JP2011092489A - Implant member - Google Patents

Abstract

An implant member is provided that can selectively capture vascular endothelial progenitor cells (EPCs) under blood flow to form an endothelialized surface.
An embedding member for capturing EPC under blood flow and endothelializing, comprising a substrate and a coating layer provided on the surface of the substrate. The coating layer is made of a water-insoluble synthetic polymer, and the polymer has a functional group for immobilizing a protein that specifically interacts with EPC.
[Selection figure] None

Description

  The present invention relates to an implant member having a coating layer that specifically captures vascular endothelial progenitor cells (hereinafter sometimes referred to as EPC) in blood and promotes endothelialization.

  For members that are implanted in the body and come into contact with blood, such as artificial blood vessels, various efforts have been made in the past to reduce occlusion and restenosis due to thrombus formation.

  As part of this, the present inventors have provided a polymer material layer containing a component having anticoagulability provided on the base material surface of the blood contact surface, and a chemical immobilization provided on the surface of the polymer material layer. A protein that is a ligand or receptor antibody that specifically acts on a receptor that is expressed only on EPC, and this biologically specific action can selectively capture EPC in the bloodstream. A human body embedded member has been proposed (Patent Document 1).

JP 2008-125682 A

  The polymer material layer included in the embedded member described in Patent Document 1 includes a component having anticoagulant ability, and the component having anticoagulant ability is gradually polymerized after the embedded member is embedded in the body. While the protein released from the layer and exhibiting anticoagulant ability and selectively immobilized on the surface of the polymer material layer in the bloodstream selectively captures EPC and endothelializes the surface of the polymer material layer, It suppresses generation. Heparin is exemplified as a component having anticoagulant ability. Specifically, the polymer material layer included in the embedded member described in Patent Document 1 is a gel layer obtained by crosslinking albumin or gelatin with water-soluble carbodiimide, and heparin contained in the gel layer is gradually released from the gel layer. Thus, thrombus formation is suppressed until the surface of the polymer material layer is endothelialized.

  However, in order to gradually release heparin contained in the gel layer from the gel layer at a ratio suitable for suppressing the formation of thrombus, it is necessary to control the structure of the gel layer, the presence state of heparin, and the like.

  Therefore, if an embedded member having a polymer material layer capable of selectively capturing EPC and endothelizing the surface of the polymer material layer without using a component having sustained release anticoagulant properties such as heparin can be provided. The present inventors thought that the utility value was high.

  Accordingly, an object of the present invention is to provide an implant capable of selectively capturing EPC in the bloodstream to form an endothelialized surface without using an anticoagulant component such as heparin as a sustained-release component. It is to provide a member.

  As a result of the study by the present inventors, the coating layer provided on the surface of the embedded member is composed of a water-insoluble synthetic polymer having a functional group for immobilizing a protein that specifically interacts with EPC. Discovered that the above-mentioned problems can be solved by immobilizing a protein (specific protein) that specifically interacts with vascular endothelial progenitor cells (EPC) in the coating layer, and completed the present invention.

The present invention is as follows.
[1]
A substrate;
An embedded member having a coating layer provided on the surface of a substrate,
The coating layer is composed of a water-insoluble synthetic polymer, and the polymer immobilizes a protein (hereinafter referred to as a specific protein) that specifically interacts with vascular endothelial precursor cells (hereinafter referred to as EPC). It has a functional group (hereinafter, a functional group for immobilization),
An implant member for capturing EPC under blood flow and for endothelialization.
[2]
The embedded member according to [1], wherein the water-insoluble synthetic polymer is a poly (ethylene / vinyl alcohol) copolymer, a poly (methyl methacrylate / acrylic acid) copolymer, or an aminated polyvinyl alcohol copolymer.
[3]
The embedded member according to [1] or [2], wherein the immobilizing functional group is a hydroxyl group, an amino group, or a carboxyl group.
[4]
The embedded member according to any one of [1] to [3], wherein at least part of the immobilizing functional group is a functional group activated with N, N′-carbonyldiimidazole or carbodiimide.
[5]
The embedded member according to any one of [1] to [4], wherein a specific protein is immobilized on the surface of the coating layer through at least a part of the functional group for immobilization.
[6]
The specific protein is at least one selected from the group consisting of vascular endothelial growth factor (VEGF), an antibody against a VEGF cell surface receptor, angiopoietin, and an antibody against an angiopoietin cell receptor [5]. Embedded member.
[7]
The implant member according to [6], wherein the cell surface receptor of VEGF is VEGFR1 or VEGFR2, and the cell receptor of angiopoietin is Tie1 or Tie2.
[8]
The embedded member according to any one of [5] to [7], wherein the coating layer has a blocking agent immobilized on a surface on which a specific protein is not immobilized.
[9]
The embedding member according to [8], wherein the blocking agent is polyethylene glycol.
[10]
The implant member according to any one of [1] to [9], wherein the base material is a constituent member of an artificial implant member that is implanted in the body and comes into contact with blood.
[11]
The implant member according to [10], wherein the constituent member is used for an artificial blood vessel, a stent, or an artificial valve.

  According to the present invention, without using an anticoagulant component such as heparin as a sustained-release component, EPC is selectively captured under the bloodstream, coated with EPC, and an endothelialized surface ( It is possible to provide an embedding member that can form EPC or a surface coated with EPC or differentiated vascular endothelial cells (EC).

The schematic diagram of the surface where the blocking agent was fix | immobilized is shown. 2 is a photograph of a stent formed in Example 1 and having a uniform thin coating layer (thickness: about 3 microns) formed thereon. 2 shows the profile of the surface concentration of a fluorescently labeled protein immobilized on a coating layer in Example 1. The measurement result of the dependence of the fluorescence intensity of the coating layer surface in Example 1 on the protein concentration is shown. It is the photograph of the cylindrical body (artificial blood vessel) produced in Example 2 in which the uniform thin film layer (film thickness of 2 microns) was formed in the lumen. It is the photograph of the cylindrical body (artificial blood vessel) produced in Example 2 in which the uniform thin film layer (film thickness of 2 microns) was formed in the lumen. The photograph of the cylindrical body (artificial blood vessel) produced and extended in Example 2 (upper figure: stent, lower figure: fluorescence image of adherent cells). The photograph (left figure: fibronectin fixed surface, right figure polyethyleneglycol blocking surface) which shows the mode of the cell adhesion of the surface blocked with the polyethyleneglycol of the one terminal amino group in Example 3 is shown. The photograph of the fluorescent albumin fixed surface in the comparative example 1 is shown. The dependence of the fluorescence intensity on the substrate surface on the protein concentration and the amount of protein immobilized upon polyethylene glycol (PEG) adsorption are shown.

  The embedding member of the present invention is an embedding member that can capture EPC on the surface of the member in blood, and can endothelize the surface. The embedding member of the present invention has a base material and a coating layer provided on the surface of the base material. Here, the surface of the base material on which the coating layer is provided refers to the surface on the side in contact with blood.

  The base material is not particularly limited as long as it is a constituent member of an artificial embedded member that is embedded in the body and comes into contact with blood. Such a component can be used for, for example, an artificial blood vessel, a stent, or an artificial valve. In the case of an artificial blood vessel, for example, it can be composed of a biocompatible material such as a tube-shaped polyester fiber or polytetrafluoroethylene resin. There are various types of artificial blood vessels, such as a large-diameter artificial blood vessel of 10 mm or more, a medium-diameter artificial blood vessel of about 8 mm, and a small-diameter artificial blood vessel of 6 mm or less, depending on the inner diameter thereof. However, in particular, a small-diameter artificial blood vessel having an inner diameter of 6 mm or less is sought after in the treatment of coronary artery disease, peripheral artery disease, etc., but has not been put into practical use due to thrombus occlusion. In the case of a stent, for example, one made of a metal or a biocompatible polymer material can be mentioned. In the case of an artificial valve, it is a so-called mechanical valve, and part or all of the parts can be made of metal or a biocompatible polymer material.

  The coating layer is made of a water-insoluble synthetic polymer, and the polymer has a functional group (an immobilizing functional group) for immobilizing a protein that specifically interacts with EPC (specific protein). Is.

  Water-insoluble synthetic polymers include, for example, vinyl alcohol copolymers such as ethylene / vinyl alcohol copolymers, acrylate systems such as hydroxy acrylate / methacrylate copolymers and poly (methyl methacrylate / acrylic acid) copolymers. It can be a biocompatible polymer such as a copolymer, polyacrylonitrile, polysulfone, and polyester polymer alloy. These materials are materials that have been demonstrated to be biocompatible, for example, for use in artificial kidneys. The water-insoluble synthetic polymer is selected from materials having a functional group (an immobilizing functional group) capable of reacting with a functional group of the protein under mild conditions because it immobilizes a specific protein on its surface. . Examples of the material having a hydroxyl group as a functional group include an ethylene / vinyl alcohol copolymer and a hydroxyacrylate / methacrylate copolymer. Examples of the material having a carboxyl group as a functional group include a poly (methyl methacrylate / acrylic acid) copolymer. Furthermore, examples of the material having an amino group as a functional group include a water-insoluble synthetic polymer made of an aminated polyvinyl alcohol copolymer (for example, an amination rate of 25 mol%).

  The water-insoluble synthetic polymer is dissolved in a suitable organic solvent, and this coating solution is applied to the substrate surface as a coating solution to volatilize the organic solvent to form a coating layer. The organic solvent is preferably a solvent that dissolves the water-insoluble synthetic polymer and can be easily volatilized and removed from the coating layer after the coating layer is formed. In particular, since the embedded member is required to have biocompatibility, the remaining organic solvent in the coating layer should be avoided. The water-insoluble polymer coating solution can be applied by using an ultrasonic spray device when the substrate is a stent, and injecting the coating solution into the lumen when the substrate is an artificial blood vessel. Although the thickness of a coating layer does not have a restriction | limiting in particular, For example, it can be set as the range of 1-500 micrometers. The thickness of the coating layer can be appropriately determined according to the type of substrate on which the coating layer is formed.

  When an ethylene-vinyl alcohol copolymer that is a water-insoluble polymer having a hydroxyl group in the polymer side chain is used, after forming the coating layer, the hydroxyl group present in at least the surface layer of the coating layer is, for example, N, N ′ The functional group for immobilization can also be activated by reacting with -carbonyldiimidazole to form imidazolyl. Specifically, the functional group for immobilization can be activated by bringing an N, N′-carbonyldiimidazole-containing solution into contact with the coating layer.

  Even when an acrylic acid copolymer, which is a water-insoluble polymer having a carboxyl group in the polymer side chain, is used, it is applied to the substrate surface, and then the carboxyl group present in at least the surface layer of the coating layer is converted into a water-soluble condensation agent. The functional group for immobilization can be activated by reacting with [WSC; carbodiimide; 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrocloride]. Specifically, the functional group for immobilization can be activated by bringing the carbodiimide-containing solution into contact with the coating layer.

  A specific protein is immobilized on the surface of the coating layer via at least a part of the functional group for immobilization. The specific protein means a protein that specifically interacts with EPC. Such specific proteins include, for example, vascular endothelial growth factor (VEGF), antibodies against VEGF cell surface receptors (eg, VEGFR1 or VEGFR2), angiopoietin, and angiopoietin cell receptors (eg, Tie1 or Mention may be made of at least one selected from the group consisting of antibodies against Tie2). These specific proteins can be used alone or in combination of two or more (two or more).

  The specific protein can be immobilized by bringing an aqueous solution (for example, a phosphate buffer) containing the specific protein into contact with the surface of the coating layer having a functional group for immobilization. The amount of specific protein immobilized on the surface of the coating layer can be determined appropriately according to the type of specific protein, etc., and when the surface of the coating layer on which the specific protein is immobilized comes into contact with blood It is appropriate to be able to be trapped via the target protein and then promoted differentiation to be coated almost uniformly with endothelial cells (EC). Compared to the size of the specific protein, the size of EPC is much larger. Therefore, even if the immobilization density of the specific protein on the coating layer surface is small, if the EPC is captured, the surface of the coating layer is EPC. Can be coated. Moreover, since the specific protein illustrated above is not necessarily cheap, it is preferable to optimize the amount of specific protein immobilized from the viewpoint of providing an embedded member at a lower cost.

  In addition, even if high-density surface immobilization of specific proteins is intended, in principle there is an unfixed part, and this part may be a source of unintended cell type adhesion and activation of the blood coagulation system. is there. That is, when the surface of the coating layer on which the specific protein is not immobilized increases, the water-insoluble polymer forming the coating layer easily interacts with cells and protein components other than EPC. In order to further suppress such interaction, the coating layer preferably has a blocking agent immobilized on the surface on which the specific protein is not immobilized. A schematic view of the surface on which the blocking agent is immobilized is shown in FIG. Examples of the blocking agent include polyethylene glycol. Polyethylene glycol having a molecular weight of, for example, about 200 to 10,000 can be used. However, the molecular weight of polyethylene glycol can be appropriately set in consideration of blocking performance. A plurality of types of polyethylene glycol can be used in combination.

  The immobilization of polyethylene glycol can be carried out by using a one-terminal aminated polyethylene glycol in which amino is introduced at one end of the polyethylene glycol and reacting with the functional group for immobilization of the coating layer. When a blocking agent is used, the ratio of the specific protein immobilization amount and the blocking agent immobilization amount can be, for example, in the range of 5: 100 to 90:10.

  According to the present invention, a coating layer that inhibits thrombus formation on the surface of an artificial device (stent and small-diameter artificial blood vessel) for reconstructing a diseased blood vessel wall and develops a permanent non-thrombogenic property equivalent to that of a living artery blood vessel is provided. An embedding member is provided. The surface of the coating layer of the implant member of the present invention can selectively capture and coat (endothelialize) vascular endothelial cells contained in a trace amount in blood flow. Highly selective capture is biologically specific interaction with an antibody of VEGF or its receptor (for example, VEGFR1 or VEGFR2), an angiopoietin or its receptor (for example, Tie1 or Tie2), etc. immobilized on the coating layer surface. It is realized by.

  Hereinafter, the present invention will be described in more detail with reference to examples.

Example 1
A 0.5% HFIP (hexafluoroisopropyl alcohol) solution of a poly (ethylene / vinyl alcohol) copolymer (polyvinyl alcohol content: 72 mol%) was coated on a stent using a Sono-Tek ultrasonic spraying apparatus. A uniform coating layer was formed by air drying (thickness of about 3 microns: FIG. 2). Subsequently, this stent was immersed in a 5% acetonitrile solution of N, N′-carbonyldiimidazole at room temperature for 2 hours to activate the hydroxyl group of the surface layer by imidazoylation. Then, unreacted carbonyldiimidazole was washed with acetonitrile, and then in a phosphate buffer solution of 0.1 mg / ml protein (in this case, fluorescently labeled albumin instead of VEGF) for 24 hours at room temperature with gentle stirring. Soaked. After immersion, the sample was thoroughly washed with a phosphate buffer, and the fluorescence intensity was measured with a fluorescence microscope.

  Fig. 3 shows the profile of the surface concentration of the fluorescently labeled protein immobilized on an ethylene-vinyl alcohol copolymer (vinyl alcohol content: 72 mol.%), And Fig. 4 shows the dependence of the fluorescence intensity on the substrate surface on the protein concentration. . It was found that a high-density protein surface layer was formed at a protein concentration of 0.1 mg / ml.

Example 2
Segmented polyurethane mesh cylindrical body (inner diameter 3.5mm, length 10cm; poly (methyl methacrylate / acrylic acid) copolymer (polyacrylic acid content 30 mol%) 2% HFIP solution And the solution was immediately drained by gravity and dried to form a uniform coating layer in the cylindrical body lumen (film thickness: 2 microns: FIGS. 5 and 6 show photographs). Subsequently, it was immersed in a phosphate buffer of VEGF (1 μg to 100 μg / ml). After 24 hours, it was washed with physiological saline. Vascular endothelium and endothelial progenitor cells adhered and spread over time (Fig. 7: Seeded with endothelial cells labeled with fluorescent lipid-labeled DiO and cultured for 3 days Upper: stent, Lower: adhesive cells Fluorescence image).

  Instead of the ethylene / vinyl alcohol copolymer and the poly (methyl methacrylate / acrylic acid) copolymer in Examples 1 and 2, for example, an aminated polyvinyl alcohol copolymer (amination rate of 25 mol%) is used. Similarly, a coating layer is formed on a stent or an artificial blood vessel, and then immersed in a phosphate buffer solution of a protein (VEGF) containing a water-soluble condensing agent (carbodiimide) to immobilize the protein on the surface of the present invention. It is also possible to produce an embedded member.

Example 3 Blocking of Unfixed Portion A surface on which polyethylene glycol (Example 3) or albumin (Comparative Example 1) that suppresses cell adhesion / protein adsorption is immobilized is used to block the unfixed portion of a specific protein. Formed. One-terminal amino group polyethylene glycol (molecular weight 2,000) The surface of the surface activated hydroxyl group material of Example 1 immersed in a 0.1% solution (24 hours) is greatly inhibited in terms of cell adhesion compared to the fibronectin-immobilized surface. The effect was remarkable (FIG. 8). On the other hand, when immersed in a fluorescent albumin solution (1 mg / ml) and then washed with a phosphate buffer, almost no fluorescence was observed (FIGS. 9 and 10).

  The present invention is useful in the field related to an implant member for a human body such as an artificial blood vessel.

Claims (11)

A substrate;
An embedded member having a coating layer provided on the surface of a substrate,
The coating layer is composed of a water-insoluble synthetic polymer, and the polymer immobilizes a protein (hereinafter referred to as a specific protein) that specifically interacts with vascular endothelial precursor cells (hereinafter referred to as EPC). It has a functional group (hereinafter, a functional group for immobilization),
An implant member for capturing EPC under blood flow and for endothelialization. The embedded member according to claim 1, wherein the water-insoluble synthetic polymer is a poly (ethylene / vinyl alcohol) copolymer, a poly (methyl methacrylate / acrylic acid) copolymer, or an aminated polyvinyl alcohol copolymer. The embedded member according to claim 1 or 2, wherein the immobilizing functional group is a hydroxyl group, an amino group, or a carboxyl group. The embedded member according to claim 1, wherein at least a part of the immobilizing functional group is a functional group activated with N, N′-carbonyldiimidazole or carbodiimide. The embedded member according to any one of claims 1 to 4, wherein a specific protein is immobilized on the surface of the coating layer via at least a part of the functional group for immobilization. 6. The specific protein is at least one selected from the group consisting of vascular endothelial growth factor (VEGF), an antibody against a VEGF cell surface receptor, angiopoietin, and an antibody against an angiopoietin cell receptor. Embedded member. The implant member according to claim 6, wherein the cell surface receptor of VEGF is VEGFR1 or VEGFR2, and the cell receptor of angiopoietin is Tie1 or Tie2. The embedded member according to any one of claims 5 to 7, wherein the coating layer has a blocking agent immobilized on a surface on which a specific protein is not immobilized. The embedding member according to claim 8, wherein the blocking agent is polyethylene glycol. The implant member according to any one of claims 1 to 9, wherein the base material is a constituent member of an artificial implant member that is implanted in the body and comes into contact with blood. The implant member according to claim 10, wherein the constituent member is used for an artificial blood vessel, a stent, or an artificial valve.