Dry hydrogel-forming article, hydrogel, and method for producing dry hydrogel-forming article

Abstract

Provided is a dry hydrogel-forming article which contains a crosslinked body of a vinyl alcohol polymer and an alcohol solvent, and which is characterized in that the content of the alcohol solvent is 0.01-15 mass% inclusive and the content of water is 0-70 mass% inclusive, based on the amount of the dry hydrogel-forming article. The dry hydrogel-forming article has a sterility assurance level SAL of 1 * 10 <-6 > or less and an aggregation degree of 10% or less.

Description

Technical Field

[0001] The present invention relates to a dry hydrogel-forming article, a hydrogel, and a method for producing a dry hydrogel-forming article. Background Art

[0002] Polyvinyl alcohol (hereinafter sometimes referred to 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 referred to as complexed hydrogels) exhibit unique functions, with applications such as enzyme immobilization supports (e.g., Non-Patent Document 1), affinity supports (e.g., Non-Patent Document 2), cell culture substrates (e.g., Non-Patent Document 3), vascular embolization particles (e.g., Patent Document 1), and cell culture supports (e.g., Patent Document 2) being proposed.

[0003] In particular, the hydrogels must be sterilized for use in the manufacture or manufacture of pharmaceuticals and medical devices for human use. If unsterilized hydrogels are used in these applications, residual bacteria can cause infection and potentially lead to fatal damage to organisms or cells.

[0004] As methods for sterilizing hydrogels, for example, the aforementioned prior art discloses irradiating the composite hydrogel with UV light for several hours or immersing it in an aqueous ethanol solution for several hours before using it for cell culture (e.g., Non-Patent Documents 3-5). Furthermore, attempts have been made to sterilize the hydrogel-forming composition before crosslinking (Patent Document 3).

[0005] The PVA contained in such a hydrogel is cross-linked. As cross-linking methods, for example, a method of covalently bonding a hydrogel chemically cross-linked by glutaraldehyde to an enzyme using carbonyldiimidazole is known (for example, non-patent document 1); a method of covalently bonding PVA into which a carboxyl group has been introduced to a cell-adhesive protein (for example, non-patent documents 3 and 4); a method of producing a hydrogel covalently bonded with a cell-adhesive peptide by cross-linking the hydrogel by initiating free radical polymerization by introducing acrylamide groups into the PVA (for example, non-patent documents 6 and 7); and a method of obtaining PVA particles by free radical polymerization and cross-linking PVA having a (meth)acryloyl group, and covalently bonding cell-adhesive gelatin to the PVA particles (for example, patent document 2).

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: Japanese Patent Application No. 2002-527206

[0009] Patent Document 2: International Publication No. 2020 / 105708

[0010] Patent Document 3: International Publication No. 2020 / 153382

[0011] Non-patent literature

[0012] Non-patent document 1: Food Chemistry, 2001, Vol. 74, pp. 281-288

[0013] Non-patent document 2: Biotechnology Techniques, 1997, Vol. 11, pp. 67-70

[0014] Non-patent document 3: Journal of Biomedical Materials Research, 2001, Vol. 57, pp. 217-223

[0015] Non-patent document 4: Journal of Polymer Engineering, 2017, Vol. 37, pp. 647-660

[0016] Non-patent document 5: Journal of Applied Biomaterials, 1991, Vol. 2, pp. 261-267

[0017] Non-patent document 6: Biomaterials, 2002, Vol. 23, pp. 4325-4332

[0018] Non-patent document 7: Biomaterials, 2012, Vol. 33, pp. 3880-3886 Summary of the Invention

[0019] Problems to be solved by the invention

[0020] As a sterility assurance level (hereinafter sometimes referred to as SAL) sufficient for human body etc., it is preferable to achieve SAL=1×10 -6 However, when the hydrogel is sterilized by UV light irradiation or immersion in an ethanol aqueous solution, even if it can be sterilized to a level that can be used in a short-term cell culture test, it is impossible to achieve SAL = 1 × 10 -6 The following sterilization methods cannot eliminate the risk of infection caused by microorganisms when used in cell culture processes or on human bodies.

[0021] In addition, in order to improve the sterilization effect, when performing treatments such as autoclave sterilization and gamma ray sterilization at high temperatures, it is known that in hydrogels complexed with physiologically active substances such as enzymes, the physiologically active substances lose their original activity under harsh sterilization conditions, and therefore such treatments are generally not performed. In addition, it is also known that if a hydrogel treated with gamma ray sterilization is used for cell culture, cell adhesion is sometimes reduced. In addition, it is known that when a harsh treatment such as gamma ray sterilization is performed in a state before cross-linking, it is difficult to obtain a hydrogel formed into a desired shape by cross-linking through the treatment. In addition, it is also known that the strength of the gel can be reduced by becoming uneven by the cross-linked portion.

[0022] During extensive research on methods for sterilizing hydrogels, the present inventors attempted to sterilize the hydrogels in a dry state. However, they found that in this case, the dried hydrogels sometimes adhered or aggregated.

[0023] The present invention has been made in view of the above-mentioned problems, and an object of the present invention is to provide a dry hydrogel-forming article that can provide a sufficiently sterilized hydrogel and in which aggregation during drying is suppressed.

[0024] In a preferred embodiment, the present invention aims to provide a dry hydrogel-forming article that can provide a hydrogel that is sufficiently sterilized and has sufficient strength and in which aggregation during drying is suppressed.

[0025] In another preferred embodiment, the present invention aims to provide a dried hydrogel product that is capable of providing a hydrogel that is sufficiently sterilized and has high cell adhesiveness and in which aggregation during drying is suppressed.

[0026] Means used to solve problems

[0027] The present inventors have conducted intensive research and found that in the state of a dry hydrogel-forming article containing an alcoholic solvent and dried so as to have an aggregation degree of 10% or less, the sterility assurance level SAL of 1×10 -6 The above-mentioned problem is solved by sterilizing under the following conditions: Such a dry hydrogel-forming article can be produced, for example, by replacing the water in the hydrogel with an alcoholic solvent, drying the article, removing the solvent, and then fully sterilizing the dry hydrogel-forming article.

[0028] That is, the present invention relates to the following [1] to

[15] .

[0029] [1] A dry hydrogel-forming article comprising a crosslinked product of a vinyl alcohol polymer and an alcoholic solvent,

[0030] Based on the amount of the dry hydrogel-forming article, the content of the alcoholic solvent is 0.01% by mass or more and 15% by mass or less, and the content of water is 0% by mass or more and 70% by mass or less,

[0031] The sterility assurance level (SAL) of the dried hydrogel-forming article is 1×10 -6 Below, the degree of agglomeration is below 10%.

[0032] [2] The dry hydrogel-forming article according to [1], which satisfies the following condition 1:

[0033] Condition 1:

[0034] When 100 parts by mass of water and 1 part by mass of a dry hydrogel-forming article are added to a glass bottle and stirred at 400 rpm for 3 hours using a stirring head having a length of at least 80% of the bottom diameter of the glass bottle, the breakage rate of the swollen hydrogel-forming article is 5% or less.

[0035] [3] The dry hydrogel-forming article according to [1] or [2], wherein the degree of polymerization of the vinyl alcohol polymer is 450 or higher.

[0036] [4] The dry hydrogel-forming article according to any one of [1] to [3], wherein the vinyl alcohol polymer has carboxyl groups, and the introduction rate of the carboxyl groups is 0.1 to 50 mol% in all the structural units constituting the vinyl alcohol polymer.

[0037] [5] The dry hydrogel-forming article according to any one of [1] to [4], wherein the vinyl alcohol polymer has an ethylenically unsaturated group, and the introduction rate of the ethylenically unsaturated group is 0.01 to 10 mol% in all the structural units constituting the vinyl alcohol polymer.

[0038] [6] The dry hydrogel-forming article according to [5], 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 norbornene group, and derivatives thereof.

[0039] [7] The dry hydrogel-forming article according to any one of [1] to [6], wherein the dry hydrogel-forming article is an amorphous particle, a spherical particle, a fine molded body, an article of any shape formed by a 3D printer, a sheet, a filament, a hollow fiber, a porous block, or a coated article.

[0040] [8] The dry hydrogel-forming article according to any one of [1] to [7], wherein the dry hydrogel-forming article is a spherical particle.

[0041] [9] The dry hydrogel-forming article according to any one of [1] to [8], wherein the physiologically active substance is complexed.

[0042]

[10] The dry hydrogel-forming article according to any one of [1] to [9], wherein the physiologically active substance is complexed with a cross-linked vinyl alcohol polymer via a covalent bond.

[0043]

[11] A hydrogel which is a swollen body of the dry hydrogel-forming article according to any one of [1] to

[10] .

[0044]

[12] The hydrogel according to

[11] , wherein the hydrogel is in the form of spherical particles having a particle size of 10 to 5000 μm.

[0045]

[13] The method for producing a dry hydrogel-forming article according to any one of [1] to

[10] , comprising: preparing a preliminary hydrogel comprising a crosslinked product of a vinyl alcohol polymer;

[0046] The step of immersing the preliminary hydrogel in an alcohol solvent and then drying it to obtain a dried preliminary hydrogel; and

[0047] Sterilize the dried prepared hydrogel to a sterility assurance level (SAL) of 1×10 -6 The following steps.

[0048]

[14] The method for producing a dry hydrogel-forming article according to

[13] , wherein radiation sterilization is performed in the sterilization step to achieve a sterility assurance level (SAL) of 1×10 -6 the following.

[0049]

[15] The method for producing a dry hydrogel-forming article according to

[14] , wherein the radiation dose during radiation sterilization is 8.2 kGy or more.

[0050] Effects of the Invention

[0051] According to the present invention, a dry hydrogel-forming article can be provided that can provide a sufficiently sterilized hydrogel and in which aggregation during drying is suppressed. DETAILED DESCRIPTION

[0052] 1. Drying the Hydrogel-forming Articles

[0053] <Dry hydrogel-forming articles>

[0054] As a first embodiment, the present invention provides a dry hydrogel-forming article comprising a crosslinked product of a vinyl alcohol polymer and an alcoholic solvent, wherein the content of the alcoholic solvent is from 0.01% to 15% by mass, and the content of water is from 0% to 70% by mass, based on the amount of the dry hydrogel-forming article, and the sterility assurance level (SAL) of the dry hydrogel-forming article is 1×10 -6 Below, the degree of agglomeration is below 10%.

[0055] <Vinyl alcohol polymer and method for producing the same>

[0056] The vinyl alcohol polymer in the crosslinked vinyl alcohol polymer contained in the dry hydrogel-forming article of the present invention can be produced by saponifying polyvinyl ester obtained by polymerizing vinyl ester monomers and converting the ester groups in the polyvinyl ester into hydroxyl groups.

[0057] Examples of the vinyl ester monomers include aliphatic vinyl esters such as vinyl formate, vinyl acetate, vinyl propionate, vinyl n-butyrate, vinyl isobutyrate, vinyl pivalate, vinyl versatate, vinyl caproate, vinyl octanoate, vinyl decanoate, vinyl laurate, vinyl myristate, vinyl palmitate, vinyl stearate, and vinyl oleate; and aromatic vinyl esters such as vinyl benzoate. These vinyl ester monomers 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 perspective of manufacturing cost. That is, the polyvinyl ester is preferably polyvinyl acetate obtained by polymerizing vinyl acetate.

[0058] In addition, the aforementioned polyvinyl ester may contain structural units derived from other monomers in addition to the vinyl ester monomers as needed, within the scope of not impairing the effects of the present invention. Examples of such other monomers include α-olefins such as ethylene, propylene, n-butene, and isobutylene; acrylic acid or its salts; alkyl acrylates such as methyl acrylate, ethyl acrylate, n-propyl acrylate, isopropyl acrylate, n-butyl acrylate, isobutyl acrylate, tert-butyl acrylate, 2-ethylhexyl acrylate, dodecyl acrylate, and octadecyl acrylate; methacrylic acid or its salts; alkyl methacrylates such as methyl methacrylate, ethyl methacrylate, n-propyl methacrylate, isopropyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, tert-butyl methacrylate, 2-ethylhexyl methacrylate, dodecyl methacrylate, and octadecyl methacrylate; acrylamide, N-methylacrylamide, N-ethylacrylamide, N,N-dimethylacrylamide, diacetoneacrylamide, acrylamidopropanesulfonic acid or its salts, acrylamidopropyldimethylamine or its salt or quaternary salt, N-hydroxymethylacrylic acid Acrylamide derivatives such as acrylamide or its derivatives; methacrylamide derivatives such as methacrylamide, N-methylmethacrylamide, N-ethylmethacrylamide, methacrylamidepropanesulfonic acid or its salts, methacrylamidopropyldimethylamine or its salts or quaternary salts, N-hydroxymethylmethacrylamide or its derivatives; N-vinylamide derivatives such as N-vinylformamide and N-vinylacetamide; vinyl ethers such as methyl vinyl ether, ethyl vinyl ether, n-propyl vinyl ether, isopropyl vinyl ether, n-butyl vinyl ether, isobutyl vinyl ether, tert-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 its salts, esters, or anhydrides; vinylsilyl compounds such as vinyltrimethoxysilane; and isopropenyl acetate. These other monomers may be used alone or in combination of two or more.

[0059] The method for saponifying the polyvinyl ester is not particularly limited and can be carried out by the same methods as those in the past. For example, alcoholysis or hydrolysis using an alkali catalyst or an acid catalyst can be applied. Among them, the saponification reaction using methanol as a solvent and caustic soda (NaOH) as a catalyst is simple and preferred.

[0060] The polymerization degree of the vinyl alcohol-based polymer is preferably 450 or more, more preferably 500 or more, further preferably 550 or more, and particularly preferably 600 or more from the viewpoint of inhibiting the embrittlement of the crosslinked body of the vinyl alcohol-based polymer at the time of sterilization treatment and at the time of water swelling. In addition, the polymerization degree of the vinyl alcohol-based polymer is preferably 10,000 or less, more preferably 5,000 or less, and further preferably 3,500 or less from the viewpoint of inhibiting the increase in the viscosity of an aqueous solution at the time of production of the crosslinked body of the vinyl alcohol-based polymer and improving the ease of processing. Note that two or more different substances having different polymerization degrees can be mixed and used as the vinyl alcohol-based polymer. The polymerization degree of the vinyl alcohol-based polymer in this specification refers to the polymerization degree measured in accordance with JIS K 6726:1994. Specifically, the intrinsic viscosity of a raw material PVA, which is saponified, can be measured in water at 30°C.

[0061] The saponification degree of the vinyl alcohol-based polymer is preferably 50 mol% or more, more preferably 60 mol% or more, and further preferably 65 mol% or more from the viewpoint of improving the water solubility of the vinyl alcohol-based polymer. The upper limit of the saponification degree is 100 mol% or less, or 99 mol% or less. In this specification, the saponification degree of the vinyl alcohol-based polymer refers to the proportion (mol%) of the number of moles of the vinyl alcohol unit with respect to the total number of moles of the structural unit capable of being converted into the vinyl alcohol unit by saponification (for example, a vinyl acetate unit) and the vinyl alcohol unit in the raw material PVA, and can be measured in accordance with JIS K 6726:1994.

[0062] The 4 mass% viscosity at 20°C of the vinyl alcohol-based polymer is preferably 0.5 to 110 mPa-s, more preferably 1 to 80 mPa-s, and further preferably 2 to 60 mPa-s. If the aforementioned viscosity is within the aforementioned range, the strength of the composite hydrogel can be improved while the ease of production of the hydrogel is improved. Note that the viscosity in this specification refers to the viscosity of a 4 mass% aqueous solution of the vinyl alcohol-based polymer, which is measured at a temperature of 20°C using a B-type viscometer (rotation speed: 12 rpm) in accordance with the rotation viscometer method of JIS K 6726:1994.

[0063]

[0064] The dried hydrogel-forming article of the present application contains the aforementioned crosslinked body of the vinyl alcohol-based polymer. The crosslinked body can be obtained by physically crosslinking the vinyl alcohol-based polymer, or can be obtained by crosslinking through a covalent bond. The total amount of the structural unit derived from vinyl alcohol and the structural unit derived from a vinyl ester with respect to the entire structural units constituting the vinyl alcohol-based polymer used in the present application is preferably 80 mol% or more, more preferably 90 mol% or more, and further preferably 95 mol% or more.

[0065] ​Methods for physically crosslinking vinyl alcohol polymers include, for example, freezing and melting an aqueous solution of the vinyl alcohol polymer; dissolving the solution in a mixed solvent of dimethyl sulfoxide and water, heating it, and cooling the heated solution to room temperature. Furthermore, methods for covalently crosslinking vinyl alcohol polymers include, for example, using a polyfunctional crosslinking agent that reacts with the side chain hydroxyl groups of the vinyl alcohol polymer; or introducing functional groups into the vinyl alcohol polymer through copolymerization or post-modification and reacting them. Crosslinking via covalent bonds is preferred from the perspective of improving the stability of the hydrogel during water swelling.

[0066] 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).

[0067] As a method for introducing functional groups into vinyl alcohol polymers by copolymerization and causing them to react, there is a method in which, during the production process of the vinyl alcohol polymer, a vinyl ester monomer is copolymerized with a monomer having a reactive substituent other than a hydroxyl group, which is a polymerizable monomer other than the vinyl ester monomer, followed by saponification to obtain a copolymerized modified polyvinyl alcohol (hereinafter sometimes referred to as "copolymerized modified PVA"), and then a multifunctional crosslinking agent that reacts with functional groups such as carboxyl groups or amino groups present in the copolymerized modified PVA is added. It should be noted that copolymerized modified PVA having carboxyl groups may be referred to as "carboxylic acid-modified PVA," and copolymerized modified PVA having amino groups may be referred to as "amino-modified PVA."

[0068] Monomers constituting carboxylic acid-modified PVA include α,β-unsaturated carboxylic acids such as (meth)acrylic acid, maleic acid, fumaric acid, and itaconic acid; alkyl (meth)acrylates such as methyl (meth)acrylate and ethyl (meth)acrylate; and α,β-unsaturated carboxylic anhydrides such as maleic anhydride and itaconic anhydride, and their derivatives. Carboxylic acid-modified PVA is produced by copolymerizing vinyl ester monomers with the monomers constituting the carboxylic acid-modified PVA, followed by saponification. Hydrogels can be obtained by combining a polyfunctional crosslinking agent reactive with the introduced carboxyl groups, such as polyfunctional epoxy compounds such as epichlorohydrin and ethylene glycol diglycidyl ether; polyfunctional amino compounds such as ethylenediamine, polyethyleneimine, and polyallylamine; and a carbodiimide condensation agent such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0069] The carboxyl groups introduced into the carboxylic acid-modified PVA can complex physiologically active substances having an amino group by covalent bonding via an amide bond (-CONH-).

[0070] Further, the amino-modified PVA can be copolymerized with a vinyl ester-based monomer and the like, and thereafter saponified, and by the reaction of the introduced amino group with a multifunctional crosslinking agent such as the aforementioned multifunctional epoxy compound, a multifunctional carboxylic acid compound such as succinic acid, maleic acid, and the like, and the aforementioned carbodiimide condensing agent, and the like, a hydrogel can be obtained.

[0071] As a method of introducing a functional group into a vinyl alcohol-based polymer by post-modification and reacting it, a method of introducing an olefinically unsaturated group into a side chain of a vinyl alcohol-based polymer can be exemplified. The introduced olefinically unsaturated group can initiate a polymerization reaction by the addition of an additive such as a radical initiator, and the like, and thereby a hydrogel can be easily obtained. The introduction of the olefinically unsaturated group is preferably performed via a side chain or a terminal functional group of the vinyl alcohol-based polymer, and more preferably the reaction of a hydroxyl group of a side chain of the vinyl alcohol-based polymer with a compound containing an olefinically unsaturated group (hereinafter sometimes simply referred to as "compound containing an olefinically unsaturated group").

[0072] As the compound containing an olefinically unsaturated group which reacts with a hydroxyl group of a side chain of a vinyl alcohol-based polymer, for example, (meth)acrylic acid, (meth)acrylic anhydride, (meth)acryloyl halide, (meth)acrylate, or the like (a (meth)acrylic acid or a derivative thereof) can be exemplified, and by causing these compounds to undergo an esterification reaction or a transesterification reaction in the presence of a base, a (meth)acryloyl group can be introduced.

[0073] Further, as the compound containing an olefinically unsaturated group which reacts with a hydroxyl group of a side chain of a vinyl alcohol-based polymer, for example, a compound containing an olefinically unsaturated group and a glycidyl group within a molecule, such as a (meth)acrylic acid glycidyl ester, an allyl glycidyl ether, or the like, can be exemplified. By causing these compounds to undergo an etherification reaction in the presence of a base, a (meth)acryloyl group and / or an allyl group can be introduced into the vinyl alcohol-based polymer.

[0074] Furthermore, as a compound containing an ethylenically unsaturated group that reacts with the 1,3-diol group of the vinyl alcohol polymer, for example, acrolein (acrolein), methacrolein (methacrolein), 5-norbornene-2-carboxaldehyde, 7-octenal, 3-vinylbenzaldehyde, and 4-vinylbenzaldehyde, compounds containing an ethylenically unsaturated group and an aldehyde group in the molecule. By subjecting these compounds to an acetalization reaction in the presence of an acid catalyst, an ethylenically unsaturated group can be introduced into the raw material PVA. More specifically, for example, by subjecting 5-norbornene-2-carboxaldehyde, 3-vinylbenzaldehyde or 4-vinylbenzaldehyde to an acetalization reaction, a norbornene group or a vinylphenyl group can be introduced into the raw material PVA. In addition, by reacting N-(2,2-dimethoxyethyl)(methyl)acrylamide and the like, a (meth)acryloylamino group can be introduced into the vinyl alcohol polymer. As a method for introducing an ethylenically unsaturated group into a vinyl alcohol-based polymer, a method other than the above-mentioned reaction may be used, and two or more reactions may be used in combination.

[0075] As a method for introducing the aforementioned ethylenically unsaturated groups, there can also be mentioned a method of reacting reactive substituents such as the carboxyl groups present in carboxylic acid-modified PVA or the amino groups present in amino-modified PVA with a compound containing an ethylenically unsaturated group. For the carboxyl groups of the carboxylic acid-modified PVA, for example, by reacting glycidyl methacrylate under acidic conditions, an ester bond can be generated to introduce a methacryloyl group. For the amino groups of the amino-modified PVA, for example, by subjecting acrylic anhydride to an amidation reaction in the presence of a base, an acryloylamino group can be introduced, for example, by subjecting divinyl adipate to an amidation reaction, thereby introducing a vinyloxycarbonyl group. The method of introducing ethylenically unsaturated groups via copolymerization-modified PVA can also use methods other than the aforementioned reactions exemplified above, or a combination of two or more reactions.

[0076] As the vinyl alcohol polymer having an ethylenically unsaturated group, from the viewpoint of ease of production, a vinyl alcohol polymer in which an ethylenically unsaturated group is introduced via a hydroxyl group of a side chain of the raw material PVA, such as a 1,3-diol group, is preferred. More preferred is a vinyl alcohol polymer obtained by an esterification reaction or transesterification reaction of a hydroxyl group of a side chain of the raw material PVA with (meth)acrylic acid or a derivative thereof, or a vinyl alcohol polymer obtained by an acetalization reaction of a 1,3-diol group of a vinyl alcohol polymer with a compound containing an ethylenically unsaturated group and an aldehyde group in the molecule.

[0077] In a preferred embodiment of the present invention, the vinyl alcohol polymer in the cross-linked body of the vinyl alcohol polymer is preferably a vinyl alcohol polymer having a carboxyl group from the perspective of appropriately maintaining the swellability of the hydrogel to a solvent (particularly water), maintaining physical properties such as gel strength, and improving the composite density of physiologically active substances. In this embodiment, the introduction rate of the carboxyl group is preferably 50 mol% or less, more preferably 30 mol% or less, and further preferably 15 mol% or less in all the structural units constituting the vinyl alcohol polymer. Moreover, from the perspective of maintaining a high composite density of physiologically active substances and performing high functions, it is preferably 0.1 mol% or more, more preferably 0.5 mol% or more, and further preferably 1.0 mol% or more. In particular, the hydrogel is expected to be circulated or stored in a dry state before use, but if the hydrogel is dried once, it is sometimes difficult to return to its original swollen state. When the vinyl alcohol polymer preferably has carboxyl groups at the above-mentioned carboxyl group introduction ratio, even when a dried hydrogel-forming article that has been dried once is swollen to obtain a hydrogel, the swelling degree can be restored to the original level, thereby producing a high-strength hydrogel. This is preferred.

[0078] In another preferred embodiment of the present invention, from the perspective of suppressing hydrogel embrittlement and promoting the crosslinking reaction, the vinyl alcohol polymer in the crosslinked vinyl alcohol polymer is preferably a vinyl alcohol polymer having an ethylenically unsaturated group. In this embodiment, the ethylenically unsaturated group is more preferably selected from at least one of a vinyl group, a (meth)acryloyl group, a (meth)acryloylamino group, a vinylphenyl group, a norbornene group, and derivatives thereof.

[0079] In the above embodiment, the introduction rate of the ethylenically unsaturated group is preferably 10 mol% or less, more preferably 5 mol% or less, and even more preferably 3 mol% or less, based on the total structural units constituting the vinyl alcohol polymer, from the perspective of suppressing embrittlement of the hydrogel. Furthermore, from the perspective of promoting the crosslinking reaction, rapidly forming the hydrogel, and increasing the elastic modulus of the resulting hydrogel, it is preferably 0.01 mol% or more, more preferably 0.1 mol% or more, and even more preferably 0.5 mol% or more.

[0080] In a more preferred embodiment of the present invention, from the same viewpoint as above, the vinyl alcohol polymer in the crosslinked vinyl alcohol polymer is more preferably a vinyl alcohol polymer having a carboxyl group and an ethylenically unsaturated group. In this case, the introduction ratios of the carboxyl group and the ethylenically unsaturated group are more preferably the introduction ratios described above.

[0081] From the perspective of improving the mechanical strength of the hydrogel and introducing functional groups different from the hydroxyl groups of PVA, the vinyl alcohol polymer may further contain monomers. 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 free radical polymerizable monomers such as vinylpyridine, hydroxyethyl (meth)acrylate, styrenesulfonic acid, and polyethylene glycol mono(meth)acrylate; and crosslinking agents 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 relative to the vinyl alcohol polymer having an ethylenically unsaturated group.

[0082] When the vinyl alcohol polymer has an ethylenically unsaturated group, the ethylenically unsaturated group introduced into the aforementioned vinyl alcohol polymer can be crosslinked by active energy rays or heat to gel, thereby obtaining a crosslinked product of the vinyl alcohol polymer. As active energy rays, for example, gamma rays, ultraviolet rays, visible rays, infrared rays (heat rays), radio waves, alpha rays, beta rays, electron rays, plasma streams, ionizing rays, particle rays, etc. can be mentioned. Among the aforementioned active energy rays, when the aforementioned vinyl alcohol polymer is crosslinked by ultraviolet rays, visible rays, infrared rays (heat rays), etc. or heat, the uncrosslinked polymer solution described later preferably contains a free radical polymerization initiator. As free radical polymerization initiators, light free radical polymerization initiators and thermal free radical polymerization initiators can be mentioned.

[0083] As a light radical polymerization initiator, as long as free radical polymerization is initiated by the irradiation of active energy rays such as ultraviolet rays, visible light, etc., there is no particular limitation. However, from the viewpoint of washing and removing the light radical polymerization initiator after cross-linking, it is preferably shown that it is water-soluble. Specifically, for example, 1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (trade name " Omnirad2959 ", IGM RESINS BV system), α-ketoglutaric acid, phenyl (2,4,6-trimethylbenzoyl) phosphonic acid lithium salt (trade name " L0290 ", Tokyo Chemical Industry (strain) system), eosin Y etc. can be enumerated.

[0084] The thermal free radical polymerization initiator is not particularly limited as long as it initiates free radical polymerization by heat, and examples thereof include azo initiators and peroxide initiators commonly used in free radical polymerization. From the perspective of improving the transparency and physical properties of the vinyl alcohol polymer, peroxide initiators that do not generate gas are preferred. As mentioned above, from the perspective of washing and removing the thermal free radical polymerization initiator after crosslinking, water solubility is preferred. Specific examples include inorganic peroxides such as ammonium persulfate, potassium persulfate, and sodium persulfate.

[0085] In addition, a redox polymerization initiator combined with a reducing agent can be used. If the redox polymerization initiator is a peroxide-based initiator, crosslinking can be achieved through stimulation by the mixture of the peroxide-based initiator and the reducing agent. As the reducing agent combined to form the redox polymerization initiator, known reducing agents can be used, among which highly water-soluble N,N,N',N'-tetramethylethylenediamine, sodium sulfite, sodium bisulfite, sodium dithionite, etc. are preferred.

[0086] The azo initiator used is also preferably 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-carboxyethyl)-2-methylpropionamidine] tetrahydrate (trade name "VA-057"), 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-cyanovaleric acid) (trade name "V-501") (all manufactured by Wako Pure Chemical Industries, Ltd.), etc.

[0087] In a preferred embodiment of the present invention, when a vinyl alcohol polymer having an ethylenically unsaturated group is used, in which the ethylenically unsaturated group is a vinyl group, a polythiol having two or more thiol groups in the molecule can be added to promote crosslinking, thereby achieving crosslinking through a thiol-ene reaction. The polythiol used is preferably water-soluble, and examples thereof include polythiols having a hydroxyl group such as dithiothreitol; and polythiols containing ether bonds, such as terminal thiolates such as 3,6-dioxa-1,8-octanedithiol, polyethylene glycol dithiol, and multi-arm polyethylene glycol.

[0088] As described above, the most suitable method can be selected as needed as the cross-linking method utilizing the covalent bond of the vinyl alcohol polymer.

[0089] For example, methods using glutaraldehyde as a crosslinking agent are conventionally known. However, glutaraldehyde is highly toxic and can cause adverse effects if even a small amount of glutaraldehyde dissolves from a chemically crosslinked hydrogel. Therefore, while it can be used for research purposes, it is difficult to apply to pharmaceutical applications. Therefore, from the perspective of improving safety, the crosslinked substance in the dry hydrogel-forming article of the present invention is preferably not a substance crosslinked with glutaraldehyde, and substances crosslinked with glutaraldehyde are preferably excluded.

[0090] From the viewpoint of the toxicity of unreacted crosslinking agent and condensing agent contained in the hydrogel and the ease of further molding, a method of introducing functional groups into the vinyl alcohol polymer by post-modification and reacting them, particularly a method of introducing ethylenically unsaturated groups into the side chains of the vinyl alcohol polymer, is preferred.

[0091] <Alcohol solvents>

[0092] The dried hydrogel-forming article of the present invention further contains an alcoholic solvent. In the dried hydrogel-forming article of the present invention, the content of the alcoholic solvent is 0.01% to 15% by mass based on the amount of the dried hydrogel-forming article. When the dried hydrogel-forming article is dried under conditions such that the alcoholic solvent content falls within the above range, aggregation of the dried hydrogel-forming article can be prevented. The content of the alcoholic solvent in the dried hydrogel-forming article is not particularly limited as long as it falls within the above range. However, from the perspective of easily reducing the degree of aggregation, it is preferably 0.05 to 15% by mass, more preferably 0.07 to 15% by mass, and even more preferably 0.1 to 15% by mass.

[0093] The alcohol solvent is not particularly limited as long as it is a water-soluble alcohol, and examples thereof include monoalcohols such as methanol, ethanol, propanol, and isopropanol, and polyols such as ethylene glycol, diethylene glycol, triethylene glycol, and glycerol. The alcohol solvent may contain one solvent or two or more solvents.

[0094] Water

[0095] In the present invention, a dry hydrogel-forming article is a hydrogel in a dry state, which may or may not contain water but can absorb additional water to swell and form a hydrogel. In the dry hydrogel-forming article of the present invention, the water content is from 0% to 70% by mass based on the amount of the dry hydrogel-forming article. The water content of the dry hydrogel-forming article is not particularly limited as long as it is within the above range. However, from the perspective that a lower water content reduces capacity and improves transport efficiency, the water content is preferably 60% by mass or less, more preferably 50% by mass or less, even more preferably 45% by mass or less, and even more preferably 40% by mass or less.

[0096] As a method for measuring the alcoholic solvent content in a dried hydrogel-forming article, for example, when the water contained in the hydrogel is replaced with an alcoholic solvent and then dried, the solvent contained in the hydrogel can be considered to be an alcoholic solvent. Therefore, one method can be used to measure the alcoholic solvent content based on the weight change after complete volatilization of the water-soluble alcohol. Specifically, the weight of the pellets after drying at 37°C is measured, and then the pellets are further dried at 120°C and the weight after drying is measured until there is no weight change. The alcoholic solvent content can be calculated using the following calculation formula (I) based on the weight difference before and after drying at 120°C.

[0097] Alcohol solvent content = ((weight after drying at 37°C) - (weight after drying at 120°C)) / (weight after drying at 37°C) (I)

[0098] The content of the alcoholic solvent in the dry hydrogel-forming article can also be adjusted together with the water content by, for example, 1 The measurement conditions are described in detail in the Examples.

[0099] In one embodiment of the present invention, the total content of water and alcoholic solvent in the dry hydrogel-forming article can be, for example, 0.01% by mass or more and 85% by mass or less, 0.05% by mass or more and 75% by mass or less, 0.07% by mass or more and 65% by mass or less, 0.1% by mass or more and 55% by mass or less. In addition, from the perspective of easily preventing aggregation, the ratio of the content of the alcoholic solvent to the total content of water and alcoholic solvent in the dry hydrogel-forming article is preferably 50% by mass or more (or more than 50% by mass), more preferably 60% by mass or more, even more preferably 70% by mass or more, even more preferably 80% by mass or more, and particularly preferably 90% by mass or more. The upper limit of this ratio is 100% by mass or less. It should be noted that the dry hydrogel-forming article of the present invention contains an alcoholic solvent, but may or may not contain water.

[0100] <Agglutination degree>

[0101] The dried hydrogel-forming article of the present invention has a degree of cohesion of 10% or less. If the degree of cohesion exceeds 10%, the fixed and / or coagulated dried hydrogel-forming article will remain in a coagulated state during swelling, resulting in uneven swelling. This can damage the surface structure of the hydrogel during collision or separation during swelling. Furthermore, the coagulated hydrogel may break apart during separation, potentially generating hydrogel fragments. If such fragments are incorporated during use, for example, when the hydrogel is used for cell culture, they may be taken up by cells during culture, making separation from the cultured cells difficult.

[0102] A certain amount of the dried hydrogel-forming article is weighed, and two or more aggregates are visually separated. The weight of the separated aggregates is measured, and the degree of aggregation is calculated according to the following formula (II).

[0103] Agglutination degree (%) = (weight of aggregate / total weight) × 100... (II)

[0104] The degree of aggregation is calculated using a measurement sample comprising a plurality of dried hydrogel-forming articles. Alternatively, for example, if the dried hydrogel-forming article is a sheet, the degree of aggregation can be measured based on the number of sheets affixed to each other, using a dried hydrogel-forming article obtained by stacking and drying a plurality of sheets.

[0105] As a method for adjusting the contents of water and alcoholic solvents and the degree of coagulation within the above ranges, the following method can be cited: after preparing a hydrogel containing water, immersing the hydrogel in an alcoholic solvent, replacing the water contained in the hydrogel with the alcoholic solvent, and then drying the obtained hydrogel. In addition, in order to achieve the sterility assurance level SAL described later of 1×10 -6 Next, it is considered that the hydrogel obtained by drying as described above is sterilized by radiation sterilization (for example, gamma ray sterilization) or the like.

[0106] <Sterility Assurance Level SAL>

[0107] The sterility assurance level SAL of the dry hydrogel-forming article of the present invention is 1×10 -6 Below. Sterility assurance level SAL exceeds 1×10 -6 When the sterilization is too slow, a hydrogel that can be used as a pharmaceutical cannot be provided. According to the dry hydrogel-forming article of the present invention, since the sterility assurance level SAL is satisfied in the state of the dry hydrogel used as a hydrogel that can swell, a high sterility can be obtained even in the final use. In the state of the dry hydrogel-forming article, the sterility assurance level SAL can be reduced to 1×10 -6 Although the reason is unclear, it is considered that the generation of free radicals can be suppressed, thereby facilitating improvement of cell adhesion.

[0108] Sterility assurance level SAL is preferably achieved through VD max Sterilization is carried out under the conditions specified by the law to ensure that VD max The details of the conditions for the method are described in the Examples, etc. The dried hydrogel-forming article of the present invention is an article that meets the above-mentioned sterility assurance level, preferably an article sterilized by radiation, more preferably an article sterilized by gamma rays, and even more preferably an article sterilized by VD maxArticles sterilized by gamma ray sterilization at a sterilization dose specified by the law. It should be noted that dry hydrogel-forming articles containing crosslinked products obtained by crosslinking vinyl alcohol polymers that have been sterilized by radiation (particularly gamma ray sterilization) before crosslinking do not have sufficient formability as hydrogels and may have insufficient strength, and are therefore preferably excluded from the scope of the present invention.

[0109] <Breakage rate>

[0110] The dried hydrogel-forming article of the present invention preferably satisfies the following condition 1.

[0111] Condition 1:

[0112] When 100 parts by mass of water and 1 part by mass of a dry hydrogel-forming article are placed in a glass bottle and stirred at 400 rpm for 3 hours using a stirring head having a length of at least 80% of the bottom diameter of the glass bottle, the breakage rate of the swollen hydrogel-forming article is 5% or less.

[0113] When the dry hydrogel-forming article satisfies condition 1, in other words, has a breakage rate of 5% or less as measured by the above method, the dry hydrogel-forming article has high strength and can be used even at a sterility assurance level (SAL) of 1×10 -6 Even when sterilized in the following manner, the strength of the resulting hydrogel can be maintained, and such dried hydrogel-forming articles are also more likely to maintain their swelling when they swell again after drying. From the perspective of improving gel strength, the breakage rate is preferably 4% or less, more preferably 3% or less, even more preferably 2% or less, and even more preferably 1% or less. The breakage rate is calculated by observing the state of the stirred solid component under a microscope under the above conditions, observing the solid component in a state where at least 60% of the field of view is visible, visually distinguishing between broken and unbroken areas, and calculating the ratio of the area of ​​the broken portion to the total area of ​​the solid component.

[0114] <Swelling degree>

[0115] The swelling degree of the dried hydrogel-forming article of the present invention is calculated from the dry weight W1g of the dried gel-forming article and the swollen weight W2g of 30 mg of the dried gel-forming substance when immersed in 5 mL of PBS (phosphate-buffered saline) overnight and allowed to swell, using the following formula (III):

[0116] Swelling degree = W2 / W1(III)

[0117] The upper limit of the swelling degree is not particularly limited, and from the viewpoint of the strength of the hydrogel, it is preferably 40 or less, more preferably 30 or less, and further preferably 20 or less.

[0118] <Shape of the dried hydrogel-forming article>

[0119] The shape of the dried hydrogel-forming article is not particularly limited, and is preferably an amorphous particle, a spherical particle, a micro-molded body, an arbitrary shape article formed by a 3D printer, a sheet, a filament, a hollow fiber, a porous block, or a coated article. From the viewpoint of inhibiting aggregation, the dried hydrogel-forming article is preferably a spherical particle, and more preferably a spherical particle having a particle diameter of 10 to 5000 μm when swollen. In addition, the particle diameter of the dried hydrogel-forming article of the spherical particle can be 1 to 5000 μm. Note that the dried hydrogel-forming article can be one dried hydrogel-forming article having the above-described shape, or can be a group of a plurality of dried hydrogel-forming articles (for example, a group of particles), and generally is a group of a plurality of dried hydrogel-forming articles, and is preferably a group of 10 or more, more preferably a group of 30 or more, further preferably a group of 50 or more, and further more preferably a group of 70 or more of dried hydrogel-forming articles.

[0120] The micro-molded body is a molded body in which a micro concave-convex or the like is implemented on the surface or inside, and the size of the microfabrication is 10 to 1000 μm. In addition, the arbitrary shape formed by a 3D printer refers to an arbitrary shape that can be formed by a 3D printer of a light molding method, an inkjet method, a nozzle extrusion type, or the like. The coated article refers to an article in which a composite hydrogel is coated on a base material having a shape such as a sheet, a tray, a filament, a hollow fiber, a porous block, a micro-molded body, an arbitrary shape article formed by a 3D printer, and the like, and the raw material of the base material can be freely selected from a raw material such as glass, polyolefin, polymethyl methacrylate, polystyrene, polyester, polyolefin, polyethylene vinyl alcohol copolymer, polyamide, polyimide, and the like. From the viewpoint of preventing aggregation, the dried hydrogel-forming article is preferably a dried hydrogel-forming particle.

[0121] <Complexation of a physiologically active substance>

[0122] In the dried hydrogel-forming article of the present application, a physiologically active substance can also be complexed in the crosslinked body of the vinyl alcohol-based polymer. In the case where a physiologically active substance is complexed, when a hydrogel that swells the dried hydrogel-forming article is used for cell culture or the like, cell adhesion can be improved. In addition, in the case of the dried hydrogel-forming article of the present application in which a physiologically active substance is complexed, a hydrogel having high cell adhesion and being sufficiently sterilized can be provided.

[0123] As physiologically active substances, for example, cell adhesion proteins or peptides such as gelatin, collagen, laminin, fibronectin, RetroNectin, HydroNectin, elastin, synthetic RGD peptide, growth factors such as fibroblast growth factor (FGF), epidermal growth factor (EGF), vascular endothelial cell growth factor (VEGF), acidic polysaccharides containing glycosaminoglycans such as heparin, hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, antibodies, hormones, serum proteins, albumin, macroglobulin, globulin, antibody binding proteins such as protein A, various pharmaceuticals, proteases, lipases, amylases, cellulases, and enzymes. The physiologically active substance preferably has a functional group capable of bonding to the dried hydrogel-forming article. As such a functional group, for example, primary or secondary amino groups, carboxyl groups, hydroxyl groups, and the like can be mentioned, with primary amino groups or carboxyl groups being preferred, and primary amino groups being more preferred. When the physiologically active substance is complexed in the dried hydrogel-forming article of the present application, the physiologically active substance can be one substance, or a combination of two or more substances.

[0124] When the dried hydrogel-forming article of the present application contains a crosslinked body of a vinyl alcohol-based polymer having a carboxyl group, the physiologically active substance is preferably a physiologically active substance having a primary or secondary amino group. The physiologically active substance can be, for example, at least one selected from the group consisting of cell adhesion proteins, cell adhesion peptides, growth factors, acidic polysaccharides, antibodies, hormones, serum proteins, antibody binding proteins, drugs, and enzymes. As physiologically active substances having an amino group (preferably a primary or secondary amino group), for example, cell adhesion proteins or peptides such as gelatin, collagen, laminin, fibronectin, RetroNectin, HydroNectin, elastin, synthetic RGD peptide, growth factors such as fibroblast growth factor (FGF), epidermal growth factor (EGF), vascular endothelial cell growth factor (VEGF), antibodies, serum proteins, albumin, macroglobulin, globulin, antibody binding proteins such as protein A, glycosaminoglycans such as heparin, hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, various pharmaceuticals, proteases, lipases, amylases, cellulases, and enzymes can be mentioned. These physiologically active substances contain an amino group, and when the dried hydrogel-forming article of the present application contains a crosslinked body of a vinyl alcohol-based polymer having a carboxyl group, these physiologically active substances are easily complexed with the crosslinked body via an amide bond, and are thus preferred. As the physiologically active substance, a physiologically active polypeptide containing two or more amino acids is preferred. Furthermore, the amino group can be either an aromatic amino group or an aliphatic amino group, with an aliphatic amino group being preferred, and the side chain amino group of lysine (Lys) of the physiologically active polypeptide or the amino group at the N terminus being preferred. Note that the amino group contained in the physiologically active substance can be either a primary amino group or a secondary amino group, and either one or both can be included. Either a primary amino group or a secondary amino group can form an amide bond with the carboxyl group possessed by the crosslinked body of a vinyl alcohol-based polymer.

[0125] <Complex>

[0126] In one embodiment of the present invention, the dry hydrogel-forming article of the present invention is a complex with a physiologically active substance. In this embodiment, the dry hydrogel-forming article of the present invention may be a dry hydrogel-forming article simply containing the aforementioned physiologically active substance, or may be a complex in which a cross-linked vinyl alcohol polymer contained in the dry hydrogel-forming article is covalently bonded to the physiologically active substance. Preferably, the dry hydrogel-forming article is a dry hydrogel-forming article in which the physiologically active substance is covalently bonded. The covalent bond between the cross-linked vinyl alcohol polymer and the physiologically active substance retains the physiologically active substance in the hydrogel, enabling stable function.

[0127] Methods for covalently bonding the hydrogel particles to the physiologically active substance include, for example, a method of activating the hydroxyl groups of PVA to react with the functional groups of the physiologically active substance to form a covalent bond; a method of using carboxylic acid-modified PVA or amino-modified PVA to react with the functional groups of the physiologically active substance to form a covalent bond; etc. From the perspective of reaction efficiency, the functional group of the physiologically active substance is preferably an amino group, a carboxylic acid group, or a thiol group, and more preferably a carboxylic acid group.

[0128] Specific methods for activating the hydroxyl groups of PVA and introducing physiologically active substances include, for example, methods using hydroxyl-activating reagents such as 1,1'-carbonyldiimidazole, bis(N-succinimidyl) carbonate, p-toluenesulfonyl chloride, 2,2,2-trifluoroethanesulfonyl chloride, and cyanuric chloride. These hydroxyl-activating reagents react with the hydroxyl groups of PVA to form covalent bonds with functional groups such as primary or secondary amino groups, carboxyl groups, and hydroxyl groups of the physiologically active substance. Alternatively, anhydride reagents such as succinic anhydride, which can form an ester bond with the hydroxyl groups of the aforementioned vinyl alcohol polymer to introduce carboxylic acids, and acetal reagents such as 2,2-dimethoxyethylamine, which can form an acetal bond with the 1,3-diol groups of the aforementioned vinyl alcohol polymer to introduce amino groups. These carboxylic acids and amino groups can form amide bonds with amino groups or carboxylic acids of the physiologically active substance using, for example, the aforementioned carbodiimide condensing agents. For example, when an acid anhydride reagent such as succinic anhydride reacts with a cross-linked vinyl alcohol polymer, a carboxyl group (COOH) derived from succinic acid is introduced at the terminal. This carboxyl group is condensed with the amino group of the physiologically active substance using a condensation agent such as 1,1'-carbonyldiimidazole, dicyclohexylcarbodiimide, or a water-soluble carbodiimide, thereby forming a complex with the cross-linked vinyl alcohol polymer via an amide bond.

[0129] As a specific method for introducing a physiologically active substance into a carboxylic acid-modified PVA or an amino-modified PVA, there can be mentioned the method of forming an amide bond with the amino group or carboxylic acid of the physiologically active substance using the aforementioned carbodiimide condensation agent, etc. Although the above examples can be used, the most suitable method including other methods can also be used depending on the application.

[0130] As described below, the complex can be formed after the crosslinking step of the uncrosslinked polymer solution, or it can be formed during the uncrosslinked polymer solution stage. In order to avoid the reduction or loss of activity of the physiologically active substance due to crosslinking, the complex is preferably formed after the crosslinking step.

[0131] 2. Method for producing dry hydrogel-forming articles

[0132] The method for producing a dry hydrogel-forming article of the present invention is not particularly limited, and examples thereof include the steps of preparing a preliminary hydrogel comprising a crosslinked product of a vinyl alcohol polymer (preliminary hydrogel preparation step), immersing the preliminary hydrogel in an alcohol solvent and then drying it to obtain a dry preliminary hydrogel (dried preliminary hydrogel preparation step), and treating the dry preliminary hydrogel with a SAL of 1×10 -6 The production method includes a sterilization step (sterilization step) as follows.

[0133] (Preparatory hydrogel preparation steps)

[0134] First, in the preliminary hydrogel preparation step, a preliminary hydrogel comprising a crosslinked product of a vinyl alcohol polymer is prepared. Specifically, the preliminary hydrogel preparation step preferably includes at least the steps of preparing an uncrosslinked polymer solution comprising the aforementioned vinyl alcohol polymer (uncrosslinked polymer solution preparation step); thereafter, shaping the uncrosslinked polymer solution (shaping step); and then, crosslinking the vinyl alcohol polymer contained in the uncrosslinked polymer solution to form a gel (crosslinking step). The present invention also provides a method for producing a dried hydrogel comprising the aforementioned steps. Each step is described in detail below.

[0135] Preparation steps of uncrosslinked polymer solution

[0136] The step of preparing an uncrosslinked polymer solution is a step of preparing an uncrosslinked polymer solution containing the aforementioned vinyl alcohol-based polymer. The uncrosslinked polymer solution can be obtained by dissolving the aforementioned 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; monohydric alcohols such as methanol, ethanol, propanol, and isopropanol; and polyhydric alcohols such as ethylene glycol, diethylene glycol, triethylene glycol, and glycerol. The water-soluble organic solvent may be used alone or as a mixture of two or more, and a water-soluble organic solvent and water may be used in combination.

[0137] When the uncrosslinked polymer solution contains the water-soluble organic solvent, its content is preferably 30% by mass or less, more preferably 20% by mass or less, and even more preferably 10% by mass or less, based on the total amount of the uncrosslinked polymer solution. 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.

[0138] 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, based on the total amount of the uncrosslinked polymer solution. Furthermore, from the perspective of suppressing the increase in viscosity of the uncrosslinked polymer solution and achieving 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 low. If the content exceeds 50% by mass, the viscosity of the uncrosslinked polymer solution is high, making molding difficult.

[0139] Molding steps

[0140] The molding step is a step of molding the uncrosslinked polymer solution into a desired shape. The method for molding the uncrosslinked polymer solution is not particularly limited, and known methods can be used to mold the uncrosslinked polymer solution into shapes such as the aforementioned amorphous particles, spherical particles, fine molded bodies, articles of any shape molded using a 3D printer, sheets, filaments, hollow fibers, porous blocks, and coated articles.

[0141] Cross-linking step

[0142] The cross-linking step is a step of cross-linking the vinyl alcohol polymer after the molding step. The cross-linking in this step can be performed by the aforementioned method. The cross-linking step can be performed using an uncross-linked polymer solution containing a solvent, or can be performed after further removing the solvent from the uncross-linked polymer solution. In the cross-linking step, when cross-linking is performed in the state of an uncross-linked polymer solution containing a solvent, the cross-linked gel shrinks violently in the drying step described later, making it difficult to form shapes such as sheets, filaments, hollow fibers, coatings, etc. In this case, it is preferred to dry the uncross-linked polymer solution first after molding and then cross-link it. The degree of drying of the uncross-linked polymer solution can be selected to a suitable state according to the molded product and the cross-linking method. From the viewpoint of easily preventing shrinkage in the drying step, it is sometimes preferred to perform cross-linking after drying so that the solvent content is preferably 50% or less, more preferably 25% or less, and even more preferably 10% or less.

[0143] The uncrosslinked polymer solution contains not only the aforementioned vinyl alcohol polymer before molding, but also the aforementioned crosslinking agent, initiator and other components required for crosslinking. As mentioned above, when the uncrosslinked polymer solution is dried first after molding, the components required for crosslinking can be included after molding. As mentioned above, the most suitable conditions are selected according to the vinyl alcohol polymer used and the crosslinking method. If crosslinking is completed to form a hydrogel, molding becomes difficult, so it is preferred to mold before forming a hydrogel. The above-mentioned crosslinking method includes a method of starting the reaction at a temperature below room temperature by simply mixing the uncrosslinked polymer solution with the components required for crosslinking. Therefore, it is desirable to mix immediately before molding. In addition, when thermal crosslinking is required, from the viewpoint of maintaining the activity of the physiologically active substance, the temperature of the crosslinking reaction is preferably 100°C or less, more preferably 60°C or less, and even more preferably 37°C or less.

[0144] (Combination step)

[0145] When a physiologically active substance is complexed in the dry hydrogel-forming article of the present invention, the preliminary hydrogel preparation step preferably further includes a complexing step. The complexing step is a step of complexing the physiologically active substance with a cross-linked vinyl alcohol polymer to produce a complexed hydrogel. However, if the complexing step is performed at the stage of an uncrosslinked polymer solution, the complexing step is generally unnecessary.

[0146] When a carboxylic acid-modified vinyl alcohol copolymer or an amino-modified vinyl alcohol polymer is used as the vinyl alcohol polymer, the residual functional group can be introduced into a physiologically active substance by the above-mentioned composite method. In addition, the hydroxyl group etc. contained in the cross-linked body of the vinyl alcohol polymer after the cross-linking step can first be introduced into the functional group for composite, and then the composite step is implemented. The method for introducing the functional group can be as described above, using a hydroxyl activating reagent, anhydride reagent or acetal reagent, etc., which can also similarly introduce a physiologically active substance into the functional group by the above-mentioned composite method.

[0147] (Drying preparatory hydrogel preparation step)

[0148] The hydrogel obtained in the preliminary hydrogel preparation step is dried by a common method such as hot air drying, vacuum drying, or freeze drying before sterilization, so that the alcohol solvent content and water content fall within the following ranges. For example, it is preferred that the preliminary hydrogel obtained in the preliminary hydrogel preparation step be immersed in an alcohol solvent, and then the water contained in the hydrogel be replaced with the alcohol solvent before drying. To avoid reduction or loss of activity of the physiologically active substance, the hot air drying temperature is preferably approximately 30-100°C, more preferably approximately 37-60°C. Drying is preferably performed such that the alcohol solvent content in the dried hydrogel-forming article after drying is preferably 0.01-15% by mass, more preferably 0.05-15% by mass, even more preferably 0.07-15% by mass, and even more preferably 0.1-15% by mass, and the water content is preferably 0-70% by mass, more preferably 0-60% by mass, even more preferably 0-50% by mass, and even more preferably 0-40% by mass or less.

[0149] The alcoholic solvent in which the pre-hydrogel is immersed is not particularly limited, and examples thereof include monoalcohols such as methanol, ethanol, propanol, and isopropanol, and polyols such as ethylene glycol, diethylene glycol, triethylene glycol, and glycerol. Alcoholic solvents may be used alone or as a mixture of two or more, or as a mixture of an alcoholic solvent and water.

[0150] (Sterilization step)

[0151] During the sterilization step, the dry prepared hydrogel was sterilized to a SAL of 1 × 10 -6 By sterilizing the hydrogel to the above extent, the hydrogel can be used in pharmaceuticals for human use or incorporated into a part of a medical device.

[0152] In the present invention, the dried hydrogel-forming article is preferably a dried hydrogel-forming article sterilized in a dry state with an alcohol solvent and water content within the above-mentioned range, and is sterilized after the drying step. When a composite hydrogel containing a large amount of water is sterilized before the drying step, for example, when a physiologically active substance is composited, the activity may be reduced. In addition, due to the generation of free radicals, when the hydrogel is used for cell culture, the cell adhesion may be reduced. It should be noted that although there are existing technologies that sterilize before cross-linking, when the SAL is 1×10 -6 In the case of sterilization under the following strict conditions, it is considered that cross-linking of the vinyl alcohol polymer proceeds during sterilization, and sufficient moldability and strength after molding cannot be obtained.

[0153] As long as SAL = 1 × 10 -6 Hereinafter, the sterilization method is not particularly limited. For example, autoclave sterilization, ethylene oxide gas sterilization, hydrogen peroxide low-temperature plasma sterilization, dry heat sterilization, chemical sterilization using glutaraldehyde, etc., radiation sterilization using gamma rays or electron beams, etc. can be used. When the composite physiologically active substances are compounded, from the viewpoint of maintaining their activity and improving the cell adhesion when the hydrogel is used for cell culture, etc., ethylene oxide gas sterilization, hydrogen peroxide low-temperature plasma sterilization, and radiation sterilization are preferably used. From the viewpoint of no residue, radiation sterilization is further preferred. The dry hydrogel-forming article of the present invention preferably does not contain ethylene oxide gas, does not contain glutaraldehyde, and does not contain hydrogen peroxide. Sterilization is preferably carried out by placing the pre-dried hydrogel-forming article in a container, sealing the container, and then performing a sterilization step. As a container, a flexible resin container, such as a resin soft bag, can be used, and sealing can be performed by heat sealing. In addition, a bottle container with a lid that can be opened after sterilization can be used as a container, and sealing can be performed by screwing a lid or ampoule with a bolt, etc., and heat sealing can be performed. For example, when performing radiation sterilization, it is preferred to use a container of a raw material having sufficient strength and radiation transmittance. As the raw material of the container, polyolefin resins such as polyethylene, polypropylene, polybutene or copolymers of more than two olefins, polyester resins such as polyethylene terephthalate, polybutylene terephthalate, polyvinyl alcohol, ethylene-vinyl acetate copolymer saponification, polyvinyl chloride, polyvinylidene chloride, vinyl resins, polycarbonate resins, polystyrene resins, silicone resins, polyamide resins, glass, etc. can be enumerated. The aforementioned container can be composed of a monolayer or laminate of these raw materials, or a metal foil or a multilayer body comprising metal foil, such as a material obtained by vapor-depositing aluminum, silicon dioxide, etc. on these raw materials, an aluminum film, an aluminum laminated film, etc., can be used.

[0154] Methods for implementing sterilization validation include the half-cycle method, the overkill method, the bioburden / BI combined method, and the absolute bioburden method. Any of these methods can be used. In the case of radiation sterilization, sterilization validation based on the absolute bioburden method described in ISO (ISO 11137-2: 2013) or JIS (JIS T 0806-2: 2014) is performed. Methods for setting the sterilization radiation dose in sterilization validation include Method 1, VD max Method 1 and Method 2 (Method 2) can both be used for sterilization when manufacturing the dry hydrogel-forming article of the present invention. max Method 1 is a method for setting the sterilization radiation dose based on the number of bacteria attached to the pre-dried hydrogel-forming article before sterilization, and Method 2 is a method for setting the sterilization radiation dose based on the radiation resistance of the bacteria attached to the pre-dried hydrogel-forming article before sterilization. max The sterilization dose can be determined by performing a bioburden test according to ISO 11737-1:2006 or JIS T11737-1:2013, etc., and measuring the number of bacteria attached to the pre-dried hydrogel-forming article before sterilization. Considering the number of samples required and the cost, method 1 and VD are more preferable. max If cost reduction is considered, VD is more preferred. max Law.

[0155] To achieve SAL=10 -6 Below, Method 1 requires 11 kilogray (kGy) or more, VD max Method 2 requires an irradiation dose of 15 kGy or more. In addition, method 2 requires an irradiation dose of 8.2 kGy or more. It was previously recognized that hydrogels (especially composite hydrogels) could not withstand this very high energy γ-ray irradiation dose. In a preferred embodiment of the present invention, by sterilizing the dried pre-dried hydrogel-forming article, for example, even high energy γ-ray irradiation of 8.2 kGy or more can prevent damage to the product, and its activity can be maintained within the desired range while suppressing the reduction of cell adhesion and the complexation of physiologically active substances. In a preferred embodiment of the manufacturing method of the present invention, it is preferred to perform radiation sterilization in the sterilization step so that the SAL reaches 1×10 -6 Hereinafter, it is more preferred that the radiation exposure during the radiation sterilization be 8.2 kGy or more.

[0156] 3. Hydrogel

[0157] The dried hydrogel-forming article of the present invention can be swollen in water and used as a hydrogel. The present invention also provides a hydrogel that is a swollen body (preferably a water-swollen body) of the dried hydrogel-forming article of the present invention. The hydrogel of the present invention usually contains water, but may contain a water-soluble organic solvent other than water, or a mixture of water and 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 water-soluble organic solvents such as polyols such as ethylene glycol, diethylene glycol, triethylene glycol, and glycerol may also be used in combination. Based on the total amount of the hydrogel, the content of the solvent (preferably water and / or water-soluble organic solvent) in the hydrogel is preferably 30 to 99.9% by mass, more preferably 40 to 99% by mass, and even more preferably 50 to 97% by mass. Based on the total amount of the hydrogel, the content of water in the hydrogel is preferably 30 to 95% by mass, more preferably 40 to 90% by mass, and even more preferably 50 to 90% by mass. The content of the water-soluble organic solvent in the hydrogel is preferably 30 to 95% by mass, more preferably 40 to 95% by mass, and even more preferably 50 to 95% by mass.

[0158] The solid content of the hydrogel of the present invention is preferably 0.1 to 70% by mass, more preferably 1 to 60% by mass, and even more preferably 3 to 50% by mass, based on the total weight of the hydrogel, from the viewpoint of strength. The solid content of the hydrogel can be calculated by the following formula (IV) based on the weight of the hydrogel before drying and the weight of the hydrogel after drying at 120°C for 5 hours.

[0159] Solid content [mass %] = (weight of hydrogel after drying / weight of hydrogel before drying) × 100 (IV)

[0160] The hydrogel of the present invention can be used in combination with cells or contain cells. When used in combination with cells or containing cells, it is preferred that water be supplied to the aforementioned article to form the hydrogel. The term "cell" as used herein is not particularly limited and preferably includes pluripotent stem cells, tissue stem cells, somatic cells, established mammalian cell lines used in the production or treatment of useful substances such as pharmaceuticals, and insect cells.

[0161] Cells include adherent cells and suspension cells. Adherent cells refer to cells that grow by attaching to a support, such as the hydrogel of the present invention, during cell culture. Suspension cells refer to cells that essentially do not require attachment to a support for growth. Suspension cells include cells that can only weakly attach to a support.

[0162] The above-mentioned pluripotent stem cells refer to stem cells that have the ability to differentiate into cells of any tissue (differentiation pluripotency), such as embryonic stem cells (ES cells), artificial pluripotent stem cells (iPS cells), embryonic germ stem cells (EG cells), germ stem cells (GS cells), etc.

[0163] The above-mentioned tissue stem cells refer to stem cells that have the ability to differentiate into various cell types (differentiation multipotency) although the tissues into which they differentiate are limited. For example, tissue stem cells include undifferentiated mesenchymal stem cells from bone marrow, skeletal muscle stem cells, hematopoietic system stem cells, neural stem cells, liver stem cells, adipose tissue stem cells, epidermal stem cells, intestinal stem cells, sperm stem cells, pancreatic stem cells (pancreatic duct epithelial stem cells, etc.), leukocyte stem cells, lymphocyte stem cells, corneal stem cells, etc.

[0164] The somatic cells mentioned above refer to cells that constitute multicellular organisms, and examples thereof include osteoblasts, chondrocytes, hematopoietic cells, epithelial cells (such as mammary epithelial cells), endothelial cells (such as vascular endothelial cells), epidermal cells, fibroblasts, mesenchymal-derived cells, cardiomyocytes, myoblasts, smooth muscle cells, skeletal muscle cells of organism origin, human tumor cells, fibrocytes, Epstein-Barr virus mutant cells, hepatocytes, kidney cells, bone marrow cells, macrophages, hepatocytes, small intestinal cells, mammary cells, salivary gland cells, thyroid cells, skin cells, plasma cells, T cells, B cells, killer cells, lymphoblasts, and pancreatic β cells, but are not limited thereto.

[0165] Examples of established 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, HE2 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, and MIA cells. 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.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, etc.

[0166] Examples of the above-mentioned insect cells include silkworm cells (BmN cells and BoMo cells, etc.), wild silkworm cells, tussah cells, pearl silkworm cells, cabbage looper cells (Sf9 cells and Sf21 cells, etc.), ecdysis moth cells, leaf roller moth cells, fruit fly cells, flesh fly cells, aedes aegypti cells, swallowtail butterfly cells, American cockroach cells and cabbage looper cells (Tn-5 cells, HIGHFIVE cells and MG1 cells, etc.), etc.

[0167] The cells can aggregate or differentiate. Aggregated cells can function as organs. The cells can be freshly collected from an organism or cultured. Cells collected from an organism can form organs.

[0168] When the hydrogel of the present invention is used to culture cells in contact with cells, it is preferred to add culture medium components. The culture medium used is not particularly limited and can be freely selected to be suitable for the cells. The culture medium can use basic culture media such as DMEM, MEM, F12 with added fetal bovine serum, reduced serum culture medium, serum-free culture medium, etc. The serum-free culture medium that can be used includes a culture medium that does not contain xenobiotics (xeno-free) and a complete synthetic culture medium that not only does not contain serum but also does not contain protein components and animal components.

[0169] Hydrogel of the present invention is a hydrogel through fully sterilizing, and, because agglutination during drying is suppressed, therefore dispersibility is good, in addition, can prevent the fragment of hydrogel etc. from sneaking into.In addition, hydrogel of the present invention preferably has following characteristics, can maintain high swelling degree, has high strength, can maintain its activity when containing physiologically active substance etc., has high adhesiveness etc. when being used for cell adhesion.Hydrogel of the present invention preferably can provide hydrophilicity, reactivity, biodegradability, bioadaptability and low toxicity etc. excellence, flexibility and intensity height, through sterilizing hydrogel, and do not use the expensive processes such as aseptic environment.Therefore, hydrogel of the present invention is useful as carrier (carrier or support).The carrier that comprises hydrogel of the present invention can be suitable for the purposes such as enzyme immobilization carrier, affinity carrier, cell culture carrier, drug delivery carrier.

[0170] The hydrogel of the present invention is obtained by swelling the dried hydrogel-forming article of the present invention. The vinyl alcohol polymer contained in the dried hydrogel-forming article of the present invention is also contained in the hydrogel of the present invention. It should be noted that, unless otherwise explicitly stated in this specification, the details, production methods, and usage of the hydrogel of the present invention are the same as those described in "1. Dried Hydrogel-Forming Article."

[0171] The hydrogel of the present invention is a substance that forms a hydrogel when the dried hydrogel-forming article of the present invention swells upon contact with an aqueous solution such as water, a buffer solution, a body fluid, or a culture medium. The hydrogel of the present invention may be a hydrogel that completely swells until the dried hydrogel-forming article no longer swells, or a hydrogel that is capable of further swelling and still has hydrogel-forming properties.

[0172] The shape of the hydrogel is not particularly limited and, similar to dry hydrogel-forming articles, can be in the form of amorphous particles, spherical particles, finely molded articles, articles of any shape formed using a 3D printer, sheets, threads, hollow fibers, porous blocks, or coated articles. The hydrogel of the present invention is preferably in the form of spherical particles, more preferably spherical particles having a particle size of 10 to 5000 μm.

[0173] In this specification, "amide bond complex hydrogel" refers to a hydrogel in which a physiologically active substance having an amino group is covalently bonded to the carboxyl group of the vinyl alcohol polymer via an amide bond on a crosslinked product of a vinyl alcohol polymer having an ethylenically unsaturated group and a carboxyl group.

[0174] Furthermore, examples of the hydrogel's morphology, size, and production method include those described in "1. Drying of Hydrogel-Forming Articles."

[0175] The hydrogel of the present invention is a swollen body of the dried hydrogel-forming article of the present invention, preferably a water-swollen body of the dried hydrogel-forming article of the present invention. A water-swollen body is a body that swells upon contact with a solution containing at least water. For example, it may swell upon contact with water or upon contact with an aqueous solution such as a buffer, body fluid, or culture medium. It should be noted that the alcoholic solvent contained in the dried hydrogel-forming article of the present invention may or may not be contained in the hydrogel of the present invention. For example, when a dried hydrogel-forming article of the present invention containing a cross-linked product of a vinyl alcohol polymer and an alcoholic solvent is placed in water and swelled, the alcoholic solvent contained in the dried hydrogel-forming article of the present invention is released into the water, and the hydrogel of the present invention may no longer contain alcohol. However, such a hydrogel can also be considered a water-swollen body of the dried hydrogel-forming article of the present invention. The hydrogel of the present invention is a swollen body of a dry hydrogel-forming article that satisfies a specific degree of cohesion and sterility assurance level SAL. Therefore, the hydrogel contains little impurities such as gel fragments and is a fully sterilized hydrogel.

[0176] The sterility assurance level SAL of the hydrogel of the present invention is not particularly limited, but preferably has the same sterility assurance level SAL as that of the dry hydrogel-forming article = 1×10 -6 the following.

[0177] <Crosslinked vinyl alcohol polymers>

[0178] The hydrogel of the present invention comprises a cross-linked product of the same vinyl alcohol polymer as the dried hydrogel-forming article of the present invention. Unless otherwise specified in this specification, the description of the cross-linked product of the vinyl alcohol polymer contained in the hydrogel also applies to the cross-linked product of the vinyl alcohol polymer contained in the dried hydrogel-forming article.

[0179] <Physiologically active substances>

[0180] When the dried hydrogel-forming article of the present invention further contains a physiologically active substance, it contains the physiologically active substance in the same manner as the hydrogel of the present invention. Unless otherwise specified in this specification, the description of the physiologically active substance that can be contained in the hydrogel also applies to the description of the physiologically active substance that can be contained in the dried hydrogel-forming article.

[0181] <Method for producing hydrogel>

[0182] The hydrogel of the present invention is a swollen body of the dry hydrogel-forming article of the present invention, and is obtained by contacting the dry hydrogel-forming article with water, the above-mentioned water-soluble organic solvent, an aqueous solution, or a mixture thereof.

[0183] Hydrogel of the present invention is a hydrogel through fully sterilizing, because aggregation during drying is suppressed, therefore can not produce the mixing of fragment, destruction on surface etc., is suitable as the hydrogel used for medical purposes etc. and uses.In addition, hydrogel of the present invention is in a preferred embodiment, owing to use the cross-linking method of hydrophilicity, reactivity, biodegradability, excellent biocompatibility, flexibility and intensity height, low toxicity, therefore also has toxicity reduction, efficiently imports the advantages such as physiologically active substance or enzyme.Therefore, hydrogel of the present invention is useful as carrier (carrier or support).The carrier comprising hydrogel of the present invention can be suitable for the purposes such as enzyme immobilization carrier, affinity carrier, cell culture carrier, drug delivery carrier.According to dry hydrogel formative article of the present invention, above-mentioned excellent hydrogel can be provided.

[0184] Example

[0185] Hereinafter, the present invention will be described in further detail with reference to Examples, but the present invention is not limited to these Examples.

[0186] [Raw materials used]

[0187] The main components used in Synthesis Examples, Examples, and Comparative Examples are shown below.

[0188] <Raw Material PVA>

[0189] PVA117: Polyvinyl alcohol (trade name "PVA117," degree of polymerization 1700, saponification degree approximately 98.0-99.0 mol%, viscosity (4%, 20°C) 25.0-31.0 mPa·s, manufactured by Kuraray Co., Ltd.)

[0190] In addition, the degree of polymerization of the raw material PVA was measured in accordance with JIS K 6726:1994.

[0191] AF-17: Polyvinyl alcohol (trade name "AF-17," 1.3 mol% itaconic acid copolymer (Scientific Reports, 2017, Vol. 7, p. 45146), degree of polymerization 1700, saponification degree 96.5 mol% or higher, viscosity (4%, 20°C) 30 ± 3 mPa·s, manufactured by Japan Vam & Poval Co., Ltd.)

[0192] <Compounds containing an ethylenically unsaturated group>

[0193] · Vinyl methacrylate: manufactured by Tokyo Chemical Industry Co., Ltd.

[0194] 5-Norbornene-2-carboxaldehyde: manufactured by Tokyo Chemical Industry Co., Ltd.

[0195] <Synthetic Reagents>

[0196] 50% glutaraldehyde solution: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.

[0197] · Triethylamine: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.

[0198] Succinic anhydride: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.

[0199] Liquid paraffin: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.

[0200] Span80: Sorbitan monooleate, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.

[0201] <Radical polymerization initiator>

[0202] Potassium persulfate: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.

[0203] L0290: Phenyl (2,4,6-trimethylbenzoyl) phosphonate lithium salt (photoradical polymerization initiator, trade name "L0290", manufactured by Tokyo Chemical Industry Co., Ltd.)

[0204] Polythiol

[0205] 3,6-Dioxa-1,8-octanedithiol: manufactured by Tokyo Chemical Industry Co., Ltd.

[0206] <Activating reagents for hydroxyl groups>

[0207] 1,1'-Carbonyldiimidazole: Made by Tokyo Chemical Industry Co., Ltd.

[0208] Succinic anhydride: manufactured by Tokyo Chemical Industry Co., Ltd. or Fujifilm Wako Pure Chemical Industries, Ltd.

[0209] <Complex reagent>

[0210] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.

[0211] N-Hydroxysuccinimide: manufactured by Tokyo Chemical Industry Co., Ltd.

[0212] <Physiologically active substances>

[0213] Gelatin (porcine origin, type A): manufactured by Sigma-Aldrich Japan Co., Ltd.

[0214] Collagen (trade name Cellmatrix, Type IC): manufactured by Nitta Gelatin Co., Ltd.

[0215] Solvents

[0216] Ion exchange water: conductivity 0.08×10 -4Ion exchange water below S / m

[0217] PBS: prepared by dissolving PBS tablets (manufactured by Takara Bio Inc.) in a predetermined amount of ion-exchanged water.

[0218] MES buffer: A 0.1 mol / L aqueous solution of 2-morpholinoethanesulfonic acid monohydrate (manufactured by Dojindo Chemical Laboratories) was prepared and neutralized with a 0.1 mol / L aqueous NaOH solution to adjust the pH to 5.6.

[0219] [Measurement Methods of Compounds Synthesized in Synthesis Examples]

[0220] <Introduction rate of ethylenically unsaturated groups and carboxyl groups (succinic acid)>

[0221] The introduction ratio of the ethylenically unsaturated group and the carboxyl group (succinic acid) of the vinyl alcohol polymer having an ethylenically unsaturated group obtained in the following synthesis example is determined by 1 H-NMR measurement: The introduction rate was determined from the ratio of the integrated value of the signal of the ethylenically unsaturated group and the carboxyl group (succinic acid methylene group) to the signal of the vinyl alcohol polymer.

[0222] Similarly, for hydrogel particles and sheets, it is also possible to try them in a swollen state in a deuterated solvent. 1 In H-NMR measurement, the introduction rate into the hydrogel particles and the sheet was determined from the ratio of the integrated value of the carboxyl group (succinic acid methylene) signal to the integrated value of the vinyl alcohol polymer signal.

[0223] [ 1 H-NMR measurement conditions]

[0224] Equipment: JEOL Ltd. nuclear magnetic resonance device "JNM-ECX400"

[0225] Temperature: 25℃

[0226] [Water content and alcohol solvent content]

[0227] The moisture content of the dried hydrogel-forming articles obtained in the following examples was measured by a heat-drying moisture meter when only water was contained as a solvent, and by a water-soluble organic solvent such as an alcohol solvent. 1 H-NMR, measured. [Heating and drying type moisture meter measurement conditions]

[0228] Device: Heat drying moisture meter manufactured by AE-ANDO-DEI Co., Ltd.

[0229] [ 1 H-NMR measurement conditions]

[0230] The content of water and alcohol solvent can be determined by 1The water content was calculated by H-NMR (deuterated DMSO solvent). The water content was determined from the ratio of the integrated 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 was subtracted.

[0231] Equipment: JEOL Ltd. nuclear magnetic resonance device "JNM-ECX400"

[0232] Temperature: 25℃

[0233] <Sterilization>

[0234] By VD max The sterilization dose specified by the law is sterilized by gamma ray to ensure that the dry hydrogel-forming articles reach the sterility assurance level SAL = 10 -6 Below. Achieve sterility assurance level SAL = 10 -6 If the sterilization rate is below 0, the sterilization rate is marked as “0”, and if it is not reached, the sterilization rate is marked as “×”.

[0235] <Agglutination degree>

[0236] The degree of aggregation of a dried hydrogel-forming article was determined by measuring 0.2 g of the dried hydrogel-forming article, visually separating two or more aggregated articles, and measuring the weight of the separated aggregates. The weight was then calculated using the following formula (II). An aggregation degree of 10% or less was evaluated as "0," while an aggregation degree exceeding 10% was evaluated as "×."

[0237] Agglutination degree (%) = (weight of aggregate / total weight) × 100... (II)

[0238] <Breakage rate>

[0239] The breakage rate of a dried hydrogel-forming article was determined by measuring the breakage of 0.1 g of the dried hydrogel-forming article, placing it in a 50 ml glass bottle (bottom diameter 25 mm) along with 10 ml of ion-exchanged water, and stirring it at 400 rpm with a 20 mm diameter stirring head for 3 hours. Specifically, the state of the solid content after stirring was observed under a microscope, with the solid content visible in at least 60% of the visual field. The breakage rate was visually distinguished between broken and unbroken areas, and the breakage rate was calculated based on the ratio of the area of ​​the broken portion to the total area of ​​the solid content. In the present examples and comparative examples, the breakage rate was calculated visually due to the very high or very low breakage rates, but the breakage rate can also be calculated by measuring the area using imaging software.

[0240] [Synthesis example]

[0241] <Synthesis of vinyl alcohol polymer having ethylenically unsaturated groups>

[0242] [Synthesis Example 1-1]

[0243] 40 g (monomer repeating unit: 908 mmol) of PVA117 (raw material PVA) was added to a separable flask with a 1 L Dimroth cooling tube, 350 mL of dimethyl sulfoxide (DMSO) was added, and stirring was started with a mechanical stirrer. After heating to 80 ° C in a water bath, stirring was continued at 80 ° C for 4 hours. After visually confirming that the raw material PVA was dissolved, heating and stirring were performed at 80 ° C, while adding 2.1 g (18.7 mmol) of vinyl methacrylate, and further stirring was performed at 80 ° C for 3 hours. After cooling, the reaction solution was injected into 2 L of methanol while stirring. Stop stirring and let it stand directly for 1 hour. After recovering the obtained solid, it was further immersed in 1 L of methanol for 1 hour and washed. This washing operation was performed a total of 3 times. The recovered solid was vacuum dried at room temperature overnight to obtain methacrylic acid PVA117. The introduction rate of the ethylenically unsaturated group (methacryloyl group) of the methacrylated PVA117 was 2.0 mol% relative to the repeating unit of the raw material PVA (hereinafter referred to as "MA-PVA117 (2.0)").

[0244] [Synthesis Example 1-2]

[0245] 60 g (monomer repeating unit: 1.36 mol) of PVA117 (raw material PVA) was added to a separable flask with a 1 L Dimroth cooling tube, 540 mL of ion exchange water was added, and stirring was started with a mechanical stirrer. After heating to 80°C in a water bath, stirring was continued at 80°C for 4 hours. After visually confirming that the raw material PVA was 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 solution of sulfuric acid were added, and the mixture was further stirred at 40°C for 4 hours. After cooling, 80 mL of a 1N NaOH aqueous solution was added and neutralized, and a dialysis membrane with a fractional molecular weight of 3,500 was added for desalination (implemented 4 times for 5 L of ion exchange water). The desalted aqueous solution was injected into 2 L of methanol while stirring and left directly for 1 hour. After recovering the obtained solid, it was further immersed in 1 L of methanol for 1 hour for washing. The recovered solid was vacuum-dried at room temperature overnight to obtain norbornene-modified (Nor) PVA 117. The introduction rate of ethylenically unsaturated groups (norbornene groups) in this norbornene-modified PVA 117 was 1.3 mol% relative to the repeating units of the starting PVA (hereinafter referred to as "Nor-PVA 117 (1.3)").

[0246] [Synthesis Example 1-3]

[0247] 10 g (monomer repeating unit: 220 mmol) of MA-PVA117 (2.0) prepared in Synthesis Example 1-1 was added to a separable flask with a 0.5 L Dimroth cooling tube, 90 mL of dimethyl sulfoxide (DMSO) was added, and stirring was started with a mechanical stirrer. After heating to 80°C in a water bath, stirring was continued at 80°C for 4 hours. After visually confirming that the aforementioned raw material PVA was dissolved, the water bath was set to 60°C. After confirming that the internal temperature reached 60°C, 1.2 g (12.1 mmol) of triethylamine and 1.1 g (11 mmol) of succinic anhydride were added, and the mixture was further stirred at 60°C for 5 hours. After cooling, the reaction solution was injected into 0.5 L of methanol while stirring. Stop stirring and let it stand for 1 hour. After recovering the obtained solid, it was further immersed in 0.5 L of methanol for 1 hour for washing. This washing operation was performed a total of 3 times. The recovered solid was vacuum-dried overnight at room temperature to obtain methacrylated PVA117 into which succinic acid had been introduced. The introduction ratio of succinic acid (SA) 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)").

[0248] <Manufacturing of molded products (pellets, sheets)>

[0249] [Synthesis Example 1-A Particles]

[0250] 440 mL of ion-exchanged water was added to 60 g of MA-PVA117 (2.0), and the mixture was stirred at 80°C for 4 hours to dissolve the mixture. After cooling to room temperature, potassium persulfate, a water-soluble thermal radical polymerization initiator, was added and dissolved in the MA-PVA117 (2.0) aqueous solution to a concentration of 0.1% by mass to prepare an uncrosslinked polymer solution.

[0251] 3300 mL of liquid paraffin and 8 g of Span80 were added to a separable flask with a 5 L Dimroth cooling tube, and the uncrosslinked polymer solution was slowly added thereto. The mixed solution was stirred at 350 rpm with a mechanical stirrer to form a W / O dispersion. After heating to 40°C in a water bath, nitrogen substitution was performed for 30 minutes. Thereafter, stirring was continued at 70°C for 3 hours. After cooling to room temperature, the liquid paraffin in which the hydrogel particles were dispersed was filtered with a mesh of 100 μm. 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 into a particle size of 180 to 300 μm using a JIS standard sieve. Furthermore, the hydrogel particles were added to 1 L of acetone and dehydrated, and then dried under reduced pressure to obtain dry hydrogel particles (hereinafter referred to as "MA-PVA117 (2.0) gel particles").

[0252] [Synthesis Example 1-B Particles]

[0253] To 60 g of Nor-PVA117(1.3) was added 440 mL of ion exchange water, and stirred at 80°C for 4 hours while dissolving. After cooling to room temperature, 3.4 g of 3,6-dioxa-l,8-octanedithiol as a polythiol was added to the Nor-PVA117(1.3) aqueous solution, and stirred. To this solution, potassium persulfate as a water-soluble thermal radical polymerization initiator was added to achieve 0.1 mass%, and dissolved, to prepare an uncrosslinked polymer solution. Using this uncrosslinked polymer solution, by the same method as in Synthesis Example 1-A, a dried hydrogel particle (hereinafter referred to as "Nor-PVA117(1.3) gel particle") was obtained.

[0254] [Synthesis Example 1-C sheet]

[0255] To 6 g of MA-PVA117(2.0)-SA(3.4) was added 44 mL of ion exchange water, and stirred at 80°C for 4 hours while dissolving. After cooling to room temperature, potassium persulfate as a water-soluble thermal radical polymerization initiator was added to the MA-PVA117(2.0)-SA(3.4) aqueous solution to achieve 0.1 mass%, and dissolved, to prepare an uncrosslinked polymer solution. The uncrosslinked polymer solution was flowed into 2 glass plates with a 0.5 mm spacer interposed therebetween under nitrogen, and cured at 40°C for 3 hours. The cured hydrogel sheet (10 x 20 cm) having a thickness of 0.5 mm was washed with a total of 1.5 L of ion exchange water. Further, it was dehydrated by immersion in 0.5 L of acetone, and dried under reduced pressure, to obtain a dried hydrogel sheet (hereinafter referred to as "MA-PVA117(2.0)-SA(3.4) gel sheet"). The introduction rate (active group introduction rate) of succinic acid (SA) to the dried PVA sheet was 3.4 mol%.

[0256] [Synthesis Example 1-D sheet]

[0257] To 6 g of PVA117 was added 44 mL of ion exchange water, and stirred at 80°C for 4 hours while dissolving. After cooling to room temperature, 7.5 mL of a 50% glutaraldehyde (GA) solution was added to the PVA117 aqueous solution after mixing, and further 6 mL of a 1 mol / L aqueous hydrochloric acid solution was added and mixed rapidly, to prepare an uncrosslinked polymer solution. The uncrosslinked polymer solution was flowed into 2 glass plates with a 0.5 mm spacer interposed therebetween, and cured at 65°C for 7 hours. The cured hydrogel sheet (10 x 20 cm) having a thickness of 0.5 mm was washed with a total of 1.5 L of ion exchange water. Further, it was dehydrated by immersion in 0.5 L of acetone, and dried under reduced pressure, to obtain a dried hydrogel sheet (hereinafter referred to as "GA-crosslinked PVA117 gel sheet").

[0258] <Activation of hydroxyl group>

[0259] [Synthesis Example 1-i Introduction of Succinic Acid into Molded Articles (Pellets)]

[0260] 1.5 g of MA-PVA117 (2.0) gel particles (monomer repeating unit: 33 mmol) prepared in Synthesis Example 1-A were added to a separable flask with a 0.5 L Dimroth cooling tube, 100 mL of dimethyl sulfoxide (DMSO) was added, and the mixture was stirred with a mechanical stirrer to swell. After stirring at 60 ° C for 1 hour in a water bath, 364 mg (3.6 mmol) of triethylamine and 330 mg (3.3 mmol) of succinic anhydride were added, and the mixture was further stirred at 60 ° C for 5 hours. After cooling, the particles were filtered out, washed twice with 100 mL of DMSO, and washed twice with 100 mL of ion exchange water. Then, the particles were immersed in 100 mL of acetone for 3 times for dehydration, and the obtained particles were dried under reduced pressure to obtain dry hydrogel particles into which succinic acid (SA) was 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 referred to as "SA-MA-PVA117(2.0) gel particles").

[0261] [Synthesis Example 1-ii: Introduction of Succinic Acid into Molded Articles (Pellets)]

[0262] Dry hydrogel particles incorporating succinic acid were obtained by the same method 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 succinic acid incorporation rate (active group incorporation rate) was 8.7 mol% relative to the repeating units of Nor-PVA117(1.3) (hereinafter referred to as "SA-Nor-PVA117(1.3) gel particles").

[0263] [Synthesis Example 1-iii: Introduction of Succinic Acid into Molded Article (Sheet)]

[0264] A dry hydrogel sheet incorporating succinic acid was obtained by the same method as in Synthesis Example 1-i, except that the dry GA-crosslinked PVA117 gel sheet prepared in Synthesis Example 1-D was used. The succinic acid incorporation rate (active group incorporation rate) was 9.8 mol% relative to the repeating units of the GA-crosslinked PVA117 (hereinafter referred to as the "SA-GA-crosslinked PVA117 gel sheet").

[0265] [Synthesis Example 1-iv: Introduction of Carbonyl Imidazolyl Groups into Molded Articles (Pellets)]

[0266] 377 mg (2.3 mmol) of carbonyldiimidazole (hereinafter referred to as "CDI") was dissolved in 7 g of dry acetonitrile, and 1.0 g of dry MA-PVA117 (2.0) gel particles (monomer repeating unit: 23 mmol) prepared in Synthesis Example 1-A were added thereto. Centrifuge at 40°C for 4 hours in a centrifugal sedimentation tube, shake the centrifugal sedimentation tube while reacting, and then wash 4 times with 50 mL of dry acetone. The particles washed with acetone were vacuum dried at room temperature overnight to obtain carbonyl imidazole-treated (CI-treated) hydrogel particles (hereinafter referred to as "CI-treated-MA-PVA117 (2.0) gel particles"). The introduction rate of carbonyl imidazole groups (active group introduction rate) relative to the repeating units of MA-PVA117 (2.0) was 13.5%.

[0267] <Combination>

[0268] [Synthesis Example 1-a]

[0269] 1 g of dried SA-MA-PVA117 (2.0) gel particles (succinic acid incorporation: approximately 1.9 mmol) prepared in Synthesis Example 1-i was swollen in MES buffer (pH = 5.6) overnight at room temperature. The swollen gel particles were dispersed in 45 mL of MES buffer and stirred with a magnetic stirrer. 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 and shaken at room temperature for 1 hour. The gel particles were washed with 30 mL of MES buffer for 10 minutes three times and then added to 70 mL of a 1 mg / mL gelatin solution in PBS. After shaking at room temperature for 3 hours, the obtained hydrogel particles were washed with 70 mL of ion exchange water (heated to 60°C) for 20 minutes, and the above operation was repeated twice 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 in an excess of PBS overnight, and the amount of gelatin immobilized per unit gel weight (100 mg) was measured by the bicinchoninic acid (BCA) method (BCA Protein Assay Kit (manufactured by Takara Bio Inc.)). The result showed that the composite density of the physiologically active substance was 48.5 μg / 100 mg of gel particles.

[0270] [Synthesis Example 1-b]

[0271] Collagen-composite hydrogel particles (hereinafter referred to as "collagen-SA-MA-PVA117 (2.0) gel particles") were obtained by the same method as in Synthesis Example 1-a, except that collagen was used instead of gelatin. The collagen-SA-MA-PVA117 (2.0) gel particles were also measured in the same manner as in Synthesis Example 1-a.

[0272] [Synthesis Example 1-c]

[0273] Using 1 g of the dried SA-Nor-PVA117 (1.3) gel particles prepared in Synthesis Example 1-ii, gelatin composite hydrogel particles (hereinafter referred to as "gelatin-SA-Nor-PVA117 (1.3) gel particles") were obtained by the same method as Synthesis Example 1-a. The gelatin immobilization amount (complexation density of physiologically active substances) of the gelatin-SA-Nor-PVA117 (2.0) gel particles was also measured by the same method as Synthesis Example 1-a.

[0274] [Synthesis Example 1-d]

[0275] A gelatin composite hydrogel sheet (hereinafter referred to as "gelatin-MA-PVA117(2.0)-SA(3.4) gel sheet") was obtained by the same method 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. The gelatin immobilization amount (complexation density of physiologically active substances) of the gelatin-MA-PVA117(2.0)-SA(3.4) gel sheet was also measured by the same method as in Synthesis Example 1-a.

[0276] [Synthesis Example 1-e]

[0277] A gelatin composite hydrogel sheet (hereinafter referred to as "gelatin-SA-GA cross-linked PVA117 gel sheet") was obtained by the same method as Synthesis Example 1-a, except that 1 g of the dried SA-GA cross-linked PVA117 gel sheet prepared in Synthesis Example 1-iii was used. The gelatin immobilization amount (complexation density of physiologically active substances) of the gelatin-SA-GA cross-linked PVA117 gel sheet was also measured by the same method as Synthesis Example 1-a.

[0278] [Synthesis Example 1-f]

[0279] 0.6 g of gelatin was dissolved in 100 mL of PBS at 60°C with a magnetic stirrer. The gelatin solution was cooled to room temperature, and the CI-MA-PVA117 (2.0) gel particles prepared in Synthesis Example 1-iv were added, and the mixture was shaken and stirred overnight at room temperature. The mixture was washed three times with 100 mL of PBS to obtain gelatinized gel particles (hereinafter referred to as "gelatin-carbamate bond-MA-PVA117 (2.0) gel particles"). The gelatin immobilization amount (complexation density of physiologically active substances) of the gelatin-carbamate bond-MA-PVA117 (2.0) gel particles was determined by the same method as in Synthesis Example 1-a.

[0280] <Drying of granules and gamma ray sterilization>

[0281] [Example 1-1]

[0282] 2 g of gelatin-SA-MA-PVA117 (2.0) gel particles (swollen with ion exchange water) prepared in Synthesis Example 1-a were repeatedly immersed in 25 mL of ethanol three times, and the dehydrated composite gel particles were vacuum dried at room temperature overnight. The water content and alcohol solvent content of the obtained dried particles were 1 Calculations were made using the above-described measurement method under H-NMR (deuterated DMSO solvent). A sterilized amide bond-complexed hydrogel was obtained by gamma ray irradiation using the same method as described in Comparative Example 1-1. The results of the measurement of water content and alcohol solvent content, as well as the evaluation results of sterilization and cohesion, are shown in Table 1. The cohesion and breakage rate of the dried hydrogel-forming article obtained in Example 1-1 were comparable to those in Example 1-2.

[0283] [Examples 1-2]

[0284] 2 g of collagen-SA-enhanced-MA-PVA117 (2.0) gel particles (swollen with ion-exchanged water) prepared in Synthesis Example 1-b were dried using the same method as in Example 1-1, and the water content and alcohol solvent content were measured and irradiated with gamma rays to obtain a sterilized amide bond-complexed hydrogel. The results of the water content and alcohol solvent content measurements are shown in Table 1. The dried hydrogel-forming article obtained in Example 1-2 had a cohesion rate of 5.3% and a breakage rate of 0%.

[0285] [Comparative Example 1-1]

[0286] The gelatin-SA-MA-PVA117 (2.0) gel particles (swelling with ion exchange water) of 2g prepared in synthesis example 1-a were spread on a small dish and dried naturally in air for 2.7 hours at room temperature. The result of measuring the water content of the obtained dried particles with a heating drying type moisture meter (A&D Company, Limited system) was 72% by mass. The dried gel particles were added to a 15mL centrifugal sedimentation tube, and further, in order to prevent the evaporation of water, they were enclosed in an aluminum vapor deposition bag and irradiated with gamma rays (25kGy) by KogaIsotope Ltd. to obtain a sterilized amide bond composite hydrogel. The measurement results of water content, etc. are shown in Table 1.

[0287] [Comparative Examples 1-2 to 1-4]

[0288] A sterilized amide-bonded composite hydrogel was obtained by measuring water content and irradiating with gamma rays using the same methods as in Comparative Example 1-1, except that 2 g of gelatin-SA-enhanced-MA-PVA117 (2.0) gel particles (swollen with ion-exchanged water) were naturally dried for 3.7, 4.5, and 5.5 hours. The results of the water content measurement are shown in Table 1.

[0289] [Comparative Examples 1-5]

[0290] 2 g of gelatin-SA-enhanced-MA-PVA117 (2.0) gel particles (swollen with ion-exchanged water) were spread on a small dish and vacuum-dried overnight at room temperature. A sterilized amide bond-complexed hydrogel was obtained by measuring the water content and irradiating with gamma rays using the same method as in Comparative Example 1-1. The results of the water content measurement are shown in Table 1. The dried hydrogel-forming article obtained in Comparative Example 1-5 had a cohesion degree of 99.8%.

[0291] [Comparative Examples 1-6 to 1-7]

[0292] A sterilized amide bond composite hydrogel was obtained by measuring water content and irradiating with gamma rays using the same methods as in Comparative Example 1-1, except that 2 g of gelatin-SA-enhanced-MA-PVA117 (2.0) gel particles (swollen with ion-exchanged water) were not naturally dried (Comparative Example 1-6) or the natural drying time was set to 0.5 hours (Comparative Example 1-7). The results of the water content measurement are shown in Table 1.

[0293] [Reference Example 1]

[0294] Using the gelatin-SA-Nor-PVA117 (1.3) gel particles (swollen with ion-exchanged water) prepared in Synthesis Example 1-c, drying, water content measurement, and gamma-ray irradiation were performed in the same manner as in Comparative Example 1-5 to obtain a sterilized amide bond complex hydrogel. The results of the water content measurement are shown in Table 1. It should be noted that for these gel particles, when dried, water content measurement, and gamma-ray irradiation are performed in the same manner as in Example 1-1 to obtain a sterilized amide bond complex hydrogel, it is believed that the alcohol solvent content, water content, aggregation degree, and cell adhesion rate are the same as those in Example 1-1.

[0295] [Reference Example 2]

[0296] A sterilized amide bond-complexed hydrogel was obtained by drying, measuring the water content, and irradiating with gamma rays using the same methods as in Comparative 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 results of the water content measurement are shown in Table 1. It is believed that the sterilized amide bond-complexed hydrogel obtained by drying, measuring the water content, and irradiating with gamma rays using the same methods as in Example 1-1 exhibited similar alcohol solvent content, water content, aggregation degree, and cell adhesion rate as in Example 1-1.

[0297] [Reference Example 3]

[0298] The gelatin-SA-GA cross-linked PVA117 gel sheet (swollen with ion-exchanged water) prepared in Synthesis Example 1-e was used. In addition, drying, water content measurement and gamma-ray irradiation were performed in the same manner as in Comparative Example 1-5 to obtain a sterilized amide bond composite hydrogel. The results of the water content measurement are shown in Table 1. It should be noted that for this gel sheet, it is believed that when the sterilized amide bond composite hydrogel is dried, the water content is measured and gamma-ray irradiation is performed in the same manner as in Example 1-1, the alcohol solvent content, water content, aggregation degree, and cell adhesion rate are the same as those in Example 1-1.

[0299] [Examples 1-3]

[0300] A sterilized composite hydrogel was obtained by drying, measuring the moisture content, and irradiating with gamma rays using the same methods as in Example 1-1, except that the gelatin-urethane bond-MA-PVA117 (2.0) gel particles (swollen with ion-exchanged water) prepared in Synthesis Example 1-f were used. The results of the moisture content measurement are shown in Table 1. The degree of aggregation and breakage rate of the dried hydrogel-forming article obtained in Example 1-3 were comparable to those in Example 1-2.

[0301] [Evaluation Method of Composite Hydrogels Obtained in Examples and Comparative Examples]

[0302] <Evaluation of cell adhesion (particles)>

[0303] Dissolve poly (2-hydroxyethyl methacrylate) in 95% ethanol at a concentration of 30 mg / mL, inject 200 μL of the solution into each well of a 24-well plate for cell culture (manufactured by IWAKI), and dry it in a clean bench. Next, immerse the sterilized amide bond composite hydrogel (spherical particles) obtained in Example 1-1 in PBS (phosphate buffered saline) for 1 hour to swell the particles. Add 200 μL of the gel particles to one well of the coated well plate, and further add 500 μL of DMEM culture medium supplemented with 10% fetal bovine serum to the same well. 1.9×10 NIH / 3T3 cells (purchased from ATCC) grown by prior culture were added thereto. 5 The cells were cultured under conditions of 5% carbon dioxide concentration, saturated water vapor pressure, and 37°C. After 24 hours of culture, the center of the culture well was photographed using an inverted microscope equipped with a camera and image linking software. The number of particles on which cells adhered and spread was counted. Cells adhered and spread on 132 of the 200 gel particles. The cell adhesion rate (%) was calculated as the percentage of the number of particles on which cells adhered and spread out of the 200 particles. The cell adhesion rate was 91% (Table 1).

[0304] The cell adhesion of the sterilized composite hydrogels (spherical particles) obtained in Examples 1-2 to 1-3, Comparative Examples 1-1 to 1-7, and Reference Example 1 was evaluated using the same method as described above for cell adhesion evaluation (particles).

[0305] <Evaluation of cell adhesion (sheet)>

[0306] The sterilized amide bond composite hydrogel (sheet) obtained in Reference Example 2 was immersed in PBS for 1 hour to swell the gel sheet. Then, a 15 mm diameter punch was used to punch the gel sheet into a circular shape and add it to the bottom surface of a 24-well plate. 500 μL of DMEM culture medium supplemented with 10% fetal bovine serum was further added to the same well. In addition, in the same plate, wells were prepared in which only 500 μL of culture medium was added without adding the gel sheet. Next, 1.9×10 NIH / 3T3 cells grown by prior culture were added to the wells with the gel sheet added and the wells without the gel sheet added. 5Each well was incubated under conditions of 5% carbon dioxide concentration, saturated water vapor pressure, and 37°C. After 24 hours of incubation, the culture medium added to the wells was removed with an aspirator, 500 μL of new culture medium was added, and 50 μL of Cell Counting Kit-8 (CCK-8, manufactured by Dojindo Chemical Research Institute) for cell growth / cytotoxicity assay was further added. At the same time, 500 μL of new culture medium and 50 μL of CCK-8 were added to unused wells to which gel sheets and cells were not added, and the well plate was incubated under conditions of 5% carbon dioxide concentration, saturated water vapor pressure, and 37°C for 1 hour. After incubation, 100 μL of culture medium from each well to which CCK-8 was added was transferred to a 96-well plate, and the absorbance at 450 nm was measured using a microplate reader (ARVO, manufactured by PerkinElmer). The absorbance obtained from the wells with only cells added was recorded as the cell adhesion rate of 100%, the absorbance obtained from the wells with only culture medium added was recorded as the cell adhesion rate of 0%, and the cell adhesion rate calculated based on the absorbance obtained from the wells with both the gel sheet and cells added was 90% (Table 1).

[0307] The sterilized amide bond composite hydrogel obtained in Reference Example 3 was evaluated for cell adhesion by the same method as in Reference Example 2. The result was that the cell adhesion rate was 87% (Table 1).

[0308] <Hydrogel Swelling Properties>

[0309] Ten parts by mass of the dried hydrogel-forming articles obtained in Examples 1-2 and Comparative Examples 1-5 were placed in 90 parts by mass of water and allowed to swell for 1.5 hours. The hydrogels were then visually inspected for signs of fragmentation and surface damage. The results were evaluated using the following criteria: A for Example 1-2 and C for Comparative Examples 1-5.

[0310] A: No hydrogel fragments or damage to the hydrogel surface.

[0311] B: The generation of hydrogel fragments and / or damage to the hydrogel surface is slightly generated but not problematic.

[0312] C: Hydrogel fragments and / or damage to the hydrogel surface

[0313] D: Significant hydrogel fragmentation and / or damage to the hydrogel surface

[0314] [Table 1]

[0315]

[0316] The results of Examples 1-1 to 1-3 and Comparative Examples 1-1 to 1-7 demonstrate that the present invention enables the production of fully sterilized dried hydrogel-forming articles in which aggregation during drying is suppressed. Furthermore, the hydrogels, which are swollen products of these dried hydrogel-forming articles, were confirmed to have higher cell adhesion rates than the hydrogels of Comparative Examples 1-6 and 1-7, which were sterilized at high water content.

[0317] The breakage rate was 0% in Example 1-2. Examples 1-1 and 1-3, Comparative Examples 1-1 to 1-7, and Examples 2-1 to 2-8 described later used the same vinyl alcohol polymer as Example 1-2 and had the same or higher degree of polymerization, and therefore are considered to have exhibited breakage rates comparable to those of Example 1-2.

[0318] [Synthesis example]

[0319] <Synthesis of vinyl alcohol polymer having ethylenically unsaturated groups>

[0320] [Synthesis example 2-1]

[0321] 40 g (monomer repeating unit: 908 mmol) of PVA117 (raw material PVA) was added to a separable flask with a 1 L Dimroth cooling tube, 350 mL of dimethyl sulfoxide (DMSO) was added, and stirring was started with a mechanical stirrer. After heating to 80 ° C in a water bath, stirring was continued at 80 ° C for 4 hours. After visually confirming that the raw material PVA was dissolved, heating and stirring were performed at 80 ° C, while adding 1.2 g (10.9 mol) of vinyl methacrylate, and further stirring was performed at 80 ° C for 3 hours. After cooling, the reaction solution was injected into 2 L of methanol while stirring. Stop stirring and let it stand directly for 1 hour. After recovering the obtained solid, it was further immersed in 1 L of methanol for 1 hour and washed. This washing operation was performed a total of 3 times. The recovered solid was vacuum dried at room temperature overnight to obtain methacrylic acid PVA117. The introduction rate of the ethylenically unsaturated group (methacryloyl group) in the methacrylated PVA117 was 1.2 mol% relative to the repeating unit of the raw material PVA (hereinafter referred to as "MA-PVA117 (1.2)").

[0322] [Synthesis Examples 2-2~2-3]

[0323] As shown in Table 2, except that the amount of vinyl methacrylate added was changed, a vinyl alcohol polymer having an ethylenically unsaturated group was produced in the same manner as in Synthesis Example 2-1.

[0324] [Synthesis example 2-4]

[0325] 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 itaconic acid was copolymerized to introduce a carboxyl group as a raw material PVA (AF-17).

[0326] [Synthesis example 2-5]

[0327] 10 g (monomer repeating unit: 220 mmol) of MA-PVA117 (2.0) prepared in Synthesis Example 2-2 was added to a separable flask with a 0.5 L Dimroth cooling tube, 90 mL of dimethyl sulfoxide (DMSO) was added, and stirring was started with a mechanical stirrer. After heating to 80°C in a water bath, stirring was continued at 80°C for 4 hours. After visually confirming that the aforementioned raw material PVA was dissolved, the water bath was set to 60°C. After confirming that the internal temperature reached 60°C, 1.2 g (12.1 mmol) of triethylamine and 1.1 g (11 mmol) of succinic anhydride were added, and the mixture was further stirred at 60°C for 5 hours. After cooling, the reaction solution was injected into 0.5 L of methanol while stirring. Stop stirring and let it stand for 1 hour. After recovering the obtained solid, it was further immersed in 0.5 L of methanol for 1 hour for washing. This washing operation was performed a total of 3 times. The recovered solid was vacuum-dried overnight at room temperature to obtain methacrylated PVA117 into which succinic acid had been introduced. The introduction ratio of succinic acid (SA) 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)").

[0328] [Synthesis example 2-6]

[0329] 60 g (monomer repeating unit: 1.36 mol) of PVA117 (raw material PVA) was added to a separable flask with a 1 L Dimroth cooling tube, 540 mL of ion exchange water was added, and stirring was started with a mechanical stirrer. After heating to 80°C in a water bath, stirring was continued at 80°C for 4 hours. After visually confirming that the raw material PVA was 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 solution of sulfuric acid were added, and the mixture was further stirred at 40°C for 4 hours. After cooling, 80 mL of a 1N NaOH aqueous solution was added and neutralized, and a dialysis membrane with a fractional molecular weight of 3,500 was added for desalination (implemented 4 times for 5 L of ion exchange water). The desalted aqueous solution was injected into 2 L of methanol while stirring and left directly for 1 hour. After recovering the obtained solid, it was further immersed in 1 L of methanol for 1 hour for washing. The recovered solid was vacuum-dried at room temperature overnight to obtain norbornene-modified PVA 117. The norbornene-modified PVA 117 had an ethylenically unsaturated group (norbornene group (Nor)) introduction ratio of 1.3 mol% relative to the repeating units of the starting PVA (hereinafter referred to as "Nor-PVA 117 (1.3)").

[0330] Table 2 summarizes the vinyl alcohol polymers having an ethylenically unsaturated group produced according to the methods described in Synthesis Examples 2-1 to 2-6.

[0331] [Table 2]

[0332]

[0333] <Manufacturing of molded products (pellets, sheets)>

[0334] [Synthesis Example 2-A Particles]

[0335] 440 mL of ion exchange water was added to 60 g of MA-PVA117 (1.2), and the mixture was stirred at 80°C for 4 hours while dissolving. After cooling to room temperature, potassium persulfate as a water-soluble thermal free radical polymerization initiator was added to the MA-PVA117 (2.0) aqueous solution in an amount of 0.1% by mass and dissolved to prepare an uncrosslinked gel solution. 3300 mL of liquid paraffin and 8 g of Span80 were added to a separable flask with a 5 L Dimroth cooling tube, and the uncrosslinked gel solution was slowly added thereto. The mixture was stirred at 350 rpm with a mechanical stirrer to prepare a W / O dispersion. After heating to 40°C in a water bath, nitrogen substitution was performed for 30 minutes. Thereafter, stirring was continued at 70°C for 3 hours. After cooling to room temperature, the liquid paraffin in which the hydrogel particles were dispersed was filtered through a mesh of 100 μm. The resulting hydrogel particles were washed with a total of 3 L of hexane to remove the liquid paraffin, and then classified using a JIS standard sieve to a particle size of 180 to 300 μm. Furthermore, the hydrogel particles were placed in 1 L of acetone for dehydration, and then dried under reduced pressure at room temperature overnight to obtain dried hydrogel particles (hereinafter referred to as "MA-PVA117 (1.2) gel particles").

[0336] [Synthesis Examples 2-B to 2-E Particles]

[0337] Methacryl-modified PVA was produced in the same manner as in Synthesis Example 2-1 except that the vinyl alcohol-based polymer having an ethylenically unsaturated group synthesized in Synthesis Examples 2-2 to 2-5 was used.

[0338] [Synthesis Example 2-F Particles]

[0339] In Synthesis Example 2-A, 440 mL of ion exchange water was added to 60 g of Nor-PVA117 (1.3), and the mixture was stirred at 80°C for 4 hours while dissolving. After cooling to room temperature, 3.4 g of 3,6-dioxa-1,8-octanedithiol as a polythiol was added to the Nor-PVA117 (1.3) aqueous solution and stirred. To this solution, potassium persulfate as a water-soluble thermal free radical polymerization initiator was added and dissolved in a manner to achieve 0.1% by mass to prepare an uncrosslinked gel solution. Using this uncrosslinked gel solution, dry hydrogel particles (hereinafter referred to as "Nor-PVA117 (1.3) gel particles") were obtained by the same method as in Synthesis Example 2-A.

[0340] [Synthesis Example 2-G Sheet]

[0341] To 6 g of MA-PVA117(2.0)-SA(3.4) was added 44 mL of ion exchange water, and dissolved while stirring at 80°C for 4 hours. After cooling to room temperature, potassium persulfate as a water-soluble thermal radical polymerization initiator was added to the MA-PVA117(2.0) aqueous solution so as to achieve 0.1 mass%, and dissolved, to prepare an uncrosslinked gel solution. The uncrosslinked gel solution was flowed into 2 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) having a thickness of 0.5 mm was washed with 300 mL of ion exchange water 5 times. After being immersed in 1 L of acetone and dehydrated, a dried hydrogel sheet (hereinafter referred to as "MA-PVA117(2.0)-SA(3.4) gel sheet") was obtained by drying under reduced pressure at room temperature overnight.

[0342] The molded articles produced according to the methods described in Synthesis Examples 2-A to 2-G are summarized in Table 3.

[0343] [Table 3]

[0344]

[0345] <Activation of hydroxyl group>

[0346] [Butanedioic acid introduction in Synthesis Example 2-i molded article (granule)]

[0347] To 6 g of MA-PVA117(2.0)-SA(3.4) was added 44 mL of ion exchange water, and dissolved while stirring at 80°C for 4 hours. After cooling to room temperature, potassium persulfate as a water-soluble thermal radical polymerization initiator was added to the MA-PVA117(2.0) aqueous solution so as to achieve 0.1 mass%, and dissolved, to prepare an uncrosslinked gel solution. The uncrosslinked gel solution was flowed into 2 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) having a thickness of 0.5 mm was washed with 300 mL of ion exchange water 5 times. After being immersed in 1 L of acetone and dehydrated, a dried hydrogel sheet (hereinafter referred to as "MA-PVA117(2.0)-SA(3.4) gel sheet") was obtained by drying under reduced pressure at room temperature overnight.

[0348] [Butanedioic acid introduction in Synthesis Example 2-i molded article (granule)]

[0349] Using the gel particles prepared in Synthesis Examples 2-B to 2-C and 2-F, a dry hydrogel particle into which succinic acid was introduced was obtained by the same method as in Synthesis Example 2-i, except for this. The results of the introduction rate of carboxyl groups (succinic acid) (introduction density of carboxyl groups) are shown in Table 4.

[0350] The introduction rate of carboxyl groups (succinic acid) in the dry hydrogel particles produced according to the method described in Synthesis Examples 2-i to 2-iv is shown in Table 4.

[0351] [Table 4]

[0352]

[0353] <Complexation>

[0354] [Example 2-1]

[0355] 1 g of the dry SA-MA-PVA117(1.2) gel particles (succinic acid introduction amount: about 1.9 mmol) prepared in Synthesis Example 2-i was swelled in MES buffer (pH = 5.6) at room temperature overnight. The swelled gel particles were dispersed in 45 mL of the MES buffer, stirred with a magnetic stirrer, while 0.78 g (6.8 mmol) of N-hydroxysuccinimide, 0.65 g (3.4 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added, and shaken at room temperature for 1 hour. The gel particles were washed with 30 mL of the MES buffer for 10 minutes, and the operation was repeated 3 times, and added to 70 mL of a 1 mg / mL gelatin PBS solution. The resulting hydrogel particles were washed with 70 mL of ion exchange water (warmed to 60°C) for 20 minutes, and the operation was repeated 2 times. Further, after 50 mL of ethanol immersion was repeated 3 times, vacuum drying was performed at room temperature overnight to obtain dry amide bond complexed hydrogel particles (hereinafter referred to as "gelatin-SA-MA-PVA117(1.2) gel particles"). A part of the gelatin-SA-MA-PVA117(1.2) gel particles was immersed in an excess amount of PBS overnight, and the results of the gelatin immobilization amount per unit gel weight (100 mg) were 48.2 μg / 100 mg of gel particles by the bicinchoninic acid (BCA) method (BCA Protein Assay Kit (manufactured by Takara Bio Inc.)). Sterilized amide bond complexed hydrogels were obtained by performing γ-ray irradiation by the same method as in Comparative Example 1-1. The water content and the content of alcohol-based solvents of the obtained dry particles were measured by the same method as in Comparative Example 1-1. 1 H-NMR (deuterated DMSO solvent) was calculated according to the above measurement method. The results of the measurement of the water content and the content of alcohol-based solvents, etc. are shown in Table 5.

[0356] [Examples 2-2 to 2-6]

[0357] Amide-bonded gelatin composite hydrogel particles were obtained by the same method 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 results of the BCA method for measuring the gelatin immobilization amount (physiologically active substance composite density), as well as the water content and alcohol solvent content, are shown in Table 5.

[0358] [Example 2-7]

[0359] Collagen-complexed PVA particles (hereinafter referred to as "collagen-SA-MA-PVA117(1.2) gel particles") were obtained by the same method as in Example 2-1, except that collagen was used instead of gelatin. The gelatin immobilization amount (complexation density of physiologically active substances) was measured by the BCA method and the result was 53.2 μg / 100 mg of gel particles.

[0360] [Example 2-8]

[0361] <Gelatin composite sheet>

[0362] An amide-bonded gelatin composite hydrogel sheet (hereinafter referred to as "gelatinized-MA-PVA117(2.0)-SA(3.4) gel sheet") was obtained by the same method as 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. The gelatin immobilization amount (the composite density of the physiologically active substance) was measured by the BCA method and the result was 20.0 μg / 100 mg of gel particles.

[0363] Comparative Example

[0364] [Comparative Example 2-1]

[0365] Particles made of commercially available dextran, which did not contain a cross-linked vinyl alcohol polymer, were used as a carrier for gel filtration. When the breakage rate was measured by the above-mentioned method, the breakage rate was 40%.

[0366] [Comparative Example 2-2]

[0367] The same commercially available glucan granules as in Comparative Example 2-1 were irradiated with gamma rays at a dose of 25 kGy or more using the same method as in Example 2-1 to obtain sterilized glucan granules. The breakage rate of the sterilized glucan granules was measured using the above method and found to be 60%.

[0368] [Comparative Example 2-3]

[0369] Commercially available cellulose particles containing no crosslinked vinyl alcohol polymer were used as a carrier for gel filtration. When the breakage rate was measured by the above-mentioned method, it was 10%.

[0370] [Comparative Examples 2-4]

[0371] The same commercially available cellulose particles as in Comparative Example 2-3 were irradiated with gamma rays at a dose of 25 kGy or more using the same method as in Example 2-1 to obtain sterilized glucan particles. The breakage rate of the sterilized glucan particles was measured using the above method and found to be 40%.

[0372] [Evaluation Method of Amide Bond Composite Hydrogels Obtained in Examples and Comparative Examples]

[0373] <Evaluation of cell adhesion (cell growth rate)>

[0374] 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 (weight when swollen / weight when dried) and the average particle size when swollen were measured, and the total surface area of ​​1 g of the average dry particles when swollen was calculated. The swelling degree of the dried gel particles obtained in Example 2-1 was 8 or more. Then, 30 mL of DMEM culture medium supplemented with 10% fetal bovine serum was added to a 125 mL rotating flask (manufactured by Corning Incorporated), and the dried SA-MA-PVA117 (1.2) gel particles obtained in Example 2-1 were immersed in PBS based on the total surface area calculated above, so that the total surface area reached 162 cm 2 Furthermore, 8.1×10 NIH / 3T3 cells (purchased from ATCC) grown in a pre-culture were added. 5 The cells were cultured in an incubator at 37°C with stirring at 60 rpm in a 5% carbon dioxide concentration and saturated water vapor pressure. On the fourth day of culture, all gel particles were recovered, washed with PBS, and then treated with trypsin to detach the cells from the gel particles. The cell density of the resulting cell suspension was counted using a hemocytometer to calculate the post-culture cell count. If the cell growth rate is defined as the post-culture cell count divided by the initial cell count, the cell growth rate of the gel particles obtained in Example 2-1 was 10.6.

[0375] The cell growth rates of the gel particles of Examples 2-2 to 2-7 were measured by the same method, and the results are shown in Table 5. The swelling degrees of these dried gel particles and the dried gel particles of Examples 1-1 to 1-3 were all 8 to 11.

[0376] The cell growth rate of the gel sheet of gelatinized-MA-PVA117(2.0)-SA(3.4) obtained in Examples 2-8 was measured by the following method. The gel sheet was immersed in PBS overnight to swell, and then punched into a circle using a punch having a diameter of 34 mm, and added to the bottom of a polystyrene 6-well plate for cell culture. To this, 3 mL / well of culture medium was added, and 4.9 x 104cells of NIH / 3T3 cells grown by pre-culture were added, and incubated in an incubator at 5% CO2concentration, saturated water vapor pressure, and 37°C. On the 4th day of the culture, the cells were peeled off and recovered from the surface of the gel sheet by trypsin treatment, and the number of cells after the culture was counted to calculate the cell growth rate. As a result, the growth rate was 4.3. 6

[0377] <Verification of sterilization (VD max method) >

[0378] [Example 3]

[0379] In a clean room of 10,000 class, all of the buffer and ion exchange water were used as sterilized substances filtered through a membrane filter having a pore size of 0.2 μm, and 30 g of the dried SA-MA-PVA117(2.0) gel particles prepared in Synthesis Example 1-i were used, and otherwise, in the same manner as in Synthesis Example 1-a, to obtain gelatin complexed hydrogel particles (hereinafter referred to simply as "gelatin-SA-MA-PVA117(2.0) gel particles (clean room)"). As a result of measuring the gelatin immobilization amount per unit gel weight (100 mg) by the bicinchoninic acid (BCA) method, the complexation density of the physiologically active substance was 56.3 μg / 100 mg of gel particles. The obtained swollen gel particles were dried in the same manner as in Example 1-1. The water content measured in the same manner as in Example 1-1 was 0.1% by weight.

[0380] The above obtained dried hydrogel particles were sterilized using the VD max 25 method. Specifically, 1 g of each of the above dried hydrogel particles was added to a sterilized 25 mL centrifuge tube, capped, and 25 bottles of the dried hydrogel particles contained in the container were prepared. As a result of measuring the average biological load of 10 of the bottles, it was 7.6. As a result of the sterility test of 10 bottles irradiated with the amount of γ-rays corresponding to the VD max 25 -1 corresponding to the average biological load of 8.0, i.e., 6.9 kGy, one of the 10 bottles was positive (SAL = 10 -6 ​​Sterilized dry hydrogel particles.

[0381] [Table 5]

[0382]

[0383] The results of Examples 2-1 to 2-8 demonstrate that the present invention enables the production of fully sterilized, dried hydrogel-forming articles whose aggregation during drying is suppressed. Furthermore, the hydrogels, which are swollen forms of the dried hydrogels, were confirmed to have a high cell growth rate. In contrast, the hydrogels of Comparative Examples 2-1 to 2-4, which do not contain crosslinked polyvinyl alcohol-based polymers, exhibited insufficient strength. Furthermore, sterilization with gamma rays further destroyed the particles, resulting in low strength.

Claims

1. A dry hydrogel-forming article comprising a crosslinked product of a vinyl alcohol polymer and an alcoholic solvent, Based on the amount of the dry hydrogel-forming article, the content of the alcoholic solvent is 0.01% by mass or more and 15% by mass or less, and the content of water is 0% by mass or more and 70% by mass or less, The sterility assurance level (SAL) of the dried hydrogel-forming article is 1×10 -6 Below, the degree of agglomeration is below 10%.

2. The dry hydrogel-forming article according to claim 1, which satisfies the following condition 1: Condition 1: When 100 parts by mass of water and 1 part by mass of a dry hydrogel-forming article are added to a glass bottle and stirred at 400 rpm for 3 hours using a stirring head having a length of at least 80% of the bottom diameter of the glass bottle, the breakage rate of the swollen hydrogel-forming article is 5% or less.

3. The dry hydrogel-forming article according to claim 1 or 2, wherein The degree of polymerization of the vinyl alcohol polymer is 450 or more.

4. The dry hydrogel-forming article according to any one of claims 1 to 3, wherein The vinyl alcohol polymer has carboxyl groups, and the introduction rate of the carboxyl groups is 0.1 to 50 mol% in all the structural units constituting the vinyl alcohol polymer.

5. The dry hydrogel-forming article according to any one of claims 1 to 4, wherein The vinyl alcohol polymer has an ethylenically unsaturated group, and the introduction rate of the ethylenically unsaturated group is 0.01 to 10 mol% in all the structural units constituting the vinyl alcohol polymer.

6. The dry hydrogel-forming article according to claim 5, 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 norbornene group, and derivatives thereof.

7. The dry hydrogel-forming article according to any one of claims 1 to 6, wherein The dry hydrogel-forming article is an amorphous particle, a spherical particle, a finely molded body, an article of any shape formed by a 3D printer, a sheet, a filament, a hollow fiber, a porous block, or a coated article.

8. The dry hydrogel-forming article according to any one of claims 1 to 7, wherein The dried hydrogel-forming articles are spherical particles.

9. The dry hydrogel-forming article according to any one of claims 1 to 8, wherein Physiologically active substances are compounded.

10. The dry hydrogel-forming article according to any one of claims 1 to 9, wherein The physiologically active substance is complexed with the cross-linked vinyl alcohol polymer via a covalent bond.

11. A hydrogel which is a swollen body of the dry hydrogel-forming article according to any one of claims 1 to 10.

12. The hydrogel according to claim 11, wherein The hydrogel is in the form of spherical particles having a particle size of 10 to 5000 μm.

13. The method for producing a dry hydrogel-forming article according to any one of claims 1 to 10, comprising: a step of preparing a preliminary hydrogel comprising a cross-linked body of a vinyl alcohol-based polymer; The step of immersing the preliminary hydrogel in an alcohol solvent and then drying the preliminary hydrogel to obtain a dried preliminary hydrogel; and Sterilize the dried prepared hydrogel to a sterility assurance level (SAL) of 1×10 -6 The following steps.

14. The method for producing a dry hydrogel-forming article according to claim 13, wherein: During the sterilization process, radiation sterilization is performed to achieve a sterility assurance level (SAL) of 1×10 -6 the following.

15. The method for producing a dry hydrogel-forming article according to claim 14, wherein: The radiation dose in radiation sterilization is 8.2 kGy or more.

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