KR102468362B1 - Artificial blood vessel and preparation method thereof - Google Patents

Abstract

Disclosed are an artificial blood vessel and a manufacturing method thereof, wherein the artificial blood vessel comprises: a porous tube substrate; a coating layer disposed on a surface of the porous tube substrate; and a fucoidan disposed on the coating layer. The coating layer comprises a first coating layer and a second coating layer disposed on the first coating layer. The first coating layer includes at least one of a catechol group-containing monomolecular compound, a derivative thereof, and a catechol group-containing monomolecular compound polymer, the second coating layer includes at least one of an amine-based polymer and an imine-based polymer, and a content of at least one of the amine-based polymer and the imine-based polymer is 2 μg/cm^2 or more and 9 μg/cm^2 or less. The artificial blood vessel has improved antithrombotic and patency rate.

Description

Artificial blood vessel and preparation method thereof {Artificial blood vessel and preparation method thereof}

It relates to an artificial blood vessel and a manufacturing method thereof.

Fabrics of polyester fibers and stretched polytetrafluoroethylene tubes are used as artificial blood vessels.

As for the stretchable polytetrafluoroethylene tube, polytetrafluoroethylene itself, which is a material, has excellent antithrombotic properties. Further, the stretched polytetrafluoroethylene tube has excellent biocompatibility of the porous structure obtained by stretching. Therefore, the elongated polytetrafluoroethylene tube has been mainly applied to artificial blood vessels compared to fabrics of polyester fibers.

However, antithrombotic properties are not sufficient even in a stretched polytetrafluoroethylene tube, and a sufficient patency rate may not be obtained in a small-diameter artificial blood vessel.

Therefore, there is still a need for artificial blood vessels having improved antithrombotic properties and patency rates.

US Patent Publication No. 2019-0365954

Vacuum 85 (2011) 1083-1086

One aspect is to provide an artificial blood vessel with improved antithrombotic properties and improved patency.

Another aspect is to provide a method for manufacturing the artificial blood vessel.

According to one aspect,

porous tube substrates;

A coating layer disposed on the surface of the porous tube substrate; and

It includes fucoidan disposed on the coating layer,

The coating layer is a first coating layer; And a second coating layer disposed on the first coating layer,

The first coating layer includes at least one of a catechol group-containing monomolecular compound, a derivative thereof, and a catechol group-containing monomolecular compound polymer,

The second coating layer includes at least one of an amine-based polymer and an imine-based polymer,

A content of at least one of the amine-based polymer and the imine-based polymer is 2 μg/cm ² or more and 9 μg/cm ² or less, and an artificial blood vessel is provided.

According to another aspect,

providing a plasma modified porous tube substrate;

introducing a coating layer on the surface of the plasma-modified porous tube substrate; and

Including; immobilizing fucoidan on the coating layer,

The coating layer includes a first coating layer and a second coating layer,

The first coating layer includes at least one of a catechol group-containing monomolecular compound, a derivative thereof, and a catechol group-containing monomolecular compound polymer,

The second coating layer includes at least one of an amine-based polymer and an imine-based polymer,

The content of at least one of the amine-based polymer and the imine-based polymer is 2 μg/cm ² or more and 9 μg/cm ² or less, and a method for preparing an artificial blood vessel is provided.

According to one aspect, an artificial blood vessel into which a coating layer having a novel composition is introduced may provide improved thrombosis resistance and patency rate.

1A to 1E are images showing contact angle measurement results for artificial blood vessel surfaces prepared in Examples 1 to 2 and Comparative Examples 1 to 3, respectively.
2a to 2e are images showing the results of quantification of toluidine blue O on the surfaces of artificial blood vessels prepared in Examples 1 to 2 and Comparative Examples 1 to 3, respectively.
3a to 3e are images showing the results of toluidine blue O quantification on the surfaces of artificial blood vessels prepared in Examples 3 to 4 and Comparative Examples 4 to 6, respectively.
4A to 4E are images showing antithrombotic ability evaluation results of artificial blood vessel surfaces prepared in Examples 3 to 4 and Comparative Examples 4 to 6, respectively.
5A to 5E are scanning electron microscope images of artificial blood vessel surfaces prepared in Examples 3 to 4 and Comparative Examples 4 to 6, respectively.
6a to 6e are cross-sectional images of four artificial blood vessels prepared in Comparative Example 4 after implantation into a porous tube substrate and rabbit carotid artery.
7A to 7F are cross-sectional images of five artificial blood vessels prepared in Example 3 after implantation into a porous tube substrate and rabbit carotid artery.

Since the present inventive concept described below may be applied with various transformations and may have various embodiments, specific embodiments are illustrated in the drawings and described in detail. However, this is not intended to limit the present creative idea to a specific embodiment, and should be understood to include all transformations, equivalents, or substitutes included in the technical scope of the present creative idea.

Like reference numerals have been used for like elements throughout the description of each figure. In the accompanying drawings, the dimensions of the structures are shown enlarged than actual for clarity of the present invention. Terms such as first and second may be used to describe various components, but the components should not be limited by the terms. These terms are only used for the purpose of distinguishing one component from another. For example, a first element may be termed a second element, and similarly, a second element may be termed a first element, without departing from the scope of the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise.

In this application, the terms "include" or "have" are intended to designate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features It should be understood that it does not preclude the possibility of the presence or addition of numbers, steps, operations, components, parts, or combinations thereof. In addition, when a part such as a layer, film, region, plate, etc. is said to be "on" another part, this includes not only the case where it is "directly on" the other part, but also the case where another part is present in the middle. Conversely, when a part such as a layer, film, region, plate, etc. is said to be "under" another part, this includes not only the case where it is "directly below" the other part, but also the case where another part is in the middle.

In this application molecular weight of a polymer means "weight average molecular weight" unless otherwise specified.

Hereinafter, an artificial blood vessel and a manufacturing method thereof according to exemplary embodiments will be described in more detail.

An artificial blood vessel according to an embodiment includes a porous tube substrate; A coating layer disposed on the surface of the porous tube substrate; and fucoidan disposed on the coating layer, wherein the coating layer comprises a first coating layer; and a second coating layer disposed on the first coating layer, wherein the first coating layer is one of a catechol group-containing unimolecular compound, a derivative thereof, and a catechol group-containing unimolecular compound polymer. Including the above, the second coating layer includes at least one of an amine-based polymer and an imine-based polymer, and the content of at least one of the amine-based polymer and the imine-based polymer is 2 μg/cm ² or more and 9 μg/cm ² or less. .

Excellent biocompatibility and low cytotoxicity are obtained by including a first coating layer including at least one of a catechol group-containing monomolecular compound, a derivative thereof, and a catechol group-containing monomolecular compound polymer. Therefore, it is suitable for application as an artificial blood vessel. Therefore, rapid generation of blood clots is suppressed by having excellent biocompatibility of the first coating layer.

Structural stability of the coating layer including the first coating layer and the second coating layer is improved when the artificial blood vessel includes a second coating layer including an amine-based monomolecular compound, a derivative thereof, or an imine-based polymer. Therefore, even if the artificial blood vessels are prevented for a long time, a high content of fucoidan fixed in the artificial blood vessels may remain. In addition, since the second coating layer includes an amine-based monomolecular compound, a derivative thereof, or an imine-based polymer, the number of reactive functional groups such as amine groups and imine groups contained in the second coating layer increases. Accordingly, the number of reaction sites at which the second coating layer can fix fucoidan increases. That is, the content of fucoidan disposed on the second coating layer is further increased. As a result, as the content of fucoidan included in the artificial blood vessel increases, the thrombotic resistance of the artificial blood vessel is improved and the patency rate is improved.

When the content of the amine-based polymer and/or the imine-based polymer disposed in the artificial blood vessel is less than 2 μg/cm ² , the content of the thrombogenesis-inhibiting substance fixed on the amine-based polymer and/or the imine-based polymer is reduced, thereby suppressing clot formation. As the ability decreases, the patency rate may decrease. If the content of the amine-based polymer and/or the imine-based polymer disposed in the artificial blood vessel is 9 μg/cm ² or more, cytotoxicity may increase, and the regenerative ability and/or hemostatic ability of the blood vessel may be deteriorated.

The content of at least one of the amine-based polymer and the imine-based polymer included in the artificial blood vessel may be, for example, 3 μg/cm ² or more and 9 μg/cm ² or less, or 3 μg/cm ² or more and 8 μg/cm ² or less. In addition to reduced cytotoxicity by including the amine-based polymer and/or imine-based polymer in the artificial blood vessel, the content of the thrombogenesis-inhibiting substance fixed on the amine-based polymer and/or the imine-based polymer increases, so excellent patency rate can be provided simultaneously.

In the present disclosure, a catechol group refers to a phenyl group containing two adjacent hydroxyl groups. For example, it means a functional group in which a hydroxyl group is connected to the 3rd and 4th positions of the phenyl group. That is, it means a functional group derived from catechol.

A hydroxyl group containing a catechol group is disposed in the direction of the surface of the porous tube substrate and can be self-assembled on the porous tube substrate. Accordingly, derivatives of unimolecular compounds containing catechol groups and polymers of unimolecular compounds containing catechol groups can be self-assembled on the porous tube substrate.

An artificial blood vessel includes a porous tube substrate.

The porous tube substrate may be, for example, a plasma modified porous tube substrate. Polar functional groups may be introduced on the surface of the porous tube substrate by plasma modification. The polar functional group introduced onto the porous tube substrate may be, for example, a hydroxy group or an amino group. By supplying oxygen (O ₂ ) gas, nitrogen (N ₂ ) gas, air, etc. at a constant rate while plasma is applied to the porous tube substrate, these supplied gases react with the surface of the porous tube substrate to form a porous tube substrate. Polar functional groups may be introduced on the surface. By disposing such a polar functional group on the surface of the porous tube substrate, hydrophilicity of the porous tube substrate is increased, and the first coating layer can be more easily introduced onto the porous tube substrate.

The porous tube substrate may include, for example, a hydrophobic polymer. The hydrophobic polymer included in the porous tube substrate is, for example, stretched polytetrafluoroethylene (ePTFE), polyurethane, etc., but is not limited thereto, and any material that can be used as a substrate for artificial blood vessels in the art is possible. Hydrophobic polymers are, for example, stretched polytetrafluoroethylene.

The artificial blood vessel includes a coating layer disposed on a porous tube substrate, and the coating layer includes a first coating layer.

The first coating layer may be, for example, a conformal layer disposed along the surface contour of the porous tube substrate. The first coating layer may be, for example, a conformal layer disposed to conform to the contour of the porous surface of the porous tube substrate. Since the first coating layer constitutes such a conformal layer, changes in the porosity of the artificial blood vessel can be suppressed by the introduction of the first coating layer. For example, when the first coating layer is coated with a conventional general polymer such as polycaprolactone, the polymer may block pores and decrease the porosity of the artificial blood vessel. In contrast, the first coating layer of the present invention is self-assembled on the surface of a porous tube substrate having catechol group-containing monomolecular compounds while and/or after self-assembly, such monomolecular compounds are self-assembled on the surface of the modified porous tube substrate. polymerized to form a polymer layer. Alternatively, the first coating layer of the present invention may form, for example, a monolayer of a plurality of monomolecular compounds self-assembled along the surface contour on the surface of a porous tube substrate modified with a catechol group-containing monomolecular compound. can Therefore, such a polymer layer or monolayer can effectively prevent clogging of pores included in the porous tube substrate.

The first coating layer includes at least one of a catechol group-containing monomolecular compound, a derivative thereof, and a catechol group-containing monomolecular compound polymer.

The molecular weight of the catechol group-containing monomolecular compound may be, for example, 300 Dalton or less, 250 Dalton or less, 200 Dalton or less, or 150 Dalton or less. The molecular weight of the monomolecular compound containing a catechol group may be, for example, 100 to 300 Daltons, 100 to 250 Daltons, 100 to 200 Daltons, or 100 to 150 Daltons.

Monomolecular compounds containing catechol groups are, for example, dopamine, 3,4-dihydroxyphenyl acetic acid, norepinephrine, ), 3,4-dihydroxybenzaldehyde (3,4-Dihydroxybenzaldehyde), 3,4-dihydroxybenzonitrile (3,4-Dihydroxybenzonitrile), 3,4-dihydroxybenzylamine hydrobromide (3,4-Dihydroxybenzylamine hydrobromide), 3-hydroxythio 3-Hydroxytyrosol, 2,3-Dihydroxybenzaldehyde, 3,4-Dihydroxybenzoic acid, Caffeic acid, Gallic acid (Gallic acid), DL-3,4-dihydroxyphenyl glycol, and 2-chloro-3',4'-dihydroxyacetophenone (2-Chloro-3′ , 4'-dihydroxyacetophenone).

A catechol group-containing unimolecular compound or a derivative thereof is self-assembled on a porous tube substrate, for example, and may be fixed on the porous tube substrate through a covalent bond. The catechol group-containing monomolecular compound or its derivative is fixed, for example, on a porous tube substrate through a covalent bond, thereby improving the structural integrity of the first coating layer including the catechol group-containing monomolecular compound or its derivative. Stability can be further improved.

Alternatively, a monomolecular compound containing a catechol group or a derivative thereof is self-assembled on the porous tube substrate, for example, and formed on the porous tube substrate through a non-covalent bond. can be fixed

In the present disclosure, a derivative of a catechol group-containing monomolecular compound refers to a product obtained by reacting at least one terminal functional group of the catechol group-containing monomolecular compound with another functional group or compound. For example, when the terminal amino group (-NH ₂ ) of a monomolecular compound containing a catechol group reacts with another compound and/or substrate having a carboxyl group (-COOH) to form an amide group (-CONH-), A compound containing such an amide group becomes a derivative of a monomolecular compound containing a catechol group.

A polymer of a catechol group-containing monomolecular compound is formed, for example, by self-assembling and polymerization of the catechol group-containing monomolecular compound on the porous tube substrate. It can be.

The polymer of the catechol group-containing monomolecular compound on the surface of the porous substrate by self-assembling and polymerization of the catechol group-containing monomolecular compound on the surface of the porous substrate. ) A coating layer containing may be formed. The structure, molecular weight, etc. of a polymer of a catechol group-containing unimolecular compound may be controlled depending on reaction conditions. The polymer of the catechol group-containing unimolecular compound may be a homopolymer having one repeating unit or a copolymer having a plurality of different repeating units.

The thickness of the first coating layer may be, for example, 1 nm to 200 nm, 1 nm to 150 nm, or 1 nm to 100 nm. When the first coating layer has a thickness within this range, structural stability of the first coating layer may be further improved. In addition, when the first coating layer has a thickness within this range, a conformal coating layer conforming to the surface contour of the porous tube substrate can be easily obtained.

The first coating layer may have a single layer or multi-layer structure. For example, the first coating layer may have a structure in which polymer layers having the same or different structures are stacked. For example, the first coating layer having a multi-layered structure may be obtained by dipping the porous tube substrate in a solution containing the coating composition a plurality of times at regular intervals. That is, the first coating layer may have a multilayer structure formed in a layer-by-layer manner.

The artificial blood vessel includes a coating layer disposed on a porous tube substrate, and the coating layer includes a first coating layer and a first coating layer disposed on the first coating layer.

The second coating layer may be, for example, a conformal layer disposed along the surface contour of the porous tube substrate and/or the first coating layer. The second coating layer may be, for example, a conformal layer disposed to conform to the contour of the surface of the porous tube substrate and/or the first coating layer. Since the second coating layer constitutes such a conformal layer, changes in the porosity of the artificial blood vessel can be suppressed by the introduction of the second coating layer.

The second coating layer includes at least one of an amine-based polymer and an imine-based polymer. An amine-based polymer is a polymer containing an amine group, and an imine-based polymer is a polymer containing an imine group. Amine-based polymers are, for example, polymerization products of monomers containing amine groups. An imine-based polymer is a polymerization product of a monomer having an imine group.

The imine-based polymer may include, for example, at least one selected from linear PEI, branched polyethyleneimine, and polyethylene diamine. The weight average molecular weight of the imine-based polymer may be, for example, 500 to 300,000 Daltons or 800 to 270,000 Daltons. When the amine-based polymer has a molecular weight within this range, a stable second coating layer may be formed.

The amine-based polymer and/or the imine-based polymer is, for example, grafted onto one or more of a catechol group-containing monomolecular compound, a derivative thereof, and a polymer of the catechol group-containing monomolecular compound. ) can be An amine-based monomolecular compound, a derivative thereof, or an imine-based polymer is, for example, a catechol group-containing monomolecular compound, a derivative thereof, and a polymer of the catechol group-containing monomolecular compound. By being grafted, the second coating layer may be firmly connected to the first coating layer through a covalent bond. As a result, structural stability of the coating layer including the first coating layer and the second coating layer may be improved. Thus, the durability of artificial blood vessels can be improved. An amine-based unimolecular compound may be grafted onto, for example, a catechol group-containing unimolecular compound. For example, an amine-based monomolecular compound may be grafted onto a polymer of a catechol group-containing monomolecular compound. For example, a straight-chain or branched imine-based polymer may be grafted onto a monomolecular compound containing a catechol group. For example, a straight-chain or branched imine-based polymer may be grafted onto a polymer of a monomolecular compound containing a catechol group.

In addition, the amine-based polymer and/or the imine-based polymer is cross-linked with at least one of a derivative of a unimolecular compound containing a catechol group and a polymer of a unimolecular compound containing a catechol group. Through this, a composite coating layer can be formed. For example, a part or all of the first coating layer and the second coating layer may form a composite layer or a hybrid layer. For example, while the second coating layer is disposed on the first coating layer, a composite layer or a hybrid layer of the first coating layer and the second coating layer is formed by a crosslinking reaction of the first coating layer in at least a part of the inside of the first coating layer ( hybrid layer) may be formed. In this case, the coating layer may include a first coating layer, a second coating layer, and a composite layer or hybrid layer disposed between the first coating layer and the second coating layer. Alternatively, the entire coating layer may be a composite layer or a hybrid layer of the first coating layer and the second coating layer. Structural stability of the coating layer may be further improved by including the composite layer or hybrid layer in the coating layer.

The second coating layer may additionally include an amine-based monomolecular compound or a derivative thereof.

Amine-based monomolecular compounds include, for example, ethylenediamine, propylenediamine, butylenediamine, pentylenediamine, hexamethylenediamine, tris(2-aminoethyl)amine, triethylenetetramine ( triethylenetetramine), 2-(aminomethyl)-2-methyl-1,3-propanediamine trihydrochloride (2-(aminomethyl)-2-methyl-1,3-propanediamine trihydrochloride), and N,N,N′, It may be at least one selected from N'-tetrakis(3-aminopropyl)-1,4-butanediamine (N,N,N',N'-tetrakis(3-aminopropyl)-1,4-butanediamine), but must be It is not limited to these, and all are possible as long as they are used as amine-based compounds in the art. The amine-based monomolecular compound may be, for example, a compound containing a plurality of amino groups. Since the amine-based monomolecular compound includes a plurality of amino groups, it can more easily bind to the first coating layer, and unreacted amino groups can provide an additional reaction site to the second coating layer.

The thickness of the coating layer including the first coating layer and the second coating layer may be, for example, 1 nm to 300 nm, 1 nm to 200 nm, 1 nm to 150 nm, or 1 nm to 100 nm. When the coating layer has a thickness within this range, a conformal coating layer conforming to the surface contour of the porous tube substrate can be easily obtained. If the thickness of the coating layer is too thin, it may be difficult to obtain a robust coating layer. If the thickness of the coating layer is excessively increased, the internal space of the artificial blood vessel may be reduced, thereby reducing the patency rate.

The thickness of the coating layer including the composite layer and/or the hybrid layer of the first coating layer and the second coating layer may be, for example, 1 nm to 300 nm, 1 nm to 200 nm, 1 nm to 150 nm, or 1 nm to 100 nm. have.

The artificial blood vessel includes fucoidan disposed on a coating layer.

The inclusion of fucoidan in the artificial blood vessel can suppress clot formation and further improve biocompatibility.

Fucoidan may be fixed to the coating layer through, for example, an amide (-C(=O)-NH-) bond. For example, an amide bond may be formed by reacting a carboxyl group (-COOH) included in fucoidan with an amino group (-NH2) included in the coating layer.

Since fucoidan is fixed on the coating layer through a covalent bond, the high content of fucoidan included in artificial blood vessels can be stably maintained after a long period of time. Therefore, the antithrombotic ability of artificial blood vessels can be maintained for a long period of time. For example, since the second coating layer grafted on the first coating layer still includes a reactive functional group such as an amino group derived from an amine-based monomolecular compound, a derivative thereof, or an imine-based polymer, the coating layer including the second coating layer Efucoidan can be fixed in high content.

Fucoidan can be immobilized on the coating layer using a coupling agent. Examples of the coupling agent include imides such as EDC (1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride) and EDC/NHS (N-hydroxysuccinimide); aldehydes such as formaldehyde and glutaraldehyde; enzymes such as transglutaminase; It may be a crosslinking agent such as genipin or catechin, but is not limited thereto, and any one used as a coupling agent in the art is possible.

The fucoidan may be, for example, a vegetable fucoidan or an animal fucoidan. The vegetable fucoidan may be, for example, extracted from one or more selected from among Fucus vesiculosus, Cladosiphon okamuranus, Laminaria japonica, and Undaria pinnatifida. Animal fucoidan may be extracted from echinoderms. Animal fucoidan may be, for example, extracted from at least one selected from sea cucumber, sea urchin, squirt, and starfish.

The content of fucoidan fixed on the surface of the artificial blood vessel is, for example, 3 μg/cm ² or more and 60 μg/cm ² or less, 4 μg/cm ² or more and 60 μg/cm ² or less, 6 μg/cm ² or more and 60 μg/cm ^{2 .} Or less, 8 μg/cm ² or more and 60 μg/cm ² or less, or 10 μg/cm ² or more and 60 μg/cm ² or less. By immobilizing fucoidan in this range of content on the surface of the artificial blood vessel, the antithrombotic ability of the artificial blood vessel can be improved, and continuous bleeding due to interference with blood coagulation can be effectively prevented. If the content of fucoidan disposed in the artificial blood vessel is too low, the patency rate may decrease due to a decrease in the ability to inhibit clot formation. If the content of fucoidan disposed in the artificial blood vessel is too high, the ability to inhibit clot formation is excessively increased, making it difficult to hemostasis at the surgical site after transplantation of the artificial blood vessel.

The content of fucoidan disposed on the surface of the artificial blood vessel may be, for example, 200% or more, 250% or more, or 300% or more compared to the content of fucoidan disposed on the surface of a bare porous tube substrate in which the coating layer is free. have. The fucoidan content disposed on the surface of the artificial blood vessel is, for example, 200% to 500%, 250% to 500%, or It may be 300% to 500%. The antithrombotic ability of the artificial blood vessel can be further improved by immobilizing such a high content of fucoidan on the surface of the artificial blood vessel.

The surface contact angle of the artificial blood vessel measured at 25° C. and 1 atm may be 75% or less or 60% or less of the surface contact angle of the bare porous tube substrate measured under the same conditions. As the artificial blood vessel has such a reduced contact angle, the surface energy of the artificial blood vessel is reduced, and hydrophilicity and/or wettability are increased. Thus, the biocompatibility of artificial blood vessels can be further improved. Water contact angle can be measured using a contact angle meter. Since the surface of the artificial blood vessel is more easily impregnated with blood by having the improved wettability of the artificial blood vessel, the adhesion and proliferation of vascular endothelial cells can proceed more easily. Therefore, it is possible to more easily induce re-endothelialization of a defect part and endothelialization of an inner surface of an artificial blood vessel, which occur when performing an anastomosis transplant of an artificial blood vessel into a normal blood vessel. As a result, the patency rate of the artificial blood vessel can be further improved because the artificial blood vessel with improved wettability exhibits additional blood resistance.

The porosity of the artificial blood vessel introduced with fucoidan may be, for example, 60% or more or 70% or more. The porosity of the artificial blood vessel introduced with fucoidan may be, for example, 60% to 90% or 70% to 80%. By including such a high porosity of the artificial blood vessel into which fucoidan is introduced, the contact area with blood is widened and biocompatibility can be further improved. The porosity measurement method is not particularly limited, but may be measured by, for example, a nitrogen adsorption method.

Thrombus formation time according to APTT assay evaluation of the artificial blood vessel into which fucoidan is introduced may be, for example, 50 seconds or more or 60 seconds or more. The thrombus formation time according to the APTT assay evaluation of the artificial blood vessel introduced with fucoidan may be, for example, 50 seconds to 300 seconds, 70 seconds to 300 seconds, 90 seconds to 300 seconds, or 100 seconds to 300 seconds. Artificial blood vessels into which fucoidan is introduced have excellent antithrombotic activity, so that the patency rate of artificial blood vessels can be further improved and hemostasis at the wound site can not be hindered.

The artificial blood vessel may have, for example, 50% or more or 60% or more of fucoidan remaining on the surface of the artificial blood vessel after storage at 25° C. for 60 days or more. After the artificial blood vessel is stored at 25° C. for 60 days or more, the residual rate of fucoidan on the surface of the artificial blood vessel may be, for example, 50% to 90% or 60% to 80%. Long-term stability of the artificial blood vessel can be further improved by having such a high fucoidan residual rate.

The artificial blood vessel may have, for example, a patency rate of 50% or more, 60% or more, or 70% or more of the luminla opening area after 8 weeks of implantation of the rabbit carotid artery. The artificial blood vessel may have a patency rate of, for example, 50% to 90%, 60% to 85%, or 70% to 80% of the luminla opening area after 8 weeks of implantation of the rabbit carotid artery. . By having such a high patency rate of artificial blood vessels, side effects such as occlusion of blood vessels can be effectively prevented.

According to another embodiment, a method for manufacturing an artificial blood vessel includes providing a plasma-modified porous tube substrate; introducing a coating layer on the surface of the plasma-modified porous tube substrate; and fixing fucoidan on the coating layer, wherein the coating layer includes a first coating layer and a second coating layer, wherein the first coating layer comprises a derivative of a monomolecular compound containing a catechol group and a catechol group. (catechol group) containing at least one polymer of a monomolecular compound, wherein the second coating layer includes at least one of an amine-based polymer and an imine-based polymer, and the content of at least one of the amine-based polymer and the imine-based polymer is 2 [mu]g/cm ² or more and 9 [mu]g/cm ² or less. The artificial blood vessels prepared in this way provide excellent blood resistance and patency rate.

First, a plasma-modified porous tube substrate is provided.

Providing a plasma modified porous tube substrate may include, for example, providing a porous tube substrate comprising a hydrophobic polymer; and plasma-treating the porous tube substrate to introduce a polar functional group onto the porous tube substrate.

A porous tube substrate is placed within the chamber.

The porous tube substrate may be, for example, a tube-shaped porous substrate made of a hydrophobic polymer such as expanded polytetrafluoroethylene (ePTFE) or polyurethane.

The placed porous tube substrate is subjected to plasma treatment.

Plasma treatment may be performed, for example, by placing a porous tube substrate in a chamber and exposing it to direct current plasma at a chamber pressure of 10 to 100 Pa and a reactor output of 10 W to 100 W for 10 seconds to 10 minutes. have.

A reactant gas may be supplied at a flow rate of 10 sccm to 100 sccm into the reactor while plasma is applied to the porous tube substrate. The reaction gas is, for example, oxygen gas, nitrogen gas, or air.

Polar functional groups may be introduced on the inner and outer surfaces of the porous tube substrate by plasma. The polar functional group is, for example, a hydroxyl group (-OH), an amino group (-NH ₂ ), etc., but is not necessarily limited thereto.

Then, a coating layer is introduced on the surface of the plasma-modified porous tube substrate.

The step of introducing a coating layer on the surface of the plasma-modified porous tube substrate may include, for example, introducing a first coating layer on the plasma-modified porous tube substrate and introducing a second coating layer on the first coating layer. includes

First, a first coating layer is introduced on the plasma-modified porous tube substrate.

Specifically, a catechol group-containing monomolecular compound, a derivative thereof, and a polymer of the catechol group-containing monomolecular compound are self-assembled on a plasma-modified porous tube substrate.

For example, a first coating layer is formed on the surface of the plasma-modified porous tube by immersing the plasma-modified porous tube in a buffer solution containing a monomolecular compound containing a catechol group for 2 to 24 hours and then taking it out. can form.

Derivatives of unimolecular compounds containing catechol groups on porous tube substrates during self-assembly or after self-assembly on the plasma-modified porous tube surface. The first coating layer may be formed by forming, forming a monolayer of a catechol group-containing monomolecular compound, or forming a polymer of a catechol group-containing monomolecular compound.

The first coating layer may have a single-layer structure or a multi-layer structure. The first coating layer is multi-layered in a layer-by-layer manner, for example, by immersing a plurality of plasma-modified porous tube substrates in one coating composition at regular intervals or sequentially immersing in two or more coating compositions. can have a structure.

Then, a second coating layer is introduced on top of the first coating layer.

Specifically, an amine-based monomolecular compound, a derivative thereof, or an imine-based polymer is grafted or crosslinked on the first coating layer.

For example, the porous tube substrate into which the first coating layer is introduced is immersed in a buffer solution containing an amine-based polymer and/or an imine-based polymer for 1 minute to 10 hours, and then taken out to obtain the first coating by grafting and/or crosslinking. A second coating layer may be formed on the coating layer. Depending on specific reaction conditions, the coating layer may have a two-layer structure of the first coating layer/second coating layer or a three-layer structure of the first coating layer/composite layer (hybrid layer)/second coating layer. The composite layer or hybrid layer may be formed by cross-linking the first coating layer and the second coating layer.

Subsequently, fucoidan is immobilized on the coating layer.

Specifically, fucoidan is covalently bonded to the coating layer through a coupling agent.

For example, fucoidan may be immobilized on the coating layer by impregnating the porous tube substrate into which the coating layer is introduced with a solution containing a coupling agent, and then further adding and mixing fucoidan to the solution.

Any coupling agent is possible as long as it is used in the art. Coupling agents include, for example, imides such as EDC (1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride) and EDC/NHS (N-hydroxysuccinimide); aldehydes such as formaldehyde and glutaraldehyde; enzymes such as transglutaminase; It may be a cross-linking agent such as genipin or catechin.

The present invention is explained in more detail through the following Examples and Comparative Examples. However, the examples are for exemplifying the present invention, and the scope of the present invention is not limited only thereto.

(Manufacture of sheet-shaped artificial blood vessels)

Example 1 (F): Fucoidan coating by first coating layer-polydopamine (PDA)/second coating layer-polyethyleneimine (PEI)/EDC/NHS

An expanded polytetrafluoroethylene (ePTFE) sheet was prepared as a substrate. An ePTFA sheet was placed in the chamber and exposed to direct current plasma at 50 Pa chamber pressure and 100 W reactor output for 60 seconds.

While applying the plasma, oxygen gas was supplied at a flow rate of 20 sccm into the reactor. A hydroxyl group (-OH) was introduced as a polar functional group to the surface of the plasma-modified ePTFE sheet.

The existence of the polar functional groups introduced on the ePTFE sheet was confirmed by the ATR-FT IR spectrum.

A dopamine solution in which 200 mg dopamine was dissolved in 100 mL of 15 mM Tris buffer was prepared.

The plasma-modified ePTFE sheet was added to the dopamine solution, stirred for 2 hours, and then the ePTFE sheet was taken out and washed three times with distilled water.

A first coating layer of self-assembled polydopamine was formed on the surface of the plasma-treated ePTFE sheet by oxidation of dopamine.

100 mL of a polyethyleneimine solution in which 2 g of branched straight-chain polyethyleneimine (PEI) was dissolved in 1 L of distilled water was prepared.

The ePTFE sheet to which the first coating layer was introduced was put into a linear polyethyleneimine (PEI) solution, stirred for 2 hours, and then the ePTFE sheet was taken out and washed three times with distilled water.

On the first coating layer, straight chain polyethyleneimine (PEI) was introduced as a second coating layer. The weight average molecular weight of the linear polyethyleneimine (PEI) was 800 Dalton.

A straight-chain polyethyleneimine (PEI) was grafted onto the first coating layer. In addition, at least a portion of the straight-chain polyethyleneimine (PEI) was cross-linked to polydopamine to form a composite layer.

EDC (1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride, 100 mmol/L) and NHS (N-hydroxysuccinimide, 150 mmol/L) were added to a solution of fucoidan (10 mg/mL) in 0.5 M MES buffer. L) was added to activate fucoidan. Subsequently, the fucoidan was fixed by introducing the ePTFE sheet having the first and second coating layers into the fucoidan solution.

Fucoidan was immobilized on the second coating layer through an amide bond (-C(=O)-NH-).

An artificial blood vessel was prepared by washing the fucoidan-immobilized ePTFE sheet with distilled water three times.

Example 2 (D): First coating layer-Polydopamine (PDA)/Second coating layer-Polyethyleneimine (PEI)/ Fucoidan coating by glutaraldehyde

Except for activating fucoidan by adding glutaraldehyde (200 mmol/L) instead of EDC (100 mmol/L) and NHS (150 mmol/L) to the fucoidan (10 mg/mL) solution, Example 1 and Artificial blood vessels were prepared in the same manner.

Comparative Example 1 (bare)

Untreated ePTFE sheets were used as is. No fucoidan was introduced.

Comparative Example 2 (A): Fucoidan coating by EDC/NHS

An artificial blood vessel was manufactured in the same manner as in Example 1, except that the step of introducing the first coating layer and the second coating layer was omitted and nitrogen gas was used instead of oxygen gas.

Therefore, fucoidan was fixed as it was on the plasma-treated ePTFE sheet through EDC/NHS.

Comparative Example 3 (B): Fucoidan coating by first coating layer-polydopamine (PDA)/catechin

Artificial blood vessels were prepared in the same manner as in Example 1, except that the step of introducing the second coating layer was omitted and fucoidan was immobilized using catechin as a coupling agent in 15 mM Tris buffer.

Fucoidan was fixed on the first coating layer through catechin.

Evaluation Example 1: Evaluation of surface energy of artificial blood vessels

The surface energy of the artificial blood vessel was measured by measuring the contact angle with respect to distilled water on the surface of the artificial blood vessel prepared in Examples 1 to 2 and Comparative Examples 1 to 3.

The contact angle was measured using a contact angle (CA) goniometer at 25° C. and 1 atm. The measurement results are shown in Table 1 and FIGS. 1A to 1E below.

contact angle [°] Example 1 85 Example 2 87 Comparative Example 1 146 Comparative Example 2 124 Comparative Example 3 107

As shown in Table 1 and FIGS. 1A to 1E, the contact angles of the artificial blood vessels of Examples 1 and 2 were significantly reduced compared to the artificial blood vessels of Comparative Examples 1 to 3. Therefore, the artificial blood vessels of Examples 1 and 2 were compared Compared to the artificial blood vessels of Examples 1 to 3, surface energy decreased and hydrophilicity increased, so biocompatibility was improved.

Evaluation Example 2: Evaluation of fucoidan content introduced into artificial blood vessels

For the artificial blood vessels prepared in Examples 1 to 2 and Comparative Examples 1 to 3, the content of fucoidan introduced into the artificial blood vessels was evaluated.

The content of fucoidan covalently bound to artificial blood vessels was measured by toluidine blue O assay. The evaluation results are shown in Table 2 and FIGS. 2A to 2E below.

2a to 2e are images showing toluidine blue O quantification results of artificial blood vessels prepared in Example 1, Example 2, Comparative Example 1, Comparative Example 2, and Comparative Example 3, respectively.

Fucoidan content [μg/cm ² ] Example 1 5.18 Example 2 4.84 Comparative Example 1 0 Comparative Example 2 0.32 Comparative Example 3 1.78

As shown in Table 2 and FIGS. 2a to 2e, the fucoidan content introduced into the artificial blood vessels of Examples 1 and 2 was 4.5 μg/cm ² or more, but the content introduced into the artificial blood vessels of Comparative Examples 2 to 3 was 2.0 μg/cm 2 . cm ² or less. In addition, as shown in FIGS. 2A to 2E , the artificial blood vessels of Examples 1 to 2 showed a purple color, but the artificial blood vessels of Comparative Examples 1 to 3 maintained an almost white color.

Accordingly, the artificial blood vessels of Examples 1 and 2 significantly improved the introduction of fucoidan compared to the artificial blood vessels of Comparative Examples 2 and 3.

Evaluation Example 3: Evaluation of antithrombotic ability of artificial blood vessels

The antithrombotic ability of the artificial blood vessels prepared in Examples 1 to 2 and Comparative Examples 1 to 3 was evaluated.

Antithrombotic ability was performed through APTT assay (Activated partial thromboplastin time assay). The evaluation results are shown in Table 3 below.

Thrombus formation time [sec] Example 1 56.37 Example 2 64.53 Comparative Example 1 35.89 Comparative Example 2 41.21 Comparative Example 3 47.72

As shown in Table 3, the thrombus formation time of the artificial blood vessels of Examples 1 and 2 was 55 seconds or more, but the thrombus formation time of the artificial blood vessels of Comparative Examples 1 to 3 was less than 50 seconds. Compared to the artificial blood vessels of Comparative Examples 1 to 3, the antithrombotic ability of the artificial blood vessels was significantly improved.

Evaluation Example 4: Evaluation of vascular endothelial cell attachment and proliferation ability of artificial blood vessels

The vascular endothelial cell adhesion and proliferation capabilities of the artificial blood vessels prepared in Examples 1 to 2 and Comparative Examples 1 to 3 were evaluated.

Vascular endothelial cell adhesion and proliferative ability was performed through MTT assay (colorimetric assay for assessing cell metabolic activity). The evaluation results are shown in Table 4 below.

The number per unit area of HUVEC (Human Umbilical Vein Endothelial Cells) was evaluated.

HUVEC
[cells/cm ² ] HUVEC
[cells/cm ² ] HUVEC
[cells/cm ² ] elapsed time 1 day 3 days 5 days Example 1 2.55×10 ⁴ 4.76×10 ⁴ 12.73×10 ⁴ Example 2 2.67×10 ⁴ 5.58×10 ⁴ 15.22×10 ⁴ Comparative Example 1 0.12×10 ⁴ 0.27×10 ⁴ 0.58×10 ⁴ Comparative Example 2 0.63×10 ⁴ 1.16×10 ⁴ 2.52×10 ⁴ Comparative Example 3 0.98×10 ⁴ 2.19×10 ⁴ 5.19×10 ⁴

As shown in Table 4, after 5 days, the number of cells attached to and proliferated in the artificial blood vessels of Examples 1 and 2 increased by 50% or more compared to the cells attached to and proliferated in the artificial blood vessels of Comparative Examples 1 to 3. Accordingly, the artificial blood vessels of Examples 1 and 2 showed markedly improved vascular endothelial cell adhesion and proliferation capabilities compared to the artificial blood vessels of Comparative Example.

(Manufacture of tubular artificial blood vessels (I))

Example 3: Fucoidan coating by first coating layer-polydopamine (PDA), second coating layer-polyethyleneimine (PEI), EDC/NHS

An expanded polytetrafluoroethylene (ePTFE) tube was prepared as a substrate. An ePTFA tube was placed in the chamber and exposed to direct current plasma at 50 Pa chamber pressure and 100 W reactor output for 10 minutes.

While applying the plasma, oxygen gas was supplied at a flow rate of 20 sccm into the reactor. A hydroxyl group (—OH) was introduced as a polar functional group to the surface of the plasma-modified ePTFE tube.

The presence of the polar functional groups introduced on the ePTFE tube was confirmed by the ATR-FT IR spectrum.

A dopamine solution in which 200 mg dopamine was dissolved in 100 mL of 15 mM Tris buffer was prepared.

Plasma-modified ePTFE tube was added to the dopamine solution, and after stirring for 24 hours, the ePTFE tube was taken out and washed three times with distilled water.

A first coating layer of self-assembled polydopamine was formed on the surface of the plasma-treated ePTFE tube by oxidation of dopamine.

100 mL of a polyethyleneimine solution in which 2 g of branched polyethyleneimine (PEI) was dissolved in 1 L of distilled water was prepared.

The ePTFE tube in which the first coating layer was introduced was put into the branched polyethyleneimine (PEI) solution, stirred for 2 hours, and then the ePTFE tube was taken out and washed three times with distilled water.

A branched polyethyleneimine (PEI) on the first coating layer was introduced as a second coating layer. The weight average molecular weight of the branched polyethyleneimine (PEI) was 25000 Dalton.

A branched polyethyleneimine (PEI) was grafted onto the first coating layer. In addition, at least a portion of the branched polyethyleneimine was cross-linked to polydopamine to form a composite layer.

EDC (1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride, 100 mmol/L) and NHS (N-hydroxysuccinimide, 150 mmol/L) were added to a solution of fucoidan (10 mg/mL) in 0.5 M MES buffer. L) was added to activate fucoidan. Subsequently, the fucoidan was immobilized by introducing the ePTFE tube into which the first and second coating layers were introduced into the fucoidan solution.

Fucoidan was immobilized on the second coating layer through an amide bond (-C(=O)-NH-).

An artificial blood vessel was prepared by washing the fucoidan-immobilized ePTFE tube with distilled water three times.

Example 4: Fucoidan coating by first coating layer-polydopamine (PDA), second coating layer-polyethyleneimine (PEI), glutaraldehyde

Except for activating fucoidan by adding glutaraldehyde (200 mmol/L) instead of EDC (100 mmol/L) and NHS (150 mmol/L) to the fucoidan (10 mg/mL) solution, Example 1 and Artificial blood vessels were prepared in the same manner.

Comparative Example 4 (bare)

Untreated ePTFE tubes were used as is. No fucoidan was introduced.

Comparative Example 5 PLCL coating/fucoidan coating

In order to introduce fucoidan into the artificial blood vessel to which the polymer coating layer was introduced, a chemical modification reaction was first performed to introduce an amine group into fucoidan.

1 g of fucoidan was dissolved in 100 mL of distilled water and reacted for 2 hours by adding 5 mL of 2.6 N NaOH and 5 mL of epichlorohydrin. Activated fucoidan was obtained by dialysis in distilled water for 3 days and freeze-drying. Activated fucoidan was dissolved in 50 mL of 30% aqueous ammonia and reacted for 90 minutes. After dialysis in distilled water for 3 days and freeze-drying, the final product, aminated fucoidan, was obtained.

100 mL of a polycaprolactone solution in which 10 g of polycaprolactone (PLCL) was dissolved in 1 L of distilled water was prepared.

Plasma-treated ePTFE tube, maleic acid (100 mmol/L) and AIBN (2,2′-Azobis(2-methylpropionitrile) solution, 5 mmol/L) were added to the polycaprolactone (PLCL) solution, After stirring for 60 minutes, the ePTFE tube was taken out, dried at 80 °C for 48 hours, and washed three times with distilled water.

A first polycaprolactone (PLCL) coating layer was introduced on the surface of the plasma-treated ePTFE tube. The ePTFE tube to which the polycaprolactone (PLCL) coating layer was introduced was introduced into 0.5 M MES buffer, and EDC (1-[3-(Dimethylamino )propyl]-3-ethylcarbodiimide hydrochloride, 100mmol/L) and NHS (N-hydroxysuccinimide, 150mmol/L) were added to activate polycaprolactone (PLCL).

Subsequently, the ePTFE tube having the activated polycaprolactone (PLCL) coating layer was introduced into a solution in which aminated fucoidan (10 mg/mL) was dissolved to fix the fucoidan on the polycaprolactone (PLCL) coating layer.

Fucoidan was immobilized on the polycaprolactone layer through an amide bond (-C(=O)-NH-) bond.

An artificial blood vessel was prepared by washing the fucoidan-immobilized ePTFE tube with distilled water three times.

Comparative Example 6: POC coating/fucoidan coating

Aminated fucoidan was prepared in the same manner as in Comparative Example 5.

100 mL of a polyoctamethylene citrate solution in which 10 g of poly(octamethyelene citrate) was dissolved in 1 L of distilled water was prepared.

Plasma-treated ePTFE tube was added to the polyoctamethylene citrate (POC) solution, stirred for 30 minutes, and then the ePTFE tube was taken out, dried at 80 °C for 48 hours, and washed three times with distilled water.

An ePTFE tube coated with polyoctamethylene citrate (POC) was added to 0.5 M MES buffer, and EDC (1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride, 100 mmol/L) and NHS (N -hydroxysuccinimide, 150 mmol/L) was added to activate polyoctamethylene citrate (POC).

Subsequently, the ePTFE tube to which the activated polyoctamethylene citrate (POC) coating layer was introduced was put into a solution in which aminated fucoidan (10 mg/mL) was dissolved to fix the fucoidan on the polyoctamethylene citrate coating layer.

Fucoidan was immobilized on the polyoctamethylene citrate layer via an amide bond (-C(=O)-NH-).

An artificial blood vessel was prepared by washing the fucoidan-immobilized ePTFE tube with distilled water three times.

Evaluation Example 5: Evaluation of fucoidan content introduced into artificial blood vessels

For the artificial blood vessels prepared in Examples 3 to 4 and Comparative Examples 4 to 6, the content of fucoidan introduced into the artificial blood vessels was evaluated.

The content of fucoidan covalently bound to artificial blood vessels was measured by toluidine blue O assay. The evaluation results are shown in Table 5 and FIGS. 3a to 3e below.

3a to 3e are images showing the results of toluidine blue O quantification of tubular artificial blood vessels prepared in Example 3, Example 4, Comparative Example 4, Comparative Example 5, and Comparative Example 6, respectively.

Fucoidan content [μg/cm ² ] Example 3 8.73 Example 4 7.50 Comparative Example 4 0 Comparative Example 5 2.31 Comparative Example 6 2.88

As shown in Table 5 and FIGS. 3a to 3e, the content of fucoidan introduced into the artificial blood vessels of Examples 3 and 4 was 7 μg/cm ² or more, but the content introduced into the artificial blood vessels of Comparative Examples 5 to 6 was 3.0 μg/cm 2 . cm ² or less. In addition, as shown in FIGS. 3A to 3E, the artificial blood vessels of Examples 3 to 4 showed a deep purple color, but the artificial blood vessels of Comparative Examples 1 to 3 were almost white or maintained a very light purple color. .

Accordingly, the artificial blood vessels of Examples 3 to 4 significantly improved the amount of fucoidan introduced into the artificial blood vessels of Comparative Examples 5 to 6.

Evaluation Example 6: Evaluation of antithrombotic ability of artificial blood vessels (I)

The antithrombotic ability of the artificial blood vessels prepared in Examples 3 to 4 and Comparative Examples 4 to 6 was evaluated.

Antithrombotic ability was performed through APTT assay (Activated partial thromboplastin time assay). The evaluation results are shown in Table 6 below.

Thrombus formation time [sec] Example 3 76.83 Example 4 68.85 Comparative Example 4 35.89 Comparative Example 5 46.20 Comparative Example 6 49.17

As shown in Table 6, the thrombus formation time of the artificial blood vessels of Examples 3 and 4 was 65 seconds or more, but the thrombus formation time of the artificial blood vessels of Comparative Examples 4 to 6 was less than 50 seconds. Compared to the artificial blood vessels of Comparative Examples 4 to 6, the antithrombotic ability of the artificial blood vessels was significantly improved.

Evaluation Example 7: Evaluation of antithrombotic ability of artificial blood vessels (II)

The antithrombotic ability of the artificial blood vessels prepared in Examples 3 to 4 and Comparative Examples 4 to 6 against rabbit blood was evaluated.

Antithrombotic ability was measured by injecting the artificial blood vessels of Examples 3 to 4 and Comparative Examples 4 to 6 into rabbit blood containing 5 mM citrate and incubating with shaking for 5 minutes (shaking incubation, 37 ° C., 100 RPM). After taking it out and washing it once with physiological saline, the surface of the artificial blood vessel was observed to evaluate its antithrombotic ability.

The antithrombotic ability evaluation results of the artificial blood vessels of Example 3, Example 4, Comparative Example 4, Comparative Example 5, and Comparative Example 6 are shown in FIGS. 4A to 4E, respectively.

As shown in FIGS. 4A to 4E , the artificial blood vessels of Examples 3 and 4 hardly formed clots on the surface of the artificial blood vessels even after contact with rabbit blood.

In contrast, in the artificial blood vessels of Comparative Examples 5 to 6, blood clots were partially formed on the surface of the artificial blood vessels even after contact with rabbit blood.

In the artificial blood vessel of Comparative Example 4, the entire artificial blood vessel was completely covered with blood clots after contact with rabbit blood.

Accordingly, the artificial blood vessels of Examples 3 to 4 showed significantly improved antithrombotic ability compared to the artificial blood vessels of Comparative Examples 4 to 6.

Evaluation Example 8: Evaluation of porosity of artificial blood vessels

The porosity of the artificial blood vessels prepared in Examples 3 to 4 and Comparative Examples 4 to 6 was evaluated.

The porosity of the artificial blood vessels of Examples 3 to 4 and Comparative Examples 4 to 6 was measured using a mercury porosimeter.

Scanning electron microscope images of the surfaces of the artificial blood vessels of Examples 3, 4, Comparative Example 4, Comparative Example 5, and Comparative Example 6 are shown in FIGS. 5A to 5E , respectively.

The porosity measurement results of the artificial blood vessels of Examples 3, 4, Comparative Example 4, Comparative Example 5, and Comparative Example 6 are shown in Table 7 below.

Porosity is the percentage of the volume occupied by pores in the total volume of an artificial blood vessel.

Porosity [%] Example 3 71.87 Example 4 72.13 Comparative Example 4 75.47 Comparative Example 5 52.33 Comparative Example 6 47.16

As shown in Table 7, the porosity of the artificial blood vessels of Examples 3 to 4 was 70% or higher, similar to that of the untreated artificial blood vessels of Comparative Example 4. In contrast, the artificial blood vessels of Comparative Examples 5 to 6 showed porosity. stomatal flow was reduced by less than 60%.

Therefore, in the artificial blood vessels of Examples 3 to 4, it was confirmed that the coating layer was conformally disposed along the pore shape of the surface of the artificial blood vessel, thereby hardly blocking the pores on the surface of the artificial blood vessel.

Evaluation Example 8: Evaluation of the residual rate of fucoidan on the surface of artificial blood vessels

The residual rate of fucoidan on the surface of artificial blood vessels prepared in Examples 3 to 4 and Comparative Examples 5 to 6 was evaluated.

The fucoidan residual rate on the artificial blood vessel surface of Examples 3 to 4 and Comparative Examples 5 to 6 was measured by immersing the fucoidan-introduced artificial blood vessel sample in physiological saline and shaking incubation (37 ° C., 100 RPM) for 1 day and 7 days. After obtaining samples on days 14, 28, 56, and 70, the ratio of fucoidan remaining on the artificial vessel surface was measured by toluidine blue O assay.

Table 8 shows the results of measuring the residual fucoidan on the surface of artificial blood vessels of Examples 3, 4, Comparative Example 5, and Comparative Example 6.

The fucoidan residual ratio is the percentage of the fucoidan content remaining on the surface of the artificial blood vessel after a certain period of time relative to the fucoidan content of the initial surface of the artificial blood vessel.

Retention rate [%] Retention rate [%] Retention rate [%] Retention rate [%] Retention rate [%] Retention rate [%] Retention rate [%] elapsed period 1 day 7 days 14 days 28 days 42 days 56 days 70 days Example 3 97.3 93.1 87.3 74.0 71.4 62.6 60.2 Example 4 96.7 92.3 86.0 73.5 74.3 66.7 62.6 Comparative Example 5 96.7 92.7 87.6 68.4 37.1 10.4 3.8 Comparative Example 6 98.0 93.3 89.2 69.8 58.2 35.7 29.5

As shown in Table 8, the fucoidan residual rate of the artificial blood vessels of Examples 3 to 4 was 60% or more after 70 days, but the fucoidan residual rate of the artificial blood vessels of Comparative Examples 5 and 6 was less than 40% after 70 days. It was confirmed that the fucoidan residual rate of the artificial blood vessels of Examples 3 to 4 was significantly improved after 30 days compared to the residual rate of fucoidan of the artificial blood vessels of Comparative Examples 5 to 6.

In the artificial blood vessels of Comparative Examples 5 to 6, it was determined that the separation of the coating layer proceeded by hydrolysis of the biodegradable polymer.

Therefore, it was confirmed that the practical applicability of the artificial blood vessels of Examples 3 to 4 was remarkably improved by remarkably improving long-term stability.

Evaluation Example 9: Evaluation of patency rate of artificial blood vessels

The artificial blood vessels prepared in Example 3 and Comparative Example 4 were transplanted into rabbit carotid arteries, and then patency rates were evaluated after 8 weeks.

The patency rates of the artificial blood vessels of Example 3 and Comparative Example 4 were measured by measuring the cross section of the artificial blood vessel before extraction and the cross section of the artificial blood vessel extracted after 8 weeks, respectively, and using software (image J program) from the measured images before extraction. The cross-sectional area of the patency of the artificial blood vessel ( A _i ) and the patency of the artificial blood vessel extracted after 8 weeks ( A _f ) were measured by a method of measuring.

Patency rate (%) = open artificial vessel cross-section area after 8 weeks ( A _f ) / initial artificial vessel cross-sectional area ( A _i ) × 100

The evaluation results of the patency rate of the artificial blood vessels of Example 3 are shown in FIGS. 6A to 6E and Table 9 below. Green is the open lumen area.

The results of evaluating the patency rate of the artificial blood vessels of Comparative Example 4 are shown in FIGS. 7A to 7F and Table 9 below. Green is the open lumen area.

The patency rate of the artificial blood vessel is the percentage of the luminla opening area of the artificial blood vessel 8 weeks after transplantation into the rabbit carotid artery relative to the initial artificial blood vessel lumen area (luminla opening area).

The patency rate of five artificial blood vessels N1 to N5 was evaluated, and the average value was obtained and shown in Table 9 below.

Openness rate [%] Example 3 (EDC) 75 Comparative Example 4 (bare) 22

As shown in Table 9, the patency rate of the artificial blood vessel of Example 3 was significantly improved compared to that of the artificial blood vessel of Comparative Example 4. The artificial blood vessel of Comparative Example 4 shown in FIGS. After transplantation into the rabbit carotid artery, the patency rate was significantly reduced. Therefore, it is unsuitable for use as an artificial blood vessel.

In contrast, as shown in FIGS. 7A to 7F , the artificial blood vessel of Example 3 maintained a high patency rate even after being transplanted into the rabbit carotid artery compared to the initial patency rate. Therefore, it was confirmed that it is suitable for use as an artificial blood vessel.

(Manufacture of tubular artificial blood vessels (II))

Examples 5 to 8

Artificial blood vessels were prepared in the same manner as in Example 3, except that the fucoidan content introduced into the artificial blood vessels was changed to 4 μg/cm ² , 6 μg/cm ² , 40 μg/cm ² , and 60 μg/cm ² , respectively. was manufactured.

The content of fucoidan covalently bound to artificial blood vessels was measured by toluidine blue O assay.

Comparative Example 4 (bare)

Untreated ePTFE tubes were used as is. No fucoidan was introduced.

Comparative Example 5

Artificial blood vessels were prepared in the same manner as in Example 3, except that the fucoidan content introduced into the artificial blood vessels was changed to 80 μg/cm ² .

Evaluation Example 11: Evaluation of hemostasis ability of artificial blood vessels

The hemostatic ability of the artificial blood vessels prepared in Examples 5 to 8 and Comparative Examples 4 to 5 was evaluated.

After transplanting the artificial blood vessels prepared in Examples 5 to 8 and Comparative Examples 4 to 5 into rabbit carotid arteries, respectively, 60 minutes later, hemostasis at the transplanted site was evaluated.

After implantation in the carotid arteries of rabbits transplanted with the artificial blood vessels prepared in Examples 5 to 8 and Comparative Example 4, respectively, bleeding at the implanted site stopped after 20 minutes.

On the other hand, after transplanting the artificial blood vessels prepared in Comparative Example 5 to the carotid arteries of rabbits, continuous bleeding occurred at the transplanted site after 60 minutes.

Therefore, the artificial blood vessel prepared in Comparative Example 5 was unsuitable for use in surgery.

The artificial blood vessel of Comparative Example 4 prevented continuous bleeding after transplantation, but as shown in Table 9, the artificial blood vessel had a low patency rate and was unsuitable for use in surgery.

(Manufacture of tubular artificial blood vessels (III))

Examples 9 to 12

Example 3, except that the content of branched polyethyleneimine (PEI) introduced into artificial blood vessels was changed to 2 μg/cm ² , 4 μg/cm ² , 6 μg/cm ² , and 8 μg/cm ² , respectively. Artificial blood vessels were prepared in the same manner as described above.

The branched polyethyleneimine (PEI) content introduced into artificial blood vessels was measured through BCA assay. The measurement results are shown in Table 10 below.

Comparative Example 6 (bare)

Untreated ePTFE tubes were used as is. No fucoidan was introduced.

Comparative Example 7

Artificial blood vessels were prepared in the same manner as in Example 3, except that the amount of branched polyethyleneimine (PEI) introduced into the artificial blood vessels was changed to 10 μg/cm ² .

Reference Examples 1 to 4

Using hexamethylenediamine instead of branched polyethyleneimine (PEI) as the second coating layer,

Except for changing the content of hexamethylenediamine (HDA) introduced into artificial blood vessels to 2 μg/cm ² , 4 μg/cm ² , 6 μg/cm ² , and 8 μg/cm ² , Example 3 and Artificial blood vessels were prepared in the same manner.

The content of hexamethylenediamine introduced into artificial blood vessels was measured through BCA assay.

Evaluation Example 12: Evaluation of cell viability of artificial blood vessels

Cell viability was evaluated for the artificial blood vessels prepared in Examples 9 to 12 and Comparative Examples 6 to 7, and the results are shown in Table 10 below.

Cell viability was evaluated by the MTT assay method.

Branched Polyethyleneimine (PEI) Content
[μg/cm ² ] cell viability
[%] Example 9 2 91.6 Example 10 4 88.4 Example 11 6 83.1 Example 12 8 68.9 Comparative Example 6 0 100 Comparative Example 7 10 45.8

As shown in Table 10, the cell viability of the artificial blood vessels of Examples 9 to 12 was 60% or more. In contrast, the cell viability of the artificial blood vessels prepared in Comparative Example 7 was less than 50%.

Therefore, it was confirmed that an excessive increase in the content of polyethyleneimine introduced into artificial blood vessels can induce cytotoxicity.

Evaluation Example 13: Evaluation of fucoidan content introduced into artificial blood vessels

For the artificial blood vessels prepared in Examples 12 to 16, the content of fucoidan introduced into the artificial blood vessels was evaluated.

The content of fucoidan covalently bound to artificial blood vessels was measured by toluidine blue O assay. The evaluation results are shown in Table 11 below.

Fucoidan content
[μg/cm ² ] Example 9 (PEI, 2 μg/cm ² ) 4.84 Example 10 (PEI, 4 μg/cm ² ) 8.73 Example 11 (PEI, 6 μg/cm ² ) 12.92 Example 12 (PEI, 8 μg/cm ² ) 15.11 Reference Example 1 (HDA, 2 μg/cm ² ) 0.60 Reference Example 2 (HDA, 4 μg/cm ² ) 1.33 Reference Example 3 (HDA, 6 μg/cm ² ) 2.65 Reference Example 4 (HDA, 8 μg/cm ² ) 2.79

As shown in Table 11, the fucoidan content introduced into the artificial blood vessels of Examples 9 to 12 was improved compared to the artificial blood vessels of Reference Examples 1 to 4. The branched polyethyleneimine included in the artificial blood vessels of Examples 9 to 12 It was determined that this was because more fucoidan binding sites were provided compared to the hexamethylenediamine contained in the artificial blood vessels of Reference Examples 1 to 4.

Claims (20)

As an artificial blood vessel,
porous tube substrates;
A coating layer disposed on the surface of the porous tube substrate; and
It includes fucoidan disposed on the coating layer,
The coating layer is a first coating layer; And a second coating layer disposed on the first coating layer,
The first coating layer includes at least one of a catechol group-containing monomolecular compound, a derivative thereof, and a catechol group-containing monomolecular compound polymer,
The second coating layer includes at least one of an amine-based polymer and an imine-based polymer,
The second coating layer includes branched polyethyleneimine,
The thickness of the second coating layer is 100 nm to 300 nm,
The weight average molecular weight of the branched polyethyleneimine is 800 to 270,000 Dalton,
The content of at least one of the amine-based polymer and the imine-based polymer is 2 μg/cm ² or more and 6 μg/cm ² or less,
The artificial blood vessel having a porosity of 60% to 80%. The method of claim 1, wherein the porous tube substrate is a plasma-modified porous tube substrate,
A polar functional group is introduced on the surface of the porous tube substrate by the plasma modification,
The artificial blood vessel wherein the polar functional group is a hydroxyl group or an amino group. The method of claim 1, wherein the porous tube substrate comprises a hydrophobic polymer,
The artificial blood vessel, wherein the hydrophobic polymer includes at least one selected from stretched polytetrafluoroethylene and polyurethane. The artificial blood vessel according to claim 1, wherein the first coating layer is a conformal layer disposed along the surface contour of the porous tube substrate. The method of claim 1, wherein the molecular weight of the catechol group (catechol group)-containing monomolecular compound is 300 Dalton or less,
The catechol group-containing monomolecular compound is dopamine, 3,4-dihydroxyphenyl acetic acid, norepinephrine, 3,4-dihydroxybenzaldehyde (3,4-dihydroxyphenyl acetic acid) -Dihydroxybenzaldehyde), 3,4-dihydroxybenzonitrile (3,4-Dihydroxybenzonitrile), 3,4-dihydroxybenzylamine hydrobromide (3,4-Dihydroxybenzylamine hydrobromide), 3-hydroxytyrosol (3 -Hydroxytyrosol), 2,3-dihydroxybenzaldehyde, 3,4-dihydroxybenzoic acid, caffeic acid, gallic acid , DL-3,4-dihydroxyphenyl glycol (DL-3,4-Dihydroxyphenyl glycol), and 2-chloro-3 ', 4'-dihydroxyacetophenone (2-Chloro-3', 4'- An artificial blood vessel containing at least one selected from dihydroxyacetophenone). The method of claim 4, wherein the derivative of the catechol group-containing monomolecular compound is self-assembled on the porous tube substrate and fixed on the porous tube substrate through a covalent bond, or ,
The polymer of the catechol group-containing monomolecular compound is formed by self-assembling and polymerization of the catechol group-containing monomolecular compound on the porous tube substrate, artificial blood vessels. The artificial blood vessel according to claim 1, wherein at least one of the amine-based polymer and the imine-based polymer includes at least one selected from linear PEI and polyethylene diamine. The method of claim 1, wherein at least one of the amine-based polymer and the imine-based polymer is a catechol group-containing monomolecular compound, a catechol group-containing monomolecular compound derivative, and the catechol group-containing monomolecular compound An artificial blood vessel grafted onto one or more polymers of a single molecular compound. The method of claim 1, wherein at least one of the amine-based polymer and the imine-based polymer is a catechol group-containing monomolecular compound, a catechol group-containing monomolecular compound derivative, and a catechol group-containing monomolecular compound. An artificial blood vessel in which a composite coating layer is formed through cross-linking with at least one polymer of a molecular compound. The artificial blood vessel according to claim 1, wherein the fucoidan is fixed to the coating layer through an amide (-C(=O)-NH-) bond. The method of claim 1, wherein the fucoidan is vegetable fucoidan or animal fucoidan,
The vegetable fucoidan is extracted from at least one selected from Fucus vesiculosus, Cladosiphon okamuranus, Laminaria japonica, and Undaria pinnatifida,
An artificial blood vessel wherein the animal fucoidan is extracted from at least one selected from sea cucumber, sea urchin, squirt and starfish. The artificial blood vessel according to claim 1, wherein the fucoidan content disposed on the surface of the artificial blood vessel is 3 μg/cm ² or more and 60 μg/cm ² or less. The artificial blood vessel according to claim 1, wherein a surface contact angle of the artificial blood vessel measured at 25° C. and 1 atmospheric pressure is 75% or less of a surface contact angle of the porous tube substrate measured under the same conditions. The artificial blood vessel according to claim 1, wherein the artificial blood vessel has a thrombus formation time of 50 seconds or more according to APTT assay evaluation. The artificial blood vessel according to claim 1, wherein the artificial blood vessel has a residual rate of fucoidan of 50% or more on the surface of the artificial blood vessel after storage at 25°C for 60 days or more. The artificial blood vessel according to claim 1, wherein the artificial blood vessel has a patency rate of 50% or more after 8 weeks of implantation of the rabbit carotid artery of the artificial blood vessel. As an artificial blood vessel manufacturing method,
providing a plasma modified porous tube substrate;
introducing a coating layer on the surface of the plasma-modified porous tube substrate; and
Including; immobilizing fucoidan on the coating layer,
The coating layer includes a first coating layer and a second coating layer,
The first coating layer includes at least one of a catechol group-containing monomolecular compound, a derivative thereof, and a catechol group-containing monomolecular compound polymer,
The second coating layer includes at least one of an amine-based polymer and an imine-based polymer,
The second coating layer includes branched polyethyleneimine,
The thickness of the second coating layer is 100 nm to 300 nm,
The weight average molecular weight of the branched polyethyleneimine is 800 to 270,000 Dalton,
The content of at least one of the amine-based polymer and the imine-based polymer is 2 μg/cm ² or more and 6 μg/cm ² or less,
The artificial blood vessel manufacturing method, wherein the artificial blood vessel has a porosity of 60% to 80%. 18. The method of claim 17, wherein providing the plasma modified porous tube substrate comprises:
providing a porous tube substrate comprising a hydrophobic polymer; and
The artificial blood vessel manufacturing method comprising the step of plasma-treating the porous tube substrate to introduce a polar functional group on the porous tube substrate. The method of claim 17, wherein the step of introducing a coating layer on the surface of the plasma-modified porous tube substrate,
introducing a first coating layer on the plasma-modified porous tube substrate; and
Including the step of introducing a second coating layer on the first coating layer, artificial blood vessel manufacturing method. The method of claim 17, wherein the step of introducing the first coating layer,
Self-assembling a catechol group-containing unimolecular compound, a derivative thereof, and a polymer of the catechol group-containing unimolecular compound on the plasma-modified porous tube substrate;
The step of introducing the second coating layer,
Grafting or crosslinking at least one of an amine-based polymer and an imine-based polymer on the first coating layer,
The step of fixing fucoidan on the coating layer,
and covalently bonding fucoidan on the coating layer through a coupling agent.

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