JP2011201031A - Polymer substrate having laminated structure and medical appliance using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a polymer substrate having a laminated structure and an excellent HCV adsorption capability for removing HCV in the blood and a medical appliance using it.SOLUTION: The laminated polymer substrate consists of: a polymer support (A); a layer (B) obtained by treating the surface of the polymer support (A) through a graft polymerization reaction with a polymerizable compound; and a laminate structure (C) capable of combining with the layer (B). The laminate structure (C) is formed by laminating, n times (wherein n is an integer of 1-10), a paired unit of: a first layer obtained by binding, with the layer (B), a compound (D) which is ionic or has a functional group capable of ionization and can combine with the layer (B); and a second layer obtained by binding, with the first layer, a sulfated polysaccharide (E) capable of combining with the first layer.

Description

The present invention relates to a polymer substrate having a laminated structure obtained by treating the surface of a polymer support. In addition, since the present polymer substrate has a function of adsorbing viruses, the present invention relates to a medical instrument that removes viruses using the polymer substrate.

Hepatitis C is caused by chronic infection with hepatitis C virus (HCV), and a combination therapy of peginterferon and ribavirin is common as a therapeutic method using drugs. In patients with genotype 1b and a large amount of virus in the blood, the therapeutic result is about 50%, and since the rate of transition to cirrhosis and liver cancer is high, development of more effective treatment methods and drugs is desired. (Non-Patent Document 1).
In general, it is known that in treatment with drugs, if the amount of virus in the blood is low, the therapeutic result is high. Therefore, there is a report that treatment results are improved when HCV in blood is removed with a porous filter and combined therapy with a drug is performed (Non-patent Document 2). This is presumed that the treatment results were improved by reducing the amount of virus in the body.

However, since this method removes important blood components having a size similar to that of viruses, such as filrinogen, these non-removable methods are desirable for patients.
In addition, once the blood cells and plasma are separated, the virus is removed from the plasma components. Therefore, a circuit configuration is complicated, and a method for removing the virus from the blood more easily is desirable.

In view of the above background, development of a method for selectively adsorbing HCV is awaited.
One of the substances that adsorb HCV is heparin (Non-patent Document 2), and the HCV adsorption / removal module using heparin as an adsorption ligand is suitable for selectively adsorbing and removing viruses from blood components from the blood. It is thought that there is.

Examples of the form of the heparin-immobilized substrate include beads and porous hollow fibers. The internal circulation type extracorporeal circulation module has an advantage that the formation of thrombus is less because it has less blood retention than the extracorporeal circulation module filled with the particulate heparin-immobilized substrate. When immobilizing heparin on the porous hollow fiber, the type of surface functional group and the immobilization density vary depending on the base material, and it is necessary to find an optimum method for each material.

On the other hand, G. Decher et al. Have reported that a functional thin film can be produced by alternately immersing a substrate in a polyanion and polycation solution (Patent Documents 1 and 2, Non-Patent Documents 4 and 5). By using this technique, it is considered that heparin can be formed on the surface with an arbitrary thickness regardless of the material and form.
In addition, although an alternate laminate using heparin has been reported, no preferred laminate for HCV removal has been reported so far (Non-Patent Documents 6, 7, 8, and 9).

Japanese Patent No. 2996695 Japanese Patent No. 3293641

Viral hepatitis-Advances in basic and clinical research-Japanese clinical volume 62 extra number 7 (2004) A. K. Fujiwara et al. Hepatol. Res. , 37, 701 (2007) Zahn, J. et al. P. Allain, J .; Gen. Virol. , 86, 677 (2005) G. Decher, Science, 277, 1232 (1997) Thin Solid Films, 210-211, 831 (1992) Z. Mao, Bioconjugate Chem. , 16, 1316 (2005) L. Liu, J .; Biomed. Mater. Res. PartB Appl. Biometer. , 87B, 244 (2008) M.M. Liu, Langmuir, 23, 9379 (2007) K. C. Wood, PNAS, 103, 10207 (2006)

In view of the above-described background art, an object of the present invention is to provide a polymer substrate having a laminated structure excellent in HCV adsorption capacity for the purpose of removing HCV in blood, and a medical instrument using the same.

The present invention binds to the layer (B) and the layer (B) obtained by treating the surface of the polymer support (A) by a graft polymerization reaction with the polymer support (A) and a polymerizable compound. A laminated polymer base material comprising a laminated structure (C) to be obtained,
The above-mentioned problem is solved by providing a laminated polymer base material in which the laminated structure (C) is constituted by being laminated n times as a set of the following layers.
First layer: a layer obtained by binding the compound (D) having an ionic or ionizable functional group capable of binding to the layer (B) to the layer (B) Second layer: binding to the first layer A layer obtained by combining a sulfated polysaccharide (E) capable of binding with the first layer (where n represents an integer of 1 to 10).

ADVANTAGE OF THE INVENTION According to this invention, the polymer base material which has the laminated structure excellent in HCV adsorption ability can be provided, and the medical device which can remove HCV by this base material, and the HCV removal method using the same are provided. be able to.

It is a schematic sectional drawing which shows the medical device provided with the laminated polymer base material of this invention. It is drawing which shows the amount of heparin immobilization.

That is, the present invention
1. Polymer support (A), layer (B) obtained by treating surface of polymer support (A) by graft polymerization reaction with polymerizable compound, and laminated structure capable of bonding to layer (B) A laminated polymer base material comprising (C),
A laminated polymer base material, wherein the laminated structure (C) is constituted by being laminated n times with each of the following layers as a set,
First layer: a layer obtained by binding the compound (D) having an ionic or ionizable functional group capable of binding to the layer (B) to the layer (B) Second layer: binding to the first layer A layer obtained by combining a sulfated polysaccharide (E) capable of binding with the first layer (where n represents an integer of 1 to 10).
2. 1. The polymer support (A) is a polymer support using a polyolefin as a substrate. Laminated polymer substrate (C) according to
3. 1. A polymer support using a polyolefin as a substrate is a hollow fiber membrane. Laminated polymer substrate (C) according to
4). 2. The polyolefin is poly-4-methylpentene Laminated polymer substrate (C) according to
5. 1. The polymerizable compound is (meth) acrylic acid, and the compound (D) is a high molecular amine compound. ~ 4. Laminated polymer base material (C) according to any one of
6). 4. The high molecular amine compound is polyallylamine. Laminated polymer substrate (C) according to
7). 1. The polymerizable compound is glycidyl (meth) acrylate, and the compound (D) is ammonia or a polyvalent amine compound. ~ 4. Laminated polymer base material (C) according to any one of
8). 6. The polyvalent amine compound is 1,2-bis (2-aminoethoxy) ethane or polyallylamine. Laminated polymer substrate (C) according to
9. 1. The polymerizable compound is (meth) acryloyloxyalkyl isocyanate, and the compound (D) is a polyvalent amine compound. ~ 4. Laminated polymer base material (C) according to any one of
10. 8. The polyvalent amine compound is 1,2-bis (2-aminoethoxy) ethane or polyallylamine. Laminated polymer substrate (C) according to
11. 1. Sulfated polysaccharide (E) is heparin -10. Laminated polymer base material (C) according to any one of
12 The binding method of the sulfated polysaccharide and the first layer is based on the reaction between the activated carbodiimide obtained by reacting the sulfated polysaccharide with carbodiimide and the compound (D). ~ 11. Laminated polymer base material (C) according to any one of
13.1. -12. A virus removal instrument comprising the polymer substrate according to any one of
14 12. The virus is hepatitis B or C virus A virus removal device according to claim 1,
15.13. Or 14. A virus removal method using the virus removal instrument according to claim 1,
About.

The layer (B) obtained by treating the surface of the polymer support (A) by a graft polymerization reaction with the polymerizable compound of the present invention is obtained by graft polymerizing the polymerizable compound on the polymer support. Layer.

The polymerizable compound used in the present invention can be used without limitation as long as it has an ethylenically unsaturated group capable of forming the layer (B) by graft polymerization to the polymer support (A). However, a (meth) acrylic compound is preferable from the viewpoint of easily forming a laminated structure with the layer (C). In particular, the (meth) acrylic compound is preferably (meth) acrylic acid, glycidyl (meth) acrylate, (meth) acryloyloxyalkyl isocyanate, or the like.

The polymer support (A) used in the present invention may be a polymer compound having a methylene group in the main chain because radicals are generated by irradiation with ionizing radiation. As such a polymer compound, various compounds having high blood compatibility can be used. For example, olefin resin, styrene resin, sulfone resin, acrylic resin, urethane resin, ester Resin, ether resin or cellulose acetate, and more specifically, polyethylene terephthalate, ethylene vinyl alcohol copolymer, polymethyl methacrylate, polysulfone, polyether sulfone, polyacrylonitrile, polyethylene, polypropylene or poly-4-methyl. An example is pentene.

The shape of the polymer support (A) is not particularly limited, and the polymer support (A) can be used in various forms such as hollow fibers, beads, and nonwoven fabrics. When applied to extracorporeal circulation, it can be used as beads or non-woven fabrics with a structure in which blood stays. It is desirable to use a hollow fiber. Further, if the hollow fiber is not used as a filtration membrane, it is not necessary to be porous, so the form of the hollow fiber can be selected according to the application.

In the present invention, the ethylenically unsaturated group of the polymerizable compound is graft-polymerized on the polymer support by radicals generated by irradiating the polymer support (A) with ionizing radiation. Examples of the ionizing radiation source used in the graft polymerization include known and commonly used α-rays, β-rays, γ-rays, accelerated electron beams, X-rays and the like, and practically preferred are γ-rays and accelerated electron beams.

The graft polymerization method is a simultaneous irradiation graft polymerization method in which the polymer support (A) and the polymerizable compound are brought into contact with each other and irradiated with ionizing radiation, and the polymer support is pre-irradiated and then pre-irradiated with the polymerizable compound. Any of the graft polymerization methods is possible and can be selected according to the purpose.

In the graft polymerization method using ionizing radiation used in the present invention, the irradiation dose and the accelerating voltage differ depending on the polymer support (A), so the range cannot be determined unconditionally, and the material of the polymer support (A) It is necessary to adjust appropriately considering the form, thickness, and the like. For example, when the irradiation amount is large, dielectric breakdown due to charging occurs, and when the irradiation amount is small, the polymerization reaction does not proceed. For this reason, in consideration of the material and form of the polymer support (A), the irradiation dose that does not cause dielectric breakdown due to charging and that allows the polymerization reaction to proceed sufficiently may be appropriately adjusted. The acceleration voltage is related to the permeability and varies depending on the thickness of the polymer support. In the case of a thin form such as a film, the acceleration voltage is generally small and may be selected depending on the form of the polymer support.

For example, in the case where the polymer support (A) is a hollow fiber having a thickness of 0.1 (μm) to 10 (μm) made of 4-methyl-1-pentene, 10 (kGy) or more and 300 (kGy) Or less, more desirably 90 (kGy) or less. The acceleration voltage is 0.1 (kV) or more and 10 (kV) or less, preferably 5 (kV) or less.

There is no particular limitation in the order of the contact step and the ionizing radiation irradiation step. For example, after the step (1) of contacting the polymeric support (A) with the polymerizable compound or the polymerizable composition containing the polymerizable compound, the step (2) of irradiating ionizing radiation in this state is performed. Alternatively, conversely, after the step (3) of first irradiating the polymer support with ionizing radiation, the polymer support is contacted with a polymerizable compound or a polymerizable composition containing the polymerizable compound. (4) may be performed.

In the irradiation graft polymerization, radicals in the substrate after irradiation are quickly inactivated by temperature increase and contact with oxygen. Therefore, after irradiation, it is preferable to store it at a low temperature in a state where oxygen is sufficiently removed, and to quickly fix it. For the above reasons, the immobilization is preferably carried out under deoxygenation or in an inert gas.
The polymer support after polymerization may be removed from the unreacted polymerizable compound by various methods such as washing with water.

In addition, when the shape of the polymer support (A) is a hollow fiber and the polymerizable compound or the polymerizable composition is fixed to the hollow fiber, either the inner surface or the outer surface of the yarn is used depending on the embodiment. Can be immobilized on either or both. For example, when blood is circulated inside the hollow fiber, the sugar chain may be fixed by bringing the polymerizable compound or polymerizable composition into contact with the inside of the hollow fiber, and conversely, the external part of the hollow fiber is externally refluxed. The sugar chain may be immobilized by bringing the polymerizable compound or polymerizable composition into contact therewith.

Next, a polymer base material having a laminated structure (C) with a layer obtained from a compound having a functional group capable of binding to the layer (B) obtained as described above will be described.

The laminated structure (C) of the present invention can be obtained by lamination with a layer obtained from a compound having a functional group capable of bonding to the layer (B) obtained as described above.

The compound having a functional group capable of binding to the layer (B) is not particularly limited as long as it has a group capable of binding to the functional group of the compound constituting the layer (A). When an acid is used, a polymer compound having an amino group which is an ionic or ionizable functional group capable of binding to a carboxyl group of (meth) acrylic acid, particularly a compound such as polyallylamine is preferable. Further, when glycidyl (meth) acrylate is used as the polymerizable compound, a polyvalent amine derivative having reactivity with a glycidyl group is preferable. More specifically, polyvalent amine derivatives such as 2-bis (2-aminoethoxy) ethane and polyallylamine can be mentioned.

In addition, when (meth) acryloyloxyalkyl isocyanate is used as the polymerizable compound, a compound having a hydroxyl group or amino group having reactivity with an isocyanate group is preferable, and a compound is particularly preferable. More specifically, polyvalent amine derivatives such as 2-bis (2-aminoethoxy) ethane and polyallylamine can be mentioned.

In order to produce the laminated structure (C), for example, when (meth) acrylic acid is used as the polymerizable compound, the polymer support is prepared after graft polymerization of (meth) acrylic acid to the polymer support. The body can be prepared by immersing the body in a polymer compound solution having an amino group. In this case, it may be heated.
In addition, when glycidyl (meth) acrylate is used as the polymerizable compound, after glycidyl (meth) acrylate is graft-polymerized to the polymer support, the polymer support is immersed in a polyvalent amine derivative solution. Can be produced. In this case, it may be heated. The degree of heating is not particularly limited as long as the amino group and glycidyl group of the polyvalent amine derivative are reacted.
After the (meth) acrylate is graft-polymerized on the polymer support, the polymer support can be produced by immersing the polymer support in a polyvalent amine derivative solution. In this case, it may be heated. The degree of heating is not particularly limited as long as the amino group of the polyvalent amine derivative reacts with the glycidyl group or the isocyanate group.

The second layer in the laminated structure (C) can be produced by bonding with a sulfated polysaccharide (E) that can bond with the compound (D) as the first layer. Here, the sulfated polysaccharide is not limited as long as it can be adsorbed to the sulfated polysaccharide by contact with a virus, but heparin can be preferably used. Although there is no particular limitation on the bonding method, a preferred method includes a method of bonding to compound (D) via a covalent bond.

As an example, a specific method by an amidation reaction is shown below, but the bonding mode performed in the present invention is not limited thereto.

Amidation reaction methods include, for example, amidation with an active ester, amidation with a condensing agent, a combination thereof, a mixed acid anhydride method, an azide method, a redox method, a DPPA method, a Woodward method, peptide synthesis, etc. What is necessary is just to perform the well-known and usual amidation reaction used.

Examples of amidation with an active ester include NHS (N-hydroxysuccinimide), nitrophenol, pentafluorophenol, DMAP (4-dimethylaminopyridine), HOBT (1-hydroxybenzotriazole), and HOAT (hydroxyazabenzotriazole). And HOSu (hydroxysuccinimide), etc., to form an active ester obtained by once condensing a group having a high leaving ability with a carboxy group, and reacting this with an amino group. Amidation with a condensing agent may be used alone or in combination with the active ester. As the condensing agent, EDC (1- (3-dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride), HONB (endo-N-hydroxy-5-norbornene-2,3-dicarboxamide), DCC (dicyclohexylcarbodiimide) , BOP (benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate), HBTU (O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyluronium hexafluorophosphate) , TBTU (O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyluronium tetrafluoroborate), HOBt (1-hydroxybenzotriazole), HOOBt (3,4-dihydro-3- Hydroxy-4-oxo- , 2,3-benzotriazine), di-p-trioylcarbodiimide, DIC (diisopropylcarbodiimide), BDP (1-benzotriazole diethyl phosphate-1-cyclohexyl-3- (2-morpholinylethyl) carbodiimide), fluorine Cyanuric chloride, cyanuric chloride, TFFH (tetramethylfluoroformamidinium hexafluorophosphophosphate), DPPA (diphenylphosphoradidate), TSTU (O- (N-succinimidyl) -N, N, N ′, N′-tetramethyl Uronium tetrafluoroborate), HATU (N-[(dimethylamino) -1-H-1,2,3-triazolo [4,5,6] -pyridin-1-ylmethylene] -N-methylmethanaminium. Hexafluorophosphate / N-oxide) BOP-Cl (bis (2-oxo-3-oxazolidinyl) phosphine chloride), PyBOP ((1-H-1,2,3-benzotriazol-1-yloxy) -tris (pyrrolidino) phosphonium tetrafluorophosphate), BrOP (bromotris (dimethylamino) phosphonium hexafluorophosphate), DEPBT (3- (diethoxyphosphoryloxy) -1,2,3-benzotriazin-4 (3H) -one), PyBrOP (bromotris (pyrrolidino) phosphonium Hexafluorophosphate) and the like.

Among these, a method in which the carboxy group is once NHS converted and then amidated by reacting with the amino group of the compound (D), and a method in which EDC is added to NHS and amidated is more preferable.

As a solvent that can be used in these amidation methods, water and an organic solvent used for peptide synthesis can be used. For example, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexaphosphoroamide, dioxane, tetrahydrofuran ( THF), ethyl acetate, and the like, and mixed solvents and aqueous solutions containing these.

The proportion of the activated ester residue introduced into the carboxy group depends on the type of activator used and the amount of reagent used. In general, compared to the reaction in solution, the polymer obtained from the polymerizable compound is bonded, and the reactivity is lowered. Therefore, in order to increase the introduction amount, it is necessary to use a considerably excessive amount of the reaction reagent. It is thought that there is. Therefore, although the ratio of the activator and the condensing agent reaction reagent relative to the amount of immobilized carboxy group cannot be defined unconditionally, it is desirable to use about 1 to 100 in molar ratio. The amount of lactose can be used in an excess of about 1 to 100 times in molar ratio with respect to the active ester residue, but it is not preferable to use an excessive amount of expensive lactose.

Unreacted active ester residues can be converted to amides by reaction with aqueous ammonia and removed. It can also be removed by amidation using a primary amine other than ammonia that can be easily removed after the reaction. In any case, it is desirable to remove it so as not to remain in the equipment.
Since the production method of the laminated polymer substrate of the present invention can be widely applied with respect to the material and form of the polymer material, various laminated polymer substrates can be obtained according to the purpose and application.

The form of the medical device provided with the laminated polymer substrate of the present invention is not particularly limited as long as it is a shape applicable to the above-mentioned use, and examples thereof include a hollow fiber module, a filtration column, and a filter. It is done. In the hollow fiber module and the filtration column, the shape and material of the container are not particularly limited, but when applied to extracorporeal circulation of body fluid (blood), a cylindrical container having an internal volume of 10 to 400 mL and an outer diameter of about 2 to 10 cm It is preferable to use a cylindrical container having an internal volume of 20 to 200 mL and an outer diameter of about 2.5 to 4 cm.

As a method of using the medical device of the present invention, if the virus in the liquid can be adsorbed and removed by contact with a liquid containing a virus (for example, an aqueous solution containing a virus or a body fluid such as blood, plasma, serum, etc.) Either method is acceptable. Examples of such a method include the following methods.
(1) A method of preparing a module having a hollow fiber as the laminated polymer substrate of the present invention, and allowing a liquid containing virus to pass through the module. (2) Although the liquid can pass through the outlet, the polymer group of the present invention Prepare a column-like container with a filter that does not allow the material to pass through, and fill the inside with a filter, and a method for allowing the virus-containing liquid to pass therethrough. (3) Prepare a container such as a storage bag, which contains the virus The method (1) or (2), in which the solution and the polymer base material of the present invention are added and mixed, and then the supernatant is recovered, is preferable in terms of simple operation. It is possible to efficiently remove viruses from body fluids in-line. Among these, a more preferable method is the method (1). In the methods (2) and (3), for example, when handling blood, it is necessary to separate only blood into blood cells and plasma to prevent coagulation, but in the method (1), Such a process is not required, the operation is the simplest, and the burden on the patient is small.

The hepatitis virus targeted in the present invention is characterized by being a hepatitis B or C virus. In the present invention, in order to evaluate the heparin derivative adsorbing ability of hepatitis virus, the adsorbing ability of HCV E2 protein (His-tag) was evaluated. The E2 protein, together with the lipid membrane, constitutes the envelope of the virus particle (envelope) and plays an important role in virus entry. By evaluating the ability to adsorb to the E2 protein, the ability to adsorb to HCV It is a protein that can be evaluated.
As shown in the following examples, the polymer base material of the present invention was confirmed to be capable of adsorbing HCV E2 protein.

The following examples illustrate the invention in more detail.

Example 1
A hollow fiber bundle made of poly-4-methylpentene (hollow fiber surface area 200 cm ² , manufactured by DIC Corporation) was put in a glass test tube, sealed with a rubber stopper, and the inside of the test tube was purged with nitrogen, and then manufactured by RDI. 90 kGy electron beam was irradiated with the acceleration voltage of 4.8 MeV with the electron beam irradiation apparatus "Dynamitron 5MeV-150kW". Subsequently, a deoxygenated solution of methacrylic acid (Tokyo Kasei) methanol solution 40 mg / mL at 23 ° C. was added to the test tube. After 4 hours, the hollow fiber bundle was taken out from the test tube, washed several times with water, dried, and then weighed. From the increase in weight, the fixation of methacrylic acid was confirmed. A methacrylic acid-immobilized hollow fiber is immersed in a 3 mg / mL aqueous solution of polyallylamine (Toyobo, PAA-L, molecular weight 15000) for 1 hour, washed several times with water, heparin 3 mg / mL, EDC 1.5 mg / mL, NHS. It was immersed in an aqueous solution containing 2 mg / mL for 1 hour and washed with water. By alternately immersing in the solution and washing with water, hollow fibers alternately laminated a predetermined number of times were obtained. The hollow fiber was immersed in an Ac ₂ O / 0.2M AcONa solution (1/2 vol) solution and stirred for 1 hour to acetylate residual amino groups. When the cross section of the hollow fiber was observed with a transmission electron microscope after drying, a film containing sulfur atoms derived from heparin was observed at a thickness of about 100 nm.

<Evaluation of HCV E2 adsorption rate of heparin-immobilized hollow fiber>
The hollow fiber on which heparin was immobilized was cut into 5 cm ² , put into a microtube, 1% BSA / PBS solution was added as a blocking solution, and left overnight at 4 ° C. The blocking solution was removed, 500 μL of 2-4 μg / mL concentration of E2 solution (Abcam) was added, rotated at room temperature for 2 hours, and the amount of E2 before and after adsorption was measured by ELISA. As a result, the E2 adsorption rate was 65. %Met.

(Example 2)
A hollow fiber bundle made of poly-4-methylpentene (hollow fiber surface area 200 cm ² , manufactured by DIC Corporation) was put in a glass test tube, sealed with a rubber stopper, and the inside of the test tube was purged with nitrogen, and then manufactured by RDI. 90 kGy electron beam was irradiated with the acceleration voltage of 4.8 MeV with the electron beam irradiation apparatus "Dynamitron 5MeV-150kW". Subsequently, a deoxygenated solution of 40 mg / mL of acrylic acid (Tokyo Kasei) aqueous solution was added to the test tube at 23 ° C. After 4 hours, the hollow fiber bundle was taken out from the test tube, washed several times with water, dried, and then weighed. From the increase in weight, it was confirmed that acrylic acid was immobilized. Acrylic acid-immobilized hollow fiber is immersed in 3 mg / mL aqueous solution of polyallylamine (Toyobo, PAA-L, molecular weight 15000) for 1 hour, washed several times with water, heparin 3 mg / mL, EDC 1.5 mg / mL, NHS 1 It was immersed in an aqueous solution containing 2 mg / mL for 1 hour and washed with water. By alternately immersing in the solution and washing with water, hollow fibers alternately laminated a predetermined number of times were obtained. The hollow fiber was immersed in an Ac ₂ O / 0.2M AcONa solution (1/2 vol) solution and stirred for 1 hour to acetylate residual amino groups. The amount of heparin immobilized was measured using a quantitative method using adsorption of a dye solution using toluidine blue.

(Example 3)
A hollow fiber bundle made of poly-4-methylpentene (hollow fiber surface area 200 cm ² , manufactured by DIC Corporation) was put in a glass test tube, sealed with a rubber stopper, and the inside of the test tube was purged with nitrogen, and then manufactured by RDI. 90 kGy electron beam was irradiated with the acceleration voltage of 4.8 MeV with the electron beam irradiation apparatus "Dynamitron 5MeV-150kW". Subsequently, a deoxygenated solution of 40 mg / mL glycidyl methacrylate (GMA, Tokyo Kasei) methanol solution was added to the test tube at 23 ° C. After 4 hours, the hollow fiber bundle was taken out from the test tube, washed several times with water, dried, weighed, and confirmed the immobilization of GMA from the weight increase. The epoxy group was ring-opened by immersion in a methanol solution of 1,2-bis (2-aminoethoxy) ethane at 40 ° C. for 4 hours for amination. It was immersed in an aqueous solution containing heparin 3 mg / mL, EDC 1.5 mg / mL, NHS, 1.2 mg / mL for 1 hour and washed with water. Polyacrylamine (Toyobo, PAA-L, molecular weight: 15000) 3 mg / mL aqueous solution is immersed in acrylic acid-immobilized hollow fiber for 1 hour, washed several times with water, then alternately immersed in the above solution and repeatedly washed with water, alternating a predetermined number of times. A laminated hollow fiber was obtained. The hollow fiber was immersed in an Ac ₂ O / 0.2M AcONa solution (1/2 vol) solution and stirred for 1 hour to acetylate residual amino groups. The amount of heparin immobilized was measured using a quantitative method using adsorption of a dye solution using toluidine blue.

Example 4
A hollow fiber bundle made of poly-4-methylpentene (hollow fiber surface area 200 cm ² , manufactured by DIC Corporation) was put in a glass test tube, sealed with a rubber stopper, and the inside of the test tube was purged with nitrogen, and then manufactured by RDI. 90 kGy electron beam was irradiated with the acceleration voltage of 4.8 MeV with the electron beam irradiation apparatus "Dynamitron 5MeV-150kW". Subsequently, at 23 ° C., a deoxygenated solution of 2-methacryloyloxyethyl isocyanate (Showa Denko, Karenz MOI) ethyl acetate solution 40 mg / mL was added to the test tube. After 4 hours, the hollow fiber bundle was taken out from the test tube, washed several times with ethyl acetate, dried, and then weighed to confirm the fixation of 2-methacryloyloxyethyl isocyanate from the weight increase. Amination was carried out by immersing in an ethyl acetate solution of 1,2-bis (2-aminoethoxy) ethane at 23 ° C. for 1 hour. It was immersed in an aqueous solution containing heparin 3 mg / mL, EDC 1.5 mg / mL, NHS, 1.2 mg / mL for 1 hour and washed with water. Polyacrylamine (Toyobo, PAA-L, molecular weight: 15000) 3 mg / mL aqueous solution is immersed in acrylic acid-immobilized hollow fiber for 1 hour, washed several times with water, then alternately immersed in the above solution and repeatedly washed with water, alternating a predetermined number of times. A laminated hollow fiber was obtained. The hollow fiber was immersed in an Ac ₂ O / 0.2M AcONa solution (1/2 vol) solution and stirred for 1 hour to acetylate residual amino groups. The amount of heparin immobilized was measured using a quantitative method using adsorption of a dye solution using toluidine blue.

(Example 5)
A hollow fiber bundle made of poly-4-methylpentene (hollow fiber surface area 200 cm ² , manufactured by DIC Corporation) was put in a glass test tube, sealed with a rubber stopper, and the inside of the test tube was purged with nitrogen, and then manufactured by RDI. 90 kGy electron beam was irradiated with the acceleration voltage of 4.8 MeV with the electron beam irradiation apparatus "Dynamitron 5MeV-150kW". Subsequently, a deoxygenated solution of 40 mg / mL glycidyl methacrylate (GMA, Tokyo Kasei) methanol solution at 23 ° C. was added to the test tube. After 4 hours, the hollow fiber bundle was taken out from the test tube, washed several times with water, dried, weighed, and confirmed the immobilization of GMA from the weight increase. The epoxy group was ring-opened by dipping in a methanol solution (10 wt%) of polyallylamine (Toyobo, PAA-L, molecular weight 15000) at 40 ° C. for 4 hours to amination. It was immersed in an aqueous solution containing heparin 3 mg / mL, EDC 1.5 mg / mL, NHS, 1.2 mg / mL for 1 hour and washed with water. Polyacrylamine (Toyobo, PAA-L, molecular weight: 15000) 3 mg / mL aqueous solution is immersed in acrylic acid-immobilized hollow fiber for 1 hour, washed several times with water, then alternately immersed in the above solution and repeatedly washed with water, alternating a predetermined number of times. A laminated hollow fiber was obtained. The hollow fiber was immersed in an Ac ₂ O / 0.2M AcONa solution (1/2 vol) solution and stirred for 1 hour to acetylate residual amino groups. The amount of heparin immobilized was measured using a quantitative method using adsorption of a dye solution using toluidine blue.

(Comparative example)
According to the procedure of Example 1, a methacrylic acid-immobilized hollow fiber was obtained. The hollow fiber was cut into 5 cm ² and measured in the same manner as described in <Evaluation of HCV E2 adsorption rate of heparin-immobilized hollow fiber> described in Example 1. The E2 adsorption rate was 30%.

The results obtained in Examples and Comparative Examples are shown in Tables 1 and 2 below and the amount of heparin immobilized (μg / cm ² ) in FIG.
From this result, it is clear that the laminated polymer substrate of the present invention has a good HCV adsorption ability. Further, it was revealed that the number of stacking times was 10 and the amount of heparin immobilized was larger than 5 times.

The polymer substrate of the present invention can be used as a virus removal instrument.

1 Outlet 2 Inlet 3 Lactose 4 Hollow fiber 5 Container 6 Partition

Claims (15)

Polymer support (A), layer (B) obtained by treating surface of polymer support (A) by graft polymerization reaction with polymerizable compound, and laminated structure capable of bonding to layer (B) A laminated polymer base material comprising (C),
A laminated polymer base material characterized in that the laminated structure (C) is laminated n times with the following first layer and second layer as a set.
First layer: a layer obtained by binding the compound (D) having an ionic or ionizable functional group capable of binding to the layer (B) to the layer (B) Second layer: binding to the first layer A layer obtained by combining a sulfated polysaccharide (E) capable of binding with the first layer (where n represents an integer of 1 to 10). The laminated polymer substrate (C) according to claim 1, wherein the polymer support (A) is a polymer support using a polyolefin as a substrate. The laminated polymer substrate (C) according to claim 2, wherein the polymer support having a polyolefin as a substrate is a hollow fiber membrane. The laminated polymer substrate (C) according to claim 3, wherein the polyolefin is poly-4-methylpentene. The laminated polymer substrate (C) according to any one of claims 1 to 4, wherein the polymerizable compound is (meth) acrylic acid and the compound (D) is a polymer amine compound. The laminated polymer substrate (C) according to claim 5, wherein the polymer amine compound is polyallylamine. The laminated polymer substrate (C) according to any one of claims 1 to 4, wherein the polymerizable compound is glycidyl (meth) acrylate, and the compound (D) is ammonia or a polyvalent amine compound. The laminated polymer substrate (C) according to claim 7, wherein the polyvalent amine compound is 1,2-bis (2-aminoethoxy) ethane or polyallylamine. The laminated polymer substrate (C) according to any one of claims 1 to 4, wherein the polymerizable compound is (meth) acryloyloxyalkyl isocyanate and the compound (D) is a polyvalent amine compound. The laminated polymer substrate (C) according to claim 9, wherein the polyvalent amine compound is 1,2-bis (2-aminoethoxy) ethane or polyallylamine. The laminated polymer substrate (C) according to any one of claims 1 to 10, wherein the sulfated polysaccharide (E) is heparin. The lamination method according to any one of claims 1 to 11, wherein the binding method of the sulfated polysaccharide and the first layer is based on a reaction between the activated carbodiimide obtained by reacting the sulfated polysaccharide with carbodiimide and the compound (D). Polymer substrate (C). The virus removal instrument provided with the polymer base material in any one of Claims 1-12. The virus removing instrument according to claim 13, wherein the virus is a hepatitis B or C virus. The virus removal method using the virus removal instrument of Claim 13 or 14.

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