in which a biocompatible polymer, a hydrophilic polymer and a fucoidan are sequentially stacked.

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