Hydrogel and sterilized dry hydrogel-forming article

Abstract

This sterilized dry hydrogel-forming article contains a cross-linked body of a vinyl alcohol-based polymer in which a bioactive substance is composited. This hydrogel contains a bioactive substance having an amino group and a cross-linked body of a vinyl alcohol-based polymer having an ethylenically unsaturated group and a carboxy group. The carboxy group of the cross-linked body and the amino group of the bioactive substance are covalently bound through an amide bond.

Description

Hydrogels and sterilized, dry hydrogel-forming articles

[0001] [Cross-reference to related applications] This application claims priority based on Japanese Patent Application Nos. 2021-133378 and 2021-133384, filed on August 18, 2021, the entire disclosures of which are incorporated herein by reference. The present invention relates to a hydrogel and a sterilized, dried hydrogel-forming article.

[0002] Polyvinyl alcohol (hereinafter sometimes abbreviated as "PVA") is a water-soluble synthetic polymer with excellent properties such as hydrophilicity, reactivity, biodegradability, biocompatibility, and low toxicity. It forms highly flexible and strong hydrogels through physical or chemical crosslinking. Furthermore, hydrogels complexed with physiologically active substances (hereinafter sometimes abbreviated as "composite hydrogels") exhibit specific functions, and applications such as enzyme immobilization carriers (e.g., Non-Patent Document 1), affinity carriers (e.g., Non-Patent Document 2), cell culture substrates (e.g., Non-Patent Document 3), particles for vascular embolization (e.g., Patent Document 1), and cell culture carriers (e.g., Patent Document 2) have been proposed. Among these applications, the composite hydrogels must be used in a sterilized state when used in components or manufacturing of pharmaceuticals and medical devices for human use. If unsterilized composite hydrogels are used for such applications, residual microorganisms may cause infection and fatal damage to the living body or cells. Therefore, for such applications, the sterility assurance level (hereinafter sometimes abbreviated as "SAL") is set to SAL = 10. -6 It is desirable to achieve the following:

[0003] It is known that physiologically active substances, including enzymes, generally lose their original activity under harsh sterilization or drying conditions, making sterilization or drying of the composite hydrogel difficult. For example, it has been known that hydrogels in which cell adhesive proteins are composited as physiologically active substances can be suitably used as cell culture substrates. In these prior art techniques, the prepared composite hydrogels are irradiated with UV light for several hours or immersed in a 70-75% ethanol aqueous solution for several hours before being used for cell culture (e.g., Non-Patent Documents 3-5). UV light irradiation or immersion in an ethanol aqueous solution is unlikely to cause damage to the physiologically active substance to the extent that it loses its activity, but the SAL=10 -6 Sterilization cannot be performed to achieve the following:

[0004] Attempts have also been made to produce sterilized composite hydrogels (e.g., Non-Patent Document 6). This study uses a sol-state composite PVA in which acrylamide groups are introduced as crosslinkable sites and RGDS peptide is introduced as a cell adhesive protein into low-molecular-weight PVA. The study discloses that a sterilized composite hydrogel can be produced by filtering and sterilizing this aqueous solution and then curing it aseptically.

[0005] Furthermore, a particularly commonly used method for chemically crosslinking hydrogels is to use glutaraldehyde as a crosslinking agent. Methods have already been disclosed, such as a method of covalently bonding an enzyme to a hydrogel chemically crosslinked with glutaraldehyde using carbonyldiimidazole (e.g., Non-Patent Document 1), and a method of covalently bonding a cell adhesive protein to PVA with carboxyl groups introduced therein (e.g., Non-Patent Documents 3 and 4). However, the crosslinking agent glutaraldehyde is highly toxic, and it cannot be denied that it elutes slightly from the chemically crosslinked hydrogel. Therefore, although glutaraldehyde can be used for research purposes, it has been difficult to put it to practical use in the above-mentioned applications.

[0006] To overcome the problems associated with chemical crosslinking of PVA using glutaraldehyde, attempts have been made to produce hydrogels in which a cell-adhesive peptides are covalently bonded by introducing acrylamide groups into PVA and inducing radical polymerization to crosslink the PVA (see, for example, Non-Patent Documents 6 and 7). Similarly, a hydrogel has been disclosed in which cell-adhesive gelatin is covalently bonded to PVA particles obtained by radical polymerization crosslinking PVA having (meth)acryloyl groups (see, for example, Patent Document 2).

[0007] Special table 2002-527206 publication WO2020 / 105708 publication

[0008] Food Chemistry, 2001, Vol. 74, pp. 281-288 Biotechnology Techniques, 1997, Vol. 11, pp. 67-70 Journal of Biomedical Materials Research, 2001, Vol. 57, pp. 217-223 Journal of Polymer Engineering, 2017, Vol. 37, p. 647-660 Journal of Applied Biomaterials, 1991, Vol. 2, pp. 261-267 Biomaterials, 2002, Vol. 23, pp. 4325-4332 Biomaterials, 2012, Vol. 33, pp. 3880-3886

[0009] Although the composite hydrogels irradiated with UV light or immersed in ethanol as described in Non-Patent Documents 3 to 5 can be used for short-term cell culture tests, the sterilization is insufficient, and therefore there is a problem that the risk of microbial infection cannot be eliminated when applied to cell culture processes or the human body.

[0010] Furthermore, the filtration sterilization described in Non-Patent Document 6 requires a sterile environment for curing and packaging after sterilization, and producing a sterilized composite hydrogel in such a sterile environment is extremely costly. Also, in order to sterilize the composite hydrogel in a sol state by filtration, it is necessary to use a low-molecular-weight PVA (average molecular weight 13,000) (a molecular weight higher than this results in a large pressure loss and makes filtration sterilization impossible), but there is also the problem that the resulting composite hydrogel is fragile and has limited uses.

[0011] In one embodiment, the present invention has been made in consideration of the above-mentioned conventional problems, and an object of the present invention is to provide a sterilized composite hydrogel-forming article without using an expensive process such as a sterile environment.

[0012] Furthermore, the methods described in Non-Patent Documents 6 and 7 solve the problem of chemical crosslinking using glutaraldehyde, but have problems with the method of introducing a physiologically active substance or enzyme. Specifically, in the prior art, a carboxyl group derived from glutamic acid or aspartic acid residues of a physiologically active substance, including an enzyme, is introduced to an amino group introduced into PVA using a carbodiimide-based condensing agent. However, because physiologically active substances typically contain not only carboxyl groups but also amino groups derived from lysine residues, self-crosslinking and other reactions simultaneously occur, making it difficult to efficiently introduce the substance into the hydrogel. Furthermore, the method using carbonyldiimidazole described in Patent Document 2 has the problem of being unable to introduce a physiologically active substance or enzyme at a density high enough to be used for the above-mentioned applications.

[0013] In one embodiment, the present invention has been made in consideration of the above-mentioned conventional problems, and an object of the present invention is to provide a hydrogel in which a physiologically active substance or enzyme is efficiently introduced by a covalent bond while using a crosslinking method with low toxicity.

[0014] As a result of extensive research, the inventors have found that a composite hydrogel-forming article can be sterilized while maintaining the activity of a physiologically active substance if the article is dried after removal of water. Furthermore, as a result of extensive research, the inventors have found that by using a vinyl alcohol-based polymer having an ethylenically unsaturated group and a carboxyl group, a hydrogel can be produced using a crosslinking method with lower toxicity than glutaraldehyde, and that a physiologically active substance having an amino group can be introduced by covalent bonding more efficiently than in a hydrogel crosslinked with glutaraldehyde. The present invention is based on these novel findings. In this specification, the term "composite hydrogel" refers to a crosslinked product of a vinyl alcohol-based polymer to which a physiologically active substance has been complexed.

[0015] That is, the present invention relates to the following items [1] to

[11] . [1] A sterilized dry hydrogel-forming article comprising a crosslinked body of a vinyl alcohol-based polymer complexed with a physiologically active substance. [2] The dry hydrogel-forming article according to [1], in which the physiologically active substance is complexed to the crosslinked body of the vinyl alcohol-based polymer by a covalent bond. [3] The dry hydrogel-forming article according to [1] or [2], in which the water content of the dry hydrogel-forming article is 75% by mass or less. [4] The dry hydrogel-forming article according to any one of [1] to [3], in which the dry hydrogel-forming article is an irregular particle, a spherical particle, a micromolded body, an arbitrarily shaped article formed by a 3D printer, a film, a thread, a hollow fiber, a porous monolith, or a coated article. [5] The dry hydrogel-forming article according to any one of [1] to [3], in which the sterility assurance level (SAL) of the dry hydrogel-forming article is 10 -3The dry hydrogel-forming article according to any one of [1] to [4], which is the following: [6] A method for producing a dry hydrogel-forming article according to any one of [1] to [5], comprising sterilizing the hydrogel-forming article in a dried state. [7] A method for producing a dry hydrogel-forming article according to [6], wherein the sterilization method is radiation sterilization. [8] A method for producing a dry hydrogel-forming article according to [7], wherein the radiation exposure dose is 8.2 kGy or more. [9] A method for producing a dry hydrogel-forming article according to [7] or [8], wherein the dry hydrogel-forming article is placed in a container and then irradiated with radiation.

[10] The dry hydrogel-forming article according to any one of [1] to [5], which is placed in a container.

[11] A hydrogel comprising a crosslinked product of a vinyl alcohol-based polymer having an ethylenically unsaturated group and a carboxy group, and a physiologically active substance having an amino group, wherein the carboxy group of the crosslinked product and the amino group of the physiologically active substance are covalently bonded via an amide bond.

[12] The hydrogel according to

[11] , wherein the ethylenically unsaturated group is at least one selected from the group consisting of a vinyl group, a (meth)acryloyl group, a (meth)acryloylamino group, a vinylphenyl group, a norbornenyl group, and derivatives thereof.

[13] The hydrogel according to

[11] or

[12] , wherein the introduction rate of the ethylenically unsaturated group is 0.01 to 10 mol % of all structural units constituting the vinyl alcohol-based polymer.

[14] The hydrogel according to any one of

[11] to

[13] , wherein the introduction rate of the carboxy group is 0.1 to 50 mol % of all structural units constituting the vinyl alcohol-based polymer.

[15] The hydrogel according to any one of

[11] to

[14] , wherein the hydrogel is an amorphous particle, a spherical particle, a finely molded body, an arbitrarily shaped article formed by a 3D printer, a film, a thread, a hollow fiber, a porous monolith, or a coated article.

[0016] In one embodiment, the present invention can provide a sterilized composite hydrogel-forming article without using an expensive process such as a sterile environment, etc. Also, in one embodiment, the present invention can provide a hydrogel that uses a hydrogel containing a crosslinked product of a vinyl alcohol-based polymer having an ethylenically unsaturated group and a carboxy group, in which a physiologically active substance having an amino group is covalently bonded to the carboxy group via an amide bond, and that has low toxicity and into which a physiologically active substance or enzyme is efficiently introduced by a covalent bond.

[0017] 1. Dry Hydrogel-Forming Article In a first embodiment, the present invention provides a sterilized dry hydrogel-forming article comprising a crosslinked vinyl alcohol-based polymer complexed with a physiologically active substance. The hydrogel contained in the dry hydrogel-forming article of the present invention is preferably a hydrogel comprising a crosslinked vinyl alcohol-based polymer having an ethylenically unsaturated group and a carboxy group, and is preferably a hydrogel in which a physiologically active substance having an amino group is covalently bonded to a carboxy group of the vinyl alcohol-based polymer via an amide bond. <Dry Hydrogel-Forming Article> The dry hydrogel-forming article of the present invention refers to a molded article containing the complexed hydrogel, in a dried state, which swells and forms a hydrogel upon contact with an aqueous solution such as water, a buffer solution, a body fluid, or a culture medium. The molded article may take the form of general irregular particles, spherical particles, films, threads, hollow fibers, porous monoliths, etc.

[0018] Furthermore, the molded article may be in the form of a micromolded body, an arbitrarily shaped article molded using a 3D printer, or an article coated with a dried hydrogel-forming article (coated article). The micromolded body is a molded body having fine irregularities on the surface or inside, with the size of the microfabrication being 10 to 1000 μm. Furthermore, an arbitrarily shaped article molded using a 3D printer refers to any shape that can be molded using, for example, a stereolithography method, an inkjet method, or a nozzle extrusion type 3D printer. The coated article refers to a composite hydrogel coated on a substrate in the form of, for example, a film, a tray, a thread, a hollow fiber, a porous monolith, a micromolded body, or an arbitrarily shaped article molded using a 3D printer. The substrate material can be freely selected from materials such as glass, polyolefin, polymethyl methacrylate, polystyrene, polyester, polyolefin, polyethylene vinyl alcohol copolymer, polyamide, and polyimide.

[0019] <Vinyl Alcohol Polymer and Production Method Thereof> The vinyl alcohol polymer used in the present invention can be produced by saponifying a polyvinyl ester obtained by polymerizing a vinyl ester monomer and converting the ester groups in the polyvinyl ester to hydroxyl groups. Examples of the vinyl ester monomer include aliphatic vinyl esters such as vinyl formate, vinyl acetate, vinyl propionate, vinyl n-butyrate, vinyl isobutyrate, vinyl pivalate, vinyl versatate, vinyl caproate, vinyl caprylate, vinyl caprate, vinyl laurate, vinyl myristate, vinyl palmitate, vinyl stearate, and vinyl oleate; and aromatic vinyl esters such as vinyl benzoate. These may be used alone or in combination of two or more. Among the vinyl ester monomers, aliphatic vinyl esters are preferred, and vinyl acetate is more preferred from the viewpoint of production costs. That is, the polyvinyl ester is preferably polyvinyl acetate obtained by polymerizing vinyl acetate.

[0020] Furthermore, the polyvinyl ester may contain structural units derived from monomers other than the vinyl ester-based monomers, if necessary, within the range that does not impair the effects of the present invention.Examples of the other monomers include α-olefins such as ethylene, propylene, n-butene, and isobutylene; acrylic acid or a salt thereof; alkyl acrylates such as methyl acrylate, ethyl acrylate, n-propyl acrylate, i-propyl acrylate, n-butyl acrylate, i-butyl acrylate, t-butyl acrylate, 2-ethylhexyl acrylate, dodecyl acrylate, and octadecyl acrylate; methacrylic acid or a salt thereof; methyl methacrylate, ethyl methacrylate, n-propyl methacrylate, and methacrylic acid; methacrylic acid alkyl esters such as i-propyl methacrylate, n-butyl methacrylate, i-butyl methacrylate, t-butyl methacrylate, 2-ethylhexyl methacrylate, dodecyl methacrylate, and octadecyl methacrylate; acrylamide, N-methylacrylamide, N-ethylacrylamide, N,N-dimethylacrylamide, diacetone acrylamide, acrylamidopropanesulfonic acid or a salt thereof, acrylamidopropyldimethylamine or a salt or quaternary salt thereof, N-methylolacrylamide or a salt thereof methacrylamide derivatives such as methacrylamide, N-methylmethacrylamide, N-ethylmethacrylamide, methacrylamidepropanesulfonic acid or a salt thereof, methacrylamidepropyldimethylamine or a salt or quaternary salt thereof, N-methylolmethacrylamide or a derivative thereof; N-vinylamide derivatives such as N-vinylformamide and N-vinylacetamide; vinyl ethers such as methyl vinyl ether, ethyl vinyl ether, n-propyl vinyl ether, i-propyl vinyl ether, n-butyl vinyl ether, i-butyl vinyl ether, t-butyl vinyl ether, dodecyl vinyl ether, and stearyl vinyl ether; nitriles such as acrylonitrile and methacrylonitrile; vinyl halides such as vinyl chloride and vinyl fluoride; vinylidene halides such as vinylidene chloride and vinylidene fluoride; allyl compounds such as allyl acetate and allyl chloride; maleic acid or a salt, ester, or acid anhydride thereof; vinylsilyl compounds such as vinyltrimethoxysilane; and isopropenyl acetate. These may be used alone or in combination of two or more.

[0021] When the polyvinyl ester contains structural units derived from other monomers, the content of the structural units derived from other monomers is preferably 20 mol % or less, more preferably 10 mol % or less, and even more preferably 5 mol % or less, based on the total structural units constituting the polyvinyl ester.

[0022] The method for saponifying the polyvinyl ester is not particularly limited, and can be a conventional method. For example, an alcoholysis method using an alkali catalyst or an acid catalyst, a hydrolysis method, or the like can be applied. Among them, a saponification reaction using methanol as a solvent and caustic soda (NaOH) as a catalyst is simple and preferable.

[0023] The degree of polymerization of the vinyl alcohol polymer is preferably 300 or more, more preferably 350 or more, even more preferably 400 or more, and particularly preferably 450 or more, from the viewpoint of suppressing embrittlement of the crosslinked vinyl alcohol polymer during swelling in water. Furthermore, from the viewpoint of suppressing high viscosity of the aqueous solution during production of the crosslinked vinyl alcohol polymer and improving ease of processing, the degree of polymerization of the vinyl alcohol polymer is preferably 10,000 or less, more preferably 5,000 or less, and even more preferably 3,500 or less. Two or more vinyl alcohol polymers with different degrees of polymerization may be mixed and used. The degree of polymerization of the vinyl alcohol polymer in this specification refers to the degree of polymerization measured in accordance with JIS K 6726:1994. Specifically, it can be determined from the intrinsic viscosity measured in water at 30°C after saponifying and purifying the raw material PVA.

[0024] The degree of saponification of the vinyl alcohol polymer is preferably 50 mol% or more, more preferably 60 mol% or more, and even more preferably 65 mol% or more, from the viewpoint of improving the water solubility of the vinyl alcohol polymer. The upper limit of the degree of saponification is 100 mol% or 99 mol%. In this specification, the degree of saponification of the vinyl alcohol polymer means the ratio (mol%) of the number of moles of vinyl alcohol units to the total number of moles of structural units (e.g., vinyl acetate units) that can be converted to vinyl alcohol units by saponification in the raw material PVA and vinyl alcohol units, and can be measured in accordance with JIS K 6726:1994.

[0025] The 4% by mass viscosity of the vinyl alcohol polymer at 20°C is preferably 0.5 to 110 mPa·s, more preferably 1 to 80 mPa·s, and even more preferably 2 to 60 mPa·s. When the viscosity is within the above range, the ease of production of the hydrogel is improved and the strength of the composite hydrogel can be improved. Note that the viscosity in this specification refers to the viscosity measured at 20°C for a 4% by mass aqueous solution of the vinyl alcohol polymer using a B-type viscometer (rotation speed: 12 rpm) in accordance with the rotational viscometer method of JIS K 6726:1994.

[0026] <Crosslinked vinyl alcohol polymer> The crosslinked vinyl alcohol polymer may be one in which the vinyl alcohol polymer is physically crosslinked or may be crosslinked by a covalent bond. The total amount of vinyl alcohol-derived structural units and vinyl ester-derived structural units relative to all structural units constituting the vinyl alcohol polymer used in the present invention is preferably 80 mol% or more, more preferably 90 mol% or more, and even more preferably 95 mol% or more.

[0027] Methods for physically crosslinking a vinyl alcohol polymer include, for example, freezing and thawing an aqueous solution of the vinyl alcohol polymer, or dissolving the vinyl alcohol polymer in a mixed solvent of dimethyl sulfoxide and water and cooling the hot solution to room temperature. Methods for covalently crosslinking a vinyl alcohol polymer include using a multifunctional crosslinking agent that reacts with the side-chain hydroxyl groups of the vinyl alcohol polymer, or introducing functional groups into the vinyl alcohol polymer by copolymerization or post-modification, followed by reaction. However, covalent crosslinking is preferred because it increases the stability of the hydrogel when swollen in water.

[0028] Methods using a polyfunctional crosslinking agent that reacts with the side chain hydroxyl groups of a vinyl alcohol polymer include a method of reacting a polyfunctional aldehyde compound such as glyoxal, malondialdehyde, or glutaraldehyde under acidic conditions (polyacetal crosslinking), a method of reacting a polyfunctional epoxy compound such as epichlorohydrin or ethylene glycol diglycidyl ether under alkaline conditions (polyether crosslinking), and a method of reacting a polyfunctional carboxylic acid compound such as maleic acid or succinic acid (polyester crosslinking).

[0029] <Modified PVA> A method for introducing functional groups into a vinyl alcohol polymer by copolymerization and reacting them includes copolymerizing a vinyl ester monomer with a polymerizable monomer other than the vinyl ester monomer, which is a monomer having a reactive substituent other than a hydroxyl group, during the production process of the vinyl alcohol polymer, followed by saponification to obtain a copolymerized modified polyvinyl alcohol (hereinafter sometimes abbreviated as "copolymerized modified PVA"). Thereafter, a polyfunctional crosslinking agent that reacts with functional groups, such as carboxy groups and amino groups, present in the copolymerized modified PVA is added. A copolymerized modified PVA having a carboxy group is sometimes referred to as a "carboxylic acid-modified PVA," and a copolymerized modified PVA having an amino group is sometimes referred to as an "amino-modified PVA."

[0030] Examples of monomers constituting carboxylic acid-modified PVA include α,β-unsaturated carboxylic acids such as (meth)acrylic acid, maleic acid, fumaric acid, and itaconic acid; (meth)acrylic acid alkyl esters such as methyl (meth)acrylate and ethyl (meth)acrylate; and α,β-unsaturated carboxylic acid anhydrides such as maleic anhydride and itaconic anhydride, and derivatives thereof. A carboxylic acid-modified PVA is produced by copolymerizing a vinyl ester monomer with a monomer constituting the carboxylic acid-modified PVA and then saponifying the copolymer. A hydrogel can be obtained by combining a polyfunctional crosslinker that reacts with the introduced carboxyl groups, such as a polyfunctional epoxy compound such as epichlorohydrin or ethylene glycol diglycidyl ether, or a polyfunctional amino compound such as ethylenediamine, polyethyleneimine, or polyallylamine, with a carbodiimide condensing agent such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. The carboxyl group introduced into the carboxylic acid-modified PVA can be conjugated with a physiologically active substance having an amino group by covalently bonding it via an amide bond (-CONH-).

[0031] Furthermore, amino-modified PVA can be obtained as a hydrogel by copolymerizing a vinyl ester monomer with N-vinylformamide or the like, followed by saponification, and then combining a polyfunctional crosslinking agent that reacts with the introduced amino groups, such as the polyfunctional epoxy compound or a polyfunctional carboxylic acid compound such as succinic acid or maleic acid, with the carbodiimide condensing agent.

[0032] An example of a method for introducing functional groups into a vinyl alcohol polymer by post-modification and reacting them is a method for introducing ethylenically unsaturated groups into the side chains of the vinyl alcohol polymer. The introduced ethylenically unsaturated groups can be easily polymerized by adding an additive such as a radical initiator to induce a polymerization reaction, thereby producing a hydrogel. The ethylenically unsaturated groups are preferably introduced via the side chains or terminal functional groups of the vinyl alcohol polymer, and more preferably, a compound containing an ethylenically unsaturated group (hereinafter sometimes abbreviated as "ethylenically unsaturated group-containing compound") is reacted with a hydroxyl group in the side chain of the vinyl alcohol polymer.

[0033] Examples of the ethylenically unsaturated group-containing compound to be reacted with the hydroxyl group in the side chain of the vinyl alcohol polymer include (meth)acrylic acid or derivatives thereof such as (meth)acrylic acid, (meth)acrylic anhydride, (meth)acrylic acid halide, and (meth)acrylic acid ester. A (meth)acryloyl group can be introduced by subjecting these compounds to an esterification reaction or an ester exchange reaction in the presence of a base.

[0034] Furthermore, examples of the ethylenically unsaturated group-containing compound to be reacted with the hydroxyl group in the side chain of the vinyl alcohol polymer include compounds containing an ethylenically unsaturated group and a glycidyl group in the molecule, such as glycidyl (meth)acrylate, allyl glycidyl ether, etc. By subjecting these compounds to an etherification reaction in the presence of a base, it is possible to introduce a (meth)acryloyl group or an allyl group into the vinyl alcohol polymer.

[0035] Furthermore, examples of ethylenically unsaturated group-containing compounds that can be reacted with the 1,3-diol groups of vinyl alcohol polymers include compounds containing an ethylenically unsaturated group and an aldehyde group in the molecule, such as acrylaldehyde (acrolein), methacrylaldehyde (methacrolein), 5-norbornene-2-carboxaldehyde, 7-octenal, 3-vinylbenzaldehyde, and 4-vinylbenzaldehyde. By subjecting these compounds to an acetalization reaction in the presence of an acid catalyst, ethylenically unsaturated groups can be introduced into the starting PVA. More specifically, by subjecting, for example, 5-norbornene-2-carboxaldehyde, 3-vinylbenzaldehyde, or 4-vinylbenzaldehyde to an acetalization reaction, norbornenyl groups or vinylphenyl groups can be introduced into the starting PVA. Furthermore, it is possible to introduce a (meth)acryloylamino group into a vinyl alcohol polymer by reacting it with N-(2,2-dimethoxyethyl)(meth)acrylamide, etc. Methods for introducing an ethylenically unsaturated group into a vinyl alcohol polymer other than the above-exemplified reactions can also be used, and two or more types of reactions may be used in combination.

[0036] Other methods for introducing the ethylenically unsaturated group include reacting a reactive substituent, such as a carboxy group present in a carboxylic acid-modified PVA or an amino group present in an amino-modified PVA, with an ethylenically unsaturated group-containing compound. The carboxy group of a carboxylic acid-modified PVA can be reacted with, for example, glycidyl methacrylate under acidic conditions to form an ester bond and introduce a methacryloyl group. The amino group of an amino-modified PVA can be introduced with an acryloylamino group by amidation reaction with, for example, acrylic anhydride in the presence of a base, or with a vinyloxycarbonyl group by amidation reaction with, for example, divinyl adipate. Methods for introducing an ethylenically unsaturated group via a copolymerized modified PVA can be other than the reactions exemplified above, and two or more reactions may be used in combination.

[0037] From the viewpoint of ease of production, the vinyl alcohol polymer having an ethylenically unsaturated group is preferably a vinyl alcohol polymer in which an ethylenically unsaturated group is introduced via a hydroxyl group in a side chain of the raw material PVA, such as a 1,3-diol group, and more preferably a vinyl alcohol polymer in which a hydroxyl group in a side chain of the raw material PVA is subjected to an esterification reaction or transesterification reaction with (meth)acrylic acid or a derivative thereof, or a vinyl alcohol polymer in which a 1,3-diol group of the vinyl alcohol polymer is subjected to an acetalization reaction with a compound containing an ethylenically unsaturated group and an aldehyde group in the molecule.

[0038] <Introduction rate of ethylenically unsaturated groups> The introduction rate of the ethylenically unsaturated groups is preferably 10 mol % or less, more preferably 5 mol % or less, and even more preferably 3 mol % or less, based on all structural units constituting the vinyl alcohol-based polymer, from the viewpoint of suppressing embrittlement of the hydrogel. From the viewpoint of accelerating the crosslinking reaction, rapidly forming the hydrogel, and improving the elastic modulus of the obtained hydrogel, the introduction rate is preferably 0.01 mol % or more, more preferably 0.1 mol % or more, and even more preferably 0.5 mol % or more.

[0039] From the viewpoint of improving the mechanical strength of the hydrogel and introducing functional groups different from the hydroxyl groups of PVA, the vinyl alcohol polymer having an ethylenically unsaturated group may further contain a monomer. Examples of such monomers include acrylamides such as acrylamide, N-isopropylacrylamide, 2-acrylamido-2-methylpropanesulfonic acid, and N,N-dimethylacrylamide; α,β-unsaturated carboxylic acids such as (meth)acrylic acid, crotonic acid, itaconic acid, maleic acid, and fumaric acid; water-soluble radically polymerizable monomers such as vinylpyridine, hydroxyethyl (meth)acrylate, styrenesulfonic acid, and polyethylene glycol mono(meth)acrylate; and crosslinkers having two or more ethylenically unsaturated groups in the molecule such as N,N'-methylenebisacrylamide, ethylene glycol di(meth)acrylate, diethylene glycol di(meth)acrylate, and polyethylene glycol di(meth)acrylate. From the viewpoint of improving the mechanical strength of the hydrogel, the content of the monomer is preferably 50% by mass or less, more preferably 30% by mass or less, and even more preferably 10% by mass or less, based on the vinyl alcohol polymer having an ethylenically unsaturated group.

[0040] <Crosslinking by Polymerization of Ethylenically Unsaturated Groups> A vinyl alcohol polymer having an ethylenically unsaturated group can be gelled by crosslinking the ethylenically unsaturated group introduced into the vinyl alcohol polymer using active energy rays or heat, thereby obtaining a crosslinked vinyl alcohol polymer of the present invention. Examples of active energy rays include gamma rays, ultraviolet rays, visible light, infrared rays (heat rays), radio waves, alpha rays, beta rays, electron beams, plasma flow, ionizing rays, and particle beams. When the vinyl alcohol polymer is crosslinked using active energy rays such as ultraviolet rays, visible light, and infrared rays (heat rays) or heat, the uncrosslinked polymer solution described below preferably contains a radical polymerization initiator. Examples of radical polymerization initiators include photoradical polymerization initiators and thermal radical polymerization initiators.

[0041] The photoradical polymerization initiator is not particularly limited as long as it initiates radical polymerization by irradiation with active energy rays such as ultraviolet light or visible light. However, from the viewpoint of washing and removing the photoradical polymerization initiator after crosslinking, a water-soluble initiator is preferred. Specific examples include 1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propan-1-one (trade name "Omnirad 2959", manufactured by IGM RESINS B.V.), α-ketoglutaric acid, lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate (trade name "L0290", manufactured by Tokyo Chemical Industry Co., Ltd.), and Eosin Y.

[0042] The thermal radical polymerization initiator is not particularly limited as long as it initiates radical polymerization by heat, and examples thereof include azo initiators, peroxide initiators, and the like that are commonly used in radical polymerization. From the viewpoint of improving the transparency and physical properties of the vinyl alcohol polymer, peroxide initiators that do not generate gas are preferred. From the viewpoint of washing and removing the thermal radical polymerization initiator after crosslinking as described above, those that are water-soluble are preferred. Specific examples include inorganic peroxides such as ammonium persulfate, potassium persulfate, and sodium persulfate.

[0043] Alternatively, a redox polymerization initiator may be used in combination with a reducing agent. The redox polymerization initiator can be crosslinked by the stimulus of mixing a peroxide initiator with a reducing agent. Known reducing agents can be used as the reducing agent to be combined with the redox polymerization initiator. Among these, highly water-soluble N,N,N',N'-tetramethylethylenediamine, sodium sulfite, sodium hydrogensulfite, sodium hydrosulfite, and the like are preferred.

[0044] Similarly, the azo initiator is preferably one that is water-soluble, and specific examples thereof include 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (trade name "VA-044"), 2,2'-azobis[2-(2-imidazolin-2-yl)propane]disulfate dihydrate (trade name "VA-044B"), 2,2'-azobis[2-methylpropionamidine]dihydrochloride (trade name "V-50"), 2,2'-azobis[N-(2- 2,2'-azobis[2-(2-imidazolin-2-yl)propane] (trade name "VA-061"), 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide] (trade name "VA-086"), 4,4'-azobis(4-cyanopentanoic acid) (trade name "V-501") (all manufactured by Wako Pure Chemical Industries, Ltd.), and the like.

[0045] <Polythiol> In the crosslinking step described below, when a vinyl alcohol polymer having a vinyl group as the ethylenically unsaturated group is used, from the viewpoint of promoting crosslinking, for example, a polythiol having two or more thiol groups in the molecule may be added to perform crosslinking using a thiol-ene reaction. The polythiol is preferably water-soluble, and examples thereof include polythiols having a hydroxyl group such as dithiothreitol; and polythiols containing an ether bond, such as terminal thiolated products such as 3,6-dioxa-1,8-octanedithiol, polyethylene glycol dithiol, and multi-arm polyethylene glycol.

[0046] As described above, the most suitable method for crosslinking a vinyl alcohol polymer by covalent bonding can be selected as needed. However, from the viewpoints of the toxicity of unreacted crosslinking agent and condensing agent contained in the hydrogel and ease of molding, a method in which functional groups are introduced into the vinyl alcohol polymer by post-modification and then reacted, particularly a method in which an ethylenically unsaturated group is introduced into the side chain of the vinyl alcohol polymer, is preferred.

[0047] <Conjugation of Physiologically Active Substances> Examples of the physiologically active substances include cell adhesive proteins or peptides such as gelatin, collagen, laminin, fibronectin, retronectin, vitronectin, elastin, and synthetic RGD peptides; growth factors such as fibroblast growth factor (FGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF); acidic polysaccharides including glycosaminoglycans such as heparin, hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparan sulfate; antibodies, hormones, serum proteins, antibody-binding proteins such as albumin, macroglobulins, globulins, and protein A; various pharmaceuticals; and enzymes such as proteases, lipases, amylases, and cellulases. The physiologically active substance preferably has a functional group capable of binding to the dried hydrogel-forming article. Examples of such functional groups include primary or secondary amino groups, carboxy groups, and hydroxy groups, preferably primary amino groups or carboxy groups, and more preferably primary amino groups.

[0048] <Complex> In a first embodiment, the dry hydrogel-forming article of the present invention is a complex with a physiologically active substance. More specifically, in this embodiment, the dry hydrogel-forming article of the present invention may be a dry hydrogel-forming article that simply contains the physiologically active substance, or may be a complex in which the dry hydrogel-forming article and the physiologically active substance are covalently bonded. However, a dry hydrogel-forming article in which the physiologically active substance is covalently bonded is preferred. By forming a covalent bond between the dry hydrogel-forming article and the physiologically active substance, the physiologically active substance can be retained in the dry hydrogel-forming article and function stably. Methods for covalently bonding the dry hydrogel-forming article and the physiologically active substance can be selected, for example, by activating the hydroxyl groups of PVA and reacting them with the functional groups of the physiologically active substance to form a covalent bond, or by using a carboxylic acid-modified PVA or an amino-modified PVA to react with the functional groups of the physiologically active substance to form a covalent bond. From the viewpoint of reaction efficiency, it is preferable to use an amino group, a carboxylic acid or a thiol group as the functional group of the physiologically active substance, with a carboxylic acid being more preferable.

[0049] Specific methods for activating the hydroxyl groups of PVA to introduce a physiologically active substance include, for example, methods using hydroxyl group activating reagents such as 1,1'-carbonyldiimidazole, di(N-succimidyl)carbonate, p-toluenesulfonyl chloride, 2,2,2-trifluoroethanesulfonyl chloride, and cyanuric acid chloride. Reacting these hydroxyl group activating reagents with the hydroxyl groups of PVA enables the formation of covalent bonds with functional groups such as primary or secondary amino groups, carboxyl groups, and hydroxyl groups of the physiologically active substance. Alternatively, acid anhydride reagents such as succinic anhydride can be used to introduce a carboxylic acid via an ester bond to the hydroxyl groups of the vinyl alcohol polymer, or acetal reagents such as 2,2-dimethoxyethylamine can be used to introduce an amino group via an acetal bond to the 1,3-diol groups of the vinyl alcohol polymer. These carboxylic acids and amino groups can form amide bonds with the amino groups and carboxylic acids of the physiologically active substance using the carbodiimide condensing agent or the like. For example, when a crosslinked vinyl alcohol polymer is reacted with an acid anhydride reagent such as succinic anhydride, a carboxy group (COOH) derived from succinic acid is introduced at the terminal, and this carboxy group and an amino group of a physiologically active substance are condensed using a condensing agent such as 1,1'-carbonyldiimidazole, dicyclohexylcarbodiimide, or water-soluble carbodiimide, whereby the physiologically active substance can be conjugated to the crosslinked vinyl alcohol polymer via an amide bond.

[0050] As a specific method for introducing a physiologically active substance into carboxylic acid-modified PVA or amino-modified PVA, as described above, an amide bond can be formed using the amino group of the physiologically active substance, carboxylic acid, and the carbodiimide condensing agent, etc. Although the above examples are possible, an optimal method, including other methods, can be used depending on the application.

[0051] The complex may be formed after the crosslinking step of the uncrosslinked polymer solution as described below, or may be complexed at the stage of the uncrosslinked polymer solution. Complexation of the physiologically active substance is preferably carried out after the crosslinking step to avoid a decrease or loss of activity of the physiologically active substance due to crosslinking.

[0052] <Method for producing dried hydrogel-forming article> There are no particular limitations on the method for producing the hydrogel-forming article of the present invention, but it is preferable to produce the article by first going through a step of preparing an uncrosslinked polymer solution containing the vinyl alcohol-based polymer (uncrosslinked polymer solution preparation step), then a step of molding the uncrosslinked polymer solution (molding step), and then a step of crosslinking the vinyl alcohol-based polymer contained in the uncrosslinked polymer solution to form a gel (crosslinking step). A specific method is described below.

[0053] <Uncrosslinked Polymer Solution Preparation Step> The uncrosslinked polymer solution preparation step in the present invention is a step of preparing an uncrosslinked polymer solution containing the vinyl alcohol-based polymer, and can be obtained by dissolving the vinyl alcohol-based polymer in a solvent. The solvent is preferably water, and may further contain a water-soluble organic solvent. Examples of water-soluble organic solvents include aprotic polar solvents such as dimethylformamide, dimethylacetamide, dimethyl sulfoxide, and N-methylpyrrolidone; monoalcohols such as methanol, ethanol, propanol, and isopropanol; and polyhydric alcohols such as ethylene glycol, diethylene glycol, triethylene glycol, and glycerin. The water-soluble organic solvent may be used alone or in combination of two or more, or a mixture of a water-soluble organic solvent and water may be used.

[0054] When the uncrosslinked polymer solution contains the water-soluble organic solvent, the content thereof is preferably 30% by mass or less, more preferably 20% by mass or less, and even more preferably 10% by mass or less. The content of the solvent in the uncrosslinked polymer solution is preferably 50% by mass or more, more preferably 55% by mass or more, and even more preferably 60% by mass or more, and is preferably 99.999% by mass or less, more preferably 99.99% by mass or less, and even more preferably 99.9% by mass or less.

[0055] The content of the vinyl alcohol polymer in the uncrosslinked polymer solution is preferably 0.001% by mass or more, more preferably 0.01% by mass or more, and even more preferably 0.1% by mass or more. From the viewpoint of suppressing the increase in viscosity of the uncrosslinked polymer solution and obtaining good moldability, the content is preferably 50% by mass or less, more preferably 45% by mass or less, and even more preferably 40% by mass or less. If the content of the vinyl alcohol polymer is less than 0.001% by mass, the strength and film thickness of the resulting gel are too small, while if it exceeds 50% by mass, the viscosity of the uncrosslinked polymer solution is high, making molding difficult.

[0056] <Molding Step> In producing the hydrogel-forming article of the present invention, the method for molding the uncrosslinked polymer solution is not particularly limited, and it can be molded into the shape of the amorphous particles, spherical particles, fine molded bodies, arbitrarily shaped articles molded with a 3D printer, films, threads, hollow fibers, porous monoliths, coated articles, and the like, using known methods.

[0057] <Crosslinking Step> The hydrogel-forming article of the present invention is preferably produced by a crosslinking step in which the vinyl alcohol polymer is crosslinked after the molding step. Crosslinking in this step can be carried out by the method described above. The crosslinking step may be carried out while the uncrosslinked polymer solution contains a solvent, or it may be carried out after removing the solvent from the uncrosslinked polymer solution. In the crosslinking step, a gel crosslinked while the uncrosslinked polymer solution contains a solvent may experience significant shrinkage during the drying step described below, making it difficult to form shapes such as films, threads, hollow fibers, and coatings. In this case, it is preferable to dry the uncrosslinked polymer solution once after molding and then crosslink it. The degree of drying of the uncrosslinked polymer solution can be selected according to the molded product and crosslinking method, but the solvent content is preferably 50% or less, more preferably 25% or less, and even more preferably 10% or less. A solvent content exceeding 50% tends to result in significant shrinkage during the drying step described below.

[0058] Before molding, the uncrosslinked polymer solution may contain not only the vinyl alcohol-based polymer but also components necessary for crosslinking, such as the crosslinking agent and initiator. As described above, if the uncrosslinked polymer solution is dried after molding, the components necessary for crosslinking may be added after molding. As described above, optimal crosslinking conditions are selected depending on the vinyl alcohol-based polymer and crosslinking method used. However, molding is preferably performed before the hydrogel is formed, since molding becomes difficult once crosslinking is completed and a hydrogel is formed. Among the above-mentioned crosslinking methods, some involve simply mixing the uncrosslinked polymer solution with the components necessary for crosslinking, which initiates the reaction at temperatures below room temperature. Therefore, mixing immediately before molding is desirable. Furthermore, in the case of crosslinking that requires heat, the temperature of the crosslinking reaction is preferably 100°C or lower, more preferably 60°C or lower, and even more preferably 37°C or lower, from the viewpoint of maintaining the activity of the physiologically active substance.

[0059] <Complexation Step> The composite step in the present invention is a step of complexing a physiologically active substance with a crosslinked vinyl alcohol polymer to obtain a composite hydrogel. Therefore, as already explained, if the complexation is performed at the uncrosslinked polymer solution stage, the composite step is basically unnecessary. When a carboxylic acid-modified vinyl alcohol copolymer or an amino-modified vinyl alcohol polymer is used as the vinyl alcohol polymer, the physiologically active substance can be introduced to the remaining functional groups by the above-mentioned composite method. Alternatively, it is also possible to first introduce functional groups for complexation to hydroxyl groups, etc., contained in the crosslinked vinyl alcohol polymer after the crosslinking step, and then perform the composite step. As a method for introducing functional groups, a hydroxyl group activating reagent, an acid anhydride reagent, an acetal reagent, etc. may be used as described above, and the physiologically active substance can also be introduced to the functional groups by the above-mentioned composite method.

[0060] <Drying Step> Before sterilization, the composite hydrogel is dried by removing water from the target product using a common method such as hot air drying, vacuum drying, or freeze drying. The hot air drying temperature is preferably about 30 to 100°C, more preferably about 37 to 60°C, to avoid a decrease or loss of activity of the physiologically active substance. The water content of the dried hydrogel-forming article of the present invention, as expressed by the following formula (1), is preferably 75% by mass or less, more preferably 60% by mass or less, even more preferably 50% by mass or less, still more preferably 40% by mass or less, and particularly preferably 25% by mass or less.

[0061]

[0062] The appropriate method for measuring the water content of the dried hydrogel-forming article of the present invention varies depending on the type of solvent contained. When the dried hydrogel-forming article contains only water as a solvent, the water content of the dried hydrogel-forming article can be calculated by directly evaporating the contained water using a heat-drying moisture meter and measuring the weight of the remaining dried composite hydrogel. When the dried hydrogel-forming article contains the water-soluble organic solvent, the composite hydrogel can be calculated by directly evaporating the contained water using a heat-drying moisture meter and measuring the weight of the remaining dried composite hydrogel. 1 The water content is measured by H-NMR. Specifically, the composite hydrogel is swollen with deuterated DMSO. 1 By measuring the H-NMR spectrum and the integral value, the contents of water and the water-soluble organic solvent can be calculated by comparing the integral values ​​of the peaks derived from the crosslinked vinyl alcohol polymer. 1 Measurement of the water content by H-NMR is effective from the viewpoint of separately evaluating the contents of the water-soluble organic solvent and water when the water-soluble organic solvent is contained. 1 In one preferred embodiment, the presence or absence of a water-soluble organic solvent is confirmed by H-NMR, and if the solution contains only water as a solvent, the water content is then measured using a heat-drying moisture meter.

[0063] <Sterilization process> Sterilization in the present invention does not necessarily mean sterilization at a SAL of 10 or less, as required for pharmaceuticals and medical devices used on the human body. -6 It does not have to be less than SAL=10 -3or less, more preferably SAL=10 -4 More preferably, SAL=10 -5 If the following can be guaranteed, it can be used for research and testing purposes. However, if it is incorporated into a pharmaceutical or medical device that is used on the human body, or if it is used to manufacture such an item, the SAL must be 10. -6 It is preferable to ensure the following:

[0064] In a typical embodiment, the composite hydrogel is sterilized after the drying process. Sterilization before the drying process, i.e., of a composite hydrogel containing a large amount of water, reduces the activity of the composited physiologically active substance. On the other hand, sterilization after drying as described above maintains the activity of the composited physiologically active substance within an acceptable range.

[0065] The sterilization method is not particularly limited, and specific examples of sterilization methods that can be used include autoclave sterilization, ethylene oxide gas sterilization, low-temperature hydrogen peroxide plasma sterilization, dry heat sterilization, chemical sterilization using glutaraldehyde or the like, and radiation sterilization using gamma rays or electron beams. Among these, ethylene oxide gas sterilization, low-temperature hydrogen peroxide plasma sterilization, and radiation sterilization are preferred from the viewpoint of easily maintaining the activity of the complexed physiologically active substance, and radiation sterilization is even more preferred because it leaves no residue. The dried hydrogel-forming article of the present invention is preferably ethylene oxide gas-free, glutaraldehyde-free, and hydrogen peroxide-free. Sterilization is preferably performed by placing the dried hydrogel-forming article in a container, sealing the container, and then performing a sterilization process. Flexible resin containers, such as resin pouches, can be used as the container, and sealing can be performed by heat sealing. In addition, bottle containers with lids that can be opened after sterilization can be used as the container, and sealing can be performed by screwing the lid on or heat sealing like an ampoule. For example, when performing radiation sterilization, it is preferable to use a container made of a material that is radiotransparent and retains sufficient strength after irradiation. Examples of container materials include polyolefin resins such as polyethylene, polypropylene, and polybutylene, or copolymers of two or more olefins, polyester resins such as polyethylene terephthalate and polybutylene terephthalate, polyvinyl alcohol, saponified ethylene-vinyl acetate copolymer, vinyl resins such as polyvinyl chloride and polyvinylidene chloride, polycarbonate resin, polystyrene resin, silicone resin, polyamide resin, and glass. The container may be composed of a single layer or a laminate of these materials. These materials may also be vapor-deposited with aluminum, silica, or the like, or multilayers containing metal foils such as aluminum film and aluminum laminate film. It is also preferable to perform sterilization on a hydrogel-forming article in a dried state.

[0066] Sterilization validation can be performed using any of the following methods: half cycle method, overkill method, bioburden / BI combined method, and absolute bioburden method. In the case of radiation sterilization, sterilization validation is performed using the absolute bioburden method described in ISO (ISO 11137-2:2013) or JIS (JIS T 0806-2:2014). Methods for setting the sterilization dose in sterilization validation include Method 1, VD max Method 1 and Method 2 are available, both of which can be used to sterilize the dried hydrogel-forming article of the present invention. max Method 1 and VD are methods for setting the sterilization dose based on the number of bacteria adhering to the dried hydrogel-forming article, and Method 2 are methods for setting the sterilization dose based on the radiation resistance of the bacteria adhering to the dried hydrogel-forming article. max To determine the sterilization dose by the VD method, a bioburden test as described in ISO 11737-1:2006 or JIS T11737-1:2013 can be performed to measure the number of bacteria adhering to the dried hydrogel-forming article before sterilization. Considering the number of required data and costs, Method 1 and VD max The VD method is more preferable, and considering cost reduction max The method is more preferable.

[0067] SAL=10 -6 To achieve the following, Method 1 requires 11 kGy or more, VD max In method 1, an irradiation dose of 15 kGy or more is required. In method 2, an irradiation dose of 8.2 kGy or more is required. It has been conventionally believed that composite hydrogels cannot withstand such extremely high-energy gamma ray irradiation doses. One preferred embodiment of the present invention is based on the discovery that, by using a dried composite hydrogel-forming article, damage to the product can be prevented even with high-energy gamma ray irradiation of 8.2 kGy or more, and a sterilized composite hydrogel-forming article can be produced in which the activity of the physiologically active substance is maintained within a desired range.

[0068] The dry hydrogel-forming article of the present invention may be used in combination with cells or may contain cells. When used in combination with cells or containing cells, it is preferable to supply water to the article to form a hydrogel. The term "cells" in this specification is not particularly limited, but preferably includes pluripotent stem cells, tissue stem cells, somatic cells, mammalian cell lines used for the production of useful substances such as pharmaceuticals, treatments, etc., and insect cells.

[0069] The cells include adherent cells and suspension cells. Adherent cells are cells that grow by adhering to a carrier such as the hydrogel of the present invention during cell culture. Suspension cells are cells that do not essentially require attachment to a carrier for cell growth. Suspension cells include cells that can weakly adhere to a carrier.

[0070] The pluripotent stem cells are stem cells that have the ability to differentiate into cells of any tissue (pluripotency), and include, for example, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic germ stem cells (EG cells), and germ stem cells (GS cells).

[0071] The above-mentioned tissue stem cells refer to stem cells that are limited in the tissues they differentiate into but have the ability to differentiate into various cell types (pluripotency). Examples of tissue stem cells include bone marrow undifferentiated mesenchymal stem cells, skeletal muscle stem cells, hematopoietic stem cells, neural stem cells, hepatic stem cells, adipose tissue stem cells, epidermal stem cells, intestinal stem cells, spermatogonial stem cells, pancreatic stem cells (pancreatic duct epithelial stem cells, etc.), leukocyte stem cells, lymphocyte stem cells, and corneal stem cells.

[0072] The somatic cells refer to cells that constitute a multicellular organism, and examples thereof include, but are not limited to, osteoblasts, chondrocytes, hematopoietic cells, epithelial cells (such as mammary epithelial cells), endothelial cells (such as vascular endothelial cells), epidermal cells, fibroblasts, mesenchymal-derived cells, cardiac muscle cells, myoblasts, smooth muscle cells, skeletal muscle cells derived from living organisms, human tumor cells, fibrocytes, EB virus mutant cells, hepatocytes, kidney cells, bone marrow cells, macrophages, hepatic parenchymal cells, small intestinal cells, mammary gland cells, salivary gland cells, thyroid cells, skin cells, plasma cells, T cells, B cells, killer cells, lymphoblasts, and pancreatic β cells.

[0073] The above mammalian-derived cell lines include CRFK cells, 3T3 cells, A549 cells, AH130 cells, B95-8 cells, BHK cells, BOSC23 cells, BS-C-1 cells, C3H10T1 / 2 cells, C-6 cells, CHO cells, COS cells, CV-1 cells, F9 cells, FL cells, FL5-1 cells, FM3A cells, G-361 cells, GP+ E-86 cells, GP+envAm12 cells, H4-II-E cells, HEK293 cells, HeLa cells, HEp-2 cells, HL-60 cells, HTC cells, HUVEC cells, IMR-32 cells, IMR-90 cells, K562 cells, KB cells, L cells, L5178Y cells, L-929 cells, MA104 cells, MDBK cells, MDCK cells, MIA PaCG-2 cells, N18 cells, Namalwa cells, NG108-15 cells, NRK cells, OC10 cells, OTT6050 cells, P388 cells, PA12 cells, PA317 cells, PC-12 cells, PER. Examples include C6 cells, PG13 cells, QGH cells, Raji cells, RPMI-1788 cells, SGE1 cells, Sp2 / O-Ag14 cells, ST2 cells, THP-1 cells, U-937 cells, V79 cells, VERO cells, WI-38 cells, ψ2 cells, and ψCRE cells.

[0074] Examples of the insect cells include silkworm cells (BmN cells, BoMo cells, etc.), mulberry cells, Anemone cells, Anemone cells, Mamestra frugiperda cells (Sf9 cells, Sf21 cells, etc.), mulberry fly cells, leafroller cells, Drosophila cells, Boettcheris caudatus cells, Aedes albopictus cells, swallowtail butterfly cells, American cockroach cells, and nettle looper cells (Tn-5 cells, HIGH FIVE cells, MG1 cells, etc.).

[0075] The cells may be aggregated together or differentiated. The aggregated cells may have the function of an organ. The cells may be immediately collected from a living body or may be cultured. The cells collected from a living body may have formed an organ.

[0076] When cells are cultured by contacting the dried hydrogel-forming article of the present invention as a hydrogel, it is preferable to add medium components. There are no particular limitations on the medium used, and any medium suitable for the cells can be freely selected and used. Examples of media that can be used include basal media such as DMEM, MEM, and F12 supplemented with fetal bovine serum, reduced serum media, and serum-free media. Usable serum-free media include xeno-free media and fully synthetic media that contain not only serum but also protein components and animal components.

[0077] The dry hydrogel-forming article of the present invention has excellent hydrophilicity, reactivity, biodegradability, biocompatibility, and low toxicity, and is flexible and strong, allowing for the provision of a sterilized composite hydrogel-forming article without the need for expensive processes such as aseptic environments. Therefore, the dry hydrogel-forming article of the present invention is useful as a carrier or support. A carrier made of the dry hydrogel-forming article of the present invention can be suitably used for applications such as an enzyme immobilization carrier, an affinity carrier, a cell culture carrier, and a drug delivery carrier.

[0078] 2. Hydrogel In a second embodiment, the present invention provides a hydrogel comprising a crosslinked product of a vinyl alcohol-based polymer having an ethylenically unsaturated group and a carboxy group, and a physiologically active substance having an amino group, wherein the carboxy group of the crosslinked product and the amino group of the physiologically active substance are covalently bonded via an amide bond. Unless otherwise specified in this specification, the details, production method, and usage of the hydrogel in the second embodiment may be the same as those of the first embodiment of the present invention described in "1. Dried Hydrogel-Forming Article."

[0079] <Hydrogel> In this embodiment, the hydrogel of the present invention refers to a composite hydrogel obtained by combining a crosslinked vinyl alcohol polymer having an ethylenically unsaturated group and a carboxyl group with a physiologically active substance having an amino group, or a dried or molded product thereof, and may be in a hydrated or dried state. The dried hydrogel swells to form a hydrogel when it comes into contact with an aqueous solution such as water, a buffer solution, a body fluid, or a culture medium. The molded product may be in the form of general irregular particles, spherical particles, a film, a thread, a hollow fiber, a porous monolith, or the like.

[0080] In this specification, the term "amide bond composite hydrogel" refers to a hydrogel in which a physiologically active substance having an amino group is covalently bonded to a crosslinked product of a vinyl alcohol polymer having an ethylenically unsaturated group and a carboxy group via an amide bond with the carboxy group of the vinyl alcohol polymer.

[0081] Furthermore, the shape, dimensions, manufacturing method, etc. of the molded product may be the same as those described above in "1. Dried hydrogel-forming article."

[0082] In the second embodiment, the hydrogel of the present invention may be in a dried state from which the solvent has been removed, or may contain a solvent. The solvent is preferably water, and the hydrogel may further contain a water-soluble organic solvent. Examples of the water-soluble organic solvent include aprotic polar solvents such as dimethylformamide, dimethylacetamide, dimethyl sulfoxide, and N-methylpyrrolidone; monoalcohols such as methanol, ethanol, propanol, and isopropanol; and polyhydric alcohols such as ethylene glycol, diethylene glycol, triethylene glycol, and glycerin. The solvent content in the hydrogel is preferably 0.1 to 99% by mass, more preferably 0.5 to 98% by mass, and even more preferably 1 to 97% by mass.

[0083] <Method for producing vinyl alcohol-based polymer> Unless otherwise specified in this specification, the method for producing the vinyl alcohol-based polymer, raw materials, viscosity measurement method, and the like in the second embodiment may be the same as those in the first embodiment of the present invention described in "1. Dried hydrogel-forming article."

[0084] <Crosslinked vinyl alcohol polymer> In the second embodiment, the hydrogel of the present invention comprises a crosslinked vinyl alcohol polymer having an ethylenically unsaturated group and a carboxy group. The total amount of vinyl alcohol-derived structural units and vinyl ester-derived structural units relative to all structural units constituting the vinyl alcohol polymer used in the present invention is preferably 80 mol % or more, more preferably 90 mol % or more, and even more preferably 95 mol % or more.

[0085] <Vinyl alcohol polymer having an ethylenically unsaturated group> In the second embodiment, the vinyl alcohol polymer of the present invention has an ethylenically unsaturated group as a crosslinkable moiety via a covalent bond. As described below, the ethylenically unsaturated group undergoes a polymerization reaction even in water under mild conditions, allowing the vinyl alcohol polymer to be crosslinked and gelled. Furthermore, the vinyl alcohol polymer is extremely useful because it can be used in various molding processes, including 3D printers.

[0086] The ethylenically unsaturated group is not particularly limited and can be freely selected, but is preferably a group that can form crosslinks between vinyl alcohol polymer chains by the action of active energy rays, heat, a redox polymerization initiator, etc., as described below. It is more preferable to use a radically polymerizable group as the ethylenically unsaturated group, and examples thereof include cyclic unsaturated hydrocarbon groups such as vinyl groups, (meth)acryloyl groups, (meth)acryloylamino groups, vinylphenyl groups, cyclohexenyl groups, cyclopentenyl groups, norbornenyl groups, and dicyclopentenyl groups, as well as derivatives thereof. These ethylenically unsaturated groups may be present in either the side chain or the end of the vinyl alcohol polymer chain. The vinyl group in the present invention includes not only ethenyl groups, but also chain unsaturated hydrocarbon groups such as allyl groups and alkenyl groups, vinyloxycarbonyl groups, etc.

[0087] Among the radical polymerizable groups, from the viewpoint of improving the mechanical strength of the hydrogel particles, at least one selected from the group consisting of a vinyl group, a (meth)acryloyl group, a (meth)acryloylamino group, a vinylphenyl group, a norbornenyl group, and derivatives thereof is preferred. Furthermore, from the viewpoint of reactivity, a functional group having a terminal unsaturated carbon bond is preferred, and a (meth)acryloyl group is more preferred.

[0088] The ethylenically unsaturated group is preferably formed via a side chain or a terminal functional group of the vinyl alcohol polymer, and more preferably by reacting a compound containing an ethylenically unsaturated group (hereinafter sometimes abbreviated as "ethylenically unsaturated group-containing compound") with a hydroxyl group in a side chain of the vinyl alcohol polymer.

[0089] Examples of the ethylenically unsaturated group-containing compound to be reacted with the hydroxyl group in the side chain of the vinyl alcohol polymer include (meth)acrylic acid or derivatives thereof such as (meth)acrylic acid, (meth)acrylic anhydride, (meth)acrylic acid halide, and (meth)acrylic acid ester, and norbornene derivatives such as 5-norbornene-2,3-dicarboxylic anhydride and 5-norbornene-2-carboxylic acid. These compounds can be subjected to an esterification reaction or an ester exchange reaction in the presence of a base or an acid to introduce a (meth)acryloyl group and a norbornene group.

[0090] In addition, examples of the ethylenically unsaturated group-containing compound to be reacted with the hydroxyl group in the side chain of the vinyl alcohol polymer include compounds containing an ethylenically unsaturated group and a glycidyl group in the molecule, such as glycidyl (meth)acrylate, allyl glycidyl ether, etc. By subjecting these compounds to an etherification reaction in the presence of a base, it is possible to introduce a (meth)acryloyl group or an allyl group into the vinyl alcohol polymer.

[0091] Furthermore, examples of the ethylenically unsaturated group-containing compound to be reacted with the 1,3-diol group of the vinyl alcohol polymer; the method for introducing the ethylenically unsaturated group into the vinyl alcohol polymer; and the vinyl alcohol polymer having the ethylenically unsaturated group include those similar to those described in "1. Dried hydrogel-forming article."

[0092] <Introduction rate of ethylenically unsaturated group> Examples of the introduction rate of the ethylenically unsaturated group; the monomer that may be further contained in the vinyl alcohol-based polymer having an ethylenically unsaturated group, include the same monomers as those described in "1. Dried hydrogel-forming article".

[0093] <Crosslinking by polymerization of ethylenically unsaturated groups> A vinyl alcohol polymer having an ethylenically unsaturated group can be gelled to obtain a crosslinked product by crosslinking the ethylenically unsaturated groups introduced into the vinyl alcohol polymer using active energy rays or heat. Examples of the active energy rays and radical polymerization initiators (photoradical polymerization initiators, thermal radical polymerization initiators; redox polymerization initiators combined with a reducing agent, azo initiators, etc.) used in this process include those similar to those described in "1. Dried hydrogel-forming article."

[0094] <Polythiol> In the crosslinking step, when a vinyl alcohol polymer having a vinyl group as the ethylenically unsaturated group is used, in order to promote curing, for example, a polythiol having two or more thiol groups in the molecule may be added to perform crosslinking utilizing a thiol-ene reaction; examples of the polythiol in this case include those similar to those described in "1. Dried hydrogel-forming article."

[0095] <Vinyl alcohol polymer having a carboxy group> In the second embodiment, the vinyl alcohol polymer of the present invention has a carboxy group. Methods for introducing a carboxy group into the vinyl alcohol polymer include, for example, activating a hydroxyl group of PVA to introduce a carboxy group, and utilizing, as is, a carboxy group contained in a carboxylic acid-modified PVA into which a monomer having a carboxy group other than a vinyl ester monomer has been copolymerized in advance when producing the vinyl alcohol polymer.

[0096] Examples of methods for activating the hydroxyl groups of PVA to introduce carboxyl groups include the use of acid anhydride reagents such as succinic anhydride, which can introduce carboxylic acids by ester linkage to hydroxyl groups, and acetal reagents such as glyoxylic acid, which can introduce carboxyl groups by acetal linkage to 1,3-diol groups of the vinyl alcohol polymer. These carboxylic acids (COOH) can be combined with amino groups of physiologically active substances to form amide bonds using condensing agents such as carbonyldiimidazole, dicyclohexylcarbodiimide, and water-soluble carbodiimides, which are used in peptide synthesis.

[0097] Examples of the monomer having a carboxy group other than the vinyl ester monomer that constitutes the carboxylic acid-modified PVA include α,β-unsaturated carboxylic acids such as (meth)acrylic acid, maleic acid, fumaric acid, itaconic acid, etc.; (meth)acrylic acid alkyl esters such as methyl (meth)acrylate, ethyl (meth)acrylate, etc.; α,β-unsaturated carboxylic acid anhydrides such as maleic anhydride, itaconic anhydride, etc., and derivatives thereof. A carboxylic acid-modified vinyl alcohol polymer (carboxylic acid-modified PVA) can be produced by copolymerizing a vinyl ester monomer and a monomer having a carboxy group other than the vinyl ester monomer that constitutes the carboxylic acid-modified PVA, followed by saponification.

[0098] The introduction rate of the carboxyl groups is preferably 50 mol % or less, more preferably 30 mol % or less, and even more preferably 15 mol % or less, of all structural units constituting the vinyl alcohol polymer, from the viewpoints of maintaining an appropriate swelling property of the hydrogel in a solvent (particularly water) and maintaining physical properties such as gel strength, etc., and is preferably 0.1 mol % or more, more preferably 0.5 mol % or more, and even more preferably 1.0 mol % or more, from the viewpoints of maintaining a high complexation density of the physiologically active substance and exhibiting high functionality.

[0099] <Physiologically active substances> Examples of physiologically active substances having an amino group (primary or secondary) include cell adhesive proteins or peptides such as gelatin, collagen, laminin, fibronectin, retronectin, vitronectin, elastin, and synthetic RGD peptides; growth factors such as fibroblast growth factor (FGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF); antibody-binding proteins such as antibodies, serum proteins, albumin, macroglobulin, globulin, and protein A; glycosaminoglycans such as heparin, hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparan sulfate; various pharmaceuticals; and enzymes such as proteases, lipases, amylases, and cellulases. These physiologically active substances contain amino groups, which can be used to conjugate the physiologically active substances via amide bonds. The physiologically active substance is preferably a physiologically active polypeptide containing two or more amino acids. The amino group may be either an aromatic amino group or an aliphatic amino group, but is preferably an aliphatic amino group, and is preferably a side chain amino group of lysine (Lys) or an N-terminal amino group of the physiologically active polypeptide. The amino group contained in the physiologically active substance may be either a primary amino group or a secondary amino group, and includes either or both of these. Either the primary amino group or the secondary amino group contained in the physiologically active substance can form an amide bond with a carboxy group of the crosslinked vinyl alcohol polymer.

[0100] <Complex> In a second embodiment, the hydrogel of the present invention is a composite of a crosslinked vinyl alcohol-based polymer having an ethylenically unsaturated group and a carboxy group, and a physiologically active substance having an amino group. More specifically, the hydrogel of the present invention is a hydrogel in which the physiologically active substance having an amino group is bound to the carboxy group of the vinyl alcohol-based polymer via a covalent bond (—CO—NH—). The formation of a covalent bond (amide bond) between the hydrogel and the physiologically active substance allows the physiologically active substance to be retained in the hydrogel and to function stably.

[0101] As a specific method for conjugating a crosslinked vinyl alcohol polymer with a physiologically active substance, as described above, the amino group of the physiologically active substance and the carboxyl group of the vinyl alcohol polymer can be covalently bonded via an amide bond using the carbodiimide condensing agent or the like, but an optimal method, including other methods, can be used depending on the application.

[0102] The complex with the physiologically active substance may be formed after the crosslinking step of the uncrosslinked gel solution, as explained below, or may be formed at the stage of the uncrosslinked gel solution.

[0103] <Method for producing a hydrogel> In the second embodiment, the method for producing a hydrogel of the present invention is not particularly limited, but it is preferable to produce the hydrogel by first carrying out a step of preparing an uncrosslinked gel solution containing the vinyl alcohol-based polymer (uncrosslinked gel solution preparation step), then a step of molding the uncrosslinked gel solution (molding step), and then a step of crosslinking the vinyl alcohol-based polymer contained in the uncrosslinked gel solution to form a gel (crosslinking step). The step of complexing a physiologically active substance can be carried out before or after the crosslinking step. Specific methods are described below.

[0104] <Uncrosslinked Gel Solution Preparation Step> The uncrosslinked gel solution preparation step in the present invention is a step of preparing an uncrosslinked gel solution containing the vinyl alcohol-based polymer, and can be obtained by dissolving the vinyl alcohol-based polymer in a solvent. In this method, the type and amount of solvent used, the content of the vinyl alcohol-based polymer in the uncrosslinked gel solution, and the like can be the same as those in the first embodiment of the present invention described in "1. Dried Hydrogel-Forming Article."

[0105] <Molding step> In the second embodiment, in the production of the hydrogel of the present invention, there are no particular limitations on the method for molding the uncrosslinked gel solution, and the methods described in "1. Dried hydrogel-forming article" can be used as appropriate.

[0106] <Crosslinking step> In the second embodiment, the hydrogel of the present invention is also preferably produced by a crosslinking step of crosslinking the vinyl alcohol polymer after the molding step. Details of the crosslinking step may be the same as those in the first embodiment of the present invention described in "1. Dried hydrogel-forming article."

[0107] <Combination Step> As already explained, if the composite is formed at the uncrosslinked gel solution stage, the composite step after the crosslinking step is basically unnecessary. Furthermore, if a carboxylic acid-modified PVA is used as the vinyl alcohol polymer, a physiologically active substance can be introduced to the remaining functional groups by the above-mentioned composite method. Alternatively, a functional group (carboxy group) for composite formation can be first introduced to the hydroxyl groups, etc., contained in the hydrogel after the crosslinking step, and then the composite step can be carried out. As a method for introducing the functional group, a hydroxyl group activating reagent, an acid anhydride reagent, an acetal reagent, etc. can be used as described above, and a physiologically active substance having an amino group can be introduced to the carboxyl group by the above-mentioned composite method.

[0108] In the second embodiment, the hydrogel of the present invention may also be used in combination with cells or may contain cells. Details of the cells, the culture method, etc. in the second embodiment are the same as those described in "1. Dried hydrogel-forming article."

[0109] In a second embodiment, the hydrogel of the present invention has excellent hydrophilicity, reactivity, biodegradability, and biocompatibility, and has high flexibility and strength. A physiologically active substance or enzyme is efficiently introduced into the hydrogel by a covalent bond using a low-toxicity crosslinking method. Therefore, the hydrogel of the present invention is useful as a carrier or support. Carriers containing the hydrogel of the present invention can be suitably used for applications such as enzyme immobilization carriers, affinity carriers, cell culture carriers, and drug delivery carriers.

[0110] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0111] [Raw Materials Used] The main components used in the Synthesis Examples, Examples, and Comparative Examples are listed below. <Raw Material PVA> PVA117: Polyvinyl alcohol (trade name "PVA117", degree of polymerization 1700, degree of saponification approximately 98.0 to 99.0 mol%, viscosity (4%, 20°C) 25.0 to 31.0 mPa s, manufactured by Kuraray Co., Ltd.) The degree of polymerization of the raw material PVA was measured in accordance with JIS K 6726:1994. AF-17: Polyvinyl alcohol (product name "AF-17", 1.3 mol% itaconic acid copolymer (Scientific Reports, 2017, Vol. 7, p. 45146), saponification degree 96.5 mol% or more, viscosity (4%, 20°C) 30±3 mPa s, manufactured by Nippon Vinyl Acetate & Poval Co., Ltd.)

[0112] <Ethylenically unsaturated group-containing compounds> Vinyl methacrylate: manufactured by Tokyo Chemical Industry Co., Ltd. 5-norbornene-2-carboxaldehyde: manufactured by Tokyo Chemical Industry Co., Ltd.

[0113] <Synthesis Reagents> 50% glutaraldehyde solution: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. Triethylamine: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. Succinic anhydride: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. Liquid paraffin: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. Span 80: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.

[0114] <Radical polymerization initiator> Potassium persulfate: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. L0290: phenyl(2,4,6-trimethylbenzoyl)phosphinic acid lithium salt (photoradical polymerization initiator, trade name "L0290", manufactured by Tokyo Chemical Industry Co., Ltd.)

[0115] <Polythiol> 3,6-dioxa-1,8-octanedithiol: manufactured by Tokyo Chemical Industry Co., Ltd.

[0116] <Hydroxyl group activation reagents> 1,1'-carbonyldiimidazole: manufactured by Tokyo Chemical Industry Co., Ltd. Succinic anhydride: manufactured by Tokyo Chemical Industry Co., Ltd. or Fujifilm Wako Pure Chemical Industries, Ltd. <Complexing reagents> 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. N-hydroxysuccinimide: manufactured by Tokyo Chemical Industry Co., Ltd. <Biologically active substances> Gelatin (porcine-derived, type A): manufactured by Sigma-Aldrich Japan K.K. Collagen (trade name "Cell Matrix", type I-C): manufactured by Nitta Gelatin Co., Ltd. <Solvents> Ion-exchanged water: electrical conductivity 0.08 x 10 -4 Ion-exchanged water of S / m or less PBS: Prepared by dissolving PBS tablets (manufactured by Takara Bio Inc.) in a specified amount of ion-exchanged water. MES buffer: Prepared a 0.1 mol / L aqueous solution of 2-morpholinoethanesulfonic acid monohydrate (manufactured by Dojindo Laboratories Ltd.), and neutralized with a 0.1 mol / L aqueous NaOH solution to adjust the pH to 5.6.

[0117] [Methods for measuring compounds synthesized in synthesis examples] <Introduction rate of ethylenically unsaturated group and carboxyl group (succinic acid)> The introduction rates of ethylenically unsaturated group and carboxyl group (succinic acid) in the vinyl alcohol polymers having ethylenically unsaturated groups obtained in the synthesis examples below were 1 The introduction rate was measured by H-NMR. The introduction rate was determined from the ratio of the integral values ​​of the signals of the ethylenically unsaturated group and carboxyl group (methylene succinate group) to the signal of the vinyl alcohol polymer. Similarly, the hydrogel particles and sheets were also measured in a state where they were swollen in a heavy solvent. 1H-NMR measurement can be carried out, and the introduction rate in the hydrogel particles and sheet can be determined from the ratio of the integrated value of the signal of the carboxy group (methylene succinate group) to the signal of the vinyl alcohol polymer. 1 H-NMR measurement conditions Equipment: Nuclear magnetic resonance apparatus “JNM-ECX400” manufactured by JEOL Ltd. Temperature: 25°C

[0118] [Water Content] The water content of the composite hydrogels obtained in the following examples was measured using a heat-drying moisture meter when the composite hydrogels contained only water as a solvent, and when the composite hydrogels contained a water-soluble organic solvent. 1 Measurement was performed by H-NMR. [Measurement conditions for heat-drying moisture meter] Apparatus: Heat-drying moisture meter manufactured by A&D Co., Ltd. [ 1 H-NMR measurement conditions] Water content is 1 It can be calculated by H-NMR (deuterated DMSO solvent). The water content can be determined from the ratio of the integral value of the water signal (3.3 ppm) to the signal of the vinyl alcohol polymer. The water originally contained in the deuterated DMSO solvent is subtracted. Apparatus: Nuclear magnetic resonance apparatus "JNM-ECX400" manufactured by JEOL Ltd. Temperature: 25°C

[0119] [Synthesis Examples] [Synthesis Example 1-1] <Synthesis of a vinyl alcohol polymer having an ethylenically unsaturated group> 40 g (monomer repeating unit: 908 mmol) of PVA117 (raw material PVA) was placed in a separable flask equipped with a 1 L Dimroth condenser, 350 mL of dimethyl sulfoxide (DMSO) was added, and stirring was initiated with a mechanical stirrer. The temperature was raised to 80°C in a water bath, and stirring was continued at 80°C for 4 hours. After visually confirming that the raw material PVA had dissolved, 2.1 g (18.7 mmol) of vinyl methacrylate was added while heating and stirring at 80°C, and stirring was continued for an additional 3 hours at 80°C. After allowing to cool, the reaction solution was poured into 2 L of methanol while stirring. Stirring was stopped and the mixture was left as is for 1 hour. The resulting solid was collected and then immersed in 1 L of methanol for another 1 hour to wash. This washing process was repeated a total of three times. The recovered solid was dried in vacuo at room temperature overnight to obtain methacryloylated PVA 117. The introduction rate of ethylenically unsaturated groups (methacryloyl groups) in this methacryloylated PVA 117 was 2.0 mol % relative to the repeating units of the raw material PVA (hereinafter abbreviated as "MA-PVA117(2.0)").

[0120] Synthesis Example 1-2: 60 g (monomer repeating unit: 1.36 mol) of PVA117 (raw PVA) was placed in a separable flask equipped with a 1 L Dimroth condenser, 540 mL of ion-exchanged water was added, and stirring was initiated with a mechanical stirrer. The mixture was heated to 80°C in a water bath, and stirring was continued at 80°C for 4 hours. After visually confirming that the raw PVA had dissolved, the temperature was lowered to 40°C. While stirring at 40°C, 2.5 g (20.5 mmol) of 5-norbornene-2-carboxaldehyde and 22 mL of a 10% by volume aqueous sulfuric acid solution were added, and the mixture was further stirred at 40°C for 4 hours. After allowing to cool, the mixture was neutralized with the addition of 80 mL of 1 N aqueous NaOH solution and desalted by passing it through a dialysis membrane with a molecular weight cutoff of 3,500 (this was carried out four times for 5 L of ion-exchanged water). The desalted aqueous solution was poured into 2 L of methanol while stirring, and allowed to stand for 1 hour. The resulting solid was collected and then further washed by immersing it in 1 L of methanol for 1 hour. The collected solid was dried in vacuum at room temperature overnight to obtain norbornenated (Nor) PVA117. The introduction rate of ethylenically unsaturated groups (norbornenyl groups) in the norbornenated PVA117 was 1.3 mol% relative to the repeating units of the raw material PVA (hereinafter referred to as "Nor-PVA117(1.3)").

[0121] Synthesis Example 1-3: 10 g (monomer repeating unit: 220 mmol) of MA-PVA117(2.0) prepared in Synthesis Example 1-1 was placed in a separable flask equipped with a 0.5 L Dimroth condenser, 90 mL of dimethyl sulfoxide (DMSO) was added, and stirring was initiated with a mechanical stirrer. The temperature was raised to 80°C in a water bath, and stirring was continued at 80°C for 4 hours. After visually confirming that the starting PVA had dissolved, the water bath was set to 60°C. After confirming that the internal temperature had reached 60°C, 1.2 g (12.1 mmol) of triethylamine and 1.1 g (11 mmol) of succinic anhydride were added, and stirring was continued for an additional 5 hours at 60°C. After allowing to cool, the reaction solution was poured into 0.5 L of methanol with stirring. Stirring was stopped and the mixture was left to stand for 1 hour. The resulting solid was collected and then immersed in 0.5 L of methanol for 1 hour to wash. This washing process was repeated three times. The collected solid was dried overnight under vacuum at room temperature to obtain methacryloylated PVA117 containing succinic acid. The succinic acid (SA) introduction rate was 3.4 mol% relative to the repeating units of MA-PVA117(2.0) (hereinafter referred to as "MA-PVA117(2.0)-SA(3.4)").

[0122] <Production of Molded Articles (Particles, Sheets)> [Synthesis Example 1-A] <Particles> 440 mL of ion-exchanged water was added to 60 g of MA-PVA117(2.0) and dissolved with stirring at 80°C for 4 hours. After cooling to room temperature, potassium persulfate, a water-soluble thermal radical polymerization initiator, was added to and dissolved in this MA-PVA117(2.0) aqueous solution to a concentration of 0.1% by mass to prepare an uncrosslinked polymer solution. 3300 mL of liquid paraffin and 8 g of Span 80 were placed in a separable flask equipped with a 5 L Dimroth condenser, and the uncrosslinked polymer solution was slowly added to the flask. This mixture was stirred at 350 rpm with a mechanical stirrer to form a W / O dispersion, which was then heated to 40°C in a water bath and purged with nitrogen for 30 minutes. Stirring was then continued at 70°C for 3 hours. After cooling to room temperature, the liquid paraffin containing the dispersed hydrogel particles was filtered through a 100 μm mesh. The obtained hydrogel particles were washed with a total of 3 L of hexane to remove the liquid paraffin, and the obtained hydrogel particles were classified using a JIS standard sieve to have a particle size of 180 to 300 μm. The hydrogel particles were further dehydrated by adding them to 1 L of acetone, and then dried under reduced pressure to obtain dried hydrogel particles (hereinafter referred to as "MA-PVA117(2.0) gel particles").

[0123] Synthesis Example 1-B Particles 60 g of Nor-PVA117(1.3) was added to 440 mL of ion-exchanged water and dissolved at 80°C for 4 hours with stirring. After cooling to room temperature, 3.4 g of 3,6-dioxa-1,8-octanedithiol (polythiol) was added to this Nor-PVA117(1.3) aqueous solution and stirred. Potassium persulfate, a water-soluble thermal radical polymerization initiator, was added to this solution to a concentration of 0.1% by mass and dissolved to prepare an uncrosslinked polymer solution. This uncrosslinked polymer solution was used to obtain dried hydrogel particles (hereinafter referred to as "Nor-PVA117(1.3) gel particles") using the same method as in Synthesis Example 1-A.

[0124] Synthesis Example 1-C Sheet: 6 g of MA-PVA117(2.0)-SA(3.4) was dissolved in 44 mL of ion-exchanged water at 80°C with stirring for 4 hours. After cooling to room temperature, potassium persulfate, a water-soluble thermal radical polymerization initiator, was added to and dissolved in this MA-PVA117(2.0)-SA(3.4) aqueous solution to a concentration of 0.1% by mass to prepare an uncrosslinked polymer solution. This uncrosslinked polymer solution was poured into two glass plates sandwiching a 0.5 mm spacer under nitrogen and cured at 40°C for 3 hours. The cured hydrogel sheet (10 x 20 cm) with a thickness of 0.5 mm was washed with a total of 1.5 L of ion-exchanged water. The sheet was then immersed in 0.5 L of acetone for dehydration and dried under reduced pressure to obtain a dried hydrogel sheet (hereinafter abbreviated as "MA-PVA117(2.0)-SA(3.4) gel sheet"). The introduction rate of succinic acid (SA) (introduction rate of active groups) in the dried PVA sheet was 3.4 mol %.

[0125] Synthesis Example 1-D Sheet: 6 g of PVA117 was dissolved in 44 mL of ion-exchanged water at 80°C for 4 hours with stirring. After cooling to room temperature, 7.5 mL of 50% glutaraldehyde (GA) solution was added to the PVA117 aqueous solution and mixed, followed by 6 mL of 1 mol / L aqueous hydrochloric acid solution, which was then rapidly mixed to prepare an uncrosslinked polymer solution. This uncrosslinked polymer solution was poured into two glass plates sandwiching a 0.5 mm spacer and cured at 65°C for 7 hours. The cured hydrogel sheet (10 x 20 cm) with a thickness of 0.5 mm was washed with a total of 1.5 L of ion-exchanged water. The sheet was then immersed in 0.5 L of acetone for dehydration and dried under reduced pressure to obtain a dried hydrogel sheet (hereinafter referred to as "GA-crosslinked PVA117 gel sheet").

[0126] <Activation of Hydroxyl Groups> [Synthesis Example 1-i] <Introduction of Succinic Acid into Molded Article (Particles)> 1.5 g of dried MA-PVA117(2.0) gel particles (monomer repeating unit: 33 mmol) prepared in Synthesis Example 1-A were placed in a separable flask equipped with a 0.5 L Dimroth condenser, and 100 mL of dimethyl sulfoxide (DMSO) was added. The mixture was stirred with a mechanical stirrer to allow swelling. After stirring in a water bath at 60°C for 1 hour, 364 mg (3.6 mmol) of triethylamine and 330 mg (3.3 mmol) of succinic anhydride were added and further stirred at 60°C for 5 hours. After allowing to cool, the particles were filtered and washed twice with 100 mL of DMSO and twice with 100 mL of ion-exchanged water. Next, the particles were dehydrated by immersion in 100 mL of acetone three times, and the resulting particles were dried under reduced pressure to obtain dried hydrogel particles into which succinic acid (SA) had been introduced. The introduction rate of succinic acid (introduction rate of active groups) was 10.9 mol % relative to the repeating units of MA-PVA117(2.0) (hereinafter abbreviated as "SA-MA-PVA117(2.0) gel particles").

[0127] [Synthesis Example 1-ii] <Introduction of succinic acid into molded article (particles)> Dried hydrogel particles into which succinic acid had been introduced were obtained in the same manner as in Synthesis Example 1-i, except that the dried Nor-PVA117(1.3) gel particles prepared in Synthesis Example 1-B were used. The introduction rate of succinic acid (introduction rate of active groups) was 8.7 mol% relative to the repeating units of Nor-PVA117(1.3) (hereinafter abbreviated as "SA-Nor-PVA117(1.3) gel particles").

[0128] Synthesis Example 1-iii: Incorporation of succinic acid into a molded article (sheet) A dried hydrogel sheet incorporating succinic acid was obtained in the same manner as in Synthesis Example 1-i, except that the dried GA-crosslinked PVA117 gel sheet prepared in Synthesis Example 1-D was used. The introduction rate of succinic acid (introduction rate of active groups) was 9.8 mol% relative to the repeating units of the GA-crosslinked PVA117 (hereinafter referred to as "SA-GA-crosslinked PVA117 gel sheet").

[0129] Synthesis Example 1-iv: Introduction of Carbonylimidazolyl Groups into Molded Articles (Particles) 377 mg (2.3 mmol) of carbonyldiimidazole (hereinafter abbreviated as "CDI") was dissolved in 7 g of dry acetonitrile, and 1.0 g of dried MA-PVA117(2.0) gel particles (monomer repeating unit: 23 mmol) prepared in Synthesis Example 1-A was added. The mixture was allowed to react in a centrifuge tube at 40°C for 4 hours while the tube was shaken, and then washed four times with 50 mL of dry acetone. The acetone-washed particles were vacuum-dried overnight at room temperature to obtain carbonylimidazolylated (CI-modified) hydrogel particles (hereinafter abbreviated as "CI-modified MA-PVA117(2.0) gel particles"). The introduction rate of carbonylimidazolyl groups (active group introduction rate) is shown in Table 1.

[0130] <Compositing> [Synthesis Example 1-i] 1 g of the dried SA-MA-PVA117(2.0) gel particles (succinic acid content: approximately 1.9 mmol) prepared in Synthesis Example 1-i was swollen overnight at room temperature in MES buffer (pH = 5.6). The swollen gel particles were dispersed in 45 mL of MES buffer, and 0.78 g (6.8 mmol) of N-hydroxysuccinimide and 0.65 g (3.4 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added while stirring with a magnetic stirrer. The mixture was then shaken at room temperature for 1 hour. The gel particles were washed three times with 30 mL of MES buffer for 10 minutes each, and then added to 70 mL of a 1 mg / mL gelatin PBS solution. The mixture was shaken at room temperature for 3 hours, and the resulting hydrogel particles were washed twice with 70 mL of ion-exchanged water (heated to 60°C) for 20 minutes to obtain gelatin-composite hydrogel particles (hereinafter referred to as "gelatin-SA-MA-PVA117(2.0) gel particles"). A portion of the gelatin-SA-MA-PVA117(2.0) gel particles was immersed overnight in excess PBS, and the amount of gelatin immobilized per 100 mg of gel weight was measured by the bicinchoninic acid (BCA) method (BCA Protein Assay Kit (Takara Bio Inc.)). The complexation density of the physiologically active substance was 48.5 μg / 100 mg of gel particles.

[0131] [Synthesis Example 1-B] Collagen composite hydrogel particles were obtained in the same manner as in Synthesis Example 1-A, except that collagen was used instead of gelatin (hereinafter abbreviated as "collagen-SA-MA-PVA117(2.0) gel particles"). The amount of collagen immobilized in the collagen-SA-MA-PVA117(2.0) gel particles (complexation density of a physiologically active substance) was also measured in the same manner as in Synthesis Example A.

[0132] [Synthesis Example 1-C] Gelatin composite hydrogel particles were obtained in the same manner as in Synthesis Example 1-A, except that 1 g of the dried SA-Nor-PVA117(1.3) gel particles prepared in Synthesis Example 1-II was used (hereinafter abbreviated as "gelatin-SA-Nor-PVA117(1.3) gel particles"). The amount of gelatin immobilized in the gelatin-SA-Nor-PVA117(2.0) gel particles (complexation density of a physiologically active substance) was also measured in the same manner as in Synthesis Example 1-A.

[0133] Synthesis Example 1-D: A gelatin-composite hydrogel sheet was obtained in the same manner as in Synthesis Example 1-A, except that 1 g of the dried MA-PVA117(2.0)-SA(3.4) gel sheet prepared in Synthesis Example 1-C was used (hereinafter abbreviated as "gelatin-MA-PVA117(2.0)-SA(3.4) gel sheet"). The amount of gelatin immobilized in the gelatin-MA-PVA117(2.0)-SA(3.4) gel sheet (composite density of a physiologically active substance) was also measured in the same manner as in Synthesis Example 1-A.

[0134] [Synthesis Example 1-E] A gelatin-composite hydrogel sheet was obtained in the same manner as in Synthesis Example 1-A, except that 1 g of the dried SA-modified-GA crosslinked PVA117 gel sheet prepared in Synthesis Example 1-III was used (hereinafter abbreviated as "gelatin-SA-modified-GA crosslinked PVA117 gel sheet"). The amount of gelatin immobilized in the gelatin-SA-modified-GA crosslinked PVA117 gel sheet (composite density of a physiologically active substance) was also measured in the same manner as in Synthesis Example 1-A.

[0135] [Synthesis Example 1-f] 0.6 g of gelatin was dissolved in 100 mL of PBS at 60°C while stirring with a magnetic stirrer. This gelatin solution was cooled to room temperature, and the CI-MA-PVA117(2.0) gel particles prepared in Synthesis Example 1-iv were added. The mixture was shaken and stirred overnight at room temperature and washed three times with 100 mL of PBS to obtain gelatinized gel particles (hereinafter referred to as "gelatin-urethane bond-MA-PVA117(2.0) gel particles"). The amount of gelatin immobilized in the gelatin-urethane bond-MA-PVA117(2.0) gel particles (complexation density of a physiologically active substance) was measured using the same method as in Synthesis Example 1-a.

[0136] <Particle Drying and Gamma-Ray Sterilization> [Example 1-1] 2 g of gelatin-SA-MA-PVA117(2.0) gel particles (swollen with ion-exchanged water) prepared in Synthesis Example 1-A were spread on a petri dish and air-dried at room temperature for 2.7 hours. The water content of the resulting dried particles was measured using a heat-drying moisture meter (manufactured by A&D Co., Ltd.) and found to be 72% by mass. The dried gel particles were placed in a 15 mL centrifuge tube and further sealed in an aluminum-coated bag to prevent water evaporation. The particles were sterilized by gamma-ray irradiation (25 kGy) at Koga Isotope Co., Ltd. to obtain an amide-bond composite hydrogel. The water content measurement results are shown in Table 1.

[0137] Examples 1-2 to 1-4: Sterilized amide bond composite hydrogels were obtained by measuring the water content and irradiating them with gamma rays in the same manner as in Example 1-1, except that 2 g of gelatin-SA-MA-PVA117(2.0) gel particles (swollen with ion-exchanged water) was allowed to air-dry for 3.7, 4.5, or 5.5 hours. The water content measurement results are shown in Table 1.

[0138] Example 1-5: 2 g of gelatin-SA-MA-PVA117(2.0) gel particles (swollen with ion-exchanged water) were spread on a petri dish and vacuum-dried overnight at room temperature. The water content was measured and gamma-ray irradiation was carried out in the same manner as in Example 1-1, yielding a sterilized amide bond composite hydrogel. The water content measurement results are shown in Table 1.

[0139] [Example 1-6] 2 g of gelatin-SA-MA-PVA117(2.0) gel particles (swollen with ion-exchanged water) prepared in Synthesis Example 1-A were immersed in 25 mL of ethanol three times, and the dehydrated composite gel particles were vacuum-dried overnight at room temperature. The water content of the resulting dried particles was 1 The water content was calculated by H-NMR (deuterated DMSO solvent). The water content was calculated from the ratio of the integrated values ​​of the water signal (3.3 ppm) and the vinyl alcohol polymer signal (water originally contained in the deuterated DMSO solvent was subtracted). Gamma ray irradiation was carried out in the same manner as in Example 1-1 to obtain a sterilized amide bond composite hydrogel. The water content measurement results are shown in Table 1.

[0140] Example 1-7 2 g of collagen-SA-MA-PVA117(2.0) gel particles (swollen with ion-exchanged water) prepared in Synthesis Example 1-B were dried, the water content was measured, and the particles were irradiated with gamma rays in the same manner as in Example 1-6 to obtain a sterilized amide bond composite hydrogel. The water content measurement results are shown in Table 1.

[0141] Example 1-8 A sterilized amide bond composite hydrogel was obtained by drying, measuring the water content, and irradiating with gamma rays in the same manner as in Example 1-5, except that the gelatin-SA-Nor-PVA117(1.3) gel particles (swollen with ion-exchanged water) prepared in Synthesis Example 1-C were used. The water content measurement results are shown in Table 1.

[0142] Example 1-9: A sterilized amide bond composite hydrogel was obtained by drying, measuring the water content, and irradiating with gamma rays in the same manner as in Example 1-5, except that the gelatin-MA-PVA117(2.0)-SA(3.4) gel sheet (swollen with ion-exchanged water) prepared in Synthesis Example 1-D was used. The water content measurement results are shown in Table 1.

[0143] Example 1-10 A sterilized amide bond composite hydrogel was obtained by drying, measuring the water content, and irradiating with gamma rays in the same manner as in Example 1-5, except that the gelatin-SA-GA crosslinked PVA117 gel sheet (swollen with ion-exchanged water) prepared in Synthesis Example 1-E was used. The water content measurement results are shown in Table 1.

[0144] [Example 1-11] A sterilized composite hydrogel was obtained by drying, measuring the water content, and irradiating with gamma rays in the same manner as in Example 1-6, except that the gelatin-urethane bond-MA-PVA117(2.0) gel particles (swollen with ion-exchanged water) prepared in Synthesis Example 1-G were used. The water content measurement results are shown in Table 1.

[0145] [Method for Evaluating Composite Hydrogels Obtained in Examples and Comparative Examples] <Evaluation of Cell Adhesion (Particles)> Poly(2-hydroxyethyl methacrylate) was dissolved in 95% ethanol at a concentration of 30 mg / mL, and 200 μL of this solution was dispensed into each well of a 24-well cell culture plate (manufactured by IWAKI) and dried in a clean bench. Next, the sterilized amide-bonded composite hydrogel (spherical particles) obtained in Example 1-1 was immersed in PBS (phosphate-buffered saline) for 1 hour to allow the particles to swell. 200 μL of these gel particles was placed in one well of the coated well plate, and 500 μL of DMEM medium supplemented with 10% fetal bovine serum was added to the same well. NIH / 3T3 cells (purchased from ATCC) that had been grown by pre-culture were added to the well at a density of 1.9 × 10 5 The cells were cultured under conditions of 5% carbon dioxide concentration, saturated water vapor pressure, and 37°C. After 24 hours of culture, photographs of the center of the culture well were taken using an inverted microscope with a camera and image linking software, and the number of particles with cells adhering and spreading was counted. Cells were observed adhering and spreading on 132 of the 200 gel particles. The cell adhesion rate (%) in this case was calculated as the percentage of the number of particles with cells adhering and spreading out among the 200 particles. The cell adhesion rate was 66% (Table 1).

[0146] The cell adhesiveness of the sterilized composite hydrogels (spherical particles) obtained in Examples 1-2 to 1-8 and 1-11 and Comparative Examples 1-1 and 1-2 was evaluated in the same manner as in the cell adhesiveness evaluation (particles) described above. The results are shown in Table 1.

[0147] <Evaluation of Cell Adhesion (Sheet)> The sterilized amide bond composite hydrogel (sheet) obtained in Examples 1-9 was immersed in PBS for 1 hour to swell the gel sheet, and then a circular shape was punched out of the gel sheet using a 15 mm diameter hole punch, which was then placed on the bottom of a 24-well plate. 500 μL of DMEM medium supplemented with 10% fetal bovine serum was added to the same well. Also, in the same plate, wells were prepared to which 500 μL of medium alone was added without a gel sheet. Next, NIH / 3T3 cells grown by pre-culture were added at 1.9 × 10 cells per well to wells containing a gel sheet and wells without a gel sheet. 5 The wells were cultured under conditions of 5% carbon dioxide, saturated water vapor pressure, and 37°C. After 24 hours of culture, the medium in the wells was removed with an aspirator, 500 μL of fresh medium was added, and 50 μL of Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Inc.), a cell proliferation / cytotoxicity measurement reagent, was added. At the same time, 500 μL of fresh medium and 50 μL of CCK-8 were added to unused wells that did not contain gel sheets or cells, and the well plate was cultured for 1 hour under conditions of 5% carbon dioxide, saturated water vapor pressure, and 37°C. After culture, 100 μL of the medium containing CCK-8 from each well was transferred to a 96-well plate, and the absorbance at 450 nm was measured using a microplate reader (ARVO, PerkinElmer). The absorbance obtained from the well containing only cells was defined as a cell adhesion rate of 100%, and the absorbance obtained from the well containing only medium was defined as a cell adhesion rate of 0%. The cell adhesion rate was calculated from the absorbance obtained from the well containing the gel sheet and cells, and was found to be 90% (Table 1).

[0148] When the sterilized amide bond composite hydrogel obtained in Example 1-10 was evaluated for cell adhesion in the same manner as in Example 1-9, the cell adhesion rate was 87% (Table 1).

[0149]

[0150] As is clear from the results of Examples 1-1 to 1-11 and Comparative Examples 1-1 and 1-2, according to the present invention, even when irradiated with high-energy gamma rays, physiologically active substances maintain their activity and can promote cell adhesion and even cell proliferation.

[0151] [Synthesis Examples] <Synthesis of a vinyl alcohol polymer having an ethylenically unsaturated group> [Synthesis Example 2-1] 40 g (monomer repeating unit: 908 mmol) of PVA117 (raw PVA) was placed in a separable flask equipped with a 1 L Dimroth condenser, 350 mL of dimethyl sulfoxide (DMSO) was added, and stirring was initiated with a mechanical stirrer. The temperature was raised to 80°C in a water bath, and stirring was continued at 80°C for 4 hours. After visually confirming that the raw PVA had dissolved, 1.2 g (10.9 mol) of vinyl methacrylate was added while heating and stirring at 80°C, and stirring was continued for an additional 3 hours at 80°C. After allowing to cool, the reaction solution was poured into 2 L of methanol while stirring. Stirring was stopped and the mixture was left as is for 1 hour. The resulting solid was collected and then immersed in 1 L of methanol for another 1 hour to wash. This washing process was repeated a total of three times. The recovered solid was dried in vacuo at room temperature overnight to obtain methacryloylated PVA 117. The introduction rate of ethylenically unsaturated groups (methacryloyl groups) in this methacryloylated PVA 117 was 1.2 mol % relative to the repeating units of the raw material PVA (hereinafter referred to as "MA-PVA117(1.2)").

[0152] Synthesis Examples 2-2 to 2-3 As shown in Table 2, vinyl alcohol polymers having an ethylenically unsaturated group were produced in the same manner as in Synthesis Example 2-1, except that the amount of vinyl methacrylate added was changed.

[0153] Synthesis Example 2-4 As shown in Table 2, a vinyl alcohol polymer having an ethylenically unsaturated group was produced in the same manner as in Synthesis Example 2-1, except that a raw material PVA (AF-17) in which itaconic acid had been copolymerized and a carboxy group had been introduced was used.

[0154] Synthesis Example 2-5: 10 g (monomer repeating unit: 220 mmol) of MA-PVA117(2.0) prepared in Synthesis Example 2-2 was placed in a separable flask equipped with a 0.5 L Dimroth condenser, 90 mL of dimethyl sulfoxide (DMSO) was added, and stirring was initiated with a mechanical stirrer. The temperature was raised to 80°C in a water bath, and stirring was continued at 80°C for 4 hours. After visually confirming that the starting PVA had dissolved, the water bath was set to 60°C. After confirming that the internal temperature had reached 60°C, 1.2 g (12.1 mmol) of triethylamine and 1.1 g (11 mmol) of succinic anhydride were added, and stirring was continued for an additional 5 hours at 60°C. After allowing to cool, the reaction solution was poured into 0.5 L of methanol with stirring. Stirring was stopped and the mixture was left to stand for 1 hour. The resulting solid was collected and then immersed in 0.5 L of methanol for 1 hour to wash. This washing process was repeated three times. The collected solid was dried overnight under vacuum at room temperature to obtain methacryloylated PVA117 containing succinic acid. The succinic acid (SA) introduction rate was 3.4 mol% relative to the repeating units of MA-PVA117(2.0) (hereinafter referred to as "MA-PVA117(2.0)-SA(3.4)").

[0155] Synthesis Example 2-6: 60 g (monomer repeating unit: 1.36 mol) of PVA117 (raw PVA) was placed in a separable flask equipped with a 1 L Dimroth condenser, 540 mL of ion-exchanged water was added, and stirring was initiated with a mechanical stirrer. The temperature was raised to 80°C in a water bath, and stirring was continued at 80°C for 4 hours. After visually confirming that the raw PVA had dissolved, the temperature was lowered to 40°C. While stirring at 40°C, 2.5 g (20.5 mmol) of 5-norbornene-2-carboxaldehyde and 22 mL of a 10% by volume aqueous sulfuric acid solution were added, and the mixture was further stirred at 40°C for 4 hours. After allowing to cool, the mixture was neutralized by adding 80 mL of 1 N aqueous NaOH solution and desalted by placing it in a dialysis membrane with a molecular weight cutoff of 3,500 (this was carried out four times for 5 L of ion-exchanged water). The desalted aqueous solution was poured into 2 L of methanol while stirring, and allowed to stand for 1 hour. The resulting solid was collected and then further washed by immersing it in 1 L of methanol for 1 hour. The collected solid was dried in vacuum at room temperature overnight to obtain norbornenated PVA 117. The introduction rate of ethylenically unsaturated groups (norbornenyl groups (Nor)) in this norbornenated PVA 117 was 1.3 mol % relative to the repeating units of the raw material PVA (hereinafter referred to as "Nor-PVA117(1.3)").

[0156] <Production of Molded Products (Particles, Sheets, Coatings, Tubes)> [Synthesis Example 2-A] <Particles> 440 mL of ion-exchanged water was added to 60 g of MA-PVA117(1.2) and dissolved with stirring at 80°C for 4 hours. After cooling to room temperature, potassium persulfate, a water-soluble thermal radical polymerization initiator, was added to and dissolved in this MA-PVA117(2.0) aqueous solution to a concentration of 0.1% by mass to prepare an uncrosslinked gel solution. 3300 mL of liquid paraffin and 8 g of Span 80 were placed in a separable flask equipped with a 5 L Dimroth condenser, and the uncrosslinked gel solution was slowly added to the flask. This mixture was stirred at 350 rpm with a mechanical stirrer to form a W / O dispersion, which was then heated to 40°C in a water bath and purged with nitrogen for 30 minutes. Stirring was then continued at 70°C for 3 hours. After cooling to room temperature, the liquid paraffin containing the dispersed hydrogel particles was filtered through a 100 μm mesh. The resulting hydrogel particles were washed with a total of 3 L of hexane to remove the liquid paraffin, and the resulting hydrogel particles were classified using a JIS standard sieve to have particle sizes of 180 to 300 μm. The hydrogel particles were then further dehydrated by adding 1 L of acetone, and then dried overnight at room temperature under reduced pressure to obtain dried hydrogel particles (hereinafter referred to as "MA-PVA117(1.2) gel particles").

[0157] [Synthesis Examples 2-B to 2-E] <Particles> As shown in Table 3, methacryloylated PVA was produced in the same manner as in Synthesis Example 2-1, except that the vinyl alcohol polymers having an ethylenically unsaturated group synthesized in Synthesis Examples 2-2 to 2-5 were used.

[0158] [Synthesis Example 2-F] <Particles> In Synthesis Example 2-A, 440 mL of ion-exchanged water was added to 60 g of Nor-PVA117(1.3), and the mixture was stirred at 80°C for 4 hours to dissolve the mixture. After cooling to room temperature, 3.4 g of 3,6-dioxa-1,8-octanedithiol (polythiol) was added to the aqueous solution of Nor-PVA117(1.3) and stirred. Potassium persulfate, a water-soluble thermal radical polymerization initiator, was added to the solution at 0.1% by mass and dissolved to prepare an uncrosslinked gel solution. This uncrosslinked gel solution was used to obtain dried hydrogel particles (hereinafter referred to as "Nor-PVA117(1.3) gel particles") using the same method as in Synthesis Example 2-A.

[0159] Synthesis Example 2-G (Sheet) 6 g of MA-PVA117(2.0)-SA(3.4) was dissolved in 44 mL of ion-exchanged water at 80°C with stirring for 4 hours. After cooling to room temperature, potassium persulfate, a water-soluble thermal radical polymerization initiator, was added to the MA-PVA117(2.0) aqueous solution to a concentration of 0.1% by mass and dissolved to prepare an uncrosslinked gel solution. This uncrosslinked gel solution was poured into two glass plates sandwiching a 0.5 mm spacer under nitrogen and cured at 40°C for 3 hours. The cured hydrogel sheet (10 x 20 cm) with a thickness of 0.5 mm was washed five times with 300 mL of ion-exchanged water. It was then dehydrated by immersion in 1 L of acetone and dried overnight under reduced pressure at room temperature to obtain a dried hydrogel sheet (hereinafter abbreviated as "MA-PVA117(2.0)-SA(3.4) gel sheet").

[0160] Synthesis Example 2-H Coating 100 mL of ion-exchanged water was added to 0.15 g of MA-PVA117(2.0)-SA(3.4), and the mixture was dissolved by stirring at 80°C for 4 hours. This solution was cooled to room temperature to prepare an uncrosslinked gel solution. This uncrosslinked gel solution was added to a 6-well polystyrene cell culture plate (adherent cell culture plate, manufactured by Sumitomo Bakelite Co., Ltd.) at 1 mL / well, and dried at room temperature for 3 days. 1 mL / well of a 0.1 wt% Omnicure 2959 methanol solution was added, and the plate was irradiated with UV light (103 mW / cm as measured by UV intensity using a metal halide lamp, UVR-T1 (UD-T36), manufactured by GS Yuasa). 2, 520 mJ / cm 2 The methanol solution was removed, and the plate was washed twice with 2 mL of ion-exchanged water to obtain a 6-well plate coated with a hydrogel (hereinafter referred to as "MA-PVA117(2.0)-SA(3.4) gel coating").

[0161] <Activation of Hydroxyl Groups> [Synthesis Example 2-i] <Introduction of Succinic Acid into Molded Article (Particles)> 1.5 g of dried MA-PVA117(1.2) gel particles (monomer repeating unit: 33 mmol) prepared in Synthesis Example 2-A were placed in a separable flask equipped with a 0.5 L Dimroth condenser, and 100 mL of dimethyl sulfoxide (DMSO) was added. The mixture was stirred with a mechanical stirrer to allow swelling. After stirring in a water bath at 60°C for 1 hour, 364 mg (3.6 mmol) of triethylamine and 330 mg (3.3 mmol) of succinic anhydride were added and further stirred at 60°C for 5 hours. After allowing to cool, the particles were filtered and washed twice with 100 mL of DMSO and twice with 100 mL of water. Next, the particles were dehydrated by immersion in 100 mL of acetone three times, and the resulting particles were dried under reduced pressure to obtain dried hydrogel particles incorporating succinic acid. The introduction rate of carboxyl groups (succinic acid) (introduction density of carboxyl groups) was 9.9 mol % relative to the repeating units of MA-PVA117(1.2) (hereinafter referred to as "SA-MA-PVA117(1.2) gel particles").

[0162] [Synthesis Examples 2-ii to 2-iv] <Introduction of succinic acid into molded article (particles)> Dried hydrogel particles into which succinic acid had been introduced were obtained in the same manner as in Synthesis Example 2-i, except that the gel particles prepared in Synthesis Examples 2-B to 2-C and 2-F were used, as shown in Table 4. Table 4 also shows the results of the introduction rate of carboxy groups (succinic acid) (introduction density of carboxy groups).

[0163] <Compositing> [Example 2-1] 1 g of dried SA-MA-PVA117(1.2) gel particles (succinic acid content: approximately 1.9 mmol) prepared in Synthesis Example 2-i were swollen overnight at room temperature in MES buffer (pH = 5.6). The swollen gel particles were dispersed in 45 mL of MES buffer, and 0.78 g (6.8 mmol) of N-hydroxysuccinimide and 0.65 g (3.4 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added while stirring with a magnetic stirrer. The mixture was then shaken at room temperature for 1 hour. The gel particles were washed three times with 30 mL of MES buffer for 10 minutes each, and then added to 70 mL of a 1 mg / mL gelatin PBS solution. The resulting hydrogel particles were shaken at room temperature for 3 hours, and then washed twice with 70 mL of ion-exchanged water (heated to 60°C) for 20 minutes. They were then immersed in 50 mL of ethanol three times and then vacuum-dried overnight at room temperature to obtain dried amide-linked gelatin composite hydrogel particles (hereafter referred to as "gelatin-SA-MA-PVA117(1.2) gel particles"). A portion of the gelatin-SA-MA-PVA117(1.2) gel particles was immersed overnight in excess PBS, and the amount of gelatin immobilized per 100 mg of gel (i.e., the bioactive substance composite density) was measured using the bicinchoninic acid (BCA) method (BCA Protein Assay Kit, manufactured by Takara Bio Inc.). The result was 48.2 μg / 100 mg of gel particles.

[0164] Examples 2-2 to 2-6 Amide-bonded gelatin composite hydrogel particles were obtained in the same manner as in Example 2-1, except that the carboxyl group-introduced hydrogel particles of Synthesis Examples 2-ii to 2-iv, 2-D, and 2-E were used. The amount of gelatin immobilized (complexation density of physiologically active substance) was measured by the BCA method, and the results are shown in Table 5.

[0165] Example 2-7 Collagen-composite PVA particles were obtained in the same manner as in Example 2-1, except that collagen was used instead of gelatin (hereinafter abbreviated as "collagen-SA-MA-PVA117(1.2) gel particles"). The amount of gelatin immobilized (composite density of the physiologically active substance) was measured by the BCA method and found to be 53.2 μg / 100 mg of gel particles.

[0166] Example 2-8 Gelatin Composite Sheet An amide-bonded gelatin composite hydrogel sheet was obtained in the same manner as in Example 2-1, except that 1 g of the dried MA-PVA117(2.0)-SA(3.4) gel sheet prepared in Synthesis Example 2-G was used (hereinafter abbreviated as "gelatinized-MA-PVA117(2.0)-SA(3.4) gel sheet"). The amount of gelatin immobilized (complexation density of the physiologically active substance) was measured by the BCA method, and was found to be 20.0 μg / 100 mg of gel particles.

[0167] Example 2-9 Gelatin Complex Coating 251 mg (2.2 mmol) of N-hydroxysuccinimide was dissolved in 25 mL of MES buffer, followed by the addition of 425 mg (2.2 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and stirring to prepare a condensing agent solution. 1 mL of this condensing agent solution was added to the MA-PVA117(2.0)-SA(3.4) gel coating prepared in Synthesis Example 2-H, and the wells were shaken at room temperature for 1 hour. The wells were washed twice with 1 mL of MES buffer for 5 minutes to remove the MES buffer. 1 mL / well of a 1 mg / mL gelatin PBS solution was added, and the wells were shaken at room temperature for 3 hours. The wells were then washed twice with 2 mL of ion-exchanged water (heated to 60°C) for 5 minutes to obtain an amide-bonded gelatin complexed hydrogel coating (hereinafter referred to as "gelatin-SA-MA-PVA117(2.0) gel coating"). The amount of gelatin immobilized (complexed density of the physiologically active substance) was measured by the BCA method and found to be 16.6 μg / well.

[0168] Comparative Example Comparative Synthesis Example 2-1 1.0 g of dried MA-PVA117(2.0) gel particles (monomer repeating unit: 23 mmol) prepared in Synthesis Example 2-B were added to 45 mL of ion-exchanged water and swollen by shaking in a centrifuge tube. 30 μL of aminoacetaldehyde dimethyl acetal (0.28 mmol) and 2 L of 17% by weight aqueous sulfuric acid solution were added, and the mixture was stirred and shaken at 40°C for 10 hours. The particles were filtered and washed five times with 100 mL of dry acetone. The particles were then washed four times with 50 mL of acetone and vacuum-dried overnight at room temperature to obtain aminated hydrogel particles. The amino group introduction rate (amino group introduction density) relative to the MA-PVA117(1.2) repeating unit was 7.3 mol% (hereinafter referred to as "aminated-MA-PVA117(2.0) gel particles").

[0169] Comparative Synthesis Example 2-2: GA Crosslinked Particles These particles were produced with reference to Journal of Applied Science, 2013, Vol. 13, pp. 2676-2681. 3,300 mL of liquid paraffin and 8 g of Span 80 were placed in a 5 L separable flask equipped with a Dimroth condenser and stirred at 200 rpm with a mechanical stirrer. Next, 440 mL of ion-exchanged water was added to 60 g of AF-17, and the mixture was dissolved with stirring at 80°C for 4 hours. After cooling to room temperature, 75 g of a 50% aqueous solution of glutaraldehyde (hereinafter abbreviated as "GA") and 60 mL of a 1 mol / L aqueous solution of hydrochloric acid were added to this AF-17 solution, and the mixture was immediately added to the liquid paraffin. The resulting mixture was heated to 65°C in a water bath and stirred for 7 hours. After cooling to room temperature, the liquid paraffin in which the hydrogel particles were dispersed was filtered through a mesh with 100 μm openings. The obtained hydrogel particles were washed with a total of 3 L of hexane to remove the liquid paraffin, and the obtained hydrogel particles were classified using a JIS standard sieve to have particle sizes of 180 to 300 μm. The hydrogel particles were then added to 1 L of acetone for dehydration, and then dried overnight under reduced pressure at room temperature to obtain dried hydrogel particles (hereinafter abbreviated as "GA crosslinked AF-17 gel particles").

[0170] Comparative Synthesis Example 2-3: Introduction of carbonylimidazolyl groups into molded article (particles) 377 mg (2.3 mmol) of carbonyldiimidazole (hereinafter abbreviated as "CDI") was dissolved in 7 g of dry acetonitrile, and 1.0 g of dried MA-PVA117(2.0) gel particles (monomer repeating unit: 23 mmol) prepared in Synthesis Example 2-B was added to the solution. The mixture was allowed to react in a centrifuge tube at 40°C for 4 hours while the tube was shaken, and then the particles were washed four times with 50 mL of dry acetone. The acetone-washed particles were vacuum-dried overnight at room temperature to obtain carbonylimidazolylated (CI-modified) hydrogel particles (hereinafter abbreviated as "CI-modified-MA-PVA117(2.0) gel particles").

[0171] Comparative Example 2-1: 1 g of dried aminated-MA-PVA117(2.0) gel particles prepared in Comparative Synthesis Example 2-1 was swollen overnight at room temperature in 45 mL of PBS buffer (pH = 7.4). Next, 70 mL of a 1 mg / mL gelatin MES buffer solution was stirred with a magnetic stirrer, and 0.78 g (6.8 mmol) of N-hydroxysuccinimide and 0.65 g (3.4 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added. The mixture was then shaken at room temperature for 1 hour to prepare an activated gelatin solution. The aminated-MA-PVA117(2.0) gel particles swollen in the PBS buffer were filtered, and the gel particles were added to the activated gelatin solution and stirred with a magnetic stirrer at room temperature for 3 hours. The resulting hydrogel particles were washed twice with 70 mL of ion-exchanged water (heated to 60°C) for 20 minutes to obtain amide-bonded gelatin composite hydrogel particles (hereinafter referred to as "gelatin-aminated-MA-PVA117(2.0) gel particles"). A portion of the gelatin-aminated-MA-PVA117(2.0) gel particles was immersed overnight in excess PBS, and the amount of gelatin immobilized (complexation density of the physiologically active substance) was measured by the BCA method, which was found to be 5.1 μg / 100 mg of gel particles.

[0172] [Comparative Example 2-2] Amide-linked gelatin composite hydrogel particles were obtained in the same manner as in Example 2-1, except that GA-crosslinked AF-17 gel particles were used instead of SA-MA-PVA117(1.2) gel particles (hereinafter abbreviated as "gelatin-GA-crosslinked PVA117 gel particles"). The amount of gelatin immobilized (complexation density of the physiologically active substance) was measured by the BCA method and found to be 1.5 μg / 100 mg of gel particles.

[0173] Comparative Example 2-3: 0.6 g of gelatin was dissolved in 100 mL of PBS at 60°C while stirring with a magnetic stirrer. This gelatin solution was cooled to room temperature, and the CI-MA-PVA117(2.0) gel particles prepared in Comparative Synthesis Example 2-3 were added. The mixture was shaken and stirred overnight at room temperature, and then washed three times with 100 mL of PBS to obtain gelatinized gel particles (hereinafter referred to as "gelatin-CI-MA-PVA117(2.0) gel particles"). The amount of gelatin immobilized (complexation density of the physiologically active substance) was measured in the same manner as in Example 2-1, and was found to be 7.2 μg / 100 mg of gel particles.

[0174] [Method for Evaluating Amide Bond Conjugated Hydrogels Obtained in Examples and Comparative Examples] <Evaluation of Cell Adhesion (Cell Proliferation Rate)> 30 mg of the dried SA-MA-PVA117(1.2) gel particles obtained in Example 2-1 were immersed in 5 mL of PBS overnight, and the swelling degree (swollen weight / dry weight) and average particle size when swollen were measured, and the total surface area when swollen per 1 g of dry particles was calculated. Next, 30 mL of DMEM medium supplemented with 10% fetal bovine serum was added to a 125 mL spinner flask (manufactured by Corning), and the dried SA-MA-PVA117(1.2) gel particles obtained in Example 2-1 were immersed in 5 mL of PBS overnight. The total surface area was then calculated based on the calculated total surface area. 2 Furthermore, NIH / 3T3 cells (purchased from ATCC) grown in advance were added to the solution at a concentration of 8.1 × 10 5The gel particles were added and cultured in an incubator with a carbon dioxide concentration of 5%, saturated water vapor pressure, and 37°C, while stirring at a paddle speed of 60 rpm. On the fourth day of culture, all gel particles were collected and washed with PBS. After that, the cells were detached from the gel particles by trypsin treatment. The cell density of the resulting cell suspension was counted using a hemocytometer, and the number of cells after culture was calculated. The cell proliferation rate was defined as the number of cells after culture divided by the initial cell number, and the cell proliferation rate of the gel particles obtained in Example 2-1 was 10.6. The cell proliferation rates of the gel particles of Examples 2-2 to 2-7 and Comparative Examples 2-1 to 2-3 were measured using a similar method, and the results are shown in Table 5.

[0175] The cell proliferation rate of the gelatinized-MA-PVA117(2.0)-SA(3.4) gel sheet obtained in Example 2-8 was measured by the following method. The gel sheet was immersed in PBS overnight to swell, and then circular shapes were punched out of the gel sheet using a 34 mm diameter hole punch. These were then placed on the bottom of a 6-well polystyrene cell culture plate. 3 mL of medium was added to each well, and 4.9 x 10 NIH / 3T3 cells that had been grown in pre-culture were added. 6 The cells were added and cultured in an incubator with a carbon dioxide concentration of 5% and saturated water vapor pressure at 37°C. On the fourth day of culture, the cells were detached and collected from the surface of the gel sheet by trypsin treatment, and the number of cells after culture was counted to calculate the cell proliferation rate, which was found to be 4.3.

[0176] The cell proliferation rate of the gelatin-SA-MA-PVA117(2.0) gel coating obtained in Example 2-9 was measured by the following method. 3 mL of medium was added to each well of a gel-coated polystyrene 6-well cell culture plate, and 4.9 × 10 NIH / 3T3 cells, which had been grown by pre-culture, were added to each well. 6 The cells were added and cultured in an incubator with a carbon dioxide concentration of 5% and saturated water vapor pressure at 37°C. On the fourth day of culture, the cells were detached and collected from the well plate by trypsin treatment, and the number of cells after culture was counted to calculate the cell proliferation rate, which was found to be 5.8.

[0177] As is clear from the results of Examples 2-1 to 2-9 and Comparative Examples 2-1 to 2-3, according to the present invention, the physiologically active polypeptide maintains its activity even when irradiated with high-energy gamma rays, and is able to promote cell proliferation.

[0178]

[0179] Sterilization Validation (VD max Example 3 Gelatin-composite hydrogel particles were obtained in the same manner as in Synthesis Example 1-A, except that the procedure was carried out in a class 10,000 clean room, all buffers and ion-exchanged water were sterilized by filtration using a 0.2 μm pore membrane filter, and 30 g of the dried SA-MA-PVA117(2.0) gel particles prepared in Synthesis Example 1-i were used (hereinafter referred to as "gelatin-SA-MA-PVA117(2.0) gel particles (clean room)"). The amount of gelatin immobilized per 100 mg of gel weight was measured using the bicinchoninic acid (BCA) method, and the bioactive substance complexation density was found to be 56.3 μg / 100 mg of gel particles. The resulting swollen gel particles were dried in the same manner as in Example 1-6. The water content measured in the same manner as in Example 1-6 was 0.1 wt%.

[0180] VD of the dried hydrogel particles obtained above max 25 Specifically, 1 g of the dried hydrogel particles was placed in a sterilized 25 mL centrifuge tube, and the tube was closed to prepare 25 tubes of dried hydrogel particles. The average bioburden of 10 tubes was measured and found to be 7.6. The VD corresponding to an average bioburden of 8.0 was max 25 When 10 tubes were irradiated with 6.9 kGy, which is a gamma radiation dose of 1000 kJ / kg, and a sterility test was performed, one of the 10 tubes was positive (SAL = 10 -1 From this result, the sterilization dose is 25 kGy, so five dried hydrogel particles placed in a container are irradiated with gamma rays of 25 kGy or more, and SAL = 10 -6 Sterilized dry hydrogel particles filled with HCl were obtained.

[0181] In a first embodiment, the dry hydrogel-forming article of the present invention is sterilized, and therefore is free from the risk of microbial infection, promotes cell adhesion, and allows efficient cell proliferation or induction into tissues or organs. Therefore, the dry hydrogel-forming article of the present invention is useful as a carrier or support. In a second embodiment, the hydrogel of the present invention efficiently complexes a physiologically active substance, and therefore allows efficient cell proliferation or induction into tissues or organs. Therefore, the hydrogel of the present invention is useful as a carrier or support. Therefore, a carrier made of the dry hydrogel-forming article of the present invention and a carrier containing the hydrogel of the present invention can be suitably used for applications such as enzyme immobilization carriers, affinity carriers, cell culture carriers, and drug delivery carriers.

Claims

1. A sterilized, dry hydrogel-forming article comprising a crosslinked vinyl alcohol polymer complexed with a physiologically active substance.

2. 2. The dry hydrogel-forming article according to claim 1, wherein the physiologically active substance is complexed with the crosslinked vinyl alcohol polymer by a covalent bond.

3. 2. The dry hydrogel-forming article according to claim 1, wherein the water content of the dry hydrogel-forming article is 75% by mass or less.

4. The dry hydrogel-forming article according to claim 1, wherein the dry hydrogel-forming article is an irregular particle, a spherical particle, a micromolded body, an arbitrarily shaped article formed by a 3D printer, a film, a thread, a hollow fiber, a porous monolith, or a coated article.

5. The sterility assurance level (SAL) of the dried hydrogel-forming article is 10 -3 2. The dried hydrogel-forming article of claim 1, wherein:

6. 6. A method for producing a dried hydrogel-forming article according to claim 1, wherein the dried hydrogel-forming article is sterilized.

7. The method for producing a dry hydrogel-forming article according to claim 6, wherein the sterilization method is radiation sterilization.

8. The method for producing a dry hydrogel-forming article according to claim 7, wherein the radiation exposure dose is 8.2 kGy or more.

9. The method for producing a dry hydrogel-forming article according to claim 7, wherein the dry hydrogel-forming article is irradiated with radiation after being placed in a container.

10. The dry hydrogel-forming article according to any one of claims 1 to 5, which is housed in a container.

11. A hydrogel comprising a crosslinked vinyl alcohol polymer having an ethylenically unsaturated group and a carboxy group, and a physiologically active substance having an amino group, wherein the carboxy group of the crosslinked polymer and the amino group of the physiologically active substance are covalently bonded via an amide bond.

12. The hydrogel according to claim 11, wherein the ethylenically unsaturated group is at least one selected from the group consisting of a vinyl group, a (meth)acryloyl group, a (meth)acryloylamino group, a vinylphenyl group, a norbornenyl group, and derivatives thereof.

13. 13. The hydrogel according to claim 11, wherein the introduction rate of the ethylenically unsaturated group is 0.01 to 10 mol % of all structural units constituting the vinyl alcohol-based polymer.

14. 13. The hydrogel according to claim 11, wherein the introduction rate of the carboxyl group is 0.1 to 50 mol % of all structural units constituting the vinyl alcohol-based polymer.

15. The hydrogel according to claim 11 or 12, wherein the hydrogel is an amorphous particle, a spherical particle, a micromolded body, an arbitrarily shaped article formed by a 3D printer, a film, a thread, a hollow fiber, a porous monolith, or a coated article.