JP4433821B2 - Modified substrate - Google Patents

Description

  The present invention relates to a modified substrate that has good blood compatibility and adsorbs and removes acidic proteins. Specifically, it is suitably used for a medical device such as an artificial blood vessel, a catheter, a blood bag, a contact lens, an intraocular lens, an artificial kidney, an artificial lung, and a surgical auxiliary instrument, and a basic protein separation substrate. In particular, it is suitably used for a substrate requiring blood compatibility, for example, a separation membrane of a blood purification module.

  Conventionally, hollow fiber membrane blood treatment devices using hollow fiber membranes have been widely used in the field of extracorporeal blood circulation, particularly in hemodialysis and plasma separation, and in recent years, particularly in the fields of dialysis membranes, blood component separation membranes and the like. A polymer hollow fiber membrane is widely used. However, it has been confirmed that the oxidative denaturation of proteins and lipids is increased in patients who have been hemodialysing for a long time due to a decrease in blood antioxidant activity. When a protein is oxidized, acidity often increases. In particular, low density lipoprotein (oxidized LDL) that has undergone oxidative degeneration is considered to play an important role in the formation of arteriosclerosis. Oxidized LDL has various biological effects. In addition to the action of suppressing nitric oxide (NO) production from endothelial cells, monocytes are transported and accumulated in the subendothelium, making them themselves macrophages, and oxidized LDL. It plays an important role in the onset and progression of arteriosclerosis, taking itself into foam cells and promoting plaque formation on the arterial wall, as well as promoting endothelial cell and smooth muscle cell damage. Therefore, it is desirable to remove oxidized LDL from the blood.

  Oxidized LDL cannot be removed with dialysis membranes represented by cellulose membranes, polymethyl methacrylate membranes, ethylene-vinyl alcohol membranes, polysulfone / polyvinylpyrrolidone blend membranes, and the like that are commercially available. Rather, the value of oxidized LDL in the blood tends to increase after dialysis due to stimulation by the dialysis membrane (kidney and dialysis separate volume, 38: 125 (1995)).

  In order to solve these problems, a membrane-type blood purifier that coats the surface of a dialysis membrane with a coating of vitamin E having various physiological functions such as in vivo antioxidant action, biological membrane stabilization action, and platelet aggregation inhibition action Although proposed (Japanese Patent Publication No. Sho 62-41738), it is not possible to remove oxidatively modified substances such as oxidized LDL in the blood of a patient once produced.

Moreover, in order to selectively adsorb and remove oxidized LDL in blood, a hollow fiber membrane containing a cationic polymer has been proposed (Japanese Patent Laid-Open No. 2002-28461), but the cation density of the substrate is high. Then, it is known that not only the platelet adhesion is caused, but also the release reaction is large. Therefore, in order to increase the adsorption removal rate of oxidized LDL, it is effective to increase the cation density, but there is a trade-off problem that the blood activity is increased accordingly.
Japanese Patent Publication No.62-41738 JP 2002-28461 A

  It is an object of the present invention to provide a modified substrate that adsorbs acidic proteins, particularly oxidized LDL, while improving the drawbacks of the prior art and achieving blood compatibility.

In order to achieve the above object, the present invention has the following configuration.
(1) A modified base material having an adhesion amount of human platelets of 10 / 4.3 × 10 ³ μm ² or less and an acid protein adsorption / removal rate of 20% or more.
(2) The modified base material according to (1), wherein the acidic protein is an oxidized low-density lipoprotein.
(3) The modified base material according to (1) or (2), wherein the base material contains a cationic group-containing substance.
(4) The modified base material according to any one of (1) to (3), wherein the cationic group-containing substance is a cationic polymer.
(5) The modified substrate according to any one of (1) to (4), wherein the elution concentration of the cationic polymer is 1000 ppm or less.
(6) The modified base material according to any one of (1) to (5), wherein the base material contains a nonionic hydrophilic polymer.
(7) The modified substrate according to any one of (1) to (6), wherein the substrate according to (1) is irradiated with radiation.
(8) The modified substrate according to any one of (1) to (7), wherein the substrate is a medical substrate.
(9) The modified substrate according to (8), wherein the medical substrate is incorporated in a blood purification module.
(10) The modified base material according to (9), wherein the medical base material is incorporated in an artificial kidney module.
(11) The modified substrate according to any one of (1) to (10), wherein the medical substrate is a separation membrane.
(12) The modified substrate according to any one of (1) to (11), wherein the medical substrate is a hollow fiber membrane.
(13) The modified substrate according to (11) or 12, wherein the separation membrane is a polysulfone polymer.

  A modified base material having good blood compatibility and capable of adsorbing and removing acidic proteins can be provided.

  The present invention relates to a modified base material that adsorbs acidic proteins and is a blood-compatible base material that suppresses platelet adhesion.

It is known that if the substrate contains a cationic group-containing substance, it not only causes platelet adhesion but also causes a release reaction. However, if a nonionic hydrophilic polymer is present in a form that covers the cationic substance present on the substrate surface, large substances such as platelets and blood cells will be nonionic on the outermost surface of the substrate. It is repelled by the hydrophilic polymer layer and is not close to the underlying cationic group-containing substance. For this reason, undesirable phenomena such as blood activation can be suppressed. On the other hand, it is considered that relatively small substances such as proteins and lipoproteins such as oxidized low density lipoprotein (oxidized LDL) can interact with the cationic group-containing substance through the nonionic hydrophilic polymer layer. .

  In the present invention, the modified base material refers to a material that hardly adheres to platelets and adsorbs acidic proteins, and a polymer material is preferable. A base material means the material before giving the above characteristics. Examples of the polymer material include polysulfone, polystyrene, polyurethane, polyethylene, and polypropylene. Examples of the shape of the substrate include, but are not limited to, fibers, films, resins, and separation membranes.

The amount of adhesion of human platelets is the number of platelets adhering to the surface of the modified substrate when the modified substrate and blood are brought into contact with each other for 1 hour, and the surface area of the modified substrate is 4.3 × 10 ³ μm ^2. The value obtained as the number of. A detailed measurement method will be described later. When the amount of human platelet adhesion exceeds 10 / 4.3 × 10 ³ μm ² , blood compatibility becomes insufficient.

The acid protein adsorption removal rate is 2 μg / ml at an initial concentration contained in the plasma when the perfusion operation is performed under the condition that the amount of plasma per 1 m ² of the modified substrate surface area is 280 ml / m ^2. This is the rate of adsorption removal of acidic proteins. A detailed measurement method will be described later. If the adsorption removal rate is not 20% or more, a sufficient adsorption effect cannot be expected when actually used for medical purposes. Moreover, the surface here is a portion where the modified base material comes into contact with plasma. For example, in the case of a hollow fiber membrane for an artificial kidney, it refers to the inner surface and does not include the surface of the fine structure portion inside the membrane.

  As used herein, acidic protein refers to proteins and lipoproteins whose isoelectric point is on the acidic side of physiological pH. When a protein is oxidized, acidity often increases. Oxidized LDL is a general term for LDL that has undergone oxidative modification. Therefore, the degree of oxidation and the site of oxidation are various, such as proteins and lipids, but generally used in experiments include copper-oxidized LDL, minimally-modified-LDL, malondialdehyde Examples include LDL, acetylated LDL, 4-hydroxy-2-nonenalized LDL, and acrolein-added LDL. In the present invention, when the acidic protein is oxidized LDL, the adsorption removal performance is particularly suitably exhibited.

  In the present invention, it is preferable that a cationic group-containing substance is contained in the base material because an acidic protein such as oxidized LDL can be selectively adsorbed to the base material by electrostatic interaction.

  The cationic group means a functional group containing nitrogen and having a positive charge in an aqueous pH 4.5 solution. Primary amino group, secondary amino group, tertiary amino group, quaternary amino group, pyrrole group A pyrazole group, an imidazole group, an indole group, a pyridine group, a pyridazine group, a quinoline group, a piperidine group, a pyrrolidine group, a thiazole group, a purine group, and an aminosulfuric group, but are not limited thereto. Two or more kinds of cationic groups may exist in the modified base material. In addition, a polymer having a cationic group is preferably used because it has a high adsorption effect on acidic proteins. This is considered to be likely to interact with an acidic protein because it has a large room for an optimal configuration with an acidic protein. The cationic polymer means a polymer having a charge of 1 meq / g or more at pH 4.5, and is not particularly limited, but polyalkyleneimine, polyvinylamine, polyallylamine, diethylaminoethyldextran, polydiallyl. Examples thereof include dimethylammonium chloride, vinyl imidazolium methochloride polymer and derivatives thereof. Further, the weight average molecular weight of the cationic polymer is not particularly limited, but 1000 or more, preferably 5000 or more, more preferably 10,000 or more is preferably used. As the vinyl imidazolium methochloride polymer, a copolymer of vinyl imidazolium methochloride and vinyl pyrrolidone is commercially available from BASF and is preferably used. These two or more kinds of cationic polymers may be used in combination.

  The elution concentration of the cationic polymer is preferably 1000 ppm or less, preferably 500 ppm, and more preferably 100 ppm or less. If the elution concentration exceeds 1000 ppm, there is a possibility that the cationic polymer is eluted in the blood when it comes into contact with blood. The elution concentration of the cationic polymer is, for example, the concentration of the cationic polymer in the filling water filling the modified base material in the blood purification module. If there are compartments in the module, such as the dialysate side and blood side, as in the artificial kidney module, and the elution concentration differs for each compartment, measure the elution concentration in the compartment in contact with platelets and acidic proteins. That's fine. Furthermore, when the module is not filled with water, that is, when the modified substrate is wet or dry, the module is filled with water and left at room temperature for 1 week to elute the cationic high What is necessary is just to measure molecular concentration. Alternatively, when the modified base material is not incorporated in the module and is wet or dry, it is eluted by immersing it in pure water in an amount 10 times the dry weight of the modified base material for 1 week at room temperature. What is necessary is just to measure a cationic polymer concentration. As used herein, the dry weight refers to a weight in a state in which the weight-change rate in one hour during drying is within 2% after the minute modified base material is dried.

  As a method for introducing a cationic group into a substrate, 1) a method in which a cationic group-containing substance is introduced as a constituent component of the substrate, and 2) a cationic group-containing substance or a cationic group itself after base molding. Examples thereof include a method of chemically bonding to a material, and 3) a method of adsorbing a cationic group-containing substance on a substrate after molding the substrate.

  The method of introducing as a constituent component of the substrate refers to a substance introduced into the substrate before the substrate is molded. For example, in the case of a blended hollow fiber membrane of polysulfone and polyvinylpyrrolidone for an artificial kidney, the constituent components polysulfone and polyvinylpyrrolidone correspond to the cationic group-containing substance. Although details of the method for producing the hollow fiber membrane will be described later, in order to introduce a cationic group-containing substance into the hollow fiber membrane as a constituent component, at least one of the membrane-forming stock solution, the injection solution, and the coagulation bath is added. It is necessary to keep. When a cationic group-containing substance is added to the stock solution, it is added as a third component in addition to polysulfone and polyvinylpyrrolidone, or modified polysulfone or polyvinylpyrrolidone having a cationic group is used instead of polysulfone or polyvinylpyrrolidone. Also good.

  The chemical bond in the present invention refers to a covalent bond or an ionic bond. As a specific method for introducing the cationic group-containing substance by chemical bonding after molding the base material, the cationic group can be introduced as a chemical bond to the base material by plasma irradiation or ion cluster beam. Alternatively, the cationic group-containing substance may be cross-linked using a chemical reaction on the substrate. At this time, a spacer may be interposed between the cationic group-containing substance and the substrate. Further, the cationic polymer may be introduced by polymer polymerization by soaking or wetting the monomer solution on the substrate. Moreover, after adsorb | sucking a cationic group containing substance to a base material, a chemical bond may be formed in a cationic group containing substance and a base material by heating or radiation irradiation.

  The term “adsorption” as used in the present invention means that the constituent component of the substrate and the cationic group-containing substance are interacted with each other through a chemical bond, and may be either chemical adsorption or physical adsorption. Moreover, you may adsorb | suck by indirect interaction. For example, the cationic group-containing substance may be adsorbed by hydrogen bonding via a constituent component of the substrate and water molecules. As a specific method for adsorbing the cationic group-containing substance on the substrate, the substrate may be immersed or wetted in the cationic group-containing substance solution. The term “wetting” as used in the present invention refers to a state where the aqueous solution in which the substrate 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.

Further, it is preferable that a nonionic hydrophilic polymer is contained in the base material. Furthermore, it is preferable that the nonionic hydrophilic polymer is present so as to cover the cationic group-containing substance present on the substrate surface. If a nonionic hydrophilic polymer is present on the outermost surface of the modified substrate, large substances such as platelets and blood cells are repelled and cannot be brought close to the underlying cationic group-containing substance. Activation can be suppressed.

  The nonionic hydrophilic polymer means a polymer that has a charge of less than 1 meq / g and is soluble in water at both pH 4.5 and pH 9.5. Here, the term “soluble in water” 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. Although not particularly limited, polyvinyl pyrrolidone, polyethylene glycol, polypropylene glycol, polyvinyl alcohol and the like, copolymers of these with other monomers, derivatives such as grafts, and the like can be mentioned. Two or more kinds of nonionic hydrophilic polymers may be used in combination.

  In order to suppress adhesion of platelets, it is important that a nonionic hydrophilic polymer is present on the outermost surface of the modified substrate. As a method for introducing the nonionic hydrophilic polymer onto the outermost surface of the modified substrate, the nonionic hydrophilic polymer may be brought into contact with the substrate into which a cationic group has been introduced. Contacting here refers to a state in which a nonionic hydrophilic polymer interacts directly or indirectly on a base material into which a cationic group has been introduced. The interaction includes chemical bonding and adsorption. As an example of indirect interaction, in the case of a chemical bond, there is a case where a nonionic hydrophilic polymer is chemically bonded to a cationic group-containing substance or a substrate via a spacer. Examples of the adsorption include a case where a nonionic hydrophilic polymer forms a hydrogen bond with a cationic group-containing substance or a substrate via a water molecule.

  As a specific example of contact, when a substrate into which a cationic group is introduced is immersed or wetted in a solution containing a nonionic hydrophilic polymer, the nonionic hydrophilic polymer is adsorbed on the substrate. . In addition, since the adsorptive power of the nonionic hydrophilic polymer is weak, when the amount of the substance introduced is small and the cationic group cannot be sufficiently covered, or when elution of the substance becomes a problem, What is necessary is just to fix a substance by the chemical reaction with the base material which introduce | transduced the cationic group.

  In addition, if the polymerization reaction is carried out by immersing or wetting the substrate into which the cationic group has been introduced in a monomer solution of a nonionic hydrophilic polymer, the cationic group-containing substance present on the substrate is covered. Thus, a nonionic hydrophilic polymer can be produced.

  When the cationic group-containing substance or the nonionic hydrophilic polymer is adsorbed on the substrate, the substrate may be immersed in each aqueous solution if the shape of the substrate is a film. Further, if the shape of the base material is a porous hollow fiber membrane, it can be adsorbed inside the porous body by using a substance smaller than the pore diameter of the porous portion. Moreover, if a substance larger than the pore diameter of the porous portion is used, it is possible to adsorb a portion as required, such as only the inner surface of the hollow fiber or only the outer surface. For example, in the case of an artificial kidney, a separation membrane is built in the module case. Accordingly, when the module is filled with a cationic group-containing substance or a nonionic hydrophilic polymer aqueous solution, the substance is adsorbed on the separation membrane. Alternatively, only the separation membrane may be once immersed in a cationic group-containing substance or a nonionic hydrophilic polymer aqueous solution to adsorb the substance, and then incorporated into the module.

  In the case of an artificial kidney in which the separation membrane is a hollow fiber membrane, the portion in contact with blood is the inner surface of the hollow fiber, so that the cationic group-containing substance and the nonionic hydrophilic polymer are present at least on the inner surface of the hollow fiber. There must be. In addition, a method of filling a cationic polymer having a weight average molecular weight of 100,000 or more or a nonionic polymer while filtering from the inside to the outside of the hollow fiber is preferable because it can be efficiently introduced only into the inner surface of the membrane. Is the method.

  Alternatively, after bringing the cationic group-containing substance or the nonionic hydrophilic polymer aqueous solution into contact with the substrate, the aqueous solution attached to the substrate may be blown into a wet state.

  A cationic group-containing substance or a nonionic hydrophilic polymer may be immobilized by adsorption, but it is preferable to immobilize by a chemical bond since there is less concern about elution. What is immobilized by adsorption can generate radicals by radiation and can easily form chemical bonds. 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 preferable to apply a dose of 20 kGy or more. In addition, radicals are generated by irradiation, and a chemical bond is formed between the cationic group-containing substance and the nonionic hydrophilic polymer with the base material, or between the cationic group-containing substance and the nonionic hydrophilic polymer. Therefore, the elution amount of these substances decreases. Therefore, irradiation with radiation of 20 kGy or more is a preferable treatment because the cationic group-containing substance and the nonionic hydrophilic polymer can be immobilized and sterilized at the same time. However, at this time, it is preferable to pay attention to the deactivation of the cationic group-containing substance and the excessive modification of the nonionic hydrophilic polymer. Excessive denaturation here refers to denaturation to such an extent that the ability to suppress adhesion of blood components such as platelets possessed by the nonionic hydrophilic polymer is lost. 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 that has the property of easily giving electrons to other molecules, but it means that a base material, a cationic group-containing substance, or a nonionic hydrophilic polymer is modified by radiation. It has the property to suppress. For example, water-soluble vitamins such as vitamin C, polyphenols, alcohols such as methanol, ethanol, propanol, ethylene glycol and glycerin, sugars such as glucose, galactose, mannose and trehalose, sodium hydrosulfite, sodium pyrosulfite, Examples include, but are not limited to, inorganic salts such as sodium dithionate, uric acid, cysteine, glutathione, oxygen, and the like. 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 base material of the present invention is suitably used as a medical base material. Examples of the medical substrate include artificial blood vessels, catheters, blood bags, contact lenses, intraocular lenses, and surgical aids, and include separation membranes used in blood purification modules and the like.

  The blood purification module of the present invention refers to a module having a function of removing waste and harmful substances in blood by adsorption, filtration, and diffusion when circulating blood outside the body. There are adsorption columns.

  In addition, as a separation membrane incorporated in an artificial kidney, there are a coil type, a flat plate type, and a hollow fiber type, but from the viewpoint of processing efficiency, the hollow fiber type is widely used at present.

  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.

  Although the material used as the separation membrane of the present invention is not particularly limited, materials used for medical use are preferable, for example, polyvinyl chloride, cellulose polymer, polystyrene, polymethyl methacrylate, polycarbonate, polysulfone, polyethersulfone, etc. And polysulfone-based polymers, polyurethane and the like. 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 substrate treatment method according to the present invention may be used before the step of incorporating the separation membrane into the module, or may be used after the separation membrane is incorporated into the module. If γ-ray 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 into a required length, bundled in a necessary number, and then put into a cylindrical case. Thereafter, 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 the cationic group-containing substance into the base material in the above, a modified polyvinyl pyrrolidone covalently bonded with an ionic group, a hydrophobic group or a physiologically active substance is used instead of the polyvinyl pyrrolidone in the stock solution, or instead of the polysulfone. This can be achieved by using a modified polysulfone to which an ionic group or a hydrophobic group or a physiologically active substance is covalently bonded. Specific examples of the modified polyvinyl pyrrolidone include cationic polyvinyl pyrrolidone commercially available from BASF as represented by the following formula (3). M and n in a formula are integers, such as 10-1000, for example.

  In addition to polysulfone and polyvinylpyrrolidone in the stock solution, a cationic group-containing substance may be added as the third component. For example, cationic polymers and physiologically active substances such as the above modified polyvinyl pyrrolidone and polyalkylenimine are preferably used. Further, the cationic group-containing substance can be introduced into the base material by adding the cationic group-containing substance to the injection solution or the coagulation bath. Further, after forming into a hollow fiber membrane, the hollow fiber membrane may be immersed or wetted in an aqueous solution of a cationic group-containing substance to adsorb the substance. Further, after modularization, the inside of the module may be filled with a cationic group-containing substance aqueous solution or wetted to adsorb the substance to the hollow fiber membrane.

  If the cationic group-containing substance is introduced before the hollow fiber membrane is incorporated into the module, the nonionic hydrophilic polymer aqueous solution may be brought into contact with the hollow fiber membrane, or after modularization, The inside of the module may be filled with a nonionic hydrophilic polymer aqueous solution or wetted. When the cationic group-containing substance is introduced after the hollow fiber membrane is incorporated in the module, the nonionic hydrophilic polymer must be introduced after the cationic group-containing substance is introduced.

    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 substance used may be a high molecular weight functional material or a high molecular weight nonionic hydrophilic polymer. 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. For example, when a cationic group-containing substance removes part of a biological component by adsorption, and the target substance is smaller than the membrane pore diameter, the cationic group-containing substance should exist up to the inside of the membrane pore. Can be removed efficiently. In order to obtain selectivity, it is preferable that the nonionic hydrophilic polymer is also present in the membrane pores.

  An example of the basic structure of an artificial kidney system 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.

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.
1. Production of Hollow Fiber Membrane Module 18 parts by weight of polysulfone (Udel (registered trademark) P-3500 manufactured by Teijin Amoco) and 9 parts by weight of polyvinylpyrrolidone (K30 manufactured by BASF) were added with 72 parts by weight of N, N′-dimethylacetamide and water 1 In addition to parts by weight of the mixed solvent, it was dissolved by heating at 90 ° C. for 14 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 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.

10000 hollow fibers obtained were inserted into a cylindrical plastic case having a dialysate inlet and a dialysate outlet, as shown in FIG. 1, and both ends were sealed with a resin to obtain an effective membrane area of 1.6 m. ^Two artificial fiber hollow fiber membrane modules were prepared.
2. Measurement Method (1) Human Platelet Adhesion Test Method for 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 adherent platelets was counted. The average value of the number of adhering platelets in 10 different visual fields near the center in the longitudinal direction of the hollow fiber 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.
(2) Rabbit platelet adhesion test method for hollow fiber membranes 30 hollow fiber separation membranes are bundled, and both ends are fixed to a glass tube module case with an epoxy potting agent so as not to block the hollow portion of the hollow fiber, and a mini module is created. did. The mini module had a diameter of about 7 mm and a length of about 10 cm. The blood inlet and the dialysate outlet of the minimodule were connected by a silicone tube, and 100 ml of distilled water was allowed to flow from the blood outlet at a flow rate of 10 ml / min to wash the hollow fiber and the inside of the module. Thereafter, physiological saline was filled, and the dialysate inlet and outlet were capped. Next, physiological saline priming was performed at a flow rate of 0.59 ml / min from the blood inlet for 2 hours, and then 3.2% trisodium citrate dihydrate aqueous solution and rabbit fresh blood were added 1: 9 (volume ratio). ) Was perfused for 1 hour at a flow rate of 0.59 ml / min. Then, it was washed with physiological saline with a 10 ml syringe, filled with 3% aqueous glutaraldehyde solution both inside the hollow fiber and on the dialysate side, and placed overnight or longer to fix glutaraldehyde. Thereafter, glutaraldehyde was washed with distilled water, a hollow fiber membrane was cut out from the minimodule and dried under reduced pressure for 5 hours or more. The hollow fiber membrane was attached to a sample stage of a scanning electron microscope with a double-sided tape, and then sliced in the longitudinal direction to expose the inner surface. Thereafter, a thin film of Pt—Pd was formed on the sample by sputtering. The inner surface of the sample was observed with a scanning electron microscope (S800 manufactured by Hitachi, Ltd.) at a magnification of 3000 times, and the number of adhering platelets in one visual field (1.12 × 10 ³ μm ² ) was counted. A value obtained by dividing the average value of the number of adhering platelets in 10 different visual fields by 1.12 was defined as the number of adhering platelets (number / 1.0 × 10 ³ μm ² ).
(3) Oxidized LDL adsorption / removal test method (a) Production of antioxidant LDL antibody It was produced 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, manufactured by 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. To the washed wells, 100 μl / well of 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. 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 1 m ^{2 of the} surface area of the hollow fiber membrane 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 (4) elution polyethyleneimine concentration measurement method Module left for 1 week at room temperature after irradiation The liquid filled on the blood side was extracted from the blood outlet 4 by natural fall. The amount of polyethyleneimine eluted was quantified by HPLC under the following conditions.
Equipment: Waters, GPC-244
Column: TSKgel GMPWXL One solvent: Mixed aqueous solution of 0.1 N acetic acid and 0.1 N sodium acetate Flow rate: 0.5 ml / min
Temperature: 30 ° C
Example 1
Polyethyleneimine (BASF, weight average molecular weight 750,000) was used as the cationic group-containing substance, and polyvinylpyrrolidone (BASF, K90) was used as the nonionic hydrophilic polymer. A polyethyleneimine 0.01% by weight aqueous solution was introduced from the blood outlet 4 of the hollow fiber membrane module and exited from the dialysate outlet 9 to introduce polyethyleneimine 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, 1 L of a polyvinylpyrrolidone 0.001% by weight aqueous solution was passed in the same manner as the polyethyleneimine aqueous solution to introduce polyvinylpyrrolidone into the hollow fiber membrane. Thereafter, the module was irradiated with γ rays. The absorbed gamma ray dose was 28 kGy. The liquid filled on the blood side of the module was extracted, and the eluted polyethyleneimine concentration was measured. Furthermore, the hollow fiber was cut out and the platelet adhesion number was evaluated. A mini-module was created using the cut out hollow fiber membrane and subjected to an oxidation LDL adsorption experiment. As a result, it was as shown in Table 1.
(Comparative Example 1)
Polyethyleneimine (BASF, weight average molecular weight 750,000) was used as the cationic group-containing substance, and polyvinylpyrrolidone (BASF, K90) was used as the nonionic hydrophilic polymer. A polyvinylpyrrolidone 0.001 wt% aqueous solution was introduced from the blood outlet 4 of the hollow fiber membrane module and exited from the dialysate outlet 9 to introduce polyvinylpyrrolidone 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, 1 L of a polyethyleneimine 0.01% by weight aqueous solution was passed in the same manner as the polyvinylpyrrolidone aqueous solution to introduce polyethyleneimine into the hollow fiber membrane. Thereafter, the module was irradiated with γ rays. The absorbed gamma ray dose was 28 kGy. The liquid filled on the blood side of the module was extracted, and the eluted polyethyleneimine concentration was measured. Furthermore, the hollow fiber was cut out and the platelet adhesion number was evaluated. A mini-module was created using the cut out hollow fiber membrane and subjected to an oxidation LDL adsorption experiment. As a result, it was as shown in Table 1.
(Comparative Example 2)
Polyethyleneimine (BASF, weight average molecular weight 750,000) was used as the cationic group-containing substance. A polyethyleneimine 0.01% by weight aqueous solution was introduced from the blood outlet 4 of the hollow fiber membrane module and exited from the dialysate outlet 9 to introduce polyethyleneimine 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, the module was irradiated with γ rays. The absorbed gamma ray dose was 28 kGy. The liquid filled on the blood side of the module was extracted, and the eluted polyethyleneimine concentration was measured. Furthermore, the hollow fiber was cut out and the platelet adhesion number was evaluated. A mini-module was created using the cut out hollow fiber membrane and subjected to an oxidation LDL adsorption experiment. As a result, it was as shown in Table 1.
(Comparative Example 3)
Polyethyleneimine (manufactured by Wako Pure Chemical Industries, Ltd., weight average molecular weight 70,000) was used as the cationic group-containing substance. A polyethyleneimine 1% by weight aqueous solution was introduced from the blood outlet 4 of the hollow fiber membrane module and exited from the blood guide inlet 3 to introduce polyethyleneimine into the hollow fiber membrane. The flow rate was 1 L. At this time, the dialysate inlet 8 and outlet 9 were plugged. Thereafter, the module was irradiated with γ rays. The absorbed gamma ray dose was 28 kGy. The liquid filled on the blood side of the module was extracted, and the eluted polyethyleneimine concentration was measured. Furthermore, the hollow fiber was cut out and the platelet adhesion number was evaluated. A mini-module was created using the cut out hollow fiber membrane and subjected to an oxidation LDL adsorption experiment. As a result, it was as shown in Table 1.
(Comparative Example 4)
Polyvinyl pyrrolidone (BA90 K90) was used as the nonionic hydrophilic polymer. A polyvinylpyrrolidone 0.001 wt% aqueous solution was introduced from the blood outlet 4 of the hollow fiber membrane module and exited from the dialysate outlet 9 to introduce polyvinylpyrrolidone 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, the module was irradiated with γ rays. The absorbed gamma ray dose was 28 kGy. The liquid filled on the blood side of the module was extracted, and the eluted polyethyleneimine concentration was measured. Furthermore, the hollow fiber was cut out and the platelet adhesion number was evaluated. A mini-module was created using the cut out hollow fiber membrane and subjected to an oxidation LDL adsorption experiment. As a result, it was as shown in Table 1.
(Comparative Example 5)
Pure water was introduced from the hollow fiber membrane module blood outlet 4 and out of the dialysate outlet 9. The flow rate was 2L. Thereafter, the module was irradiated with γ rays. The absorbed gamma ray dose was 28 kGy. The liquid filled on the blood side of the module was extracted, and the eluted polyethyleneimine concentration was measured. Furthermore, the hollow fiber was cut out and the platelet adhesion number was evaluated. A mini-module was created using the cut out hollow fiber membrane and subjected to an oxidation LDL adsorption experiment. As a result, it was as shown in Table 1.

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

Claims (10)

A cationic polymer having a charge of 1 meq / g or more at pH 4.5 and one or more nonionic hydrophilic polymers selected from polyvinyl pyrrolidone, polyethylene glycol, polypropylene glycol, polyvinyl alcohol and derivatives thereof are contained. and which, modified substrate, wherein said non-ionic hydrophilic polymer is introduced into the outermost surface. The modified base material according to claim 1, wherein the cationic polymer substance is introduced on the surface of the base material, and the nonionic hydrophilic polymer is introduced on the outermost surface. Human platelet adhesion amount is 10 / 4.3 × 10 ^Three μm ² The modified base material according to claim 1 or 2, wherein the rate of adsorption and removal of acidic protein is 20% or more. The modified base material according to any one of claims 1 to 3, wherein the acidic protein is an oxidized low-density lipoprotein. The modified base material according to claim 1, wherein an elution concentration of the cationic polymer is 1000 ppm or less. The modified base material according to claim 1, wherein the base material according to claim 1 is irradiated with radiation. The modified base material according to claim 1, wherein the base material is a medical base material. The modified base material according to claim 7, wherein the medical base material is built in a blood purification module. The modified base material according to claim 1, wherein the medical base material is a hollow fiber membrane. The modified base material according to claim 9, wherein the hollow fiber membrane is a polysulfone-based polymer.

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