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  • C08J3/00—Processes of treating or compounding macromolecular substances
  • C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
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  • C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
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  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
  • C08K5/00—Use of organic ingredients
  • C08K5/04—Oxygen-containing compounds
  • C08K5/05—Alcohols; Metal alcoholates
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L29/00—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an alcohol, ether, aldehydo, ketonic, acetal or ketal radical; Compositions of hydrolysed polymers of esters of unsaturated alcohols with saturated carboxylic acids; Compositions of derivatives of such polymers
  • C08L29/02—Homopolymers or copolymers of unsaturated alcohols
  • C08L29/04—Polyvinyl alcohol; Partially hydrolysed homopolymers or copolymers of esters of unsaturated alcohols with saturated carboxylic acids
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  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J2329/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an alcohol, ether, aldehydo, ketonic, acetal, or ketal radical; Hydrolysed polymers of esters of unsaturated alcohols with saturated carboxylic acids; Derivatives of such polymer
  • C08J2329/02—Homopolymers or copolymers of unsaturated alcohols
  • C08J2329/04—Polyvinyl alcohol; Partially hydrolysed homopolymers or copolymers of esters of unsaturated alcohols with saturated carboxylic acids

Definitions

• the present invention relates to a dry hydrogel-forming article, a hydrogel, and a method for producing a dry hydrogel-forming article.
• Polyvinyl alcohol (hereinafter sometimes abbreviated as "PVA”) is a water-soluble synthetic polymer with excellent characteristics such as hydrophilicity, reactivity, biodegradability, biocompatibility, and low toxicity, and forms a hydrogel with high flexibility and strength by physical crosslinking or chemical crosslinking.
• hydrogels complexed with physiologically active substances exhibit specific functions, and applications such as enzyme immobilization carriers (e.g., Non-Patent Document 1), affinity carriers (e.g., Non-Patent Document 2), cell culture substrates (e.g., Non-Patent Document 3), particles for vascular embolization (e.g., Patent Document 1), and cell culture carriers (e.g., Patent Document 2) have been proposed.
• enzyme immobilization carriers e.g., Non-Patent Document 1
• affinity carriers e.g., Non-Patent Document 2
• cell culture substrates e.g., Non-Patent Document 3
• particles for vascular embolization e.g., Patent Document 1
• cell culture carriers e.g., Patent Document 2
• the hydrogel When used as parts or in the manufacture of pharmaceuticals and medical devices for use on the human body, the hydrogel must be used in a sterilized state. If unsterilized hydrogel is used for such purposes, residual bacteria may cause infection and cause fatal damage to the living body or cells.
• Non-Patent Documents 3 to 5 discloses irradiating composite hydrogels with UV light for several hours or immersing them in an ethanol aqueous solution for several hours before using them for cell culture.
• Patent Document 3 There have also been attempts to sterilize hydrogel-forming compositions before crosslinking.
• the PVA contained in such hydrogels is crosslinked, and known crosslinking methods include, for example, a method of covalently bonding an enzyme to a hydrogel that has been chemically crosslinked with glutaraldehyde using carbonyldiimidazole (e.g., Non-Patent Document 1), a method of covalently bonding a cell adhesive protein to PVA with a carboxyl group introduced therein (e.g., Non-Patent Documents 3 and 4), a method of crosslinking PVA by introducing an acrylamide group and inducing radical polymerization to produce a hydrogel with a cell adhesive peptide covalently bonded thereto (e.g., Non-Patent Documents 6 and 7), and a method of covalently bonding cell adhesive gelatin to PVA particles obtained by radical polymerization crosslinking PVA with (meth)acryloyl groups (e.g., Patent Document 2).
• SAL sterility assurance level
• the present invention has been made in consideration of the above problems, and aims to provide a dried hydrogel-forming article that can provide a sufficiently sterilized hydrogel and that suppresses aggregation during drying.
• the present invention aims to provide a hydrogel that is sufficiently sterilized and has sufficient strength, and that is capable of forming a dried hydrogel in which aggregation during drying is suppressed.
• the present invention aims to provide a hydrogel that is sufficiently sterilized and has high cell adhesiveness, and to provide a dried hydrogel-forming article in which aggregation during drying is suppressed.
• the present inventors have found that the above-mentioned problems can be solved by carrying out sterilization under conditions in which the sterility compensation level SAL is 1 ⁇ 10 ⁇ 6 or less in a state of a dried hydrogel-forming article that is dried to have a degree of aggregation of 10% or less by containing an alcohol-based solvent.
• a dried hydrogel-forming article can be produced, for example, by replacing the water content in the hydrogel with an alcohol-based solvent, drying the hydrogel, removing the solvent, and carrying out sufficient sterilization in the state of the dried hydrogel-forming article.
• a dry hydrogel-forming article comprising a crosslinked vinyl alcohol polymer and an alcohol solvent,
• the content of the alcohol-based 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, based on the amount of the dried hydrogel-forming article;
• the dry hydrogel-forming article has a sterility assurance level SAL of 1 ⁇ 10 ⁇ 6 or less and a degree of aggregation of 10% or less.
• the present invention provides a dried hydrogel-forming article that can provide a sufficiently sterilized hydrogel and that inhibits aggregation during drying.
• the present invention provides a dry hydrogel-forming article comprising a crosslinked product of a vinyl alcohol-based polymer and an alcohol-based solvent, the content of the alcohol-based solvent being 0.01% by mass or more and 15% by mass or less, and the content of water being 0% by mass or more and 70% by mass or less, based on the amount of the dry hydrogel-forming article, the sterility compensation level SAL of the dry hydrogel-forming article being 1 x 10-6 or less, and the degree of aggregation being 10% or less.
• the vinyl alcohol polymer in the crosslinked product of the vinyl alcohol polymer contained in the dry hydrogel-forming article of the present invention can be produced by saponifying a polyvinyl ester obtained by polymerizing a vinyl ester monomer, and converting the ester group in the polyvinyl ester to a hydroxyl group.
• the vinyl ester monomer examples include aliphatic vinyl esters such as vinyl formate, vinyl acetate, vinyl propionate, vinyl n-butyrate, vinyl isobutyrate, vinyl pivalate, vinyl versatate, vinyl caproate, vinyl caprylate, vinyl caprate, vinyl laurate, vinyl myristate, vinyl palmitate, vinyl stearate, and vinyl oleate; and aromatic vinyl esters such as vinyl benzoate. These may be used alone or in combination of two or more.
• the vinyl ester monomers aliphatic vinyl esters are preferred, and vinyl acetate is more preferred from the viewpoint of production costs. That is, the polyvinyl ester is preferably polyvinyl acetate obtained by polymerizing vinyl acetate.
• the polyvinyl ester may contain structural units derived from other monomers than the vinyl ester monomers, if necessary, within the scope of the invention.
• examples of such other monomers include ⁇ -olefins such as ethylene, propylene, n-butene, and isobutylene; acrylic acid or a salt thereof; acrylic acid alkyl esters such as methyl acrylate, ethyl acrylate, n-propyl acrylate, i-propyl acrylate, n-butyl acrylate, i-butyl acrylate, t-butyl acrylate, 2-ethylhexyl acrylate, dodecyl acrylate, and octadecyl acrylate; methacrylic acid or a salt thereof; methyl methacrylate, ethyl methacrylate, n-propyl methacrylate, methacrylic acid, and the like.
• methacrylic acid alkyl esters such as i-propyl methacrylate, n-butyl methacrylate, i-butyl methacrylate, t-butyl methacrylate, 2-ethylhexyl methacrylate, dodecyl methacrylate, and octadecyl methacrylate; acrylamide, N-methylacrylamide, N-ethylacrylamide, N,N-dimethylacrylamide, diacetone acrylamide, acrylamidopropanesulfonic acid or a salt thereof, acrylamidopropyldimethylamine or a salt or a quaternary salt thereof, N-methylolacrylamide or a salt thereof, methacrylamide derivatives such as methacrylamide, N-methyl methacrylamide, N-ethyl methacrylamide, methacrylamide propanesulfonic acid or a salt thereof, methacrylamide propyl dimethylamine or a salt or a
• the method for saponifying the polyvinyl ester is not particularly limited, and can be the same as in the past.
• the alcoholysis method using an alkali catalyst or an acid catalyst, the hydrolysis method, etc. can be applied.
• the saponification reaction using methanol as a solvent and caustic soda (NaOH) as a catalyst is simple and preferable.
• the degree of polymerization of the vinyl alcohol polymer is preferably 450 or more, more preferably 500 or more, even more preferably 550 or more, and particularly preferably 600 or more, from the viewpoint of suppressing embrittlement of the crosslinked vinyl alcohol polymer during sterilization and water swelling.
• the degree of polymerization of the vinyl alcohol polymer is preferably 10,000 or less, more preferably 5,000 or less, and even more preferably 3,500 or less. Two or more types of vinyl alcohol polymers having different degrees of polymerization may be mixed and used.
• the degree of polymerization of the vinyl alcohol polymer in this specification refers to the degree of polymerization measured in accordance with JIS K 6726:1994. Specifically, it can be determined from the limiting viscosity measured in water at 30°C after saponifying and purifying the raw material PVA.
• the degree of saponification of the vinyl alcohol polymer is preferably 50 mol% or more, more preferably 60 mol% or more, and even more preferably 65 mol% or more.
• the upper limit of the degree of saponification can be 100 mol% or less, or 99 mol% or less.
• the degree of saponification of the vinyl alcohol polymer means the ratio (mol %) of the number of moles of vinyl alcohol units to the total number of moles of structural units (e.g. vinyl acetate units) that can be converted to vinyl alcohol units by saponification in the raw material PVA and vinyl alcohol units, and can be measured in accordance with JIS K 6726:1994.
• the 4% by mass viscosity of the vinyl alcohol polymer at 20°C is preferably 0.5 to 110 mPa ⁇ s, more preferably 1 to 80 mPa ⁇ s, and even more preferably 2 to 60 mPa ⁇ s.
• the viscosity in this specification refers to the viscosity of a 4% by mass aqueous solution of the vinyl alcohol polymer measured at a temperature of 20°C using a B-type viscometer (rotation speed 12 rpm) in accordance with the rotational viscometer method of JIS K 6726:1994.
• the dry hydrogel-forming article of the present invention contains a crosslinked product of the vinyl alcohol polymer.
• the crosslinked product may be a vinyl alcohol polymer that is physically crosslinked or may be a vinyl alcohol polymer that is crosslinked by a covalent bond.
• the total amount of the vinyl alcohol-derived structural units and the vinyl ester-derived structural units relative to the total structural units constituting the vinyl alcohol polymer used in the present invention is preferably 80 mol % or more, more preferably 90 mol % or more, and even more preferably 95 mol % or more.
• Methods for physically crosslinking vinyl alcohol polymers include, for example, freezing and thawing an aqueous solution of vinyl alcohol polymers, or dissolving and heating in a mixed solvent of dimethyl sulfoxide and water, and then cooling the heated solution to room temperature.
• Methods for crosslinking vinyl alcohol polymers by covalent bonds include using a multifunctional crosslinking agent that reacts with the side chain hydroxyl groups of vinyl alcohol polymers, or introducing functional groups into vinyl alcohol polymers by copolymerization or post-modification, and then reacting them. From the viewpoint of the stability of the hydrogel when it swells in water, it is preferable for the polymers to be crosslinked by covalent bonds.
• Methods that use a multifunctional crosslinking agent that reacts with the side chain hydroxyl groups of vinyl alcohol polymers include reacting with multifunctional aldehyde compounds such as glyoxal, malondialdehyde, and glutaraldehyde under acidic conditions (polyacetal crosslinking), reacting with multifunctional epoxy compounds such as epichlorohydrin and ethylene glycol diglycidyl ether under alkaline conditions (polyether crosslinking), and reacting with multifunctional carboxylic acid compounds such as maleic acid and succinic acid (polyester crosslinking).
• multifunctional aldehyde compounds such as glyoxal, malondialdehyde, and glutaraldehyde under acidic conditions
• polyether crosslinking reacting with multifunctional epoxy compounds such as epichlorohydrin and ethylene glycol diglycidyl ether under alkaline conditions
• polyether crosslinking reacting with multifunctional carboxylic acid compounds such as maleic acid and succinic acid
• a method for introducing functional groups into vinyl alcohol polymers by copolymerization and reacting them involves copolymerizing a vinyl ester monomer with a polymerizable monomer other than a vinyl ester monomer that has a reactive substituent other than a hydroxyl group during the production process of the vinyl alcohol polymer, followed by saponification to obtain copolymerized modified polyvinyl alcohol (hereinafter sometimes abbreviated as "copolymerized modified PVA"). After that, a multifunctional crosslinking agent that reacts with functional groups such as carboxy groups present in the copolymerized modified PVA and amino groups present in the copolymerized modified PVA is added. Copolymerized modified PVA with carboxy groups is sometimes called “carboxylic acid modified PVA” and copolymerized modified PVA with amino groups is sometimes called "amino modified PVA".
• Monomers constituting carboxylic acid modified PVA include ⁇ , ⁇ -unsaturated carboxylic acids such as (meth)acrylic acid, maleic acid, fumaric acid, itaconic acid, etc.; (meth)acrylic acid alkyl esters such as methyl (meth)acrylate, ethyl (meth)acrylate, etc.; ⁇ , ⁇ -unsaturated carboxylic acid anhydrides such as maleic anhydride, itaconic anhydride, etc., and derivatives thereof.
• carboxylic acids such as (meth)acrylic acid, maleic acid, fumaric acid, itaconic acid, etc.
• (meth)acrylic acid alkyl esters such as methyl (meth)acrylate, ethyl (meth)acrylate, etc.
• ⁇ , ⁇ -unsaturated carboxylic acid anhydrides such as maleic anhydride, itaconic anhydride, etc., and derivatives thereof.
• Carboxylic acid modified PVA is produced by copolymerizing a vinyl ester monomer with the monomers constituting the carboxylic acid modified PVA and then saponifying it, and hydrogels can be obtained by combining a multifunctional crosslinking agent that reacts with the introduced carboxyl groups, for example, a multifunctional epoxy compound such as epichlorohydrin, ethylene glycol diglycidyl ether, a multifunctional amino compound such as ethylenediamine, polyethyleneimine, polyallylamine, etc., with a carbodiimide condensing agent such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, etc.
• a multifunctional crosslinking agent that reacts with the introduced carboxyl groups
• a multifunctional epoxy compound such as epichlorohydrin, ethylene glycol diglycidyl ether
• a multifunctional amino compound such as ethylenediamine, polyethyleneimine, polyallylamine, etc.
• the carboxyl groups introduced into the carboxylic acid-modified PVA can be complexed with biologically active substances that have amino groups by covalently bonding them via amide bonds (-CONH-).
• amino-modified PVA can be obtained as a hydrogel by copolymerizing a vinyl ester monomer with N-vinylformamide or the like, followed by saponification, and then combining a polyfunctional crosslinking agent that reacts with the introduced amino groups, such as the polyfunctional epoxy compound, succinic acid, maleic acid, or other polyfunctional carboxylic acid compound, with the carbodiimide condensing agent.
• a polyfunctional crosslinking agent that reacts with the introduced amino groups, such as the polyfunctional epoxy compound, succinic acid, maleic acid, or other polyfunctional carboxylic acid compound, with the carbodiimide condensing agent.
• An example of a method for introducing functional groups into a vinyl alcohol polymer by post-modification and reacting them is a method for introducing an ethylenically unsaturated group into the side chain of the vinyl alcohol polymer.
• the introduced ethylenically unsaturated group can be easily used to obtain a hydrogel by adding an additive such as a radical initiator to induce a polymerization reaction.
• the introduction of the ethylenically unsaturated group is preferably carried out via the side chain or terminal functional group of the vinyl alcohol polymer, and it is more preferable to react a compound containing an ethylenically unsaturated group (hereinafter sometimes abbreviated as "ethylenically unsaturated group-containing compound") with a hydroxyl group in the side chain of the vinyl alcohol polymer.
• ethylenically unsaturated group-containing compound a compound containing an ethylenically unsaturated group
• Examples of ethylenically unsaturated group-containing compounds that can be reacted with the hydroxyl groups on the side chains of vinyl alcohol polymers include (meth)acrylic acid or derivatives thereof, such as (meth)acrylic acid, (meth)acrylic anhydride, (meth)acrylic acid halide, and (meth)acrylic acid ester. By subjecting these compounds to an esterification reaction or ester exchange reaction in the presence of a base, a (meth)acryloyl group can be introduced.
• examples of ethylenically unsaturated group-containing compounds that are reacted with the hydroxyl groups that are side chains of the vinyl alcohol polymer include compounds that contain an ethylenically unsaturated group and a glycidyl group in the molecule, such as glycidyl (meth)acrylate and allyl glycidyl ether. By subjecting these compounds to an etherification reaction in the presence of a base, it is possible to introduce a (meth)acryloyl group or an allyl group into the vinyl alcohol polymer.
• examples of ethylenically unsaturated group-containing compounds to be reacted with the 1,3-diol group of the vinyl alcohol polymer include compounds containing an ethylenically unsaturated group and an aldehyde group in the molecule, such as acrylaldehyde (acrolein), methacrylaldehyde (methacrolein), 5-norbornene-2-carboxaldehyde, 7-octenal, 3-vinylbenzaldehyde, and 4-vinylbenzaldehyde.
• an ethylenically unsaturated group can be introduced into the raw material PVA.
• a norbornenyl group or a vinylphenyl group can be introduced into the raw material PVA.
• a norbornenyl group or a vinylphenyl group can be introduced into the raw material PVA.
• a (meth)acryloylamino group into a vinyl alcohol polymer by reacting with N-(2,2-dimethoxyethyl)(meth)acrylamide, etc.
• Methods for introducing an ethylenically unsaturated group into a vinyl alcohol polymer can be used other than the above-mentioned reactions, and two or more types of reactions may be used in combination.
• ethylenically unsaturated group examples include reacting a reactive substituent such as a carboxy group present in a carboxylic acid-modified PVA or an amino group present in an amino-modified PVA with an ethylenically unsaturated group-containing compound.
• a reactive substituent such as a carboxy group present in a carboxylic acid-modified PVA or an amino group present in an amino-modified PVA
• carboxy group of a carboxylic acid-modified PVA for example, glycidyl methacrylate can be reacted under acidic conditions to generate an ester bond to introduce a methacryloyl group.
• an acryloylamino group can be introduced by amidating acrylic anhydride in the presence of a base, and for example, a vinyloxycarbonyl group can be introduced by amidating divinyl adipate.
• Methods for introducing an ethylenically unsaturated group via a copolymerized modified PVA can be other than the above-mentioned reactions, and two or more types of reactions may be used in combination.
• vinyl alcohol polymers having ethylenically unsaturated groups are preferably vinyl alcohol polymers in which ethylenically unsaturated groups have been introduced via hydroxyl groups in the side chains of the raw material PVA, such as 1,3-diol groups, and more preferably vinyl alcohol polymers in which (meth)acrylic acid or a derivative thereof has been subjected to an esterification reaction or ester exchange reaction with the hydroxyl groups in the side chains of the raw material PVA, or vinyl alcohol polymers in which a compound containing an ethylenically unsaturated group and an aldehyde group in the molecule has been subjected to an acetalization reaction with the 1,3-diol groups of the vinyl alcohol polymer.
• the vinyl alcohol polymer in the crosslinked vinyl alcohol polymer is preferably a vinyl alcohol polymer having a carboxy group, from the viewpoint of maintaining the appropriate swelling property of the hydrogel in the solvent (particularly water), maintaining physical properties such as gel strength, and improving the complexation density of the physiologically active substance.
• the introduction rate of the carboxy group is preferably 50 mol% or less, more preferably 30 mol% or less, and even more preferably 15 mol% or less, of the total structural units constituting the vinyl alcohol polymer.
• the hydrogel is preferably 0.1 mol% or more, more preferably 0.5 mol% or more, and even more preferably 1.0 mol% or more.
• the hydrogel will be distributed or stored in a dry state until it is used, but once the hydrogel is dried, it may be difficult to return to its original swollen state.
• the vinyl alcohol polymer is a vinyl alcohol polymer having a carboxy group, preferably at the above-mentioned introduction rate of the carboxy group, even when a hydrogel is obtained by swelling a dried hydrogel-forming article that has been dried once, the degree of swelling returns to the original level, and a high-strength hydrogel can be produced, which is preferable.
• the vinyl alcohol polymer in the crosslinked product of the vinyl alcohol polymer is preferably a vinyl alcohol polymer having an ethylenically unsaturated group from the viewpoint of suppressing embrittlement of the hydrogel and promoting the crosslinking reaction.
• the ethylenically unsaturated group is at least one selected from the group consisting of a vinyl group, a (meth)acryloyl group, a (meth)acryloylamino group, a vinylphenyl group, a norbornenyl group, and derivatives thereof.
• the introduction rate of the ethylenically unsaturated groups is preferably 10 mol% or less, more preferably 5 mol% or less, and even more preferably 3 mol% or less, of all structural units constituting the vinyl alcohol-based polymer, from the viewpoint of suppressing embrittlement of the 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.
• the vinyl alcohol polymer in the crosslinked vinyl alcohol polymer is, from the same viewpoint as above, more preferably a vinyl alcohol polymer having a carboxy group and an ethylenically unsaturated group.
• the introduction rates of the carboxy group and the ethylenically unsaturated group are the introduction rates described above.
• the vinyl alcohol polymer may further contain a monomer.
• monomers include acrylamides such as acrylamide, N-isopropylacrylamide, 2-acrylamido-2-methylpropanesulfonic acid, and N,N-dimethylacrylamide; ⁇ , ⁇ -unsaturated carboxylic acids such as (meth)acrylic acid, crotonic acid, itaconic acid, maleic acid, and fumaric acid; water-soluble radical polymerizable monomers such as vinylpyridine, hydroxyethyl (meth)acrylate, styrenesulfonic acid, and polyethylene glycol mono(meth)acrylate; and crosslinkers having two or more ethylenically unsaturated groups in the molecule, such as N,N'-methylenebisacrylamide, ethylene glycol di(meth)acrylate, diethylene glycol di(meth)acrylate, and poly
• 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.
• the ethylenically unsaturated group introduced into the vinyl alcohol polymer can be crosslinked by active energy rays or heat to form a gel, thereby obtaining a crosslinked vinyl alcohol polymer.
• active energy rays include gamma rays, ultraviolet rays, visible light rays, infrared rays (heat rays), radio waves, alpha rays, beta rays, electron beams, plasma flow, ionizing rays, and particle beams.
• the uncrosslinked polymer solution described below contains a radical polymerization initiator.
• radical polymerization initiators include photoradical polymerization initiators and thermal radical polymerization initiators.
• the photoradical polymerization initiator there are no particular limitations on the photoradical polymerization initiator, so long as it initiates radical polymerization by irradiation with active energy rays such as ultraviolet light or visible light.
• active energy rays such as ultraviolet light or visible light.
• the thermal radical polymerization initiator is not particularly limited as long as it initiates radical polymerization by heat, and examples thereof include azo-based initiators and peroxide-based initiators that are commonly used in radical polymerization. From the viewpoint of improving the transparency and physical properties of the vinyl alcohol polymer, peroxide-based initiators that do not generate gas are preferred. From the viewpoint of washing and removing the thermal radical polymerization initiator after crosslinking as described above, those that are water-soluble are preferred. Specific examples include inorganic peroxides such as ammonium persulfate, potassium persulfate, and sodium persulfate.
• a redox polymerization initiator may be used in combination with a reducing agent.
• a redox polymerization initiator crosslinking can be achieved by the stimulation of mixing a peroxide initiator with a reducing agent.
• Known reducing agents can be used as reducing agents to be combined with redox polymerization initiators, but among these, highly water-soluble N,N,N',N'-tetramethylethylenediamine, sodium sulfite, sodium hydrogensulfite, sodium hydrosulfite, etc. are preferred.
• azo initiators that are water-soluble are preferred.
• Specific examples 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-cyanopentanoic acid) (trade name "
• the vinyl alcohol polymer has an ethylenically unsaturated group
• a vinyl alcohol polymer in which the ethylenically unsaturated group is a vinyl group from the viewpoint of promoting crosslinking, for example, a polythiol having two or more thiol groups in the molecule may be added and crosslinked using a thiol-ene reaction.
• polythiol one that is water-soluble is preferable, and examples thereof include polythiols having a hydroxyl group such as dithiothreitol; polythiols containing an ether bond such as terminal thiolated products such as 3,6-dioxa-1,8-octanedithiol, polyethylene glycol dithiol, and multi-arm polyethylene glycol.
• the most suitable method for cross-linking vinyl alcohol polymers by covalent bonding can be selected according to the needs.
• the crosslinked body in the dried hydrogel-forming article of the present invention is preferably not crosslinked with glutaraldehyde, and is not crosslinked with glutaraldehyde.
• a method of introducing functional groups into the vinyl alcohol polymer by post-modification and reacting these groups particularly a method of introducing ethylenically unsaturated groups into the side chains of the vinyl alcohol polymer, is preferred.
• the dry hydrogel-forming article of the present invention further contains an alcohol-based solvent.
• the content of the alcohol-based solvent in the dry hydrogel-forming article of the present invention based on the amount of the dry hydrogel-forming article is 0.01% by mass or more and 15% by mass or less.
• the content of the alcohol-based solvent in the dry hydrogel-forming article is not particularly limited as long as it is within the above range, but from the viewpoint 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.
• the alcohol-based solvent is not particularly limited as long as it is a water-soluble alcohol, but examples include monoalcohols such as methanol, ethanol, propanol, and isopropanol, and polyhydric alcohols such as ethylene glycol, diethylene glycol, triethylene glycol, and glycerin.
• the alcohol-based solvent may contain one type of solvent or two or more types of solvents.
• the dry hydrogel-forming article is a hydrogel in a dry state, which may or may not contain water, but is an article that can absorb additional water and swell to form a hydrogel.
• the water content in the dry hydrogel-forming article of the present invention based on the amount of the dry hydrogel-forming article is 0% by mass or more and 70% by mass or less.
• the water content in the dry hydrogel-forming article is not particularly limited as long as it is within the above range, but from the viewpoint that the lower the water content, the smaller the volume and the higher the transport efficiency, it 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.
• the solvent contained in the hydrogel can be considered to be an alcohol-based solvent, so a method for measuring the content of alcohol-based solvent from the change in weight after the water-soluble alcohol has been completely evaporated can be mentioned.
• the weight of particles dried at 37°C is measured, and then the particles are further dried at 120°C until there is no weight change, after which the weight is measured, and the content of alcohol-based solvent can be calculated from the weight difference before and after drying at 120°C using the following calculation formula (I).
• Alcohol-based solvent content ((Weight after drying at 37°C)-(Weight after drying at 120°C))/(Weight after drying at 37°C) (I)
• the content of the alcohol-based solvent in the dried hydrogel-forming article can also be measured, for example, together with the content of water, by 1 H-NMR. Details of the measurement conditions are as described in the Examples.
• the total content of water and alcohol-based solvent in the dry hydrogel-forming article may 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, etc.
• the ratio of the content of the alcohol-based solvent to the total content of water and alcohol-based solvent in the dry hydrogel-forming article may be 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, from the viewpoint of easily preventing aggregation.
• the upper limit of the ratio may be 100% by mass or less.
• the dry hydrogel-forming article of the present invention contains an alcohol-based solvent, but may or may not contain water.
• the degree of aggregation of the dry hydrogel-forming article of the present invention is 10% or less. If the degree of aggregation exceeds 10%, the fixed and/or aggregated dry hydrogel-forming article may remain aggregated even when swollen and may not swell uniformly, or the surface structure of the hydrogel may be destroyed when it is separated upon impact or swelling. In addition, the aggregated hydrogel may be damaged when separated, resulting in fragments of the hydrogel. If such fragments are mixed in during use, for example, when the hydrogel is used for cell culture, they may be taken up by cells during culture, or separation from the cultured cells may be difficult.
• the degree of aggregation is calculated by weighing a certain amount of dried hydrogel-forming article, visually separating aggregates that are composed of two or more particles, measuring the weight of the separated aggregates, and then using the following formula (II).
• Coagulation degree [%] (weight of aggregates / total weight) ⁇ 100 (II)
• the degree of aggregation is calculated using a measurement sample containing a plurality of dry hydrogel-forming articles. For example, when the dry hydrogel-forming articles are sheets, the degree of aggregation may be measured from the number of sheets that are adhered to each other using dry hydrogel-forming articles obtained by stacking and drying a plurality of sheets.
• a method for adjusting the contents of water and alcohol-based solvent and the degree of cohesion within the above ranges a method may be mentioned in which a hydrogel containing water is produced, the hydrogel is immersed in an alcohol-based solvent to replace the water contained in the hydrogel with the alcohol-based solvent, and the resulting hydrogel is then dried.
• the hydrogel dried as described above may be sterilized by radiation sterilization (e.g., gamma ray sterilization) or the like.
• the dried hydrogel forming article of the present invention has a sterility compensation level SAL of 1 ⁇ 10 ⁇ 6 or less.
• the sterility compensation level SAL exceeds 1 ⁇ 10 ⁇ 6 , the hydrogel can be used for pharmaceutical applications, etc.
• the above-mentioned sterility assurance level SAL is satisfied in the state of a dry hydrogel that can be swollen and used as a hydrogel, so that the sterility assurance level SAL can be satisfied at the time of final use. It is possible to obtain high sterilization even when the hydrogel-forming article is in a dried state.
• the sterility assurance level SAL is set to 1 ⁇ 10 ⁇ 6 or less, and although the reason is unclear, It is believed that this can suppress the generation of radicals and facilitate the enhancement of cell adhesiveness.
• the sterility compensation level SAL can be preferably guaranteed by sterilization under conditions defined by the VD max method. Details of the conditions set by the VD max method are as described in the Examples.
• the dry hydrogel-forming article of the present invention is an article that satisfies the above-mentioned sterility compensation level, and is preferably an article that has been sterilized by radiation, more preferably an article that has been sterilized by gamma rays, and even more preferably an article that has been sterilized by gamma rays at a sterilization dose defined by the VD max method.
• a dry hydrogel-forming article containing a crosslinked body obtained by crosslinking a vinyl alcohol-based polymer that has been sterilized by radiation (particularly gamma ray sterilization) before crosslinking may not have sufficient moldability as a hydrogel and may not have sufficient strength, and is therefore preferably excluded from the scope of the present invention.
• the dry hydrogel-forming article of the present invention preferably 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 placed in a glass vial and stirred for 3 hours at a speed of 400 rpm using a stirrer tip whose length is 80% or more of the bottom diameter of the glass vial, the breakage rate of the swollen hydrogel-forming article is 5% or less.
• the broken rate is 5% or less as measured by the above method, the strength of the dried hydrogel-forming article is high, and even if the article is sterilized so that the sterility compensation level SAL is 1 ⁇ 10 ⁇ 6 or less, the strength of the resulting hydrogel can be maintained, and such a dried hydrogel-forming article is likely to maintain its swelling degree when it is re-swollen after drying.
• the broken 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 broken rate is calculated by observing the state of the solid content after stirring under the above conditions with a microscope, observing the state in which the solid content is visible in 60% or more of the visual field, visually distinguishing between broken and unbroken solid content, and calculating the ratio of the area of the broken parts to the area of the entire solid content.
• the swelling degree is preferably not less than 5, more preferably not less than 6, and even more preferably not less than 8.
• the upper limit of the swelling degree is not particularly limited, but from the viewpoint of the strength of the hydrogel, it is preferably not more than 40, more preferably not more than 30, and even more preferably not more than 20.
• the shape of the dry hydrogel-forming article is not particularly limited, but is preferably an amorphous particle, a spherical particle, a fine molded body, an article of any shape formed by a 3D printer, a sheet, a thread, a hollow fiber, a porous monolith, or a coating article.
• the dry hydrogel-forming article is preferably a spherical particle, more preferably a spherical particle having a particle diameter of 10 to 5000 ⁇ m when swollen.
• the particle diameter of the dry hydrogel-forming article of the spherical particle may be 1 to 5000 ⁇ m.
• the dry hydrogel-forming article may be a single dry hydrogel-forming article having the above-mentioned shape, or may be a group of a plurality of dry hydrogel-forming articles (e.g., a particle group), but is usually a group of a plurality of dry hydrogel-forming articles, preferably 10 or more, more preferably 30 or more, even more preferably 50 or more, and even more preferably 70 or more dry hydrogel-forming articles.
• the micromolded body is a molded body with fine irregularities on the surface or inside, and the size of the microprocessing is 10 to 1000 ⁇ m.
• the arbitrary shape molded by a 3D printer is, for example, an arbitrary shape that can be molded by a stereolithography method, an inkjet method, or a nozzle extrusion type 3D printer.
• the coated article refers to a composite hydrogel coated on a substrate of a shape such as a sheet, tray, thread, hollow fiber, porous monolith, micromolded body, or an arbitrary shape object molded by a 3D printer, and the substrate material can be freely selected from materials such as glass, polyolefin, polymethylmethacrylate, polystyrene, polyester, polyolefin, polyethylene vinyl alcohol copolymer, polyamide, and polyimide. From the viewpoint of preventing aggregation, the dry hydrogel-forming article is preferably a dry hydrogel-forming particle.
• the crosslinked vinyl alcohol polymer may be complexed with a physiologically active substance.
• a physiologically active substance it becomes possible to increase cell adhesiveness when the hydrogel obtained by swelling the dry hydrogel-forming article is used for applications such as cell culture.
• a physiologically active substance it becomes possible to provide a hydrogel that has high cell adhesiveness and is sufficiently sterilized.
• physiologically active substances include cell adhesive proteins or peptides such as gelatin, collagen, laminin, fibronectin, retronectin, vitronectin, elastin, and synthetic RGD peptides; growth factors such as fibroblast growth factor (FGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF); acidic polysaccharides including glycosaminoglycans such as heparin, hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparan sulfate; antibodies, hormones, serum proteins, antibody-binding proteins such as albumin, macroglobulins, globulins, and protein A; various pharmaceuticals; and enzymes such as proteases, lipases, amylases, and cellulases.
• FGF fibroblast growth factor
• EGF epidermal growth factor
• VEGF vascular endothelial growth factor
• acidic polysaccharides including
• the physiologically active substance preferably has a functional group capable of binding to the dried hydrogel-forming article.
• functional groups include primary or secondary amino groups, carboxy groups, and hydroxyl groups, and preferably primary amino groups or carboxy groups, and more preferably primary amino groups.
• the physiologically active substance may be one type of substance or a combination of two or more types of substances.
• the physiologically active substance is preferably a physiologically active substance having a primary or secondary amino group.
• the physiologically active substance may be, for example, at least one selected from the group consisting of cell adhesive proteins, cell adhesive peptides, growth factors, acidic polysaccharides, antibodies, hormones, serum proteins, antibody-binding proteins, pharmaceuticals, and enzymes.
• physiologically active substances having an amino group include cell adhesive proteins or peptides such as gelatin, collagen, laminin, fibronectin, retronectin, vitronectin, elastin, and synthetic RGD peptides; growth factors such as fibroblast growth factor (FGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF); antibody, serum protein, albumin, macroglobulin, globulin, and protein A; glycosaminoglycans such as heparin, hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparan sulfate; various pharmaceuticals; and enzymes such as protease, lipase, amylase, and cellulase.
• FGF fibroblast growth factor
• EGF epidermal growth factor
• VEGF vascular endothelial growth factor
• antibody serum protein, albumin, macroglobulin, globulin, and protein A
• physiologically active substances contain amino groups, and when the dried hydrogel-forming article of the present invention contains a crosslinked product of a vinyl alcohol polymer having a carboxyl group, these physiologically active substances are preferred because they can be easily complexed with the crosslinked product through an amide bond.
• a physiologically active substance a physiologically active polypeptide containing two or more amino acids is also preferred.
• the amino group may be either an aromatic amino group or an aliphatic amino group, but is preferably an aliphatic amino group, and is preferably a side chain amino group or an N-terminal amino group of lysine (Lys) of a physiologically active polypeptide.
• the amino group contained in the physiologically active substance may be either a primary amino group or a secondary amino group, and may include one or both of these. Either the primary amino group or the secondary amino group can form an amide bond with a carboxy group that may be possessed by the crosslinked vinyl alcohol polymer.
• the dry hydrogel-forming article of the present invention is a complex with a physiologically active substance.
• the dry hydrogel-forming article of the present invention may be a dry hydrogel-forming article that simply contains the physiologically active substance, or may be a complex in which a crosslinked vinyl alcohol-based polymer contained in the dry hydrogel-forming article and a physiologically active substance are covalently bonded, but is preferably a dry hydrogel-forming article in which a physiologically active substance is covalently bonded.
• Methods for covalently bonding hydrogel particles and a physiologically active substance include, for example, activating the hydroxyl groups of PVA and reacting them with the functional groups of the physiologically active substance to form covalent bonds, or using carboxylic acid-modified PVA or amino-modified PVA to react with the functional groups of the physiologically active substance to form covalent bonds. From the standpoint of reaction efficiency, it is preferable to use amino groups, carboxylic acids, or thiol groups as the functional groups of the physiologically active substance, and carboxylic acids are more preferable.
• Specific methods for activating the hydroxyl groups of PVA to introduce a physiologically active substance include, for example, a method using a hydroxyl group activating reagent such as 1,1'-carbonyldiimidazole, di(N-succimidyl)carbonate, p-toluenesulfonic acid chloride, 2,2,2-trifluoroethanesulfonic acid chloride, or cyanuric acid chloride.
• a hydroxyl group activating reagent such as 1,1'-carbonyldiimidazole, di(N-succimidyl)carbonate, p-toluenesulfonic acid chloride, 2,2,2-trifluoroethanesulfonic acid chloride, or cyanuric acid chloride.
• an acid anhydride reagent such as succinic anhydride that can introduce a carboxylic acid by ester bonding to the hydroxyl groups of the vinyl alcohol polymer
• an acetal reagent such as 2,2-dimethoxyethylamine that can introduce an amino group by acetal bonding to the 1,3-diol group of the vinyl alcohol polymer.
• carboxylic acids and amino groups can form an amide bond using the amino group of the physiologically active substance, the carboxylic acid, and the carbodiimide condensing agent.
• a crosslinked vinyl alcohol polymer is reacted with an acid anhydride reagent such as succinic anhydride
• an acid anhydride reagent such as succinic anhydride
• a carboxy group (COOH) derived from succinic acid is introduced at the end, and this carboxy group and the amino group of a physiologically active substance are condensed using a condensing agent such as 1,1'-carbonyldiimidazole, dicyclohexylcarbodiimide, or water-soluble carbodiimide, thereby complexing the physiologically active substance with the crosslinked vinyl alcohol polymer via an amide bond.
• a condensing agent such as 1,1'-carbonyldiimidazole, dicyclohexylcarbodiimide, or water-soluble carbodiimide
• the complex may be formed after the crosslinking step of the uncrosslinked polymer solution as described below, or the complex may be formed at the uncrosslinked polymer solution stage. It is preferable to form the complex with the biologically active substance after the crosslinking step in order to avoid a decrease or loss of activity of the biologically active substance due to crosslinking.
• Manufacturing method of a dry hydrogel-forming article There is no particular limitation on the manufacturing method of the dry hydrogel-forming article of the present invention, but examples include a manufacturing method including a step of preparing a preliminary hydrogel containing a crosslinked body of a vinyl alcohol-based polymer (preliminary hydrogel preparation step), a step of immersing the preliminary hydrogel in an alcohol-based solvent and then drying to obtain a dry preliminary hydrogel (dry preliminary hydrogel preparation step), and a step of sterilizing the dry preliminary hydrogel until the SAL becomes 1 ⁇ 10 -6 or less (sterilization step).
• the preliminary hydrogel preparation step a preliminary hydrogel containing a crosslinked vinyl alcohol polymer is prepared.
• the preliminary hydrogel preparation step preferably includes at least a step of preparing an uncrosslinked polymer solution containing the vinyl alcohol polymer (uncrosslinked polymer solution preparation step), a step of molding the uncrosslinked polymer solution (molding step), and a step of crosslinking the vinyl alcohol polymer contained in the uncrosslinked polymer solution to gel (crosslinking step).
• the present invention also provides a method for producing a dry hydrogel including the above steps. Each step is specifically described below.
• the uncrosslinked polymer solution preparation step is a step of preparing an uncrosslinked polymer solution containing the vinyl alcohol polymer, and the uncrosslinked polymer solution can be obtained by dissolving the vinyl alcohol polymer in a solvent.
• the solvent is preferably water, and may further contain a water-soluble organic solvent.
• the water-soluble organic solvent include aprotic polar solvents such as dimethylformamide, dimethylacetamide, dimethylsulfoxide, and N-methylpyrrolidone; monoalcohols such as methanol, ethanol, propanol, and isopropanol; and polyhydric alcohols such as ethylene glycol, diethylene glycol, triethylene glycol, and glycerin.
• the water-soluble organic solvent may be used alone or in a mixture of two or more kinds, or may be used by mixing the water-soluble organic solvent with water.
• the 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, 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.
• the content of the vinyl alcohol-based 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. From the viewpoint of suppressing the increase in viscosity of the uncrosslinked polymer solution and obtaining good moldability, the content is preferably 50% by mass or less, more preferably 45% by mass or less, and even more preferably 40% by mass or less. If the content of the vinyl alcohol-based polymer is less than 0.001% by mass, the strength and film thickness of the obtained gel are too small, and if it exceeds 50% by mass, the viscosity of the uncrosslinked polymer solution is high, making molding difficult.
• the molding process is a process of molding the uncrosslinked polymer solution into a desired shape.
• the method of molding the uncrosslinked polymer solution can be molded into the shape of the amorphous particles, spherical particles, fine molded bodies, any shaped articles molded by 3D printers, sheets, threads, hollow fibers, porous monoliths, coating articles, etc. using known methods.
• the crosslinking step is a step of crosslinking the vinyl alcohol polymer after the molding step.
• the crosslinking in this step can be performed by the method described above.
• the crosslinking step may be performed while the uncrosslinked polymer solution contains a solvent, or may be performed after removing the solvent from the uncrosslinked polymer solution.
• the crosslinked gel may shrink significantly in the drying step described below, and it may be difficult to form a shape such as a sheet, thread, hollow fiber, coating, etc. In this case, it is preferable to dry the uncrosslinked polymer solution once after molding and then crosslink it.
• the degree of drying of the uncrosslinked polymer solution can be selected according to the molded product and the crosslinking method, but from the viewpoint of easily preventing shrinkage in the drying step, it may be preferable to crosslink after drying until the solvent content is preferably 50% or less, more preferably 25% or less, and even more preferably 10% or less.
• the uncrosslinked polymer solution may contain not only the vinyl alcohol polymer but also components necessary for crosslinking, such as the crosslinking agent and initiator. As described above, if the uncrosslinked polymer solution is dried once after molding, the components necessary for crosslinking may be added after molding. As described above, the crosslinking conditions are optimally selected depending on the vinyl alcohol polymer and crosslinking method used, but since molding becomes difficult when crosslinking is completed and hydrogel is formed, it is preferable to mold before the hydrogel is formed. Some of the above-mentioned crosslinking methods start the reaction at a temperature below room temperature simply by mixing the uncrosslinked polymer solution with the components necessary for crosslinking, so it is desirable to mix them just before molding.
• 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, from the viewpoint of maintaining the activity of the physiologically active substance.
• the preliminary hydrogel preparation step further includes a complexing step.
• the complexing step is a step of complexing a physiologically active substance with a crosslinked body of a vinyl alcohol-based polymer to obtain a complexed hydrogel.
• the complexing step is basically unnecessary.
• a physiologically active substance can be introduced into the remaining functional group by the above-mentioned complexing method.
• a hydroxyl group activating reagent As a method for introducing a functional group, a hydroxyl group activating reagent, an acid anhydride reagent, an acetal reagent, or the like may be used as described above, and a physiologically active substance can be introduced into the functional group by the above-mentioned complexing method in the same manner.
• the hydrogel obtained in the preliminary hydrogel preparation step is dried by a general method such as hot air drying, vacuum drying, or freeze drying before sterilization so that the alcohol-based solvent content and water content are within the following ranges.
• a general method such as hot air drying, vacuum drying, or freeze drying before sterilization so that the alcohol-based solvent content and water content are within the following ranges.
• the temperature of the hot air drying is preferably about 30 to 100°C, more preferably about 37 to 60°C.
• the content of the alcohol-based solvent in the dried hydrogel-forming article after drying is preferably 0.01 to 15% by mass, more preferably 0.05 to 15% by mass, even more preferably 0.07 to 15% by mass, and even more preferably 0.1 to 15% by mass, and the content of water is preferably 0 to 70% by mass, more preferably 0 to 60% by mass, even more preferably 0 to 50% by mass, and even more preferably 0 to 40% by mass or less.
• the alcohol-based solvent in which the preliminary hydrogel is immersed is not particularly limited, and examples thereof include monoalcohols such as methanol, ethanol, propanol, and isopropanol, and polyhydric alcohols such as ethylene glycol, diethylene glycol, triethylene glycol, and glycerin.
• the alcohol-based solvent may be used alone or in a mixture of two or more types, or may be used in a mixture of an alcohol-based solvent and water.
• the dried preliminary hydrogel is sterilized until the SAL becomes 1 ⁇ 10 ⁇ 6 or less.
• the hydrogel can be used for applications in which it is incorporated as part of medicines or medical devices to be used on the human body.
• the dried hydrogel-forming article is preferably a dried hydrogel-forming article sterilized in a dried state in which the content of the alcohol-based solvent and water is within the above-mentioned range, and is sterilized after the drying process. If a composite hydrogel containing a large amount of water is sterilized before the drying process, for example, if a physiologically active substance is composited, its activity may decrease. In addition, the generation of radicals may also decrease the adhesiveness of cells when the hydrogel is used for cell culture or the like.
• the sterilization method is not particularly limited.
• autoclave sterilization ethylene oxide gas sterilization, hydrogen peroxide low-temperature plasma sterilization, dry heat sterilization, chemical sterilization using glutaraldehyde or the like, radiation sterilization using gamma rays or electron beams, etc.
• ethylene oxide gas sterilization hydrogen peroxide low-temperature plasma sterilization, and radiation sterilization
• radiation sterilization is more preferable because there is no residue.
• the dried hydrogel-forming article of the present invention is preferably ethylene oxide gas-free, glutaraldehyde-free, and hydrogen peroxide-free. Sterilization is preferably performed by placing the pre-dried hydrogel-forming article in a container, sealing the container, and then performing a sterilization process.
• a container a flexible resin container, for example, a resin pouch, can be used, and sealing can be performed by heat sealing.
• a bottle container with a lid that can be opened after sterilization can be used as the container, and sealing can be performed by screwing the lid on or by heat sealing like an ampoule.
• Examples of the material for the container include polyolefin resins such as polyethylene, polypropylene, and polybutylene, or copolymers of two or more olefins, polyester resins such as polyethylene terephthalate and polybutylene terephthalate, polyvinyl alcohol, saponified ethylene-vinyl acetate copolymer, vinyl resins such as polyvinyl chloride and polyvinylidene chloride, polycarbonate resins, polystyrene resins, silicone resins, polyamide resins, and glass.
• polyolefin resins such as polyethylene, polypropylene, and polybutylene, or copolymers of two or more olefins
• polyester resins such as polyethylene terephthalate and polybutylene terephthalate
• polyvinyl alcohol such as polyethylene terephthalate and polybutylene terephthalate
• vinyl resins such as polyvinyl chloride and polyvinylidene chloride
• the container may be made of a single layer or a laminate of these materials, and may be made of these materials with aluminum, silica, or the like vapor-deposited, or a metal foil or a multi-layered body containing a metal foil such as an aluminum film or an aluminum laminate film.
• Sterilization validation can be performed by any of the following methods: half cycle method, overkill method, bioburden/BI combined method, and absolute bioburden method.
• sterilization validation is performed by the absolute bioburden method described in ISO (ISO 11137-2:2013) or JIS (JIS T 0806-2:2014).
• Methods for setting the sterilization dose in sterilization validation include Method 1, VD max method, and Method 2, all of which can be used for sterilization in producing the dried hydrogel-forming article of the present invention.
• Method 1 and VD max method are methods for setting the sterilization dose based on the number of bacteria adhering to the pre-dried hydrogel-forming article before sterilization
• Method 2 is a method for setting the sterilization dose based on the radiation resistance of bacteria adhering to the pre-dried hydrogel-forming article before sterilization.
• a bioburden test described in ISO 11737-1: 2006 or JIS T11737-1: 2013, etc. can be performed to measure the number of bacteria adhering to the pre-dried hydrogel-forming article before sterilization.
• Method 1 and the VD max method are more preferable, and in consideration of cost control, the VD max method is even more preferable.
• an irradiation dose of 11 kGray (Gy) or more is required in method 1, and an irradiation dose of 15 kGy or more is required in the VD max method. Also, an irradiation dose of 8.2 kGy or more is required in method 2. It has been conventionally recognized that hydrogels (particularly composite hydrogels) cannot withstand such a very high energy gamma ray irradiation dose.
• sterilization is performed on a dried pre-dried hydrogel-forming article, so that damage to the product can be prevented even with high energy gamma ray irradiation of, for example, 8.2 kGy or more, and a decrease in cell adhesiveness can be suppressed, and in the case where a physiologically active substance is composited, the activity can be maintained within a desired range.
• radiation sterilization is preferably performed in the step of sterilization until the SAL becomes 1 ⁇ 10 ⁇ 6 or less, and the radiation dose in the radiation sterilization is more preferably 8.2 kGy or more.
• the dry hydrogel-forming article of the present invention can be swollen in water and used as a hydrogel.
• the present invention also provides a hydrogel which is a swollen body (preferably a water-swollen body) of the dry 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 may contain a mixture of water and a water-soluble organic solvent.
• water-soluble organic solvent a mixture of water-soluble organic solvents such as aprotic polar solvents such as dimethylformamide, dimethylacetamide, dimethylsulfoxide, and N-methylpyrrolidone; monoalcohols such as methanol, ethanol, propanol, and isopropanol; and polyhydric alcohols such as ethylene glycol, diethylene glycol, triethylene glycol, and glycerin may be used.
• the content of the solvent (preferably water and/or a 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 water content in the hydrogel is preferably 30 to 95 mass %, more preferably 40 to 90 mass %, and even more preferably 50 to 90 mass %, based on the total amount of the hydrogel.
• the water-soluble organic solvent content in the hydrogel is preferably 30 to 95 mass %, more preferably 40 to 95 mass %, and even more preferably 50 to 95 mass %.
• the solid content of the hydrogel of the present invention is preferably 0.1 to 70 mass%, more preferably 1 to 60 mass%, and even more preferably 3 to 50 mass%, based on the total amount of the hydrogel.
• the solid content of the hydrogel can be calculated from the weight of the hydrogel before drying and the weight of the hydrogel after drying, for example, at 120° C. for 5 hours, according to the following formula (IV).
• Solid content [mass%] (weight of hydrogel after drying / weight of hydrogel before drying) x 100 (IV)
• the hydrogel of the present invention may be used in combination with cells or may contain cells. When used in combination with cells or to contain cells, it is preferable to supply water to the article to form a hydrogel.
• the term "cells" in this specification is not particularly limited, but preferably includes pluripotent stem cells, tissue stem cells, somatic cells, mammalian cell lines used for the production of useful substances such as pharmaceuticals, treatments, etc., and insect cells.
• Cells include adherent cells and suspension cells.
• Adherent cells are cells that grow by adhering to a carrier such as the hydrogel of the present invention during cell culture.
• Suspension cells are cells that do not fundamentally require attachment to a carrier for cell growth.
• Suspension cells include cells that can weakly adhere to a carrier.
• pluripotent stem cells are stem cells that have the ability to differentiate into cells of any tissue (pluripotency), and examples of such cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic germ stem cells (EG cells), and germline stem cells (GS cells).
• ES cells embryonic stem cells
• iPS cells induced pluripotent stem cells
• EG cells embryonic germ stem cells
• GS cells germline stem cells
• tissue stem cells refer to stem cells that are limited in the tissues they differentiate into but have the ability to differentiate into various cell types (multipotency).
• tissue stem cells include bone marrow undifferentiated mesenchymal stem cells, skeletal muscle stem cells, hematopoietic stem cells, neural stem cells, hepatic stem cells, adipose tissue stem cells, epidermal stem cells, intestinal stem cells, spermatogonial stem cells, pancreatic stem cells (pancreatic ductal epithelial stem cells, etc.), leukocyte stem cells, lymphocyte stem cells, and corneal stem cells.
• the somatic cells mentioned above refer to cells that constitute a multicellular organism, and examples thereof include, but are not limited to, osteoblasts, chondrocytes, hematopoietic cells, epithelial cells (such as mammary epithelial cells), endothelial cells (such as vascular endothelial cells), epidermal cells, fibroblasts, mesenchymal-derived cells, cardiac muscle cells, myoblasts, smooth muscle cells, skeletal muscle cells derived from living organisms, human tumor cells, fibrocytes, EB virus mutant cells, hepatic cells, kidney cells, bone marrow cells, macrophages, hepatic parenchymal cells, small intestinal cells, mammary cells, salivary gland cells, thyroid cells, skin cells, plasma cells, T cells, B cells, killer cells, lymphoblasts, and pancreatic ⁇ cells.
• epithelial cells such as mammary epithelial cells
• endothelial cells such as vascular endothelial cells
• the above mammalian cell lines include CRFK cells, 3T3 cells, A549 cells, AH130 cells, B95-8 cells, BHK cells, BOSC23 cells, BS-C-1 cells, C3H10T1/2 cells, C-6 cells, CHO cells, COS cells, CV-1 cells, F9 cells, FL cells, FL5-1 cells, FM3A cells, G-361 cells, GP+E-86 cells, GP+envAm12 cells, H4-II-E cells, HEK293 cells, HeLa cells, HEp-2 cells, HL-60 cells, HTC cells, HUVEC cells, IMR-32 cells, IMR-90 cells, K562 cells, KB cells, L cells, L5178Y cells, L-929 cells, MA104 cells, MDBK cells, MDCK cells, MIA PaCG-2 cells, N18 cells, Namalwa cells, NG108-1 5 cells, NRK cells, OC10 cells, OTT6050 cells, P388 cells, PA12 cells, PA317 cells,
• Examples include C6 cells, PG13 cells, QGH cells, Raji cells, RPMI-1788 cells, SGE1 cells, Sp2/O-Ag14 cells, ST2 cells, THP-1 cells, U-937 cells, V79 cells, VERO cells, WI-38 cells, ⁇ 2 cells, and ⁇ CRE cells.
• the above insect cells include silkworm cells (BmN cells, BoMo cells, etc.), mulberry cells, Anemone cells, Anemone cells, Scutellaria cells, Mammalian armyworm cells (Sf9 cells, Sf21 cells, etc.), mulberry fly cells, leafroller cells, Drosophila cells, Boettcheris peregrina cells, Aedes albopictus cells, Swallowtail butterfly cells, American cockroach cells, and nettle looper cells (Tn-5 cells, HIGH FIVE cells, MG1 cells, etc.).
• the cells may be aggregated together or may be differentiated.
• the aggregated cells may have the function of an organ.
• the cells may be freshly harvested from a living organism or may be cultured.
• the cells harvested from a living organism may have formed an organ.
• the medium may be a basic medium such as DMEM, MEM, or F12 supplemented with fetal bovine serum, a serum-reduced medium, or a serum-free medium.
• DMEM fetal bovine serum
• serum-free media examples include xeno-free media and fully synthetic media that not only do not contain serum, but also protein components and animal components.
• the hydrogel of the present invention is a sufficiently sterilized hydrogel, and has good dispersibility due to suppression of aggregation during drying, and is prevented from being mixed with hydrogel fragments.
• the hydrogel of the present invention preferably has characteristics such as maintaining a high degree of swelling, having high strength, maintaining the activity of a physiologically active substance or the like when it is contained, and having high adhesiveness when used for cell adhesion.
• the hydrogel of the present invention preferably has excellent hydrophilicity, reactivity, biodegradability, biocompatibility, and low toxicity, and has high flexibility and strength, and can provide a sterilized hydrogel without using an expensive process such as a sterile environment. Therefore, the hydrogel of the present invention is useful as a carrier or support.
• a carrier made of the hydrogel of the present invention can be suitably used for applications such as an enzyme immobilization carrier, an affinity carrier, a cell culture carrier, and a carrier for drug delivery.
• the hydrogel of the present invention is a hydrogel obtained by swelling the dry hydrogel-forming article of the present invention, and the vinyl alcohol-based polymer contained in the dry hydrogel-forming article of the present invention is also included in the hydrogel of the present invention. Unless otherwise specified in this specification, the details, manufacturing method, and usage of the hydrogel of the present invention are the same as those described in "1. Dry hydrogel-forming article.”
• the hydrogel of the present invention is formed by swelling the dry hydrogel-forming article of the present invention 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 has completely swollen to the point where the dry hydrogel-forming article no longer swells, or it may be a hydrogel that can swell further and still has hydrogel-forming properties.
• the shape of the hydrogel is not particularly limited, and like the dried hydrogel-forming article, it may be an amorphous particle, a spherical particle, a fine molded body, an article of any shape formed by a 3D printer, a sheet, a thread, a hollow fiber, a porous monolith, or a coated article.
• the hydrogel of the present invention is preferably a spherical particle, more preferably a spherical particle having a particle diameter of 10 to 5000 ⁇ m.
• amide bond composite hydrogel refers to a hydrogel in which a physiologically active substance having an amino group is covalently bonded to a crosslinked product of a vinyl alcohol polymer having an ethylenically unsaturated group and a carboxy group via an amide bond with the carboxy group of the vinyl alcohol polymer.
• the shape, dimensions, manufacturing method, etc. of the hydrogel may be the same as those described above in "1. Dry hydrogel-forming articles.”
• the hydrogel of the present invention is a swollen body of the dry hydrogel-forming article of the present invention, and is preferably a water-swollen body of the dry hydrogel-forming article of the present invention.
• the water-swollen body is a swollen body that swells upon contact with a solution containing at least water.
• it may be a swollen body that swells upon contact with water, or may be a swollen body that swells upon contact with an aqueous solution such as a buffer solution, a body fluid, or a culture medium.
• the alcohol-based solvent contained in the dry hydrogel-forming article of the present invention may or may not be contained in the hydrogel of the present invention.
• the dry hydrogel-forming article of the present invention which contains a crosslinked vinyl alcohol polymer and an alcohol-based solvent
• the alcohol-based solvent contained in the dry hydrogel-forming article of the present invention may be released into the water, and the hydrogel of the present invention may no longer contain alcohol.
• a hydrogel can also be said to be a water-swollen body of the dry hydrogel-forming article of the present invention.
• the hydrogel of the present invention is a swollen body of a dry hydrogel-forming article that meets a specific degree of cohesion and sterility compensation level SAL, so the hydrogel contains few contaminants such as gel fragments and is a sufficiently sterilized hydrogel.
• the sterility compensation level SAL of the hydrogel of the present invention is not particularly limited, but preferably has a sterility compensation level SAL of 1 ⁇ 10 ⁇ 6 or less, which is equivalent to that of a dry hydrogel-forming article.
• the hydrogel of the present invention contains a crosslinked vinyl alcohol polymer similar to that of the dry hydrogel-forming article of the present invention. Unless otherwise specified in this specification, the description of the crosslinked vinyl alcohol polymer contained in the hydrogel is similarly applied to the crosslinked vinyl alcohol polymer contained in the dry hydrogel-forming article.
• the hydrogel of the present invention also comprises a biologically active substance.
• the description of the biologically active substance that may be contained in the dry hydrogel-forming article similarly applies to the biologically active substance that may be contained in the hydrogel.
• the hydrogel of the present invention is a swollen body of the dry hydrogel-forming article of the present invention, and can be obtained by contacting the dry hydrogel-forming article with water, the above-mentioned water-soluble organic solvent, an aqueous solution, or a mixture thereof, etc.
• the hydrogel of the present invention is a sufficiently sterilized hydrogel, and since aggregation during drying is suppressed, there is no inclusion of debris or surface destruction, and it is suitable for use as a hydrogel for medical applications.
• the hydrogel of the present invention also has the advantages of being excellent in hydrophilicity, reactivity, biodegradability, and biocompatibility, having high flexibility and strength, being less toxic because a low-toxicity crosslinking method is used, and being efficiently introduced with a physiologically active substance or enzyme. Therefore, the hydrogel of the present invention is useful as a carrier.
• a carrier containing the hydrogel of the present invention can be suitably used for applications such as an enzyme immobilization carrier, an affinity carrier, a cell culture carrier, and a carrier for drug delivery.
• the dried hydrogel-forming article of the present invention can provide the above-mentioned excellent hydrogel.
• PVA117 Polyvinyl alcohol (product name "PVA117", polymerization degree 1700, saponification degree about 98.0 to 99.0 mol%, viscosity (4%, 20°C) 25.0 to 31.0 mPa ⁇ s, manufactured by Kuraray Co., Ltd.)
• the degree of polymerization of the raw material PVA was measured in accordance with JIS K 6726:1994.
• AF-17 Polyvinyl alcohol (product name "AF-17”, 1.3 mol% itaconic acid copolymer (Scientific Reports, 2017, Vol. 7, p. 45146), polymerization degree 1700, saponification degree 96.5 mol% or more, viscosity (4%, 20°C) 30 ⁇ 3 mPa ⁇ s, manufactured by Nippon Vinyl Acetate & Poval Co., Ltd.)
• Ion-exchanged water Ion-exchanged water with an electric conductivity of 0.08 ⁇ 10 ⁇ 4 S/m or less
• PBS Prepared by dissolving a PBS tablet (manufactured by Takara Bio Inc.) in a specified amount of ion-exchanged water.
• MES buffer A 0.1 mol/L aqueous solution of 2-morpholinoethanesulfonic acid monohydrate (manufactured by Dojindo Laboratories, Ltd.) was prepared and neutralized with a 0.1 mol/L aqueous NaOH solution to adjust the pH to 5.6.
• 1 H-NMR measurements can be performed on hydrogel particles and sheets in a state swollen in a heavy solvent, and the introduction rates in the hydrogel particles and sheets can be determined from the ratio of the integral values of the signals of the carboxyl groups (methylene succinate groups) to the signal of the vinyl alcohol polymer.
• Apparatus Nuclear magnetic resonance apparatus "JNM-ECX400" manufactured by JEOL Ltd. Temperature: 25°C
• the damage rate of the dried hydrogel-forming article was evaluated by measuring 0.1 g of the dried hydrogel-forming article, placing it in a 50 ml glass vial (bottom diameter 25 mm) together with 10 ml of ion-exchanged water, and stirring with a stirrer tip of 20 mm diameter at a speed of 400 rpm for 3 hours. That is, the state of the solid content after stirring was observed under a microscope, and observed in a state where the solid content was visible in 60% or more of the visual field, and visually distinguished between damaged and undamaged solid content, and the damage rate was calculated from the ratio of the area of the damaged parts to the area of the entire solid content. In this embodiment and the comparative example, the damage rate was very high or very low, so the ratio of the area was calculated visually, but the above area may be measured using image software to calculate the damage rate.
• the introduction rate of ethylenically unsaturated groups (methacryloyl groups) in the methacryloylated PVA117 was 2.0 mol % relative to the repeating units of the raw material PVA (hereinafter, abbreviated as "MA-PVA117(2.0)").
• the obtained hydrogel particles were washed with a total of 3 L of hexane to remove liquid paraffin, and the obtained hydrogel particles were classified into particles of 180 to 300 ⁇ m using a JIS standard sieve.
• the hydrogel particles were further dehydrated by putting them in 1 L of acetone, and then dried under reduced pressure to obtain dried hydrogel particles (hereinafter abbreviated as "MA-PVA117(2.0) gel particles").
• the cured hydrogel sheet (10 ⁇ 20 cm) having a thickness of 0.5 mm was washed with a total of 1.5 L of ion-exchanged water. It was further immersed in 0.5 L of acetone for dehydration and dried under reduced pressure to obtain a dried hydrogel sheet (hereinafter abbreviated as "MA-PVA117(2.0)-SA(3.4) gel sheet”).
• MA-PVA117(2.0)-SA(3.4) gel sheet The introduction rate of succinic acid (SA) (introduction rate of active groups) in the dried PVA sheet was 3.4 mol %.
• the cured hydrogel sheet (10 ⁇ 20 cm) with a thickness of 0.5 mm was washed with a total of 1.5 L of ion-exchanged water. It was further immersed in 0.5 L of acetone to dehydrate, and dried under reduced pressure to obtain a dried hydrogel sheet (hereinafter abbreviated as "GA crosslinked PVA117 gel sheet").
• CI-MA-PVA117(2.0) gel particles carbonylimidazolylated hydrogel particles
• gelatin-composite hydrogel particles (hereinafter, abbreviated as "gelatin-SA-MA-PVA117(2.0) gel particles").
• gelatin-SA-MA-PVA117(2.0) gel particles was immersed overnight in excess PBS, and the amount of gelatin immobilized per gel weight (100 mg) was measured by the bicinchoninic acid (BCA) method (BCA Protein Assay Kit (manufactured by Takara Bio Inc.)), and the complexation density of the physiologically active substance was 48.5 ⁇ g/100 mg of gel particles.
• BCA bicinchoninic acid
• Collagen composite hydrogel particles were obtained in the same manner as in Synthesis Example 1-A, except that collagen was used instead of gelatin (hereinafter, abbreviated as "collagen-SA-MA-PVA117(2.0) gel particles").
• collagen-SA-MA-PVA117(2.0) gel particles The amount of collagen immobilized in the collagen-SA-MA-PVA117(2.0) gel particles (composite density of a physiologically active substance) was also measured in the same manner as in Synthesis Example A.
• Example 1-1 2 g of gelatin-SA-MA-PVA117(2.0) gel particles (swollen with ion-exchanged water) prepared in Synthesis Example 1-A was immersed in 25 mL of ethanol three times, and the dehydrated composite gel particles were vacuum-dried overnight at room temperature. The water content and alcohol-based solvent content of the obtained dried particles were calculated by 1 H-NMR (deuterated DMSO solvent) according to the above-mentioned measurement method. Gamma ray irradiation was performed in the same manner as in Comparative Example 1-1 described later, to obtain a sterilized amide bond composite hydrogel.
• Example 1-1 The measurement results of the water content and the alcohol-based solvent content, and the evaluation results of sterility and cohesion are shown in Table 1.
• the degree of cohesion and the breakage rate of the dried hydrogel-forming article obtained in Example 1-1 are similar to those in Example 1-2.
• Example 1-2 2 g of collagen-SA-MA-PVA117(2.0) gel particles (swollen with ion-exchanged water) prepared in Synthesis Example 1-B were dried, the water content was measured, the alcohol solvent content was measured, and the gel was irradiated with gamma rays in the same manner as in Example 1-1 to obtain a sterilized amide bond composite hydrogel.
• the results of the water content and alcohol solvent content are shown in Table 1.
• the degree of cohesion of the dried hydrogel-forming article obtained in Example 1-2 was 5.3%, and the breakage rate was 0%.
• Examples 1-3 A sterilized composite hydrogel was obtained by carrying out drying, water content measurement, and gamma ray irradiation in the same manner 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 measurement results of water content, etc. are shown in Table 1. The degree of cohesion and breakage rate of the dried hydrogel-forming article obtained in Example 1-3 were similar to those in Example 1-2.
• 1.9 ⁇ 10 5 NIH/3T3 cells grown by pre-culture were added to the wells containing the gel sheet and the wells not containing the gel sheet, and cultured under conditions of carbon dioxide concentration 5%, saturated water vapor pressure, and 37°C. After 24 hours of culture, the medium in the wells was removed with an aspirator, 500 ⁇ L of new medium was added, and 50 ⁇ L of cell proliferation/cytotoxicity measurement reagent Cell Counting Kit-8 (CCK-8, manufactured by Dojindo Laboratories, Inc.) was added.
• CCK-8 cell proliferation/cytotoxicity measurement reagent Cell Counting Kit-8
• the sterilized amide bond composite hydrogel obtained in Reference Example 3 was evaluated for cell adhesion using the same method as in Reference Example 2, and the cell adhesion rate was 87% (Table 1).
• Example 1-2 was rated A, and Comparative Example 1-5 was rated C.
• A No hydrogel fragmentation or damage to the hydrogel surface.
• B Hydrogel fragmentation and/or slight damage to the hydrogel surface occurs, but this is not a problem.
• C Hydrogel fragmentation and/or damage to the hydrogel surface occurs.
• D Significant hydrogel fragmentation and/or damage to the hydrogel surface occurs.
• the present invention was able to produce a dry hydrogel-forming article that was sufficiently sterilized and in which aggregation during drying was suppressed. It was also confirmed that the hydrogel, which is the swollen body of the dry hydrogel-forming article, had a higher cell adhesion rate than the hydrogels of Comparative Examples 1-6 and 1-7, etc., which were sterilized in a state with a high water content.
• Example 1-2 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 below use the same vinyl alcohol polymer as Example 1-2 and have the same or higher degree of polymerization, so they are considered to show a breakage rate similar to that of Example 1-2.
• the introduction rate of ethylenically unsaturated groups (methacryloyl groups) in the methacryloylated PVA117 was 1.2 mol % relative to the repeating units of the raw material PVA (hereinafter, abbreviated as "MA-PVA117(1.2)").
• Table 2 summarizes the vinyl alcohol polymers having ethylenically unsaturated groups produced according to the methods described in Synthesis Examples 2-1 to 2-6.
• the cured hydrogel sheet (10 ⁇ 20 cm) having a thickness of 0.5 mm was washed five times with 300 mL of ion-exchanged water. After that, it was put into 1 L of acetone for dehydration, and then dried under reduced pressure at room temperature overnight to obtain a dried hydrogel sheet (hereinafter abbreviated as "MA-PVA117(2.0)-SA(3.4) gel sheet").
• the introduction rate of the carboxy group (succinic acid) (introduction density of the carboxy group) was 9.9 mol % relative to the repeating unit of MA-PVA117(1.2) (hereinafter, abbreviated as "SA-MA-PVA117(1.2) gel particles").
• the gel particles were washed with 30 mL of MES buffer for 10 minutes three times, and then added to 70 mL of 1 mg/mL gelatin PBS solution. The mixture was shaken at room temperature for 3 hours, and the resulting hydrogel particles were washed with 70 mL of ion-exchanged water (heated to 60 ° C.) for 20 minutes twice. Further, the gelatin particles were immersed in 50 mL of ethanol three times, and then vacuum-dried overnight at room temperature to obtain dried amide-bonded gelatin composite hydrogel particles (hereinafter, abbreviated as "gelatin-SA-MA-PVA117(1.2) gel particles").
• gelatin-SA-MA-PVA117(1.2) gel particles was immersed overnight in excess PBS, and the amount of gelatin immobilized (complexation density of bioactive substance) per gel weight (100 mg) was measured by the bicinchoninic acid (BCA) method (BCA Protein Assay Kit (manufactured by Takara Bio Inc.)), which was 48.2 ⁇ g/100 mg gel particles.
• BCA bicinchoninic acid
• Gamma ray irradiation was performed in the same manner as in Comparative Example 1-1 to obtain a sterilized amide-bonded composite hydrogel.
• the water content and alcohol-based solvent content of the obtained dried particles were calculated by 1 H-NMR (deuterated DMSO solvent) according to the above-mentioned measurement method. The measurement results of the water content and the alcohol-based solvent content are shown in Table 5.
• Example 2-2 to 2-6 Amide-bonded gelatin composite hydrogel particles were obtained in the same manner as in Example 2-1, except that the carboxyl group-introduced hydrogel particles of Synthesis Examples 2-ii to 2-iv, 2-D, and 2-E were used.
• Collagen-composite PVA particles were obtained in the same manner as in Example 2-1, except that collagen was used instead of gelatin (hereinafter, abbreviated as "collagen-SA-MA-PVA117(1.2) gel particles").
• the amount of gelatin immobilized was measured by the BCA method and found to be 53.2 ⁇ g/100 mg of gel particles.
• Example 2-8 ⁇ Gelatin composite sheet> An amide-bonded gelatin composite hydrogel sheet was obtained in the same manner as in Example 2-1, except that 1 g of the dried MA-PVA117(2.0)-SA(3.4) gel sheet prepared in Synthesis Example 2-G was used (hereinafter, abbreviated as "gelatinized-MA-PVA117(2.0)-SA(3.4) gel sheet"). The amount of gelatin immobilized (composite density of physiologically active substance) was measured by the BCA method, and was found to be 20.0 ⁇ g/100 mg of gel particles.
• Comparative Example 2-2 Particles made of commercially available dextran similar to that of Comparative Example 2-1 were irradiated with gamma rays at an exposure dose of 25 kGy or more in the same manner as in Example 2-1 to obtain sterilized dextran particles.
• the breakage rate of the obtained sterilized dextran particles was measured according to the above-mentioned method, and was found to be 60%.
• Comparative Example 2-4 Particles made of commercially available cellulose similar to those in Comparative Example 2-3 were irradiated with gamma rays at an exposure dose of 25 kGy or more in the same manner as in Example 2-1 to obtain sterilized dextran particles.
• the breakage rate of the obtained sterilized dextran particles was measured according to the above-mentioned method, and the breakage rate was 40%.
• Example 2-1 30 mL of DMEM medium supplemented with 10% fetal bovine serum was added to a 125 mL spinner flask (manufactured by Corning), and the dried SA-MA-PVA117(1.2) gel particles obtained in Example 2-1 were weighed and added based on the calculated total surface area so that the total surface area was 162 cm2 . Further, 8.1 x 10 5 NIH/3T3 cells (purchased from ATCC) grown by pre-culture were added and cultured in an incubator with a carbon dioxide concentration of 5%, saturated water vapor pressure, and 37°C while stirring at a paddle rotation speed of 60 rpm.
• the cell proliferation rate is defined as the number of cells after culture/the initial cell number, the cell proliferation rate of the gel particles obtained in Example 2-1 was 10.6.
• the cell proliferation rate of the gelatinized-MA-PVA117(2.0)-SA(3.4) gel sheet obtained in Example 2-8 was measured by the following method. After the gel sheet was immersed in PBS overnight and swollen, the gel sheet was punched into a circle using a hole punch with a diameter of 34 mm, and this was placed on the bottom of a 6-well polystyrene cell culture plate. 3 mL/well of medium was added thereto, and 4.9 x 10 6 NIH/3T3 cells grown by pre-culture were added, and cultured in an incubator with a carbon dioxide concentration of 5%, saturated water vapor pressure, and 37°C. On the fourth day of culture, the cells were peeled off and collected from the surface of the gel sheet by trypsin treatment, and the number of cells after culture was counted to calculate the cell proliferation rate, which was 4.3.
• Example 3 The experiment was carried out in a class 10,000 clean room, and all of the buffers and ion-exchanged water were sterilized by filtration using a membrane filter with a pore size of 0.2 ⁇ m. Except for using 30 g of dried SA-MA-PVA117(2.0) gel particles prepared in Synthesis Example 1-i, gelatin composite hydrogel particles were obtained in the same manner as in Synthesis Example 1-a (hereinafter, abbreviated as "gelatin-SA-MA-PVA117(2.0) gel particles (clean room)").
• the amount of gelatin immobilized per gel weight (100 mg) was measured by the bicinchoninic acid (BCA) method, and the composite density of the physiologically active substance was 56.3 ⁇ g/100 mg 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.
• the present invention allows the manufacture of a dry hydrogel-forming article that is sufficiently sterilized and in which aggregation during drying is suppressed. It was also confirmed that the hydrogel, which is a swollen body of the dry hydrogel, has a high cell proliferation rate. In contrast, in the case of the particles of Comparative Examples 2-1 to 2-4, which do not contain a crosslinked body of a polyvinyl alcohol-based polymer, the strength of the hydrogel is insufficient, and further, when sterilization was performed using gamma rays, the particles were further destroyed and the strength was confirmed to be low.

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