JP2006280460A - Reformed base material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a reformed base material which includes a functional group of some of a nitrogenous group, a sulfonic acid group, a carboxyl group, a phosphate group and a hydrocarbon group in an area of 5μm from its surface in its depth direction and which includes an area of 200 nm thickness where concentration of the functional group is higher in an area of 200 nm to 5μm from the surface than the concentration of the functional group in an area of 0 to 200 nm from the surface, wherein it is widely used for a base material which requires nonionic hydrophilicity for its surface without deactivating characteristic of the functional group of the nitrogenous group, the sulfonic acid group, the carboxyl group, the phosphate group, the hydrocarbon group. <P>SOLUTION: The reformed material includes the nitrogenous group, the sulfonic acid group, the carboxyl group, a phosphate group and a hydrocarbon group in its surface, and includes a functional group of the nonionic hydrophilicity on its surface. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

In the present invention, a functional group of any one of a nitrogen-containing group, a sulfonic acid group, a carboxyl group, a phosphoric acid group, and a hydrocarbon group exists in a region having a depth of 5 μm from the surface, and the depth from the surface is 200 nm. The modification is characterized in that a region having a thickness of 200 nm is present in a region having a high concentration of the functional group in a region having a thickness of 200 nm to 5 μm in the depth direction from the surface than the concentration of the functional group in the thickness region. It relates to a substrate.

    Without deactivating the properties of the functional group, it can be widely used for materials that require nonionic properties on the surface. Specifically, medical devices such as artificial blood vessels, catheters, blood bags, contact lenses, intraocular lenses, artificial kidneys, artificial lungs, and surgical aids that require biocompatibility, or proteins and organic substances It is suitably used for substrates that require non-adhesion, membranes for water purifiers, water purification membranes, sewage purification membranes, separation membranes such as RO membranes, protein chips, DNA chips, and biosensors. In particular, it is suitably used for a separation membrane of a blood purification module.

Many attempts have been made to introduce various functional groups into a substrate to remove a specific substance or activate a specific substance. For example, dextran sulfate is an example of an LDL adsorbing ligand. However, a negatively charged surface with many anionic groups such as dextran sulfate increases the production of bradykin, promotes thrombus formation, and adsorbs anticoagulants with positive charges such as fusan. There is. Therefore, a method (Japanese Examined Patent Publication No. 62-56782) in which dextran sulfate is immobilized on porous beads with controlled pore diameters to remove low density lipoprotein (LDL) is disclosed. In addition, a highly hydrophobic functional group such as a hexadecyl group adsorbs coagulation-related proteins in addition to nonspecifically adsorbing various proteins, thereby causing blood activation. Accordingly, a method (artificial organ 27 (2), 571-577, 1998) in which a hexadecyl group is immobilized on a porous bead having a controlled pore diameter to remove β ₂ -microglobulin is disclosed. However, this method has a problem that the form of the substrate on which these substances are immobilized is limited.

    In addition, polyethyleneimine having an amino group interacts with oxidized low density lipoprotein (oxidized LDL) (Japanese Patent Laid-Open No. 2002-028461). However, it is known that amino groups not only cause platelet adhesion but also have a large release reaction.

As described above, in order to remove the target substance, as described above, a functional group according to the purpose is introduced, and the density of the nitrogen-containing group, sulfonic acid group, carboxyl group, phosphoric acid group, hydrocarbon group However, there is a problem that the blood activity is increased and the shape of the base material is limited.
Japanese Examined Patent Publication No. 62-56782 JP 2002-28461 A Artificial organ 27 (2), 571-577, 1998

  The object of the present invention is to improve the drawbacks of such prior art and to prevent nonionic ions on the surface without deactivating the characteristics of functional groups such as nitrogen-containing groups, sulfonic acid groups, carboxyl groups, phosphoric acid groups and hydrocarbon groups. Another object of the present invention is to provide a modified base material having a hydrophilic property introduced therein.

  In order to achieve the above object, the present invention has the following configuration.

  According to the present invention, a modified base material that requires nonionic hydrophilicity on the surface without deactivating characteristics of functional groups such as nitrogen-containing groups, sulfonic acid groups, carboxyl groups, phosphoric acid groups, and hydrocarbon groups Can be provided.

In the present invention, a functional group of any one of a nitrogen-containing group, a sulfonic acid group, a carboxyl group, a phosphoric acid group, and a hydrocarbon group is present in a region having a depth of 5 μm from the surface. The region of 200 nm thickness in which the concentration of the functional group is higher exists in the region of 200 nm to 5 μm in the depth direction from the surface than the concentration of the functional group in the region of 0 to 200 nm thickness. The modified base material. In particular, in the region having a thickness of 200 nm from the surface, the presence of any one of a hydroxyl group, an ether group, an amide group, and an ester group can suppress adhesion of platelets and blood cells. For this reason, undesirable phenomena such as blood activation can be suppressed. On the other hand, the functions of a nitrogen-containing group, a sulfonic acid group, a carboxyl group, a phosphoric acid group, and a hydrocarbon group can be exhibited. For example, oxidized low density lipoprotein (oxidized LDL), low density lipoprotein (LDL), β ₂ -microglobulin and the like are smaller than blood cell components. For this reason, it is considered that it can sufficiently interact with a functional group such as a nitrogen-containing group in the lower layer through a thickness region of 200 nm from the surface.

Here, the nitrogen-containing group refers to a functional group containing nitrogen, specifically an amino group, pyrrole group, pyrazole group, imidazole group, indole group, pyridine group, pyridazine group, quinoline group, piperidine group. Pyrrolidine group, thiazole group, purine group and the like. The amino group may be any of primary to quaternary. The main feature of the nitrogen-containing group is that it adsorbs anionic substances such as oxidized LDL and endotoxin in order to be cationic. In particular, amino group-containing polymers such as polyalkyleneimines, polyvinylamines, polyallylamines, diethylaminoethyldextran, polydiallyldimethylammonium chloride, vinyl imidazolium methochloride polymers, The combination and the like are particularly suitable for adsorption of oxidized LDL and endotoxin. This is because, if a substance having a functional group is a polymer, each functional group can take an optimal arrangement for interacting with oxidized LDL and endotoxin, so that the affinity is increased. Conceivable. Although not particularly limited, the weight average molecular weight is preferably 1000 or more, more preferably 5000 or more, and further preferably 10,000 or more. In addition, the main characteristics of the sulfonic acid group, carboxyl group, and phosphoric acid group include adsorption of a cationic substance such as fibrinogen because of its anionic property. The hydrocarbon group is preferably a hydrocarbon group having 6 to 50 carbon atoms, and may be a chain, a ring, or an aromatic ring. Examples of the hydrocarbon group having 6 to 50 carbon atoms include: The main characteristics of the hydrocarbon group include adsorption of β ₂ -microglobulin, cytokines, coagulation-related proteins, and the like.

    Further, there may be a plurality of these functional groups. For example, if an amino group and a hydrocarbon group coexist, the oxidized LDL is adsorbed by the modified base material. This is considered to be because two forces of electrostatic interaction due to amino groups and hydrophobic interaction due to hydrocarbon groups act. Further, it may be a physiologically active substance such as protein or peptide.

    Further, in the region having a thickness of 200 nm from the surface, nonionic hydrophilic functional groups such as a hydroxyl group, an ether group, an amide group, and an ester group are present to suppress adhesion of platelets and blood cells. Can do. This is probably because coagulation-related proteins and the like are difficult to adhere to nonionic hydrophilic portions, and platelets and blood cells that recognize them are difficult to adhere. In particular, a nonionic hydrophilic polymer such as polyvinyl alcohol, polyalkylene glycol, polyvinyl pyrrolidone, dextran, or pullulan is preferably present in a region having a depth of 200 nm from the surface. These polymers can further suppress the adhesion of platelets and blood cells due to the excluded volume effect. Although not particularly limited, in order to exert the excluded volume effect, the weight average molecular weight is preferably 1000 or more, more preferably 5000 or more, and further preferably 10,000 or more. Used. In addition, these polymers may be derivatives or copolymers as long as they do not prevent them from being nonionic and hydrophilic. Nonionic means that the charge is less than 1 meq / g at both pH 4.5 and pH 9.5. Further, the hydrophilic property means that the solubility in water at 25 ° C. is preferably 0.001% by weight or more, more preferably 0.01% by weight or more, and further preferably 0.1% by weight or more.

  In the modified base material referred to in the present invention, a polymer material is preferably used as the base material. Examples of the polymer material include polysulfone, polystyrene, polyurethane, polyethylene, and polypropylene. Examples of the shape of the modified substrate include, but are not limited to, fibers, films, resins, and separation membranes.

  Concentration measurement in the depth region of functional groups such as nitrogen-containing groups, sulfonic acid groups, carboxyl groups, phosphoric acid groups, and hydrocarbon groups can be known by measurement using an analytical electron microscope. Although details will be described later, first, the entire cross section of the modified base material sliced in the depth direction is scanned to grasp the outline of the functional group concentration distribution. Since the scan results using the analytical electron microscope lack quantitative accuracy, point analysis is performed thereafter. Concentration analysis in the depth direction is performed on a region having an arbitrary width of 10 μm. That is, point analysis is performed on arbitrary 10 points in the region of 0 to 200 nm and width of 10 μm in the depth direction from the surface. After that, in the region of 200 nm to 5 μm in the depth direction, arbitrary 10 point analysis is performed in the region of 10 μm wide and 200 nm thick in the depth direction, which seems to be the highest concentration qualitatively from the scan result. At this time, the magnification of the observation image is suitably 10,000 times to 50,000 times. This is analyzed in three different cross sections, and the average value of a total of 30 points is taken as the concentration in the depth region.

  In addition, in order to raise the detection sensitivity of an analytical electron microscope, or in order to distinguish the target functional group from other functional groups, you may use for a measurement, after modifying a functional group. Modification means that a certain kind of labeling substance is chemically bonded or complexed.

  Here, when the surface is uneven in the depth direction from the surface, the average line of the surface roughness is 0 nm. Also, if the surface of the modified base material is poor and the boundary area is unclear, the median value between the highest contrast part inside the modified base material and the lowest contrast part of the non-modified base part The surface.

    In addition, when the surface of the modified substrate has an inner surface and an outer surface like a hollow fiber membrane, a region having a high functional group exists in the depth direction from either surface. Just do it. For example, in a hollow fiber membrane for artificial kidneys, when a certain functional group has a function of removing unnecessary substances from blood components, it is in the region of 200 nm to 5 μm in the depth direction from the inner surface side of the hollow fiber. In addition, a region having a thickness of 200 nm having a high concentration of the functional group may be present.

    The concentration of the functional group is preferably 1.5 times or more higher than the concentration of the functional group in the region of 0 to 200 nm, more preferably 2 times or more higher.

    When the functional group is present in a region deeper than 5 μm, the function cannot be sufficiently exhibited. Further, when the functional group concentration is highest in the region of 0 to 200 nm, the influence of the functional group on the surface becomes large. For example, in the case of a hydrocarbon group, blood compatibility deteriorates because platelets and the like adhere to the hydrocarbon group. Therefore, it is preferable that the concentration of the hydrocarbon group increases in a region deeper than 200 nm.

    The concentration of functional groups such as nitrogen-containing groups, sulfonic acid groups, carboxyl groups, phosphoric acid groups and hydrocarbon groups in the 0 to 200 nm region is preferably 50 mol% or less, more preferably 1% or less and 10 mol% or less.

    A region having a thickness of 200 nm having a high concentration of a functional group such as a nitrogen-containing group, a sulfonic acid group, a carboxyl group, a phosphoric acid group, or a hydrocarbon group exists in a region of 200 nm to 5 μm in the depth direction from the surface. There are mainly the following three methods for obtaining a modified substrate. That is, 1) after introducing functional groups such as nitrogen-containing groups, sulfonic acid groups, carboxyl groups, phosphoric acid groups, and hydrocarbon groups on the surface of the substrate, and further nonionic properties such as hydroxyl groups, ether groups, amide groups, ester groups, etc. A method of obtaining a modified base material by taking two steps of introducing a hydrophilic functional group of 2) Functionality such as nitrogen-containing group, sulfonic acid group, carboxyl group, phosphoric acid group, hydrocarbon group in the base material forming stock solution A method of kneading a substance having a group, forming a substrate, and then introducing a nonionic hydrophilic functional group to obtain a modified substrate, 3) after forming the substrate, a nitrogen-containing group, a sulfonic acid group, And a method in which a substance having a functional group such as a carboxyl group, a phosphate group, or a hydrocarbon group is pushed into the base material to obtain a modified base material.

    Among the above, as a method of introducing a functional group into the substrate, a method of directly introducing various functional groups into the substrate by plasma irradiation, a nitrogen-containing group, a sulfonic acid group, a carboxyl group, a phosphoric acid group There is a method in which a substance having a functional group such as a hydrocarbon group is introduced into a substrate by chemical bonding or adsorption.

    As a specific example of chemically bonding a substance having the above functional group to a base material, when the base has a reactive active group, for example, a chloroacetamidomethyl group, a substance having an amino group such as polyethyleneimine By mixing them in an organic solvent or in water, they can be chemically bonded immediately and amino groups can be introduced into the substrate.

    As a specific method for adsorbing the substance having the functional group to the base material, the base material may be immersed or wetted in the substance solution having the functional group. Further, after being immersed in the solution, it may be pulled up and dried. The term “wet” as used herein refers to a state where the aqueous solution in which the base material is immersed is removed and not dried. Although not particularly limited, it preferably contains 3% by weight or more of moisture with respect to the dry weight of the substrate. As used herein, the dry weight refers to the weight in a state where the base material is dried and the weight change rate within 24 hours during drying is within 2%.

    Next, in order to form a chemical bond with a nonionic hydrophilic functional group on a base material into which a functional group such as a nitrogen-containing group, a sulfonic acid group, a carboxyl group, a phosphoric acid group, or a hydrocarbon group has been introduced, A similar method can be used. In addition, if the nonionic hydrophilic substance is a polymer, a substrate into which a functional group such as a nitrogen-containing group, a sulfonic acid group, a carboxyl group, a phosphoric acid group, or a hydrocarbon group has been introduced can be used as a nonionic hydrophilic substance. If the polymerization reaction is carried out by immersing or wetting in a polymer monomer solution, a nonionic hydrophilic polymer can be produced in such a manner as to cover the functional group introduced into the substrate. Furthermore, the method similar to the above can also be used for adsorbing nonionic hydrophilic polymers such as polyvinyl alcohol, polyalkylene glycol, polyvinyl pyrrolidone, dextran, and pullulan.

    In the above 2), in the base material in which a substance having a functional group such as a nitrogen-containing group, a sulfonic acid group, a carboxyl group, a phosphoric acid group, or a hydrocarbon group is kneaded, the substance is chemically bonded to the base material. Whether it is adsorbed or not.

    The method 3) is preferably used when a nonionic hydrophilic polymer diffused layer is present on the surface of the substrate or when the substrate has a porous structure. As a specific method, a substance solution having a functional group such as a nitrogen-containing group, a sulfonic acid group, a carboxyl group, a phosphoric acid group, or a hydrocarbon group is immersed or wetted in a substrate, and then a gas or A solution not containing the substance is pressed against the substrate. Thus, the substance can be moved into the nonionic hydrophilic polymer diffused layer or the base material porous structure. Here, the solution not containing the substance means a solution having a concentration of the substance of less than 0.0001% by weight, preferably less than 0.00001% by weight. For example, in the case where the base material is a separation membrane made of polysulfone and polyvinylpyrrolidone, the concentration of the substance is deeper than 200 nm from the surface by immersing the separation membrane in the substance solution and filtering with water. A high area can be created.

    A substance having a functional group such as a nitrogen-containing group, a sulfonic acid group, a carboxyl group, a phosphoric acid group or a hydrocarbon group or a substance having a nonionic hydrophilic functional group may be introduced into the substrate by adsorption. Chemical bonding is preferable because there is less concern about elution. Depending on the type of substance and the base material, there are those in which a chemical bond is formed on both of them after being adsorbed by heating or irradiation. In such a case, it is a preferable method because it is simple.

    As the radiation, α rays, β rays, γ rays, X rays, ultraviolet rays, electron beams and the like are used. In addition, medical devices such as artificial kidneys need to be sterilized, and in recent years, radiation sterilization methods are frequently used from the viewpoint of low residual toxicity and simplicity, and γ rays and electron beams are particularly preferably used. It has been. For example, in order to sterilize a blood purification module with γ rays, it is considered that irradiation with a dose of 15 kGy or more is preferable. Substances having functional groups such as nitrogen-containing groups, sulfonic acid groups, carboxyl groups, phosphoric acid groups, hydrocarbon groups, and substrates and nonionic hydrophilic functional groups that generate radicals upon irradiation. A chemical bond is formed between the substrate and the substance having a functional group such as a nitrogen-containing group, sulfonic acid group, carboxyl group, phosphoric acid group or hydrocarbon group and a substance having a nonionic hydrophilic functional group. The Therefore, in the case of a substance introduced by adsorption, this is a preferable method because the elution amount of the substance decreases. That is, irradiation with radiation of 15 kGy or more is a preferable treatment because it can simultaneously fix and sterilize a substance introduced by adsorption by chemical bonding. However, at this time, it is preferable to pay attention to modification of a functional group such as a nitrogen-containing group, a sulfonic acid group, a carboxyl group, a phosphoric acid group, or a hydrocarbon group, or a nonionic hydrophilic functional group. An antioxidant may be added at the same time in order to prevent denaturation and deactivation during irradiation. The term “antioxidant” as used herein refers to a molecule having the property of easily giving electrons to other molecules, such as water-soluble vitamins such as vitamin C, polyphenols, methanol, ethanol, propanol, ethylene glycol, and glycerin. Examples thereof include, but are not limited to, alcohols, sugars such as glucose, galactose, mannose and trehalose, inorganic salts such as sodium hydrosulfite, sodium pyrosulfite and sodium dithionate. These antioxidants may be used alone or in combination of two or more. When the method of the present invention is used for a medical device, it is necessary to consider its safety, and therefore, an antioxidant having low toxicity is preferably used. About the density | concentration of the aqueous solution containing an antioxidant, since it changes with kinds, antioxidant dose, etc. of the contained antioxidant, what is necessary is just to use it by the optimal density | concentration suitably.

  The modified substrate of the present invention is preferably used as a medical substrate. Medical base materials include those used in artificial blood vessels, catheters, blood bags, contact lenses, intraocular lenses, surgical aids, biological component separation modules, blood purification modules, and the like.

  The modified substrate of the present invention is preferably used for a biological component separation membrane. The biological component separation membrane refers to a membrane that separates a biological substance by filtration or dialysis and collects a part thereof. For example, if a modified separation membrane with many nonionic hydrophilic functional groups on the nitrogen-containing group is used, acidic proteins can be suppressed while suppressing nonspecific adsorption of biological components and activation of blood components. It can be separated efficiently.

  A blood purification module is a module that has the function of removing waste and harmful substances in the blood by adsorption, filtration, and diffusion when circulating blood outside the body, such as artificial kidneys and exotoxin adsorption columns. There is.

  The form of the separation membrane incorporated in the blood purification module is not particularly limited, and is used in the form of a flat membrane, a hollow fiber membrane or the like. However, the hollow fiber membrane type is preferable in consideration of the processing efficiency, that is, securing the surface area in contact with blood.

  The medical substrate is preferably a separation membrane. The material to be the separation membrane is not particularly limited, but is preferably a material used for medical purposes, for example, polyvinyl chloride, cellulose polymer, polystyrene, polymethyl methacrylate, polycarbonate, polysulfone, such as polysulfone and polyethersulfone. Examples thereof include polymers and polyurethane. Of these, polysulfone is particularly suitable because it is easy to mold and has excellent material permeation performance when formed into a membrane.

  The polysulfone polymer used in the present invention has an aromatic ring, a sulfonyl group and an ether group in the main chain. For example, polysulfone represented by the following chemical formulas (1) and (2) is preferably used. However, the present invention is not limited to these. N in the formula is an integer such as 50 to 80.

  Specific examples of polysulfone include Udel polysulfone P-1700, P-3500 (manufactured by Solvay), Ultrason S3010, S6010 (manufactured by BASF), Victrex (Sumitomo Chemical), Radel A (manufactured by Solvay), Ultra Polysulfone such as Son E (manufactured by BASF) is exemplified. In addition, the polysulfone used in the present invention is preferably a polymer composed only of the repeating unit represented by the above formula (1) and / or (2). However, it does not interfere with the effects of the present invention. It may be polymerized. Although it does not specifically limit, it is preferable that another copolymerization monomer is 10 weight% or less.

  There are various methods for producing a blood purification module according to the present invention, depending on its use. As a rough process, a process for producing a separation membrane for blood purification and incorporating the separation membrane into the module is described. Can be divided into processes. The separation membrane treatment method in the present invention may be used before the step of incorporating the separation membrane into the module or after the separation membrane is incorporated into the module. If irradiation is performed after modularization, sterilization can be performed at the same time, which is preferable.

  There are various methods for manufacturing a blood purification module depending on its application, but the rough process is divided into a process for manufacturing a separation membrane for blood purification and a step of incorporating the separation membrane into the module. Can do.

An example about the manufacturing method of the hollow fiber membrane module used for an artificial kidney is shown. As a method for producing a hollow fiber membrane incorporated in an artificial kidney, there are the following methods. That is, a solution obtained by dissolving polysulfone and polyvinylpyrrolidone in a good solvent or a mixed solvent containing a good solvent is used as a stock solution. The polymer concentration is preferably 10 to 30% by weight, and more preferably 15 to 25% by weight. The weight ratio of polysulfone and polyvinylpyrrolidone is preferably 20: 1 to 1: 5, and more preferably 5: 1 to 1: 1. As the good solvent, N, N-dimethylacetamide, dimethylsulfoxide, dimethylformamide, N-methylpyrrolidone, dioxane and the like are preferable. The undiluted solution is discharged from the outer pipe of the double annular die, and after running through the dry section, it is led to the coagulation bath. An injection solution or a gas for forming a hollow portion is discharged from a tube inside the double annular base. At this time, the humidity of the dry part has an effect, so that the phase separation behavior near the outer surface is accelerated by replenishing moisture from the outer surface of the membrane while the dry part is running. It is also possible to reduce the diffusion resistance. However, when the relative humidity is too high, the solid solution coagulation on the outer surface becomes dominant, and the pore diameter is rather reduced, and as a result, the permeation / diffusion resistance during dialysis tends to increase. Therefore, the relative humidity is preferably 60 to 90%. Moreover, as an injection liquid composition, it is preferable to use what consists of a composition based on the solvent used for the undiluted | stock solution from process aptitude. For example, when dimethylacetamide is used as an injection solution concentration, an aqueous solution of 45 to 80% by weight, more preferably 60 to 75% by weight, is preferably used.

  The method of incorporating the hollow fiber membrane in the module is not particularly limited, but an example is as follows. First, the hollow fiber membrane is cut to a required length, bundled in a necessary number, and then put into a cylindrical case. Then, a temporary cap is put on both ends, and a potting agent is put on both ends of the hollow fiber membrane. At this time, the method of adding the potting agent while rotating the module with a centrifuge is a preferable method because the potting agent is uniformly filled. After the potting agent is solidified, both ends are cut so that both ends of the hollow fiber membrane are open, and a hollow fiber membrane module is obtained.

  In order to introduce a functional group such as a nitrogen-containing group, sulfonic acid group, carboxyl group, phosphoric acid group, hydrocarbon group in the above, by adding a substance having the functional group to a stock solution, an injection solution or a coagulation bath, Can be achieved. Further, after forming into a hollow fiber membrane, the hollow fiber membrane can be immersed or wetted in the substance solution to adsorb and introduce the substance. Further, after modularization, the inside of the module can be filled or wetted with the substance solution, and the substance can be adsorbed and introduced into the hollow fiber membrane.

  In addition, in the case of a substance larger than the pore diameter of the hollow fiber membrane, it is an effective method for increasing the surface density because it is concentrated on the surface of the membrane when filled through filtration through the hollow fiber membrane. Furthermore, since the filtered and filled material is strongly pressed against the membrane surface and becomes difficult to release, it is a method that is preferably used. The molecular weight of the substance used is preferably 5000 or more, more preferably 50,000 or more. These substances may be either a substance having a functional group such as a nitrogen-containing group, a sulfonic acid group, a carboxyl group, a phosphoric acid group, or a hydrocarbon group, or a substance having a nonionic hydrophilic functional group.

On the other hand, in the case of a substance smaller than the pore diameter of the hollow fiber membrane, the substance can be brought into contact with the inside of the membrane pore. The molecular weight of the substance used is preferably 5000 or less, more preferably 1000 or less. For example, in the hollow fiber membrane for artificial kidney, β ₂ -microglobulin can enter the membrane pores. Therefore, when β ₂ -microglobulin is to be adsorbed, it is more efficient to use the inside of the membrane pores. On the other hand, platelets cannot enter the membrane pores. Therefore, in order to efficiently adsorb β ₂ -microglobulin, suppress adhesion of platelets, and improve biocompatibility, a substance having a hydrocarbon group inside the pore of the membrane, such as β ₂ -micro A substance having a molecular weight of 5000 or less that interacts with globulin may be introduced by filtration, and a nonionic hydrophilic functional group may be introduced on the membrane surface.

  An example of the basic structure of an artificial kidney using the hollow fiber membrane module obtained as described above is shown in FIG. A bundle of hollow fiber membranes 5 is inserted into a cylindrical plastic case 7, and both ends of the hollow fiber are sealed with a resin 10. The case 7 is provided with an inlet 8 and an outlet 9 for dialysate, and dialysate, saline, filtered water and the like flow outside the hollow fiber membrane 5. An inlet port portion 1 and an outlet port portion 2 are provided at the ends of the case 7, respectively. The blood 6 is introduced from the blood introduction port 3 provided in the inlet side port portion 1 and is guided into the hollow fiber membrane 5 by the funnel-shaped port portion 1. The blood 6 filtered by the hollow fiber membrane 5 is collected by the outlet side port portion 2 and discharged from the blood outlet 4. A blood circuit 11 is connected to the blood inlet 3 and the blood outlet 4.

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto.
1. Preparation of hollow fiber membrane module (1) Polysulfone hollow fiber membrane 18 parts by weight of polysulfone (Udel (registered trademark) P-3500 manufactured by Solvay) was added to a mixed solvent of 81 parts by weight of N, N′-dimethylacetamide and 1 part by weight of water. In addition, it was dissolved by heating at 90 ° C. for 10 hours to obtain a film-forming stock solution. This film-forming stock solution was discharged from an outer tube of an orifice type double cylindrical die having an outer diameter of 0.3 mm and an inner diameter of 0.2 mm. A solution consisting of 60 parts by weight of N, N′-dimethylacetamide and 40 parts by weight of water was discharged from the inner tube as the core liquid. The discharged film-forming stock solution passed through a dry length of 350 mm, and was then introduced into a 100% water coagulation bath to obtain a hollow fiber.

Insert the resulting hollow fiber membranes into a cylindrical plastic case having a dialysate inlet and a dialysate outlet as shown in FIG. 1, seal both ends with resin, A wet type hollow fiber membrane module for artificial kidney 1 having an inner surface area of 1.6 m ² was prepared by filling the outside with water.
(2) Polysulfone / polyvinylpyrrolidone mixed hollow fiber membrane 18 parts by weight of polysulfone (Udelpolysulfone (registered trademark) P-3500 manufactured by Solvay) and 9 parts by weight of polyvinylpyrrolidone (BAS K30) were added to N, N′-dimethylacetamide In addition to a mixed solvent of 72 parts by weight and 1 part by weight of water, the mixture was dissolved by heating at 90 ° C. for 14 hours to obtain a stock solution. This film-forming stock solution was discharged from an outer tube of an orifice type double cylindrical die having an outer diameter of 0.3 mm and an inner diameter of 0.2 mm. A solution consisting of 58 parts by weight of N, N′-dimethylacetamide and 42 parts by weight of water was discharged from the inner tube as the core liquid. The discharged film-forming stock solution passed through a dry length of 350 mm, and was then introduced into a 100% water coagulation bath to obtain a hollow fiber.

A hollow fiber membrane module 2 for wet type artificial kidney having a hollow fiber inner surface area of 1.6 m ² as shown in FIG. 1 was prepared in the same manner as the hollow fiber membrane module 1 using 10,000 obtained hollow fiber membranes.
2. Method for Introducing Nitrogen-Containing Group (1) Polysulfone Hollow Fiber Membrane As a substance having a nitrogen-containing group, 0.01% by weight aqueous solution of polyethyleneimine (manufactured by BASF, weight average molecular weight 750,000) is used as the blood guide of the hollow fiber membrane module 1 The gas was introduced from the outlet 4, exited from the dialysate outlet 9, and introduced into the hollow fiber membrane. The flow rate was 1 L. At this time, the blood inlet 3 and the dialysate inlet 8 were plugged. Thereafter, a polyethylene glycol (manufactured by Wako Pure Chemical Industries, Ltd., weight average molecular weight 20000) 0.01 wt% aqueous solution as a substance having a nonionic hydrophilic functional group was passed through 1 L in the same manner as the polyethyleneimine aqueous solution, and the hollow fiber membrane Introduced. Thereafter, 25 kGy of γ-rays were irradiated.
(2) Polysulfone / polyvinylpyrrolidone mixed hollow fiber membrane As a substance having a nitrogen-containing group, a 0.01% by weight aqueous solution of polyethyleneimine (manufactured by BASF, weight average molecular weight 750,000) is used as the blood outlet 4 of the hollow fiber membrane module 2. From the dialysis fluid outlet 9 and introduced into the hollow fiber membrane. The flow rate was 1 L. At this time, the blood inlet 3 and the dialysate inlet 8 were plugged. Thereafter, 5 L of pure water was introduced into the hollow fiber membrane in the same manner as the polyethyleneimine aqueous solution for the purpose of pushing polyethyleneimine into the diffused layer of polyvinylpyrrolidone on the membrane surface. Thereafter, 25 kGy of γ-rays were irradiated.
3. Measurement Method and Test Method (1) Polyethyleneimine Concentration of Polysulfone Hollow Fiber Membrane A substance having a nonionic hydrophilic functional group is polyethylene glycol, a substance having a nitrogen-containing group is polyethyleneimine, and a hollow fiber membrane as a base material is polysulfone Therefore, amino groups were quantified by analyzing the amount of nitrogen.

    The blood side of the hollow fiber membrane module obtained in 2 (1) above was washed with 1 L or more of ultrapure water. Thereafter, the hollow fiber was cut out from the module, the hollow fiber membrane was frozen, and then dried at 0.5 Torr or less for 24 hours or more.

The hollow fiber membrane was embedded with an epoxy resin and sliced to a thickness of 70 nm in the longitudinal direction of the hollow fiber with a microtome. About this sample, the distribution of nitrogen was measured by energy dispersive X-ray spectroscopy (EDS) using an analytical electron microscope (JEM2100F, manufactured by JEOL Ltd.). The observation magnification was 40,000 times. Thereafter, point analysis was performed to determine the amount of nitrogen.
(2) Amino group concentration of polysulfone / polyvinylpyrrolidone mixed hollow fiber membrane A nitrogen-containing substance is polyethyleneimine and has a nitrogen atom, and polyvinylpyrrolidone has a nitrogen atom in the hollow fiber membrane. There is no distinction between the two. In such a case, analysis may be performed after modifying the amino group. Since polyethyleneimine is known to chelate divalent copper ions strongly, it was possible to examine the distribution of copper with an analytical electron microscope after chelating copper ions with a hollow fiber membrane.

After washing the hollow fiber membrane module in the same manner as in 3 (1) above, 1 L of a 0.1 mol / L copper sulfate aqueous solution was passed through the blood side and the dialysate side of the hollow fiber membrane. Thereafter, the blood side and the dialysate side of the hollow fiber membrane were similarly washed with 1 L each with pure water. Thereafter, the hollow fiber was taken out and freeze-dried, and then subjected to analytical electron microscope analysis to quantify copper. The observation magnification at this time was 40,000 times.
(3) Human platelet adhesion test method of hollow fiber membrane A double-sided tape was affixed to an 18 mmφ polystyrene circular plate, and the hollow fiber membrane was fixed thereto. The attached hollow fiber membrane was cut into a semicylindrical shape with a single blade to expose the inner surface of the hollow fiber membrane. If dirt, scratches, folds, etc. are present on the inner surface of the hollow fiber, platelets will adhere to the part and may not be evaluated correctly. The circular plate was attached to a Falcon (registered trademark) tube (18 mmφ, No. 2051) cut into a cylindrical shape so that the surface on which the hollow fiber membrane was attached was inside the cylinder, and the gap was filled with parafilm. The cylindrical tube was washed with physiological saline and then filled with physiological saline. Immediately after collecting human venous blood, heparin was added to 50 U / ml. After discarding the physiological saline in the cylindrical tube, 1.0 ml of the blood was placed in the cylindrical tube and shaken at 37 ° C. for 1 hour within 10 minutes after blood collection. Thereafter, the hollow fiber membrane was washed with 10 ml of physiological saline, blood components were fixed with 2.5% glutaraldehyde physiological saline, and washed with 20 ml of distilled water. The washed hollow fiber membrane was dried under reduced pressure at room temperature of 0.5 Torr for 10 hours. This film was attached to a sample stage of a scanning electron microscope with double-sided tape. Thereafter, a thin film of Pt—Pd was formed on the surface of the hollow fiber membrane by sputtering to prepare a sample. The inner surface of the hollow fiber membrane was observed with a field emission type scanning electron microscope (S800, manufactured by Hitachi, Ltd.) at a magnification of 1500 times. In one field of view (4.3 × 10 ³ μm ² ) The number of adhered platelets was counted. In the vicinity of the center in the longitudinal direction of the hollow fiber, the average value of the number of adhering platelets in 10 different visual fields was defined as the number of adhering platelets (pieces / 4.3 × 10 ³ μm ² ). This is because the end portion in the longitudinal direction of the hollow fiber is likely to form a blood pool.

In addition, in the platelet adhesion test, positive control and negative control are put into the standard for each experiment in order to confirm whether the test is properly performed. A positive control is a known sample known to have a high platelet adhesion. A negative control is a known sample known to have low platelet adhesion. The positive control is a hollow fiber membrane of Toray's artificial kidney “Filtizer” BG-1.6U, and the negative control is a hollow fiber membrane of Kawasumi Chemical's artificial kidney PS-1.6UW. Under the above experimental conditions, the platelet adhesion number was 40 (pieces / 4.3 × 10 ³ μm ² ) or more as a positive control and 5 (pieces / 4.3 × 10 ³ μm ² ) or less as a negative control. When there is, the measured value is adopted. If the control platelet adhesion number is out of the above range, blood freshness may be lacking or excessive activation of blood may occur, so the test is repeated.

In this experiment, if the platelet adhesion number is 20 (pieces / 4.3 × 10 ³ μm ² ) or less, it is considered that blood compatibility is good.
(4) Oxidized Low Density Lipoprotein (Oxidized LDL) Adsorption Removal Test Method Oxidized LDL is an oxidized low density lipoprotein (LDL) that is oxidatively denatured. Oxidized LDL has been pointed out to be related to arteriosclerosis and is a substance to be removed from the body.
(A) Preparation of antioxidant LDL antibody The antibody was prepared according to the method of Itabe et al. (H. Itabe et al., J. Biol. Chem. 269: 15274, 1994). That is, human atherosclerotic lesion homogenate was injected into a mouse for immunization, a hybridoma was prepared from the spleen of the mouse, and one that reacted with copper sulfate-treated LDL was selected to obtain an antioxidant LDL antibody. The antibody class of the obtained antioxidant LDL antibody is mouse IgM and does not react with untreated LDL, acetyl LDL, or malondialdehyde LDL. On the other hand, the antioxidant LDL antibody reacts with several phosphatidylcholine peroxidation products including aldehyde derivatives of phosphatidylcholine and hydroperoxide. The antioxidant LDL antibody dissolved in 10 mM borate buffer (pH 8.5) containing 150 mM NaCl was used (protein concentration 0.60 mg / ml).
(B) Preparation of oxidized LDL Commercially available LDL (HUMAN, Biomedical Technologies Inc.) was applied to a desalting column (HiTrap Desalting, manufactured by Pharmacia) to remove ethylenediaminetetraacetic acid (EDTA) and 0.2 mg / ml. The solution was diluted with a phosphate buffer (hereinafter abbreviated as PBS). Thereafter, 2 ml each was dispensed into a Falcon (registered trademark) tube (18 mmφ, No. 2051). Incubated at 37 ° C. for 3 minutes. 2 wt% of 0.5 mM copper sulfate aqueous solution was added and reacted at 37 ° C. for 5 hours. At this time, a 0.5 mM aqueous copper sulfate solution was prepared. During the reaction at 37 ° C. for 5 hours, the tube was not covered, but was in contact with air, and pipetting was performed every 2 hours. A solution obtained by adding 25 mM ethylenediaminetetraacetic acid (EDTA) to 1 wt%, 10 wt% sodium azide to 0.02 wt% to the obtained solution was used as an oxidized LDL preparation. The total protein amount of this oxidized LDL was 0.171 mg / ml, and the malondialdehyde amount was 86.3 nM / mg LDL.
(C) Measurement of oxidized LDL concentration The above-mentioned oxidized LDL antibody was diluted with PBS to 5 μg / ml and dispensed into a 96-well plate at 100 μl / well. The mixture was shaken at room temperature for 2 hours and then allowed to stand at 4 ° C. overnight or longer to allow the antibody to be adsorbed on the wall.

  The antibody solution in the well was discarded, and Tris-HCl buffer (pH 8.0) containing 1% Bovine Serum Albumin (BSA, Fraction V, Seikagaku Corporation) was dispensed at 200 μl / well. After blocking the wall by shaking for 2 hours at room temperature, the BSA solution in the well was discarded, and 100 μl / well of plasma containing oxidized LDL and a standard for preparing a calibration curve were dispensed. Then, after shaking at room temperature for 30 minutes, it was left at 4 ° C. overnight.

After returning to room temperature, the solution in the well was discarded, and the well was washed 3 times with Tris-HCl buffer (pH 8.0) containing 0.05% Tween®-20. 100 μl / well of sheep anti-apo B antibody diluted 2000 times with PBS was dispensed into the washed wells. After shaking at room temperature for 2 hours, the anti-apo B antibody in the well was discarded, and the well was washed three times with Tris-HCl buffer (pH 8.0) containing 0.05% Tween-20. Alkaline phosphatase-labeled donkey anti-sheep IgG antibody diluted 2000 times with Tris-HCl buffer (pH 8.0) containing 2% Block Ace (Dainippon Pharmaceutical Co., Ltd.) was dispensed at 100 μl / well into the washed wells. Shake for 2 hours at room temperature. Thereafter, the labeled antibody in the wells is discarded, and the wells are washed three times with Tris-HCl buffer (pH 8.0) containing 0.05% Tween-20, and further 2 with Tris-HCl buffer (pH 8.0). Washed twice. Subsequently, a 1 mg / ml solution of p-nitrophenyl phosphate (0.0005 M MgCl 2, 1 M diethanolamine buffer, pH 9.8) was dispensed at 100 μl / well. After reacting at room temperature for an appropriate time, absorbance at a wavelength of 415 nm was measured with a plate reader. A calibration curve was drawn from the standard results to determine the oxidized LDL concentration.
(D) Measurement of adsorption removal rate of oxidized LDL Healthy plasma (Japanese, 30 years old, LDL (β lipoprotein) concentration of 275 mg / dl, HDL-cholesterol concentration of 70 mg / dl) and oxidized LDL at a concentration of 2 μg / ml It added so that it might become.

  70 hollow fiber membranes were bundled and inserted into a glass tube module case having a diameter of about 7 mm and a length of 12 cm. Both ends of the hollow fiber membrane were fixed with an epoxy potting agent so as not to block the hollow portion of the hollow fiber membrane, and a mini module (inner surface area 53 cm 2) was prepared. The mini module was washed with ultrapure water at 37 ° C. for 30 minutes. Then, the inner diameter of 0.8 mm, the outer diameter of 1 mm, and the length of 37 cm are connected to both ends of the mini-module through a silicone tube (product name: ARAM (registered trademark)) having an inner diameter of 7 mm (outer diameter of 10 mm) and a length of 2 cm and a deformed connector. A silicone tube (product name: ARAM (registered trademark)) was connected, and 1.5 ml of the plasma was perfused into the hollow fiber membrane at a flow rate of 0.5 ml / min at 25 ° C. for 4 hours under a nitrogen atmosphere. The amount of plasma per square meter of hollow fiber membrane surface area was 2.8 × 10 2 ml / m 2. Furthermore, the perfusion operation was also performed only with the silicone tube without attaching the mini module. By quantifying the oxidized LDL concentration in plasma before and after perfusion, each adsorption removal rate was calculated by the following equation.

Oxidized LDL adsorption / removal rate (%) = Oxidized LDL adsorption / removal rate (%) in the mini module-Oxidized LDL adsorption / removal rate (%) only in the silicone tube
Oxidized LDL adsorption removal rate (%) = 100 × (concentration before perfusion−concentration after perfusion) / concentration before perfusion (Example 1)
A hollow fiber was cut out from the hollow fiber membrane module obtained in 2 (1) and subjected to analysis electron microscope analysis. As a result, the concentration of nitrogen at a depth of 200 to 400 nm from the surface was twice that of nitrogen within 0 to 200 nm, and a structure in which polyethylene glycol was present on top of polyethyleneimine could be confirmed. Furthermore, human platelet adhesion test and oxidized LDL adsorption removal test method were conducted. As a result, as shown in Table 1, adsorption of oxidized LDL by the amino group derived from polyethyleneimine and suppression of platelet adhesion by polyethylene glycol were compatible.
(Example 2)
A hollow fiber was cut out from the hollow fiber membrane module obtained in 2 (2) above and subjected to analysis electron microscope analysis. As a result, the concentration of copper ions (representing amino groups) at a depth of 400 to 600 nm from the surface is three times that of copper ions (representing amino groups) within 0 to 200 nm, and polyethyleneimine is present inside the polyvinylpyrrolidone diffuse layer. It was confirmed that the structure was embedded in Furthermore, human platelet adhesion test and oxidized LDL adsorption removal test method were conducted. As a result, as shown in Table 1, it was possible to achieve both adsorption of oxidized LDL by an amino group derived from polyethyleneimine and suppression of platelet adhesion by polyvinylpyrrolidone.
(Comparative Example 1)
A hollow fiber membrane module was prepared by the same method except that the order of introduction of polyethyleneimine and polyethylene glycol was reversed in 2 (1). A hollow fiber was cut out from the hollow fiber membrane module and subjected to analytical electron microscope analysis. As a result, the concentration of nitrogen at a depth of 0 to 200 nm was the highest compared to the concentration of nitrogen in a deeper region. Furthermore, human platelet adhesion test and oxidized LDL adsorption removal test method were conducted. As a result, the oxidized LDL was adsorbed due to the presence of an amino group derived from polyethyleneimine, but platelets also adhered because the amino group was abundant at a depth of 0 nm to 200 nm from the surface.
(Comparative Example 2)
A hollow fiber membrane module was prepared in the same manner as in 2 (2) except that pure water was not passed. A hollow fiber was cut out from the hollow fiber membrane module and subjected to analytical electron microscope analysis. As a result, the concentration of copper ions (representing amino groups) at a depth of 0 to 200 nm was the highest compared to the concentration of copper ions (representing amino groups) in a deeper region. Furthermore, human platelet adhesion test and oxidized LDL adsorption removal test method were conducted. As a result, the oxidized LDL was adsorbed due to the presence of an amino group derived from polyethyleneimine, but platelets also adhered because the amino group was abundant at a depth of 0 nm to 200 nm from the surface.
(Comparative Example 3)
A hollow fiber membrane module was prepared in the same manner as in 2 (1) except that pure water was used instead of the polyethyleneimine aqueous solution and polyethylene glycol. A hollow fiber was cut out from the hollow fiber membrane module and subjected to analytical electron microscope analysis. As a result, a very small amount of copper ions was observed in any depth region. This appears to be physically trapped in the membrane. Furthermore, human platelet adhesion test and oxidized LDL adsorption removal test method were conducted. As a result, oxidized LDL did not adsorb because there was no amino group, and platelets also adhered because there was no nonionic hydrophilic functional group.

1 shows an embodiment of an artificial kidney used in the present invention.

Claims (11)

A functional group of any one of a nitrogen-containing group, a sulfonic acid group, a carboxyl group, a phosphoric acid group and a hydrocarbon group exists in a region of 5 μm in the depth direction from the surface, and 0 to 200 nm in the depth direction from the surface. The modification is characterized in that a region having a thickness of 200 nm is present in a region having a high concentration of the functional group in a region having a thickness of 200 nm to 5 μm in the depth direction from the surface than the concentration of the functional group in the thickness region. Base material. The nitrogen-containing group is selected from amino group, pyrrole group, pyrazole group, imidazole group, indole group, pyridine group, pyridazine group, quinoline group, piperidine group, pyrrolidine group, thiazole group and purine group. 2. The modified base material according to 1. 3. The modification according to claim 1, wherein any one of a hydroxyl group, an ether group, an amide group and an ester group exists in a region having a thickness of 0 to 200 nm in the depth direction from the surface. Base material. The modification according to any one of claims 1 to 3, wherein the hydroxyl group is derived from polyvinyl alcohol, the ether group is derived from polyalkylene glycol, the amide group is derived from polyvinyl pyrrolidone, and the ester group is derived from dextran or pullulan. Base material. The modified base material according to any one of claims 1 to 4, wherein the modified base material according to claim 1 is irradiated with radiation. The modified base material according to claim 1, wherein the modified base material is a medical base material. The modified base material according to claim 6, wherein the medical base material is built in a blood purification module. The modified base material according to claim 6 or 7, wherein the medical base material is built in an artificial kidney. The modified substrate according to any one of claims 6 to 8, wherein the medical substrate is a separation membrane. The modified substrate according to any one of claims 6 to 9, wherein the medical substrate is a hollow fiber membrane. The modified base material according to claim 9 or 10, wherein the separation membrane is a polysulfone-based polymer.

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