Polymers, water-insoluble resin particles, medical materials, materials for biochemical experiments, and immobilized biologically active substances

Abstract

To provide a new polymer that can be used as a biomaterial such as materials for medical use and materials for biochemical experiments.SOLUTION: The polymer includes: a structure (A) that is derived from glycerin carbonate (meth)acrylate; and a structure (B) that is derived from a chemical compound which has at least one functional group (B-1) selected from the group consisting of a sulfobetaine group, a carbobetaine group and a phosphobetaine group; and a (meth)acryloyl group and / or an N-(meth)acryloyl amide group (B-2), as essential constitutional units.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a polymer, a water-insoluble resin particle, a medical material, a material for biochemical experiments, and an immobilized physiologically active substance. [Background technology]

[0002] Biomaterials is a general term for materials generally used in the fields of medicine and biotechnology, and a wide variety of biomaterials are known and used for various purposes, including materials that are used in contact with biological substances, cells, tissues, etc., which are components of living organisms, for diagnosis and treatment, or to assist or replace damaged parts of living organisms, carriers for fixing biological substances such as enzymes so that they can be easily used in tests, and fluorescent substances for giving fluorescence to biological substances. Furthermore, these substances are sometimes used in research and development in the field of biochemistry.

[0003] There are many materials obtained by organic synthesis for such applications. Unlike ordinary resins, these biomaterials are required to have not only physical and chemical properties, but also biological properties such as interaction with living organisms and biocompatibility. Therefore, molecular design different from that in the general resin field is required.

[0004] For example, it is desirable that artificial organs and medical devices are made of materials that are compatible with the substances that originally constitute the living body and are not easily stained. When a living body comes into contact with an external object such as an artificial material, the biological system recognizes the artificial material as a foreign body, and various foreign body reactions such as thrombus formation, immune reaction, and inflammatory reaction are induced over time. Therefore, when using medical devices such as artificial organs, drugs such as anticoagulants such as heparin and immunosuppressants must be used in combination. However, when using the above-mentioned anticoagulants, there is a risk of various side reactions such as liver damage and allergic reactions.

[0005] Compounds having functional groups that bind to proteins, peptides, amino acids, etc. are often used in materials for testing. Biomaterials can be used for various purposes such as testing and research by binding to these bioactive compounds. Specifically, compounds having epoxy groups are often used as functional groups that cause such binding, but immobilization of enzymes on polymers having carbonate groups has also been considered (Non-Patent Documents 1, 2).

[0006] On the other hand, glycerin carbonate (meth)acrylate has been developed and studied as a resin raw material (Patent Documents 1 and 2, etc.), but has not been studied as a medical material as described above. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2015-229770 A [Patent Document 2] JP 2017-044928 A [Patent Document 3] Patent No. 6150071 [Patent Document 4] Patent No. 6353923 [Patent Document 5] International Publication No. 2016 / 039293 [Non-patent literature]

[0008] [Non-Patent Document 1] Ecotoxlcology and Environmental Safety 170(2019) 453-460 [Non-Patent Document 2] Polym. Chem.,2019,10, 3571-3584 Summary of the Invention [Problem to be solved by the invention]

[0009] In view of the above, an object of the present invention is to obtain a novel polymer that can be used as a biomaterial such as a medical material or a material for biochemical experiments. [Means for solving the problem]

[0010] The present invention relates to Structure (A) derived from glycerin carbonate (meth)acrylate; and At least one functional group (B-1) selected from the group consisting of a sulfobetaine group, a carbobetaine group, and a phosphobetaine group, and a (meth)acryloyl group and / or an N-(meth)acryloylamide group (B-2). Structure (B) derived from a compound having the formula The polymer is characterized in that it contains the following as an essential structural unit.

[0011] The structure (B) is preferably a structure derived from sulfopropyl betaine (meth)acrylate. The polymer is preferably in the form of water-insoluble resin particles. The present invention also relates to water-insoluble resin particles comprising a base particle made of a water-insoluble resin particle and a coating layer containing the above-mentioned polymer formed on the base particle.

[0012] The present invention also relates to a medical material comprising the above polymer and / or the above water-insoluble resin particles. The present invention also relates to a material for biochemical experiments, which comprises the above polymer and / or the above water-insoluble resin particles. The present invention also relates to an immobilized physiologically active substance characterized in that the immobilized physiologically active substance is obtained by immobilizing the above-mentioned polymer and / or the above-mentioned water-insoluble resin particles. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The polymer of the present invention comprises a structure (A) derived from glycerin carbonate (meth)acrylate, and a structure (B) derived from a compound (b) having at least one functional group (B-1) selected from the group consisting of a sulfobetaine group, a carbobetaine group, and a phosphobetaine group, and a (meth)acryloyl group and / or an N-(meth)acryloylamide group (B-2). is an essential constituent unit.

[0014] Glycerol carbonate (meth)acrylate has been studied for applications such as paints and pattern-forming compositions, but has not been studied as a biomaterial such as a medical material or a biochemical experimental material. On the other hand, as disclosed in Non-Patent Documents 1 and 2, cyclic carbonate groups react with amino groups in proteins, peptides, and amino acids. The reactivity of the amino groups of enzymes and antibodies with carbonate groups is higher than that of epoxy groups, and a high immobilization amount can be obtained. Furthermore, when a physiologically active substance is immobilized, the activity of the immobilized physiologically active substance can be maintained at a high level. In this respect, it has excellent performance as a biomaterial.

[0015] Furthermore, when using a polymer having a cyclic carbonate group, it is desirable to use a material having biocompatibility. For this reason, the present inventors have also made the structural unit derived from the compound (b) having at least one functional group selected from the group consisting of a sulfobetaine group, a carbobetaine group, and a phosphobetaine group and a (meth)acryloyl group, which is a component capable of improving biocompatibility, an essential unit. This makes it possible to obtain medical materials and materials for biochemical experiments that can be used in a wide range of applications.

[0016] The glycerin carbonate (meth)acrylate has a chemical structure represented by the following general formula (1).

[0017] [ka] (In the formula, R 1represents a hydrogen atom or a methyl group.

[0018] This compound has previously been considered for use in fields such as coating compositions and modification of resins used in pattern formation, but its use in the medical field or biochemical experiments has not been considered. The present inventors have found that polymers partially having a structure derived from such compounds can be suitably used in the medical field, and have thus completed the present invention.

[0019] The compound represented by the above general formula (1) can easily undergo polymerization reaction with other monomers, and therefore, by adjusting the composition, it is easy to adjust the resin properties according to the purpose of use. Therefore, it can be suitably used as a building block of the above-mentioned medical materials and biochemical experimental materials.

[0020] Glycerin carbonate (meth)acrylate is a known compound and can be produced according to a known production method. Alternatively, commercially available products can be used.

[0021] The polymer used in the present invention preferably contains a structure derived from glycerin carbonate (meth)acrylate in a proportion of 1 to 65% by weight based on the total weight of the polymer, which is particularly preferred in that the above-mentioned object can be suitably achieved by containing the structure in the above proportion. The lower limit is more preferably 10% by weight, and further preferably 20% by weight.

[0022] The polymer used in the present invention further has a structure (B) derived from a compound (b) having at least one functional group (B-1) selected from the group consisting of a sulfobetaine group, a carbobetaine group, and a phosphobetaine group, and a (meth)acryloyl group and / or an N-(meth)acryloylamide group (B-2). Such a structure is preferable in that physical adsorption between the resin and the protein can be suppressed.

[0023] Such a structure can be represented by the following general formula.

Chemical formula

[0024] The above sulfobetaine group, carbobetaine group, and phosphobetaine group are each a functional group having both a sulfonic acid group, a carboxylic acid group, and a phosphate group and a quaternary amino group. Among these, the sulfobetaine group is most preferred. Further, as the sulfobetaine group, sulfopropylbetaine (meth)acrylate is preferred.

[0025] Sulfopropylbetaine (meth)acrylate has a sulfopropylbetaine structure represented by the following general formula (2)

[0026]

Chemical formula

[0027] and is a compound having both structures derived from (meth)acrylic acid or (meth)acrylamide in the molecule or an inner salt thereof. Specific examples of such a compound include compounds represented by the following general formula (3) or (4).

[0028]

Chemical formula

[0029] [ka] (In the formula, R a represents an alkyl group having 2 to 5 carbon atoms. R b represents an alkylene group having 2 to 9 carbon atoms. R c represents hydrogen or a methyl group R d each represents an alkyl group having 1 to 2 carbon atoms, which may be the same or different.) R e represents hydrogen or an alkyl group having 1 to 9 carbon atoms.

[0030] More preferred examples include compounds represented by the following general formula (5).

[0031] [ka] (In the formula, R c represents hydrogen or a methyl group)

[0032] The content of the above structure (B) is preferably 1 to 25% by weight based on the total amount of the polymer. By blending within this range, good biocompatibility can be obtained. The above lower limit is more preferably 5% by weight, and further preferably 10% by weight.

[0033] The polymer of the present invention may further contain a structural unit based on another monomer as a copolymerization component. Such another monomer may have a functionality preferred for the application of the present invention, or may not have any particular functionality.

[0034] The copolymerization component may, for example, be tetraphenylethylene methacrylate. The tetraphenylethylene-based methacrylate is a compound having a tetraphenylethylene skeleton and an acrylic group. The tetraphenylethylene group is a skeleton having aggregation-induced fluorescence (AIE). In the fields of medicine and biochemistry, fluorescent substances are widely used in tests and analyses. Therefore, in such medical materials, a polymer having a tetraphenylethylene group is used to impart fluorescence.

[0035] Such monomers are not particularly limited, and the aggregated luminescent materials constituting the fluorescent particles for diagnostic agents are not particularly limited, but examples thereof include ketoimine boron complex derivatives, diimine boron complex derivatives, tetraphenylethylene derivatives, aminomaleimide derivatives, aminobenzopyroxanthene derivatives, triphenylamine derivatives, hexaphenylbenzene derivatives, hexaphenylsilole derivatives, and the like.

[0036] The polymer of the present invention may further contain other monomers having an unsaturated group as copolymerization components. Such other monomers are not particularly limited, and examples thereof include the following. (Other examples of monomers) Polymerizable unsaturated aromatics such as styrene, chlorostyrene, α-methylstyrene, divinylbenzene, vinyltoluene, vinylnaphthalene, divinylnaphthalene, α-naphthyl (meth)acrylate, β-naphthyl (meth)acrylate, etc.; polymerizable unsaturated carboxylic acids such as (meth)acrylic acid, itaconic acid, maleic acid, fumaric acid, etc.; polymerizable unsaturated sulfonic acids or their salts such as sodium styrenesulfonate, etc.; methyl (meth)acrylate, ethyl (meth)acrylate, n-butyl (meth)acrylate, 2-hydroxyethyl (meth)acrylate, (meth) Polymerizable carboxylic acid esters such as glycidyl acrylate, ethylene glycol-di-(meth)acrylic acid ester, and tribromophenyl (meth)acrylate; unsaturated carboxylic acid amides such as (meth)acrylonitrile, (meth)acrolein, (meth)acrylamide, N-methylol (meth)acrylamide, methylene bis (meth)acrylamide, butadiene, isoprene, vinyl acetate, vinylpyridine, N-vinylpyrrolidone, vinyl chloride, vinylidene chloride, and vinyl bromide, polymerizable unsaturated nitriles, vinyl halides, and conjugated dienes.

[0037] Also, various polyfunctional acrylate compounds may be used as copolymerization components. As described in detail below, the polymer of the present invention can also be made into water-insoluble resin particles. Such resin particles are preferably polymers having a crosslinked structure. From this viewpoint, various known polyfunctional acrylate compounds can be used.

[0038] Examples of (meth)acrylates having a functionality of 2 include 1,4-butanediol di(meth)acrylate, 1,3-butanediol di(meth)acrylate, 1,6-hexanediol di(meth)acrylate, ethylene glycol di(meth)acrylate, diethylene glycol di(meth)acrylate, triethylene glycol di(meth)acrylate, polyethylene glycol di(meth)acrylate, dipropylene glycol di(meth)acrylate, tripropylene glycol di(meth)acrylate, polypropylene glycol di(meth)acrylate, neopentyl glycol di(meth)acrylate, Acrylate, hydroxypivalic acid neopentyl glycol di(meth)acrylate, 1,6-hexanediol di(meth)acrylate, 1,9-nonanediol di(meth)acrylate, 1,10-decanediol di(meth)acrylate, glycerin di(meth)acrylate, dimethylol tricyclodecane di(meth)acrylate (DCP-A), EO adduct diacrylate of bisphenol A (Kyoeisha Chemical Co., Ltd.; Light Acrylate BP-4EA, BP-10EA), PO adduct diacrylate of bisphenol A (Kyoeisha Chemical Co., Ltd.; BP-4PA, BP-10PA, etc.). Among them, PO adduct diacrylate of bisphenol A (Kyoeisha Chemical Co., Ltd.; BP-4PA), dimethylol tricyclodecane di(meth)acrylate (DCP-A), etc. can be preferably used.

[0039] Examples of the (meth)acrylate having three functional groups include trimethylolmethane tri(meth)acrylate, trimethylolpropane tri(meth)acrylate, trimethylolpropane ethylene oxide modified tri(meth)acrylate, trimethylolpropane propylene oxide modified tri(meth)acrylate, pentaerythritol tri(meth)acrylate, glycerin propoxy tri(meth)acrylate, tris(2-(meth)acryloyloxyethyl)isocyanurate, etc. Among them, trimethylolpropane trimethacrylate, pentaerythritol trimethacrylate, etc. can be preferably used.

[0040] Examples of the (meth)acrylate having 4 functional groups include dipentaerythritol tetra(meth)acrylate, pentaerythritol tetra(meth)acrylate, pentaerythritol ethylene oxide modified tetra(meth)acrylate, pentaerythritol propylene oxide modified tetra(meth)acrylate, ditrimethylolpropane tetra(meth)acrylate, etc. Among them, ditrimethylolpropane tetra(meth)acrylate, pentaerythritol tetra(meth)acrylate, etc. can be preferably used.

[0041] Examples of (meth)acrylates having 4 or more functional groups include polyfunctional (meth)acrylates such as pentaerythritol tetra(meth)acrylate, pentaerythritol ethylene oxide-modified tetra(meth)acrylate, ditrimethylolpropane tetra(meth)acrylate, dipentaerythritol tetra(meth)acrylate, dipentaerythritol penta(meth)acrylate, ditrimethylolpropane penta(meth)acrylate, propionic acid-modified dipentaerythritol penta(meth)acrylate, dipentaerythritol hexa(meth)acrylate, ditrimethylolpropane hexa(meth)acrylate, and caprolactone-modified dipentaerythritol hexa(meth)acrylate.

[0042] The polymer of the present invention may be a solvent-soluble polymer or a water-insoluble particle.The insoluble particle may be obtained by copolymerization of glycerin carbonate (meth)acrylate and sulfopropyl betaine (meth)acrylate, or may be obtained by a method of preparing a composition containing insoluble particle, glycerin carbonate (meth)acrylate and sulfopropyl betaine (meth)acrylate, and polymerizing this composition to produce a resin particle having a cyclic carbonate group and a sulfopropyl betaine group on the particle surface.

[0043] The method for producing the polymer of the present invention is not particularly limited, and can be carried out by a known general method such as emulsion polymerization, solution polymerization, suspension polymerization, bulk polymerization, etc. The polymerization initiator, emulsifier, etc. that can be used in the polymerization are also not particularly limited, and any known ones can be used.

[0044] The present invention also relates to a medical material or a biochemical experimental material comprising the above-mentioned polymer. That is, the above-mentioned polymer of the present invention can be used mainly in the medical field or biochemical experimental field.

[0045] The specific method of using the polymer of the present invention is not particularly limited, and it is expected to be used as a medical material used in various tests, and further as artificial organs, artificial bones, artificial joints, etc. It can also be used as a base material for immobilizing an immobilized physiologically active substance. An immobilized physiologically active substance obtained by immobilizing a physiologically active substance on the polymer of the present invention is also one aspect of the present invention. Furthermore, these materials may be used for research and development.

[0046] Examples of the immobilized physiologically active substance include immobilized enzymes, immobilized antigens, immobilized antibodies, etc. By immobilizing various physiologically active substances to the polymer of the present invention in this way, enzyme reactions, antigen-antibody reactions, etc. can be carried out on the stationary phase, and mixtures after various reactions or biochemical reactions can be separated, etc. Examples of the compound to be immobilized include proteins, peptides, amino acids, etc.

[0047] Furthermore, the polymer can be used as a carrier particle for separating an antibody from a culture solution containing the antibody during the production of an antibody drug. An antibody is immobilized on the polymer of the present invention and packed in a column. Then, the reaction mixture obtained by the production of the antibody drug is passed through the column. Then, the antibody is captured on the carrier particle, and impurities pass through the column. This makes it possible to remove the impurities. Then, the antibody is dissociated from the carrier particle by passing it through an acidic solution, etc., and thus an antibody with high purity can be obtained. Furthermore, it can also be used as a material in immunoassay in immunochromatography. EXAMPLES

[0048] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0049] (Synthesis example) (Synthesis of glycerin carbonate methacrylate) A 500 mL four-neck flask equipped with a condenser, stirrer, thermometer, and dropping funnel was charged with 93 g of glycerol carbonate (Tokyo Chemical Industry Co., Ltd.), 65 g of toluene, and 11.1 g of triethylamine, and stirred at 30°C. Using the dropping funnel, 120 g of methacrylic anhydride was added dropwise over 1 hour, while taking care not to generate heat. After the addition was complete, the temperature was raised to 80°C and the reaction was continued for 6 hours to complete the synthesis. After the reaction was complete, the product was neutralized and washed with water, and the target compound, glycerol carbonate methacrylate, was obtained as a pale yellow transparent liquid. The yield was 112.5 g (78.2%). The pale yellow transparent liquid obtained was, 1 H-NMR (DMSO-d6, 400 MHz): δ (ppm) = 6.04 (s, 1 H); 5.73 (s,1 H); 5.09 (m, 1 H); 4.59 (t, 1 H), 4.38 (t, 1 H); 4.29-4.35 (m,2 H), 1.88 (s, 3 H). This supported the proposed structure.

[0050] (Synthesis of N,N-dimethyl-N-(2-methacrylooxyethyl)-N-(3-sulfopropyl)ammonium betaine (hereinafter referred to as sulfopropylbetaine (SPB)) The synthesis was carried out according to the following procedure. A 1L four-neck flask equipped with a condenser, stirrer, thermometer, and dropping funnel was charged with 120g of Light Ester DM (dimethylaminoethyl methacrylate) and 260g of acetone, and stirred at room temperature. Next, 132g of acetone was mixed with 93.2g of 1,3-propane sultone, and the mixture was added dropwise using the dropping funnel for 2 hours to react. After the addition was complete, the mixture was stirred at room temperature for an additional 3 hours to complete the synthesis. The reaction solution was cooled in ice water and allowed to stand, and the precipitate that had separated out from the cooled reaction solution was collected by suction filtration and further washed with a large amount of acetone to obtain a white solid. After drying, the weight was 190g (yield 89%). The obtained white solid was, 1 H-NMR (D2O, 400MHz) δ: 6.13 (s, 1H), 5.76 (s, 1H), 4.83-4.66 (t, 2H), 3.86-3.84 (t, 2H), 3.65-3.59 (m, 2H), 3.24 (s, 6H), 3.05-2.98 (t, 2H), 2.33-2.12 (m, 2H), 1.96 (2, 3H). This supported the structure of the compound.

[0051] (Synthesis of Crosslinked Particle Polymers) Comparative Example 1 The polymerization operation was carried out according to the following procedure. 31.75 ml of ion-exchanged water and 0.45 g of sodium lauryl sulfate (SDS) as an emulsifier were charged into a 100 ml four-neck flask equipped with a condenser, stirrer, and thermometer, and completely dissolved by stirring at room temperature. After confirming that it was completely dissolved, 14.7 g of Light Ester M (methyl methacrylate) and 0.30 g of diethylene glycol dimethacrylate (Light Ester 2EG) were charged and stirred at 70 ° C for 1 hour. Then, 0.30 g of ammonium persulfate as a polymerization initiator was dissolved in 5.0 g of ion-exchanged water and added to the reaction vessel. The reaction was carried out at 70 ° C for 5 hours to complete the polymerization. After the reaction, the solid content concentration in the system was dried at 105 ° C for 3 hours and then weighed, and it was 28.5 wt.% (theoretical solid content: 30 wt.% (conv. = 95.0%).

[0052] Comparative Example 2 In a 100 ml four-neck flask equipped with a condenser, stirrer, and thermometer, 31.75 ml of ion-exchanged water, 0.45 g of sodium lauryl sulfate (SDS) as an emulsifier, and 0.75 g of sulfopropyl betaine were charged and completely dissolved by stirring at room temperature. After confirming that the mixture was completely dissolved, 13.95 g of methyl methacrylate and 0.30 g of Light Ester 2EG were charged and stirred at 70 ° C for 1 hour. Then, 0.30 g of ammonium persulfate as a polymerization initiator was dissolved in 5.0 g of ion-exchanged water and added to the reaction vessel. The mixture was reacted at 70 ° C for 5 hours to complete the polymerization. After the reaction, the solid concentration in the system was dried at 105 ° C for 3 hours and then weighed, and it was 29.3 wt. % (theoretical solid content: 30 wt. % (conv. = 97.7%).

[0053] Comparative Example 3 Polymerization was carried out in the same manner as in Example 2, except that the amounts of sulfopropyl betaine and methyl methacrylate were changed to 1.5 g and 13.2 g, respectively. After the reaction was completed, the solid content in the system was dried at 105° C. for 3 hours and then weighed, and was found to be 29.8 wt.% (theoretical solid content: 30 wt.% (conv.=99.3%).

[0054] Example 1 In a 100 ml four-neck flask equipped with a condenser, stirrer, and thermometer, 36.25 ml of ion-exchanged water, 0.45 g of sodium lauryl sulfate (SDS) as an emulsifier, and 2.1 g of sulfopropyl betaine were charged and completely dissolved by stirring at room temperature. After confirming that the mixture was completely dissolved, 10.5 g of methyl methacrylate, 2.1 g of GC-MA, and 0.3 g of Light Ester 2EG were charged and stirred at 70 ° C for 1 hour. Then, 0.30 g of ammonium persulfate was dissolved in 5.0 g of ion-exchanged water as a polymerization initiator and added to the reaction vessel. The reaction was carried out at 70 ° C for 5 hours to complete the polymerization. After the reaction, the solid content concentration in the system was weighed after drying at 105 ° C for 3 hours and was found to be 28.5 wt. % (theoretical solid content: 26.5 wt. % (conv. = 95.9%).

[0055] Comparative Example 4 In a 100 ml four-neck flask equipped with a condenser, stirrer, and thermometer, 31.75 ml of ion-exchanged water and 0.45 g of sodium lauryl sulfate (SDS) as an emulsifier were charged and completely dissolved by stirring at room temperature. After confirming that it was completely dissolved, 14.7 g of styrene and 0.30 g of Light Ester 2EG were charged and stirred at 70°C for 1 hour. Then, 0.30 g of ammonium persulfate as a polymerization initiator was dissolved in 5.0 g of ion-exchanged water and added to the reaction vessel. The reaction was carried out at 70°C for 5 hours to complete the polymerization. After the reaction was completed, the solid concentration in the system was dried at 105°C for 3 hours and then weighed, which was 28.5 wt.% (theoretical solid content: 30 wt.% (conv. = 95.0%).

[0056] Comparative Example 5 In a 100 ml four-neck flask equipped with a condenser, stirrer, and thermometer, 31.75 ml of ion-exchanged water, 0.45 g of sodium lauryl sulfate (SDS) as an emulsifier, and 0.75 g of sulfopropyl betaine were charged and completely dissolved by stirring at room temperature. After confirming that the mixture was completely dissolved, 13.95 g of styrene and 0.30 g of Light Ester 2EG were charged and stirred at 70°C for 1 hour. Then, 0.30 g of ammonium persulfate as a polymerization initiator was dissolved in 5.0 g of ion-exchanged water and added to the reaction vessel. The mixture was reacted at 70°C for 5 hours to complete the polymerization. After the reaction, the solid content concentration in the system was dried at 105°C for 3 hours and then weighed, and was found to be 29.0 wt.% (theoretical solid content: 30 wt.% (conv. = 96.7%).

[0057] Example 2 In a 100 ml four-neck flask equipped with a condenser, stirrer, and thermometer, 28.0 ml of ion-exchanged water, 0.36 g of sodium lauryl sulfate (SDS) as an emulsifier, and 3.6 g of sulfopropyl betaine were charged and completely dissolved by stirring at room temperature. After confirming that the mixture was completely dissolved, 4.74 g of styrene, 3.6 g of GC-MA, and 0.06 g of Light Ester 2EG were charged and stirred at 70°C for 1 hour. Then, 0.24 g of ammonium persulfate as a polymerization initiator was dissolved in 5.0 g of ion-exchanged water and added to the reaction vessel. The mixture was reacted at 70°C for 5 hours to complete the polymerization. After the reaction, the solid concentration in the system was dried at 105°C for 3 hours and then weighed, and was found to be 28.5 wt.% (theoretical solid content: 30 wt.% (conv. = 95.0%).

[0058] Comparative Example 6 In a 100 ml four-neck flask equipped with a condenser, stirrer, and thermometer, 28.0 ml of ion-exchanged water and 0.36 g of sodium lauryl sulfate (SDS) as an emulsifier were charged and completely dissolved by stirring at room temperature. After confirming that the mixture was completely dissolved, 8.82 g of styrene, 3.78 g of GC-MA, and 0.06 g of Light Ester 2EG were charged and stirred at 70°C for 1 hour. Then, 0.24 g of ammonium persulfate as a polymerization initiator was dissolved in 5.0 g of ion-exchanged water and added to the reaction vessel. The mixture was reacted at 70°C for 5 hours to complete the polymerization. After the reaction, the solid concentration in the system was dried at 105°C for 3 hours and then weighed, and was found to be 28.0 wt.% (theoretical solid content: 30 wt.% (conv. = 93.3%).

[0059] (Recovery of crosslinked polymer microparticles) Particle recovery The emulsions of Examples 1 to 8 synthesized above were dropped into a large amount of acetone or methanol to cause emulsion destruction and precipitate crosslinked polymer microparticles. The precipitated particles were collected by filtration, and the emulsifier on the particle surface was washed with a large amount of ion-exchanged water, and then the particles were dried and collected. The particles collected by this procedure were used for the following evaluations.

[0060] (Fluorescent protein adsorption experiment) Fluorescently labeled bovine serum albumin (FITC-BSA) was prepared in 10 mM phosphate buffer (pH 7.2) to a concentration of 50 μg / ml. 0.05 g of the crosslinked particles synthesized in the Comparative Examples and Examples, 0.5 to 1.0 ml of the prepared FITC-BSA solution, and 10 mM phosphate buffer (pH 7.2) were added to make a total of 1.4 ml, and incubated at 37°C for 30 minutes. The particles were then precipitated using a tabletop centrifuge, the supernatant was collected, and 0.8 ml of 10 mM phosphate buffer (pH 7.2) was added to the precipitated particles for washing. The collected particles were visually confirmed. The measurement results are shown in Table 2.

[0061] [Table 1] MMA: Methyl methacrylate Styrene: 2EG: Diethylene glycol dimethacrylate SPB: Sulfopropyl betaine methacrylate GC-MA: glycerin carbonate methacrylate

[0062] [Table 2]

[0063] As a result of the fluorescent protein adsorption test, the particles of Comparative Examples 1 and 2 showed nonspecific adsorption, which is thought to be due to hydrophobic interactions between the fluorescent protein and the particles. In contrast, it was found that the particles in which sulfopropyl betaine (SPB) was blended, as in Comparative Examples 2 and 3 and Examples 1 and 2, suppressed protein adsorption to the particle surface due to hydrophobic interactions. Furthermore, even when sulfopropyl betaine (SPB) was blended in the particles, the adsorption of fluorescent protein to the particles was observed by blending GC-MA. This suggests that the protein is covalently adsorbed (bound) via the carbonate moiety, which is the skeleton of GC-MA, and the amino group in the fluorescent protein.

[0064] In order to directly evaluate the protein adsorbed on the particle surface, evaluation was performed using a fluorescence spectrophotometer. New particles of Example 3 and Comparative Example 7 were prepared according to the particle synthesis methods in the Comparative Examples and Examples listed above. The compositions are shown in Table 3.

[0065] (Fluorescent protein adsorption experiment 2) Fluorescently labeled bovine serum albumin (FITC-BSA) was prepared in 10 mM phosphate buffer (pH 7.2) to a concentration of 50 μg / ml. 0.05 g of crosslinked particles synthesized in the comparative examples and examples, 0.5 to 1.0 ml of the prepared FITC-BSA solution, and 10 mM phosphate buffer (pH 7.2) were added to make a total of 1.4 ml, and the mixture was incubated at 37°C for 30 minutes (A) and 37°C for 18 hours (B). After that, the particles were precipitated using a tabletop centrifuge, the supernatant was collected, and 0.8 ml of 10 mM phosphate buffer (pH 7.2) was added to the precipitated particles to wash them. This operation was repeated three times. Fluorescence intensity was measured using the washed particles (JASCO: model name FP-6200). Excitation was performed at 490 nm, and fluorescence intensity (scattered light) was measured at a wavelength of 520 nm. For the blanks, particles without FITC-BSA were used under the above experimental conditions. The measurement results are shown in Table 3.

[0066] [Table 3]

[0067] As a result, in the case of short incubation times, no adsorption of fluorescent proteins to particles was observed in either Comparative Example 2 or Example 3. Furthermore, in the case of longer incubation times to promote specific binding, it was suggested that while nonspecific adsorption was suppressed to some extent, specific binding was not hindered due to the function of GC-MA, as shown in Example 3. This result is extremely effective when carrying specific proteins, antigens, etc., and this function is thought to contribute to improved sensitivity and stability.

[0068] (Enzyme loading experiment) To confirm the loading of a physiologically active substance onto the particles, enzyme loading (immobilized enzyme) was performed using invertase, a sucrose decomposition enzyme. Enzyme activity was measured using Glucose CII-Test Wako (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

[0069] 0.10 g of the particles having the composition of Example 3 was weighed out, 5.0 ml of 10 mM phosphate buffer (pH 7.2) and 1.0 ml of invertase solution (derived from yeast) were added thereto, and the particles were dispersed using an ultrasonic cleaner. The mixture was further incubated at 37°C for 18 hours. After incubation, the particles were collected using a centrifuge, washed three times with 10 mM phosphate buffer (pH 7.2), and then collected by centrifugation to obtain enzyme-carrying particles.

[0070] 5.0 ml of 1 wt.% sucrose solution was added to the enzyme-carrying particles collected by the above method, the particles were dispersed in an ultrasonic cleaner, and the reaction was carried out at 37°C for 3 hours. After the reaction, the particles were precipitated by centrifugation, and 0.05 ml of the supernatant was collected. 1.0 ml of a color-developing reagent was mixed therein and the reaction was carried out at room temperature for 5 minutes. After washing the particles collected by centrifugation, 5.0 ml of 1 wt.% sucrose solution was added again, the particles were dispersed in an ultrasonic cleaner, and the reaction was carried out at 37°C for 3 hours. Similarly, after the reaction, the particles were precipitated by centrifugation, and 0.05 ml of the supernatant was collected. 1.0 ml of a color-developing reagent was mixed therein and the reaction was carried out at room temperature for 5 minutes. This operation was repeated to confirm whether the enzyme-carrying particles could be used repeatedly.

[0071] The results are shown in the table below. For those particles with enzymes supported on them, the reaction proceeded through an enzymatic reaction, and glucose was observed to be produced, turning red in color. On the other hand, for particles that did not support enzymes, no coloring of the color reagent occurred. From this, it was possible to create immobilized enzymes that exhibit the above reaction by supporting the enzyme on the particles via GC-MA. Furthermore, it was confirmed that the enzymatic reaction proceeded without any problems even when used repeatedly.

[0072] [Table 4] [Industrial Applicability]

[0073] The polymer of the present invention can be used as a biomaterial such as various materials used in medical examinations and experiments in biochemical research.

Claims

1. Structure (A) derived from glycerin carbonate (meth)acrylate; and At least one functional group (B-1) selected from the group consisting of a sulfobetaine group, a carbobetaine group, and a phosphobetaine group, and a (meth)acryloyl group and / or an N-(meth)acryloylamide group (B-2). Structure (B) derived from a compound having the formula is the essential building block, A polymer, characterized in that the content of the structure (A) is 10 to 30% by weight based on the entire polymer, and the content of the structure (B) is 10 to 30% by weight based on the total amount of the polymer.

2. 2. The polymer according to claim 1, wherein the structure (B) is a structure derived from sulfopropyl betaine (meth)acrylate.

3. 3. The polymer according to claim 1, which is a water-insoluble resin particle.

4. 3. A water-insoluble resin particle comprising a base particle made of a water-insoluble resin particle and a coating layer containing the polymer of claim 1 or 2 formed on said base particle.

5. 5. A medical material comprising the polymer according to claim 1 and / or the water-insoluble resin particles according to claim 4.

6. A material for biochemical experiments, comprising the polymer according to any one of claims 1 to 3 and / or the water-insoluble resin particles according to claim 4.

7. 5. An immobilized physiologically active substance, characterized in that the immobilized physiologically active substance is obtained by immobilizing the polymer according to any one of claims 1 to 3 and / or the water-insoluble resin particle according to claim 4.

Images