JP7507586B2 - Polymer of 1-acryloyl imidazolidin-2-one compound and cell culture material using same - Google Patents

Description

The present invention relates to a copolymer that is expected to be used as a cell culture material.

In recent years, with the development of regenerative medicine technology, there is a demand for technology to safely and efficiently culture large quantities of mesenchymal stem cells that have the ability to differentiate into osteoblasts, adipocytes, muscle cells, cartilage cells, etc., and induced pluripotent stem cells (iPS cells) that can be induced to differentiate into all tissues and organs that make up the human body. As a material for such cell culture, a culture bed has been developed in which a polymer of N-isopropylacrylamide (NIPAAM) is immobilized on the surface of a container. In the case of a homopolymer of NIPAAM, the lower critical solution temperature (LCST) is 32°C, which is close to body temperature. For example, at the cell culture temperature (about 36°C), the surface of the culture bed on which the polymer of NIPAAM is immobilized is hydrophobic, allowing cells to adhere and grow, and when the temperature of the culture bed is controlled to a temperature lower than the LCST, the surface of the culture bed becomes hydrophilic, making it easier for the cultured cells to peel off, making it possible to recover cells with less damage to the cells (Non-Patent Document 1).

As a culture bed with the NIPAAM polymer immobilized thereon, a culture bed in which NIPAAM is graft-polymerized by electron beam irradiation (UpCell manufactured by CellSeed Co., Ltd.) is commercially available, but since a large-scale electron beam irradiation device is required, it is not suitable for mass production. In addition, Patent Document 1 shows that by using a substrate in which a block copolymer having a structure in which a water-insoluble polymer segment and a temperature-responsive polymer segment are bonded to the substrate surface is coated, it is possible to immobilize a temperature-responsive polymer on the substrate surface without the need for a large-scale device such as electron beam irradiation. However, the block copolymer disclosed in Patent Document 1 has a problem that it is insufficient in water resistance, and there is a problem that it dissolves from the substrate surface, and there is a problem that the cell detachability is insufficient. Furthermore, if the amount of the block copolymer immobilized on the substrate surface is large, the cell detachability is good, but the cell adhesion is poor. Conversely, if the amount of immobilization is small, the cell adhesion is good, but the cell detachability is poor, and the amount of NIPAAM polymer immobilized on the substrate surface greatly affects the performance of the cell culture bed.

As described above, there is room for improvement in the culture beds on which the NIPAAM polymer is immobilized in terms of stable cell culture and recovery efficiency, and there is a demand for temperature-responsive polymer materials to replace the NIPAAM polymer, as well as block copolymers that utilize these materials.

Patent No. 5846584

Polymer Journal, Vol. 75 (No. 2), pp. 174-186 (2018)

The objective of the present invention is to provide a copolymer suitable as a cell culture material.

The present inventors have conducted extensive research to solve the above problems and have completed the present invention. That is, one aspect of the present invention is a compound represented by the following general formula (1):

(wherein R ¹ is a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a benzyl group, or a group represented by the following general formula (2):

(In the formula, p represents an integer of 2 to 4, and R ⁴ represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a 2-methoxyethyl group, or an acetyl group.) R ² represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. R ³ represents a hydrogen atom or a methyl group.) and a hydrophobic block chain.

In another embodiment of the present invention, the hydrophobic block chain is represented by the following general formula (3):

(wherein R ⁵ represents an alkyl group having 1 to 6 carbon atoms, a cyclohexyl group, a benzyl group, or a phenylethyl group; and R ⁶ represents a hydrogen atom or a methyl group; provided that when R ⁶ is a hydrogen atom, R ⁵ is a methyl group, an ethyl group, a cyclohexyl group, or a benzyl group).

Another aspect of the present invention is a coating agent comprising the copolymer described above.

Another aspect of the present invention is a cell culture material formed by applying the above-mentioned coating agent to a substrate.

Another aspect of the present invention is a cell culture method using the above-mentioned cell culture material.

The present invention is described in further detail below.

Examples of the alkyl group having 1 to 4 carbon atoms for ^R1 in the general formula (1) include a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, and a cyclobutyl group. The same applies to the alkyl group having 1 to 4 carbon atoms for ^R2 .

The same applies to the alkyl group having 1 to 4 carbon atoms represented by R ⁴ in the general formula (2).

The chemical structure of the block chain having the repeating unit represented by the general formula (1) constituting the copolymer (hereinafter sometimes referred to as the imidazolidinone block chain) is naturally determined by the chemical structure of the imidazolidinone monomer (4) used in the polymerization, but there are no particular limitations on the combination as long as it is within the range of the substituents described above.

(In the formula, R ¹ , R ² and R ³ have the same meanings as above.)
In terms of controlling the temperature response of imidazolidinone block chains and cell adhesion/detachment properties, 1-acryloyl imidazolidin-2-one, 1-acryloyl-3-methyl imidazolidin-2-one, 1-acryloyl-3-ethyl imidazolidin-2-one, 1-acryloyl-3-propyl imidazolidin-2-one, 1-acryloyl-3-isopropyl imidazolidin-2-one, 1-acryloyl-3-cyclopropyl imidazolidin-2-one, 1-Acryloyl-3-benzylimidazolidin-2-one, 1-Acryloyl-3-(2-hydroxyethyl)imidazolidin-2-one, 1-Acryloyl-3-(2-methoxyethyl)imidazolidin-2-one, 1-Acryloyl-3-(3-methoxypropyl)imidazolidin-2-one, 1-Acryloyl-3-(4-methoxybutyl)imidazolidin-2-one, 1-Acryloyl-3-(2-ethoxyethyl)imidazolidin-2-one Block chains obtained by polymerizing one or more of 1-acryloyl-3-(2-(2-methoxyethoxy)ethyl)imidazolidin-2-one, 1-acryloyl-3-(2-propyloxyethyl)imidazolidin-2-one, 1-acryloyl-3-(2-(2-methoxyethoxy)ethyl)imidazolidin-2-one, and 1-acryloyl-4,4-dimethylimidazolidin-2-one are preferred, and among these, 1-acryloyl-3-(2-(2-methoxyethoxy)ethyl)imidazolidin-2-one, 1-acryloyl-4,4-dimethylimidazolidin-2-one, 1-acryloyl-3-(2-propyloxyethyl)imidazolidin-2-one, 1-acryloyl-3-(2-(2-methoxyethoxy)ethyl)imidazolidin-2-one, and 1-acryloyl-4,4-dimethylimidazolidin-2-one are preferred. Particularly preferred are block chains obtained by polymerizing one or more of 1-acryloyl-3-ethylimidazolidin-2-one, 1-acryloyl-3-isopropylimidazolidin-2-one, 1-acryloyl-3-benzylimidazolidin-2-one, 1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one, and 1-acryloyl-4,4-dimethylimidazolidin-2-one.

The imidazolidinone block chain may contain the repeating unit represented by the general formula (1), but in addition to the imidazolidine monomer, acrylic acid, methyl acrylate, ethyl acrylate, propyl acrylate, butyl acrylate, phenyl acrylate, benzyl acrylate, 2-hydroxyethyl acrylate, 2-methoxyethyl acrylate, 2-(N,N-dimethylamino)ethyl acrylate, methacrylic acid, methyl methacrylate, ethyl methacrylate, propyl methacrylate, butyl methacrylate, phenyl methacrylate, benzyl methacrylate, 2-phenylethyl methacrylate, 2-hydroxyethyl methacrylate, 2-methoxyethyl methacrylate, 2-methacryloyloxyethyl phosphonate, etc. may also be used to fine-tune the hydrophobicity, hydrophilicity, solubility, film-forming property, heat resistance, film durability, adhesion to the substrate, cell adhesion, etc. A small amount of vinyl monomer such as arylcholine, acrylamide, N,N-dimethylacrylamide, N,N-diethylacrylamide, N-isopropylacrylamide, N-tert-butylacrylamide, N-phenylacrylamide, N-hydroxymethylacrylamide, N-hydroxyethylacrylamide, N-butoxymethylacrylamide, N-(2-hydroxyethyl)acrylamide, N'-[3-(N,N-dimethylamino)propyl]acrylamide, acryloylmorpholine, N-methylmethacrylamide, N,N-dimethylmethacrylamide, N-isopropylmethacrylamide, N-tert-butylmethacrylamide, N-phenylmethacrylamide, N-methoxymethylmethacrylamide, vinyl acetate, styrene, chloromethylstyrene, 2-vinylpyridine, and acrylonitrile may be allowed to coexist. In order not to significantly impair the properties derived from the imidazolidine monomer, the amount of vinyl monomer added is preferably 10% by weight or less, more preferably 5% by weight or less, relative to the imidazolidine monomer.

The hydrophobic block chain refers to a polymer consisting of only the hydrophobic block chain that is not soluble in water or has extremely low solubility. Examples of such polymers include polyethylene, polypropylene, polybutadiene, polyacrylonitrile, polyvinyl ether, polystyrene, poly(meth)acrylic acid ester, poly(meth)acrylamide, polyester, polycarbonate, polyamide, polyimide, polyurethane, polysulfone, polyphenylene oxide, polysiloxane, polysilarylenesiloxane, and derivatives thereof. Among these, from the viewpoint of film-forming properties and cell adhesiveness, a hydrophobic block chain having at least one of the melting point and glass transition temperature of 15°C or higher is preferred.

As the hydrophobic block chain, a block chain having a repeating unit represented by the general formula (3) is preferred in terms of ease of controlling the structure of the copolymer. Examples of the alkyl group having 1 to 6 carbon atoms represented by ^R5 in the general formula (3) include a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, a neopentyl group, an isopentyl group, a tert-pentyl group, a 1-methylbutyl group, a 1-ethylpropyl group, a cyclobutylmethyl group, a cyclopentyl group, a hexyl group, a 1-methylpentyl group, a 4-methylpentyl group, a 1-ethylbutyl group, a 2-ethylbutyl group, and a cyclohexyl group. In terms of controlling the adhesiveness/detachability of cells, specifically, block chains obtained by polymerizing one or more of methyl acrylate, ethyl acrylate, cyclohexyl acrylate, benzyl acrylate, methyl methacrylate, ethyl methacrylate, propyl methacrylate, isopropyl methacrylate, butyl methacrylate, isobutyl methacrylate, sec-butyl methacrylate, tert-butyl methacrylate, cyclohexyl methacrylate, benzyl methacrylate, and 2-phenylethyl methacrylate are preferred.

The copolymer may be in the form of a linear block copolymer, a branched block copolymer, a star block copolymer, a graft copolymer, or the like.

There is no particular limit to the molecular weight of the copolymer, and the molecular weight may be weight-average molecular weight, number-average molecular weight, viscosity-average molecular weight, or the like, depending on the measurement method. The weight-average molecular weight (Mw) is preferably 1,000 to 1,000,000, and more preferably 5,000 to 500,000 from the viewpoints of control of the properties of the polymer and processability. There is no particular limit to the molecular weight distribution (PD) expressed as Mw/Mn, but it is preferably in the range of about 1 to 10, and more preferably in the range of 1 to 3 from the viewpoint of the uniformity of the polymer. Examples of methods for calculating the molecular weight include the size exclusion chromatography (SEC) method, which converts based on standard samples such as polystyrene and polyethylene glycol, the viscosity method, and the light scattering method. When graft polymerization is performed on the surface of a substrate, it is difficult to directly determine the molecular weight of the polymer, but the number-average molecular weight (Mn) of the graft chain can be estimated, for example, from the density of reaction sites on the surface of the substrate and the polymerization conversion rate.

Examples of the method for producing the copolymer include the following methods.
(Method 1) A method in which an imidazolidinone block chain and a hydrophobic block chain are separately synthesized, and then the imidazolidinone block chain and the hydrophobic block chain are reacted to produce the copolymer of the present invention. (Method 2) A method in which an imidazolidinone block chain or a hydrophobic block chain is synthesized by utilizing living polymerization or the like, and then the other block chain is synthesized from the terminal of the synthesized block chain to produce the copolymer of the present invention. (Method 3) A method in which a polymerizable functional group is introduced into the terminal of an imidazolidinone block chain, and the imidazolidinone block chain is polymerized to produce the copolymer of the present invention. (Method 4) A method in which an imidazolidinone block chain is synthesized from the surface of a polymer solid substrate by surface-initiated polymerization of an imidazolidinone monomer using electromagnetic waves such as ultraviolet light, or radiation such as electron beams or gamma rays, to produce the copolymer of the present invention. Among these methods, Method 2 is preferably used because it allows precise control of the copolymer structure and is easily applicable to coating agents, etc., which will be described later.

When producing a block copolymer using method 2, it is possible to use a known living radical polymerization method, such as iniferter polymerization, nitroxide-mediated radical polymerization, atom transfer radical polymerization, or reversible addition-fragmentation chain transfer (RAFT) polymerization. For details of these polymerization methods, see "Radical Polymerization Handbook," pp. 161-225 (2010), published by NTS Corporation. RAFT polymerization is particularly preferred because it leaves no residue of heavy metals and is easy to control the molecular weight.

The manufacturing method using the RAFT polymerization method can be roughly divided into two manufacturing steps. The first step is a step of RAFT polymerization of monomer A in the presence of a radical polymerization initiator and a RAFT agent to produce a polymer in which a RAFT agent residue is bonded to the end of polymer A (this is called a macro RAFT agent of polymer A). The second step is a step of RAFT polymerization of monomer B in the presence of the macro RAFT agent produced in the first step and a radical polymerization initiator to produce a block copolymer (diblock copolymer) in which polymer B obtained by polymerization of monomer B is bonded to one end of polymer A. In the present invention, a monomer consisting of one or more imidazolidinone monomers and a vinyl monomer that may be added as necessary corresponds to monomer A or B, and a monomer consisting of one or more (meth)acrylic acid alkyl esters corresponds to monomer B or A.

This diblock copolymer has a RAFT agent residue bonded to the end, which acts as a macro RAFT agent. By using this macro RAFT agent instead of a RAFT agent in the first step, it is possible to produce a triblock copolymer in which the respective chains are linked, for example, polymer A-polymer B-polymer A. By repeating the process in the same manner, it is possible to produce a multiblock copolymer in which any chain is linked.

The degree of polymerization can be controlled by the ratio [M]/[I.] of the molar amount of monomer [M] to the molar amount of radical species generated from the radical polymerization initiator [I.]. It is sufficient to charge an amount of monomer that satisfies the values of x and y of the block copolymer, and to control the monomer conversion rate to satisfy the values of x and y. The amount of RAFT agent used [RAFT] should be the same as or greater than [I.], and generally, [RAFT]/[I.] is adjusted to 1 to 10, preferably 1.5 to 5. The monomer conversion rate can be freely controlled by the type of RAFT agent, the value of [RAFT]/[I.], the presence or absence or type of solvent, the initial concentration of monomer, the viscosity of the solution, the polymerization temperature, the polymerization time, etc.

Examples of radical polymerization initiators used in the first and second steps include inorganic peroxides such as potassium persulfate and ammonium persulfate. When used in combination with amine compounds such as N,N,N',N'-tetramethylethylenediamine and N,N-dimethyl-p-toluidine, which are called polymerization accelerators, rapid polymerization can be achieved at low temperatures. Further, as a radical initiator, organic peroxides such as dilauroyl peroxide, benzoyl peroxide, di-t-butyl peroxide, t-butyl hydroperoxide, and cumene hydroperoxide, as well as 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile), 2,2'-azobisisobutyronitrile, 2,2'-azobis(2-methylbutyronitrile), 1,1'-azobis(cyclohexane-1-carbonitrile), 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, and 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride may be used. Examples of azo compounds include 2,2'-azobis[2-(2-imidazolin-2-yl)propane] disulfate dihydrate, 2,2'-azobis[2-(2-imidazolin-2-yl)propane], 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2,2'-azobis[N-(2-carboxyethyl)-2-methylpropionamidine] n-hydrate, 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], 2,2'-azobis[N-(2-propenyl)-2-methylpropionamide], 2,2'-azobis(N-butyl-2-methylpropionamide), dimethyl 2,2'-azobis(isobutyrate), and 4,4'-azobis(4-cyanopentanoic acid). Among these, azo compounds are preferably used from the viewpoint of suppressing side reactions.

The RAFT agent used in the first step is not particularly limited as long as it can controllably polymerize monomer A and controllably polymerize monomer B using the macro RAFT agent of polymer A, and any known RAFT agent used in the RAFT polymerization of non-conjugated monomers can be used. Dithiocarbamic acid ester compounds, dithiocarboxylic acid ester compounds, trithiocarbonic acid ester compounds, etc. are generally known, and more specifically, 4-[(2-carboxyethylsulfanylthiocarbonyl)sulfanyl-4-cyanopentanoic acid, 2-{[(2-carboxyethyl)sulfanylthiocarbonyl]sulfanyl}propanoic acid, 4-chloro-3,5-dimethylpyrazole-1-dithiocarboxylic acid 2'-cyanobutan-2'-yl, 3,5-dimethylpyrazole-1-dithiocarboxylic acid 2'-cyanobutan-2'-yl, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, 2-cyano-2-[(dodecylsulfanylthio Examples of RAFT agents include 3,5-dimethylpyrazole-1-dithiocarboxylate cyanomethyl, 4-cyano-4-[(thiobenzoyl)sulfanyl]pentanoic acid, S,S-dibenzyl trithiocarbonate, 2-[(dodecylsulfanylthiocarbonyl)sulfanylpropanoic acid, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid methyl, (dodecylsulfanylthiocarbonyl)sulfanylacetonitrile, and 2-methyl-2-[(dodecylsulfanylthiocarbonyl)sulfanyl]propanoic acid, but the selection of the RAFT agent may vary depending on the structure and combination of monomers, and further on the order of polymerization. For the selection of the RAFT agent, for example, Macromolecules, Vol. 45, pp. 5321-5342 (2012), and other known documents may be referred to.

A solvent can be used to facilitate the polymerization reaction in the first and second steps. Examples of solvents that can be used include water, methanol, ethanol, propanol, isopropyl alcohol, butanol, isobutanol, hexanol, 2-methoxyethanol, 2-butoxyethanol, ethylene glycol, pentane, cyclopentane, hexane, cyclohexane, heptane, octane, benzene, toluene, xylene, chlorobenzene, dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, acetone, methyl ethyl ketone, diethyl ether, cyclopentyl methyl ether, tetrahydrofuran, dioxane, 1,2-dimethoxyethane, ethyl acetate, propyl acetate, butyl acetate, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), ammonia, trimethylamine, diethylamine, triethylamine, diisopropylamine, butylamine, dibutylamine, tributylamine, pyridine, picoline, etc., but are not limited thereto. Two or more of these solvents can also be mixed and used. The polymerization reaction can be easily controlled by lowering the oxygen concentration in the reaction vessel to a level that does not inhibit the radical reaction, such as under vacuum or in an inert gas atmosphere. The polymerization reaction usually proceeds smoothly at temperatures in the range of 0°C to 150°C.

A portion of the imidazolidinone monomer (4) can be produced by reacting the corresponding imidazolidinone derivative (5b) with acrylic acid chloride in the presence of base 1, as shown in the following formula:

(In the formula, R ^1b represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a benzyl group, or a group represented by the following general formula (2b):

(wherein p is as defined above, and R ^4b is an alkyl group having 1 to 4 carbon atoms or a 2-methoxyethyl group.) R ² and R ³ are as defined above.)
In this case, a tertiary amine or a pyridine derivative can be used as base 1. Specifically, trimethylamine, triethylamine, tripropylamine, tributylamine, triisobutylamine, diethylmethylamine, ethyldipropylamine, ethyldiisopropylamine, pyridine, α-picoline, β-picoline, γ-picoline, 4-(N,N-dimethylamino)pyridine, quinoline, etc. can be used alone or in combination of two or more. Among these, triethylamine is preferred because of its good yield and low cost. The reaction proceeds smoothly when a solvent is used, and for example, n-pentane, cyclopentane, n-hexane, cyclohexane, n-heptane, n-octane, benzene, toluene, xylene, chlorobenzene, dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, acetone, methyl ethyl ketone, diethyl ether, cyclopentyl methyl ether, tetrahydrofuran, dioxane, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, etc. can be used alone or in combination. The reaction is usually carried out at a temperature of 100° C. or less, and preferably at 50° C. or less in terms of suppressing decomposition of the product.

After the reaction is completed, the imidazolidinone monomer (4) or (4b) can be produced by combining conventional purification methods such as extraction, filtration, column chromatography, recrystallization, reprecipitation, and distillation.

The raw material imidazolidinone derivative (5b) can be easily obtained by adding the corresponding diamine derivative (6b) and urea and heating to about 100 to 200°C.

(In the formula, R ^1b , R ² and R ³ have the same meanings as defined above.)
Also, the imidazolidinone derivative (5c) can be produced by reacting imidazolidin-2-one with 1 equivalent of base 2 as an anionizing agent to abstract the hydrogen on the N atom to form an anion, and then reacting the anion with an alkylating agent represented by X-R ^1c .

(In the formula, R ^1c represents an alkyl group having 1 to 4 carbon atoms, a benzyl group, or an oxyalkyl group represented by general formula (2b). X represents a chlorine atom, a bromine atom, an iodine atom, or a tosyl group.)
In this case, examples of base 2 include sodium hydride, potassium hydride, phenyllithium, lithium diisopropylamide, n-butyllithium, etc. The reaction proceeds smoothly when a solvent is used in the above reaction, and for example, hexane, cyclohexane, toluene, diethyl ether, tetrahydrofuran (THF), 1,4-dioxane, cyclopentyl methyl ether (CPME), N,N-dimethylformamide (DMF), N,N-dimethylacetamide, etc. can be used alone or in combination of two or more kinds.

In the case where R ¹ in the imidazolidinone monomer (4) is a 2-hydroxyethyl group or a 2-acetoxyethyl group, and R ² and R ³ are both hydrogen atoms, the imidazolidinone monomer (4) can be synthesized, for example, by the route shown in the following formula. That is, urea is added to 2-(2-aminoethylamino)ethanol and heated to about 100 to 200°C to obtain 1-(2-hydroxyethyl)imidazolidin-2-one, which is then treated with acetic anhydride to obtain 1-(2-acetoxyethyl)imidazolidin-2-one. By reacting this with acryloyl chloride in the presence of base 1, 1-acryloyl-3-(2-acetoxyethyl)imidazolidin-2-one is obtained. By hydrolyzing this in the presence of base 3, 1-(2-hydroxyethyl)imidazolidin-2-one is obtained.

In this case, as base 3, weak bases such as amine or pyridine derivatives, sodium bicarbonate, and sodium carbonate can be used. Specific examples of amine derivatives include ammonia, butylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, tripropylamine, tributylamine, triisobutylamine, diethylmethylamine, ethyldipropylamine, and ethyldiisopropylamine. Specific examples of pyridine derivatives include pyridine, α-picoline, β-picoline, γ-picoline, 4-(N,N-dimethylamino)pyridine, and quinoline. As base 3, these weak bases can be used alone or in combination of two or more. Among these, ammonia, trimethylamine, triethylamine, sodium bicarbonate, and sodium carbonate are preferred in terms of good yield and low cost. The reaction proceeds smoothly when a solvent is used, and for example, n-pentane, cyclopentane, n-hexane, cyclohexane, n-heptane, n-octane, benzene, toluene, xylene, chlorobenzene, dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, acetone, methyl ethyl ketone, diethyl ether, cyclopentyl methyl ether, tetrahydrofuran, dioxane, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, methanol, ethanol, propanol, isopropyl alcohol, butanol, etc. can be used alone or in combination. The reaction is usually carried out at a temperature of 60°C or less, and is preferably carried out at a temperature of 30°C or less in order to suppress decomposition of the product. After completion of the reaction, 1-acryloyl-3-(2-acetoxyethyl)imidazolidin-2-one and 1-(2-hydroxyethyl)imidazolidin-2-one can be produced by combining ordinary purification methods such as extraction, filtration, column chromatography, recrystallization, and reprecipitation, respectively.

The above-mentioned copolymer can be dissolved in a solvent to prepare a coating agent. The solvent used for the coating agent is not particularly limited, and examples of the solvent that can be used include water, methanol, ethanol, propanol, isopropyl alcohol, butanol, hexanol, pentane, cyclopentane, hexane, cyclohexane, heptane, octane, benzene, toluene, xylene, chlorobenzene, dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, acetone, methyl ethyl ketone, diethyl ether, cyclopentyl methyl ether, tetrahydrofuran, dioxane, acetonitrile, dimethyl sulfoxide, DMF, N,N-dimethylacetamide, N-methylpyrrolidin-2-one, ethylene glycol, 2-methoxyethanol, and 2-butoxyethanol, but is not limited to these. Two or more of these solvents can also be mixed and used.

There are no particular limitations on the concentration of the coating agent as long as it does not interfere with the coating, but by adjusting the concentration of the copolymer to 0.01 to 20% by weight, a suitable cell culture material can be obtained.

The above-mentioned cell culture material can be prepared by treating the surface of carriers of various shapes with a coating agent. In this specification, the term "cell culture equipment" is synonymous with cell culture material. There is no particular limit to the material of the carrier, and examples of the material that can be used include styrene resins such as polystyrene, polyolefin resins such as polypropylene, polyurethane resins, polycarbonate, polyethylene terephthalate, polysulfone resins, fluorine resins, natural polysaccharide polymers such as cellulose, inorganic materials such as glass and ceramics, and metal materials such as stainless steel and titanium. There is also no particular limit to the shape of the carrier, and examples of the shape of general cell culture equipment such as petri dishes, plates, and flasks, spherical, bag-like, sponge-like porous, and fibrous shapes such as nonwoven fabric and woven fabric can be used. There is also no particular limit to the method of treating the coating agent, and known methods can be used, such as dipping, spraying, casting, spin coating, and inkjet printing. Furthermore, annealing may be performed using heat or a solvent. For example, when performing thermal annealing, the treatment may be performed at 40 to 100°C for several minutes to several hours. There is no particular limit to the thickness of the copolymer film as long as it can function as a cell culture material, and it is, for example, 1 nm to 1000 nm.

The cells to be cultured using the cell culture material are not particularly limited as long as they can adhere to the surface of the cell culture substrate before the stimulation by temperature drop is applied. For example, in addition to various cultured cell lines such as bone marrow-derived mesenchymal stem cells of mammals such as humans, adipose tissue-derived mesenchymal stem cells of mammals such as humans, fibroblasts of mammals such as humans, Chinese hamster ovary-derived cells (CHO cells), mouse connective tissue L929 cells, human fetal kidney-derived cells HEK293 cells, and human cervical cancer-derived HeLa cells, for example, epithelial cells and endothelial cells that constitute various tissues and organs in the body, skeletal muscle cells that exhibit contractility, smooth muscle cells, cardiac muscle cells, neuron cells that constitute the nervous system, glial cells, fibroblasts, hepatic parenchymal cells, non-parenchymal liver cells and adipocytes that are involved in the metabolism of the body, and cells that have the ability to differentiate, such as stem cells present in various tissues and cells induced to differentiate from them, can be used. Other examples include cells (living cells) contained in blood, lymph, cerebrospinal fluid, sputum, urine, or feces, as well as microorganisms, viruses, protozoa, etc. present in the body or in the environment.

There are no particular limitations on the medium used for culture. For example, when using mesenchymal stem cells, Dulbecco-Voigt modified Eagle's minimum essential medium (10 vol% FBS/DMEM) containing 10% fetal bovine serum can be used. In addition to serum and basal medium, the medium may contain antibiotics and extracellular matrices such as fibronectin and laminin.

Cells can be cultured on the cell culture material at temperatures above the LCST of the imidazolidinone block chain. Since general cells are sensitive to high temperatures, the temperature is preferably 45°C or lower, and more preferably 37°C to 40°C.

Cells cultured on the cell culture material can be detached from the cultureware and collected by cooling to a temperature below the LCST of the imidazolidinone block chain. There are no particular limitations on the cooling method, and the medium may be replaced with a cooled medium, or the cells may be cooled in a cold place.

The present invention makes it possible to easily control properties such as hydrophilicity, hydrophobicity, and temperature responsiveness, and is expected to find applications as biocompatible materials, cell culture materials, and even temperature responsive polymers.

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

The chemical structure and copolymer composition ratio of the product were determined from the results of ¹ H-NMR measurement using AVANCEIII-400 manufactured by Bruker-Biospin.

The molecular weight of the obtained polymer was determined from the results of SEC. The SEC system used was a GL-7400 manufactured by GL Sciences (detector: GL-7456, four columns: TSKgel Super H5000, H4000 x 2, H2000, column temperature: 40°C, developing solvent: 0.01 M LiCl in DMF, standard polystyrene equivalent).

An optical cell was filled with a 1% by weight aqueous solution (dispersion) of the polymer, placed in a thermostatic chamber, and the transmitted light intensity of 650 nm light was measured using an illuminometer when the temperature was changed. The temperature at which the temperature change in transmitted light intensity was maximum was defined as the lower critical solution temperature (LCST).

Reference Example 1
Synthesis of 1-acryloyl imidazolidin-2-one

A solution of 2.2 mL (27 mmol) of acryloyl chloride in chloroform (20 mL) was added dropwise to a solution of 2.23 g (25.9 mmol) of imidazolidin-2-one and 7.0 mL (50 mmol) of triethylamine in chloroform (60 mL) cooled in an ice bath under an argon atmosphere, and the mixture was stirred for 4 hours while returning the temperature to room temperature. The reaction solution was concentrated under reduced pressure, and THF was added to the residue to remove the precipitate. The filtrate was concentrated and purified by silica gel column chromatography (developing solution: hexane/ethyl acetate=1/2) to obtain 1.18 g (yield: 32.6%) of 1-acryloylmethylimidazolidin-2-one as a colorless solid.
^1H -NMR (400MHz, _CDCl3 , ppm), δ: 3.51-3.55 (2H, m), 4.01-4.05 (2H, m), 5.11 (1H, brs), 5.80 (1H, dd, J = 2.0, 10.4 Hz), 6.49 (1H, dd, J = 2.0, 17.1 Hz), 7.59 (1H, dd, J = 10.4, 17.1 Hz).
Reference Example 2
Synthesis of 1-acryloyl-3-methylimidazolidin-2-one

13.0 mL (149 mmol) of N-methylethylenediamine, 9.01 g (150 mmol) of urea, and 10 mL of ethylene glycol were added and stirred for 8 hours at 130° C. Ethylene glycol was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (developing solution: chloroform/methanol=10/1) to obtain 10.6 g (yield: 71.1%) of 1-methylimidazolidin-2-one as a colorless solid.
^1H -NMR (400MHz, _CDCl3 , ppm), δ: 2.79 (3H, s), 3.41 (2H, t, J=2.6Hz), 3.42 (2H, J=2.6Hz), 5.10 (1H, brs).
A solution of 1.5 mL (18 mmol) of acryloyl chloride in chloroform (12 mL) was added dropwise to a solution of 1.84 g (18.4 mmol) of 1-methylimidazolidin-2-one and 5.1 mL (37 mmol) of triethylamine in chloroform (62 mL) cooled in an ice bath under an argon atmosphere, and the mixture was stirred for 12 hours while returning the temperature to room temperature. The reaction solution was concentrated and purified by silica gel column chromatography (developing solution: hexane/ethyl acetate=2/1) to obtain 1.54 g (yield: 54.3%) of 1-acryloyl-3-methylimidazolidin-2-one as a colorless solid.
^1H -NMR (400MHz, _CDCl3 , ppm), δ: 2.90 (3H, s), 3.45 (2H, t, J = 8.0 Hz), 3.91 (2H, t, J = 8.0 Hz), 5.78 (1H, dd, J = 2.0, 10.5 Hz), 6.48 (1H, dd, J = 2.0, 17.1 Hz) 7.64 (1H, dd, J = 10.5, 17.1 Hz).
Reference Example 3
Synthesis of 1-acryloyl-3-ethylimidazolidin-2-one

26.0 mL (248 mmol) of N-ethylethylenediamine, 14.4 g (240 mmol) of urea, and 18 mL of ethylene glycol were added and stirred for 6 hours at 130° C. Ethylene glycol was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (developing solution: chloroform/methanol=10/1) to obtain 13.2 g (yield: 48%) of 1-ethylimidazolidin-2-one as a colorless solid.
^1H -NMR (400MHz, _CDCl3 , ppm), δ: 1.12 (3H, t, J = 3.3Hz), 3.24 (2H, q, J = 3.3Hz), 3.42 (4H, s), 5.38 (1H, brs).
Under an argon atmosphere, a solution of 7.2 mL (88 mmol) of acryloyl chloride in 60 mL of chloroform was dropped into a solution of 9.14 g (80.1 mmol) of 1-ethylimidazolidin-2-one and 16.7 mL (120 mmol) of triethylamine in chloroform (180 mL), and the mixture was stirred for 4 hours. The reaction solution was concentrated under reduced pressure, and THF was added to the residue to remove the precipitate. The filtrate was concentrated and purified by silica gel column chromatography (developing solution: hexane/ethyl acetate=1/1), to obtain 10.8 g (yield: 79.9%) of 1-acryloyl-3-ethylimidazolidin-2-one as a pale yellow liquid.
^1H -NMR (400MHz, _CDCl3 , ppm), δ: 1.18 (3H, t), 3.36 (2H, q, J = 6.6 Hz), 3.45 (2H, t, J = 8 Hz), 3.91 (2H, t, J = 8 Hz), 5.78 (1H, dd, J = 2.0, 10.5 Hz), 6.47 (1H, dd, J = 2.0, 17.1 Hz), 7.64 (1H, dd, J = 10.5, 17.1 Hz).
Reference Example 4
Synthesis of 1-acryloyl-3-isopropylimidazolidin-2-one

10 mL (81 mmol) of N-isopropylethylenediamine, 4.9 g (81 mmol) of urea, and 5.4 mL of ethylene glycol were added, and the mixture was stirred at 130° C. for 1 hour and then at 180° C. for 4 hours. The ethylene glycol was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (developing solution: chloroform/methanol=10/1) to obtain 7.7 g (yield: 73%) of 1-isopropylimidazolidin-2-one as a colorless solid.
^1H -NMR (400MHz, _CDCl3 , ppm), δ: 1.13 (6H, d, J=6.8Hz), 3.39 (4H, m), 4.14 (1H, sep, J=6.8Hz), 5.27 (1H, brs).
A solution of 7.7 mL (94 mmol) of acryloyl chloride in 60 mL of chloroform was added dropwise to a solution of 10.5 g (82.1 mmol) of 1-isopropylimidazolidin-2-one and 17.0 mL (122 mmol) of triethylamine in chloroform (170 mL) cooled in an ice bath under an argon atmosphere, and the mixture was stirred for 4 hours. The reaction solution was concentrated under reduced pressure, and THF was added to the residue to remove the precipitate. The filtrate was concentrated and purified by silica gel column chromatography (developing solution: hexane/ethyl acetate=1/1) to obtain 12.0 g (yield: 80.0%) of colorless solid 1-acryloyl-3-isopropylimidazolidin-2-one.
^1H -NMR (400MHz, _CDCl3 , ppm), δ: 1.19 (6H, d, J = 6.8Hz), 3.38-3.42 (2H, m), 3.88-3.92 (2H, m), 4.24 (1H, sep, J = 6.8Hz), 5.77 (1H, dd, J = 2.0, 10.5Hz), 6.47 (1H, dd, J = 2.0, 17.1Hz), 7.64 (1H, dd, J = 10.5, 17.1Hz).
Reference Example 5
Synthesis of 1-acrylolyl-3-(2-methoxyethyl)imidazolidin-2-one

10 mL (0.11 mol) of 2-chloroethyl methyl ether was added dropwise to 37 mL (0.55 mol) of ethylenediamine at room temperature, and the mixture was stirred under reflux for 13 hours. Saturated saline and diethyl ether were added to the reaction solution to extract the organic layer. After concentration, the mixture was purified by distillation under reduced pressure to obtain 8.79 g (yield: 67.6%) of N-(2-methoxyethyl)ethylenediamine as a colorless liquid.
¹ H-NMR (400 MHz, CDCl ₃ , ppm), δ: 1.36 (3H, bs), 2.69 (2H, t, J=6.0 Hz), 2.78-2.81 (4H, m), 3.37 (3H, s), 3.50 (2H, t, J=6.0 Hz).
8.79 g (74.4 mmol) of N-(2-methoxyethyl)ethylenediamine, 4.45 g (74.4 mmol) of urea, and 5.0 mL of ethylene glycol were added, and the mixture was stirred at 130° C. for 1 hour and at 180° C. for 4 hours. The ethylene glycol was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (developing solution: chloroform/methanol=1/1) to obtain 10.4 g (yield: 96.5%) of 1-(2-methoxyethyl)imidazolidin-2-one as a colorless solid.
¹ H-NMR (400 MHz, CDCl ₃ , ppm), δ: 3.36-3.43 (7H, m), 3.51-3.57 (4H, m), 4.90 (1H, bs).
A solution of 2.8 mL (34 mmol) of acryloyl chloride in chloroform (23 mL) was added dropwise to a solution of 4.95 g (34.3 mmol) of 1-(2-methoxyethyl)imidazolidin-2-one and 9.5 mL (69 mmol) of triethylamine in chloroform (115 mL) cooled in an ice bath under an argon atmosphere, and the mixture was stirred for 14 hours while returning to room temperature. The reaction solution was poured into a saturated aqueous solution of sodium bicarbonate, extracted with chloroform, and concentrated. The residue was purified by alumina column chromatography (developing solution: hexane/ethyl acetate=3/1), and further purified by recrystallization from a hexane solution to obtain 4.27 g (yield: 62.8%) of 1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one as a colorless solid.
^1H -NMR (400MHz, _CDCl3 , ppm), δ: 3.36 (3H, s), 3.46-3.49 (2H, m), 3.55-3.56 (4H, m), 3.90 (2H, t, J = 8.2 Hz), 5.79 (1H, dd, J = 2.0, 10.4 Hz), 6.48 (1H, dd, J = 2.0, 17.1 Hz), 7.64 (1H, dd, J = 10.4, 17.1 Hz).
Reference Example 6
Synthesis of 1-acryloyl-4,4-dimethylimidazolidin-2-one

Under argon, 8.82 g (100 mmol) of 1,2-diamino-2-methylpropane and 6.06 g (100 mmol) of urea were dissolved in 6 mL of ethylene glycol, and the mixture was stirred at 130° C. for 1 hour, then heated and stirred at 150° C. for 1 hour and at 180° C. for 3 hours. After distilling off the ethylene glycol under reduced pressure, the residue was purified by silica gel column chromatography (chloroform/methanol=10/1) to obtain 9.53 g (83.5 mmol) (yield: 83.4%) of 4,4-dimethylimidazolidin-2-one as a colorless solid. ¹ H-NMR (400 MHz, CDCl ₃ , ppm): δ 1.32 (6H, s), 3.25 (2H, s), 4.90 (1H, brs), 4.96 (1H, brs).
Under argon, 50 mL of a chloroform solution of 6.00 mL (73.6 mmol) of acryloyl chloride was slowly dropped into 50 mL of a chloroform solution of 5.74 g (50.1 mmol) of 4,4-dimethylimidazolidin-2-one and 14.0 mL (100 mmol) of triethylamine in an ice bath, and the mixture was stirred for 4 hours. After the reaction, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate/hexane=1/1) to obtain 1.57 g (9.36 mmol) (yield: 46.7%) of 1-acryloyl-4,4-dimethylimidazolidin-2-one as a colorless solid.
^1H -NMR (400MHz, _CDCl3 , ppm): δ 1.37 (6H,s), 3.73 (2H,s), 5.11 (1H,brs), 5.80 (1H,dd,J=2.0,10.4Hz), 6.49 (1H,dd,J=2.0,17.1Hz), 7.59 (1H,dd,J=10.4,17.1Hz).
Reference Example 7
Synthesis of poly(butyl methacrylate) by RAFT polymerization-1 (PBMA(1))
5.68 g (40.0 mmol) of butyl methacrylate, 55.6 mg (199 μmol) of 4-cyano-4-[(thiobenzoyl)sulfanyl]pentanoic acid, and 13.1 mg (79.7 μmol) of 2,2'-azobis(isobutyronitrile) (AIBN) were dissolved in 2 mL of DMF. This DMF solution was freeze-degassed, sealed, and stirred at 70°C for 18 hours. The reaction solution was diluted with THF and precipitated and purified in an excess amount of methanol to obtain 5.62 g (yield: 98.8%) of a pink solid polybutyl methacrylate (PBMA (1)). As a result of SEC measurement, Mn was 18,700, Mw was 22,900, and PD was 1.23.

Reference Example 8
Synthesis of poly(butyl methacrylate) by RAFT polymerization-2 (PBMA(2))
5.68 g (40.0 mmol) of butyl methacrylate, 111.6 mg (399 μmol) of 4-cyano-4-[(thiobenzoyl)sulfanyl]pentanoic acid, and 13.1 mg (79.8 μmol) of AIBN were dissolved in 2 mL of DMF. This DMF solution was freeze-degassed, sealed, and stirred at 70° C. for 18 hours. The reaction solution was diluted with THF and purified by precipitation in an excess amount of methanol to obtain 5.69 g (yield: 99.9%) of a pink solid polybutyl methacrylate (PBMA (2)). As a result of SEC measurement, Mn was 8,300, Mw was 10,700, and PD was 1.28.

Reference Example 9
Synthesis of poly(butyl methacrylate) by RAFT polymerization-3 (PBMA(3))
5.71 g (40.2 mmol) of butyl methacrylate, 55.6 mg (199 μmol) of 4-cyano-4-[(thiobenzoyl)sulfanyl]pentanoic acid, and 13.1 mg (84.7 μmol) of AIBN were dissolved in 2 mL of DMF. This DMF solution was freeze-degassed, sealed, and stirred at 70° C. for 18 hours. The reaction solution was diluted with THF and purified by precipitation in an excess amount of methanol to obtain 5.12 g (yield: 89.5%) of polybutyl methacrylate (PBMA (3)) as a pink solid. As a result of SEC measurement, Mn was 18,500, Mw was 22,400, and PD was 1.22.

Reference Example 10
Synthesis of poly(benzyl methacrylate) by RAFT polymerization-1 (PBnMA (1))
6.80 mL (40.1 mmol) of benzyl methacrylate, 55.6 mg (199 μmol) of 4-cyano-4-[(thiobenzoyl)sulfanyl]pentanoic acid, and 13.2 mg (80.6 μmol) of AIBN were dissolved in 2 mL of DMF. This DMF solution was freeze-degassed, sealed, and stirred at 70° C. for 18 hours. The reaction solution was diluted with THF and purified by precipitation in an excess amount of methanol to obtain 7.01 g (yield: 99.2%) of pink solid polybenzyl methacrylate (PBnMA (1)). As a result of SEC measurement, Mn was 24,800, Mw was 30,600, and PD was 1.23.

Reference Example 11
Synthesis of poly(benzyl methacrylate) by RAFT polymerization-2 (PBnMA (2))
3.40 mL (20.0 mmol) of benzyl methacrylate, 20.5 mg (73.5 μmol) of 4-cyano-4-[(thiobenzoyl)sulfanyl]pentanoic acid, and 4.95 mg (30.1 μmol) of AIBN were dissolved in 1 mL of DMF. This DMF solution was freeze-degassed, sealed, and stirred at 70° C. for 18 hours. The reaction solution was diluted with THF and purified by precipitation in an excess amount of methanol to obtain 3.44 g (yield: 97.5%) of pink solid polybenzyl methacrylate (PBnMA (2)). As a result of SEC measurement, Mn was 31,000, Mw was 37,500, and PD was 1.20.

Reference Example 12
Synthesis of poly(benzyl methacrylate) by RAFT polymerization-3 (PBnMA (3))
3.40 mL (20.0 mmol) of benzyl methacrylate, 42.0 mg (150 μmol) of 4-cyano-4-[(thiobenzoyl)sulfanyl]pentanoic acid, and 9.53 mg (58.0 μmol) of AIBN were dissolved in 1 mL of DMF. This DMF solution was freeze-degassed, sealed, and stirred at 70° C. for 18 hours. The reaction solution was diluted with THF and purified by precipitation in an excess amount of methanol to obtain 3.70 g (yield: 99.1%) of pink solid polybenzyl methacrylate (PBnMA (3)). As a result of SEC measurement, Mn was 15,600, Mw was 19,200, and PD was 1.23.

Reference Example 13
Synthesis of poly(benzyl methacrylate) by RAFT polymerization-4 (PBnMA(4))
3.40 mL (20.0 mmol) of benzyl methacrylate, 83.5 mg (300 μmol) of 4-cyano-4-[(thiobenzoyl)sulfanyl]pentanoic acid, and 19.3 mg (118 μmol) of AIBN were dissolved in 1 mL of DMF. This DMF solution was freeze-degassed, sealed, and stirred at 70° C. for 18 hours. The reaction solution was diluted with THF and purified by precipitation in an excess amount of methanol to obtain 3.47 g (yield: 98.4%) of pink solid polybenzyl methacrylate (PBnMA (4)). As a result of SEC measurement, Mn was 6,150, Mw was 8,360, and PD was 1.36.

Reference Example 14
Synthesis of poly(butyl methacrylate-ran-benzyl methacrylate) by RAFT polymerization-1 (P(BMA-ran-BnMA) (1))

Butyl methacrylate 3.20 mL (20.1 mmol), benzyl methacrylate 3.4
0 mL (20.0 mol), 56.7 mg (203 μmol) of 4-cyano-4-[(thiobenzoyl)sulfanyl]pentanoic acid, and 13.4 mg (82.8 μmol) of AIBN were dissolved in 2 mL of DMF. This DMF solution was freeze-degassed, sealed, and stirred at 70° C. for 18 hours. The reaction solution was diluted with THF and precipitated and purified in an excess amount of methanol to obtain 6.34 g (yield: 99.4%) of pink solid poly(butyl methacrylate-ran-benzyl methacrylate) (P(BMA-ran-BnMA)(1)). As a result of SEC measurement, Mn was 22,200, Mw was 27,000, and PD was 1.22. From the integral value in the ¹ H-NMR spectrum, p/q=51/49(%).

Reference Example 15
Synthesis of poly(benzyl acrylate) by RAFT polymerization-1 (PBnA(1))
3.10 mL (20.2 mmol) of benzyl acrylate, 27.8 mg (99.5 μmol) of 4-cyano-4-[(thiobenzoyl)sulfanyl]pentanoic acid, and 6.71 mg (40.9 μmol) of AIBN were dissolved in 1 mL of DMF. This DMF solution was freeze-degassed, sealed, and stirred at 70° C. for 18 hours. The reaction solution was diluted with THF and purified by precipitation in an excess amount of methanol to obtain 2.17 g (yield: 66.3%) of pink solid polybenzyl methacrylate (PBnA(1)). As a result of SEC measurement, Mn was 18,900, Mw was 25,400, and PD was 1.34.

Example 1
Synthesis of polybutyl methacrylate-block-poly[(1-acryloyl-3-isopropylimidazolidin-2-one)-ran-(1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one)]-1 (Block copolymer (1))

1.02 g (5.60 mmol) of 1-acryloyl-3-isopropylimidazolidin-2-one synthesized by the method of Reference Example-4, 477 mg (2.41 mmol) of 1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one synthesized by the method of Reference Example-5, 1.14 g (7.99 mmol in terms of monomer units) of PBMA (1) synthesized in Reference Example-7, and 5.02 mg (16.3 μmol) of 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (V-70) were dissolved in 6 mL of DMF. This DMF solution was freeze-degassed, sealed, and stirred at 40 ° C. for 18 hours. The reaction solution was diluted with DMF and purified by precipitation in an excess amount of a mixed solvent of methanol / water (= 1 / 1). The precipitate was collected, dried, and washed with diethyl ether to obtain 2.28 g (yield: 86.6%) of a pale pink block copolymer (1). SEC measurement revealed that Mn was 45,200, Mw was 80,100, and PD was 1.77. The integral values in the ¹ H-NMR spectrum revealed that x/y was 68/32 (%) and m/n was 52/48 (%).

Example 2
Synthesis of polybutyl methacrylate-block-poly[(1-acryloyl-3-isopropylimidazolidin-2-one)-ran-(1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one)]-2 (Block copolymer (2))

1.53 g (8.25 mmol) of 1-acryloyl-3-isopropylimidazolidin-2-one synthesized by the method of Reference Example-4, 714 mg (3.60 mmol) of 1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one synthesized by the method of Reference Example-5, 569 mg (4.19 mmol in terms of monomer units) of PBMA (2) synthesized in Reference Example-8, and 10.5 mg (33.9 μmol) of V-70 were dissolved in 6 mL of DMF. This DMF solution was freeze-degassed, sealed, and stirred at 40° C. for 18 hours. The reaction solution was diluted with DMF, and precipitation purification was performed in an excess amount of a mixed solvent of methanol/water (=1/1). The precipitate was collected and dried, and then washed with diethyl ether to obtain 2.51 g (yield: 89.2%) of a light pink block copolymer (2). As a result of SEC measurement, Mn was 45,200, Mw was 85,100, and PD was 1.88. From the integrated values in the ¹ H-NMR spectrum, x/y was 68/32(%), and m/n was 28/72(%).

Example 3
Synthesis of polybutyl methacrylate-block-poly[(1-acryloyl-3-isopropylimidazolidin-2-one)-ran-(1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one)]-3 (Block copolymer (3))

1.02 g (5.60 mmol) of 1-acryloyl-3-isopropylimidazolidin-2-one synthesized by the method of Reference Example-4, 476 mg (2.40 mmol) of 1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one synthesized by the method of Reference Example-5, 1.14 g (8.00 mmol in terms of monomer units) of PBMA (2) synthesized in Reference Example-8, and 21.5 mg (69.6 μmol) of V-70 were dissolved in 6 mL of DMF. This DMF solution was freeze-degassed, sealed, and stirred at 40° C. for 18 hours. The reaction solution was diluted with DMF, and precipitation purification was performed in an excess amount of a mixed solvent of methanol/water (=1/1). The precipitate was collected, dried, and then washed with diethyl ether to obtain 2.05 g (yield: 77.7%) of a light pink block copolymer (3). As a result of SEC measurement, Mn was 21,500, Mw was 44,500, and PD was 1.53. From the integrated values in the ¹ H-NMR spectrum, x/y was 68/32(%), and m/n was 50/50(%).

Example 4
Synthesis of poly(butyl methacrylate)-block-poly(1-acryloyl-4,4-dimethylimidazolidin-2-one)-1 (Block copolymer (4))

2.02 g (12.0 mmol) of 1-acryloyl-4,4-dimethylimidazolidin-2-one synthesized by the method of Reference Example-6, 568 mg (4.00 mmol in terms of monomer units) of PBMA (1) synthesized by Reference Example-7, and 4.74 mg (15.4 μmol) of V-70 were dissolved in 6 mL of DMF. This DMF solution was freeze-degassed, sealed, and stirred at 40° C. for 18 hours. The reaction solution was diluted with DMF, and precipitation purification was performed in an excess amount of a mixed solvent of methanol/water (=1/1). The precipitate was collected, dried, and washed with diethyl ether to obtain 2.29 g (yield: 88.4%) of a pale pink block copolymer (4). As a result of SEC measurement, Mn was 69,000, Mw was 165,500, and PD was 2.40. The integral value in the ¹ H-NMR spectrum gave m/n=30/70(%).

Example 5
Synthesis of poly(butyl methacrylate)-block-poly(1-acryloyl-4,4-dimethylimidazolidin-2-one)-2 (Block copolymer (5))

2.02 g (12.0 mmol) of 1-acryloyl-4,4-dimethylimidazolidin-2-one synthesized by the method of Reference Example-6, 569 mg (4.00 mmol in terms of monomer units) of PBMA (2) synthesized by Reference Example-8, and 10.7 mg (34.7 μmol) of V-70 were dissolved in 6 mL of DMF. This DMF solution was freeze-degassed, sealed, and stirred at 40° C. for 18 hours. The reaction solution was diluted with DMF, and precipitation purification was performed in an excess amount of a mixed solvent of methanol/water (=1/1). The precipitate was collected, dried, and washed with diethyl ether to obtain 2.21 g (yield: 85.4%) of a pale pink block copolymer (5). As a result of SEC measurement, Mn was 46,100, Mw was 77,500, and PD was 1.68. The integral value in the ¹ H-NMR spectrum gave m/n=30/70(%).

Example 6
Synthesis of polybutyl methacrylate-block-poly[(1-acryloyl-3-isopropylimidazolidin-2-one)-ran-(1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one)]-5 (Block copolymer (6))

1.97 g (10.8 mmol) of 1-acryloyl-3-isopropylimidazolidin-2-one synthesized by the method of Reference Example-4, 238 mg (1.20 mmol) of 1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one synthesized by the method of Reference Example-5, 570 mg (4.01 mmol in terms of monomer units) of PBMA (3) synthesized in Reference Example-9, and 4.60 mg (14.9 μmol) of V-70 were dissolved in 12 mL of DMF. This DMF solution was freeze-degassed, sealed, and stirred at 40° C. for 18 hours. The reaction solution was diluted with DMF, and precipitation purification was performed in an excess amount of a mixed solvent of methanol/water (=1/1). The precipitate was collected, dried, and then washed with diethyl ether to obtain 1.88 g (yield: 67.7%) of a light pink block copolymer (6). As a result of SEC measurement, Mn was 78,300, Mw was 140,700, and PD was 1.80. From the integrated values in the ¹ H-NMR spectrum, x/y was 89/11(%), and m/n was 35/65(%).

Example 7
Synthesis of poly(butyl methacrylate)-block-poly(1-acryloyl-3-isopropylimidazolidin-2-one)-1 (Block copolymer (7))

2.19 g (12.0 mmol) of 1-acryloyl-3-isopropylimidazolidin-2-one synthesized by the method of Reference Example-4, 570 mg (4.01 mmol in terms of monomer units) of PBMA (3) synthesized in Reference Example-9, and 4.91 mg (15.9 μmol) of V-70 were dissolved in 12 mL of DMF. This DMF solution was freeze-degassed, sealed, and stirred at 40° C. for 18 hours. The reaction solution was diluted with DMF, and precipitation purification was performed in an excess amount of a mixed solvent of methanol/water (=1/1). The precipitate was collected, dried, and washed with diethyl ether to obtain 2.07 g (yield: 75.2%) of a pale pink block copolymer (7). As a result of SEC measurement, Mn was 74,300, Mw was 132,200, and PD was 1.78. The integral value in the ¹ H-NMR spectrum gave m/n=34/66(%).

Example 8
Synthesis of polybenzyl methacrylate-block-poly[(1-acryloyl-3-isopropylimidazolidin-2-one)-ran-(1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one)]-1 (Block copolymer (8))

1.53 g (8.40 mmol) of 1-acryloyl-3-isopropylimidazolidin-2-one synthesized by the method of Reference Example-4, 715 mg (3.61 mmol) of 1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one synthesized by the method of Reference Example-5, 704 mg (4.00 mmol in terms of monomer units) of PBnMA (1) synthesized in Reference Example-10, and 4.62 mg (15.0 μmol) of V-70 were dissolved in 16 mL of DMF. This DMF solution was freeze-degassed, sealed, and stirred at 40° C. for 18 hours. The reaction solution was diluted with DMF, and precipitation purification was performed in an excess amount of a mixed solvent of methanol/water (=1/1). The precipitate was collected, dried, and then washed with diethyl ether to obtain 2.11 g (yield: 71.5%) of a light pink block copolymer (8). As a result of SEC measurement, Mn was 72,200, Mw was 187,600, and PD was 2.60. From the integral values in the ¹ H-NMR spectrum, x/y was 69/31(%), and m/n was 33/67(%).

Example 9
Synthesis of polybenzyl methacrylate-block-poly[(1-acryloyl-3-isopropylimidazolidin-2-one)-ran-(1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one)]-2 (Block copolymer (9))

1.53 g (8.40 mmol) of 1-acryloyl-3-isopropylimidazolidin-2-one synthesized by the method of Reference Example-4, 715 mg (3.61 mmol) of 1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one synthesized by the method of Reference Example-5, 704 mg (4.00 mmol in terms of monomer units) of PBnMA (1) synthesized in Reference Example-10, and 4.69 mg (15.2 μmol) of V-70 were dissolved in 16 mL of DMF. This solution was freeze-degassed, sealed, and stirred at 40° C. for 18 hours. The reaction solution was diluted with DMF, and precipitation purification was performed in an excess amount of a mixed solvent of methanol/water (=1/3). The precipitate was collected and dried, and then washed with diethyl ether to obtain 1.99 g (yield: 67.4%) of a light pink block copolymer (9). As a result of SEC measurement, Mn was 79,300, Mw was 153,300, and PD was 1.93. From the integrated values in the ¹ H-NMR spectrum, x/y was 68/32(%), and m/n was 33/67(%).

Example 10
Synthesis of polybenzyl methacrylate-block-poly[(1-acryloyl-3-isopropylimidazolidin-2-one)-ran-(1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one)]-3 (Block copolymer (10))

1.33 g (7.28 mmol) of 1-acryloyl-3-isopropylimidazolidin-2-one synthesized by the method of Reference Example-4, 618 mg (3.12 mmol) of 1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one synthesized by the method of Reference Example-5, 988 mg (5.61 mmol in terms of monomer units) of PBnMA (2) synthesized in Reference Example-11, and 4.51 mg (14.6 μmol) of V-70 were dissolved in 16 mL of 2-methoxyethanol. This solution was freeze-degassed, sealed, and stirred at 40° C. for 18 hours. The reaction solution was diluted with 2-methoxyethanol and precipitated in an excess amount of water for purification. The precipitate was collected, dried, and then washed with diethyl ether to obtain 2.45 g (yield: 83.4%) of a light pink block copolymer (10). As a result of SEC measurement, Mn was 70,900, Mw was 127,300, and PD was 1.80. From the integrated values in the ¹ H-NMR spectrum, x/y was 68/32(%), and m/n was 39/61(%).

Example 11
Synthesis of polybenzyl methacrylate-block-poly[(1-acryloyl-3-isopropylimidazolidin-2-one)-ran-(1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one)]-4 (Block copolymer (11))

1.74 g (9.53 mmol) of 1-acryloyl-3-isopropylimidazolidin-2-one synthesized by the method of Reference Example-4, 809 mg (4.08 mmol) of 1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one synthesized by the method of Reference Example-5, 424 mg (2.41 mmol in terms of monomer units) of PBnMA (3) synthesized in Reference Example-12, and 4.22 mg (13.7 μmol) of V-70 were dissolved in 18 mL of 2-methoxyethanol. This solution was freeze-degassed, sealed, and stirred at 40° C. for 18 hours. The reaction solution was diluted with 2-methoxyethanol and precipitated in an excess amount of water for purification. The precipitate was collected, dried, and then washed with diethyl ether to obtain 1.91 g (yield: 64.3%) of a light pink block copolymer (11). As a result of SEC measurement, Mn was 82,400, Mw was 175,100, and PD was 2.13. From the integrated values in the ¹ H-NMR spectrum, x/y was 67/33(%), and m/n was 21/79(%).

Example 12
Synthesis of polybenzyl methacrylate-block-poly[(1-acryloyl-3-isopropylimidazolidin-2-one)-ran-(1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one)]-5 (Block copolymer (12))

1.89 g (10.4 mmol) of 1-acryloyl-3-isopropylimidazolidin-2-one synthesized by the method of Reference Example-4, 880 mg (4.44 mmol) of 1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one synthesized by the method of Reference Example-5, 212 mg (1.20 mmol in terms of monomer units) of PBnMA (4) synthesized in Reference Example-13, and 5.40 mg (17.5 μmol) of V-70 were dissolved in 20 mL of 2-methoxyethanol. This solution was freeze-degassed, sealed, and stirred at 40° C. for 18 hours. The reaction solution was diluted with 2-methoxyethanol and precipitated in an excess amount of warm water for purification. The precipitate was collected, dried, and then washed with diethyl ether to obtain 1.69 g (yield: 56.7%) of a light pink block copolymer (12). As a result of SEC measurement, Mn was 87,600, Mw was 152,900, and PD was 1.75. From the integrated values in the ¹ H-NMR spectrum, x/y was 68/32(%), and m/n was 11/89(%).

Example 13
Synthesis of polybenzyl methacrylate-block-poly[(1-acryloyl-3-isopropylimidazolidin-2-one)-ran-(1-acryloyl-3-ethylimidazolidin-2-one)] (Block copolymer (13))

1.53 g (8.40 mmol) of 1-acryloyl-3-isopropylimidazolidin-2-one synthesized by the method of Reference Example-4, 613 mg (3.64 mmol) of 1-acryloyl-3-ethylimidazolidin-2-one synthesized by the method of Reference Example-3, 705 mg (4.00 mmol in terms of monomer units) of PBnMA (1) synthesized in Reference Example-10, and 4.41 mg (14.3 μmol) of V-70 were dissolved in 16 mL of 2-methoxyethanol. This 2-methoxyethanol solution was freeze-degassed, sealed, and stirred at 40° C. for 18 hours. The reaction solution was poured into an excess amount of a mixed solvent of methanol/water (=1/3), and the resulting precipitate was collected, dried, and washed with diethyl ether to obtain 2.04 g (yield: 71.7%) of a light pink block copolymer (13). As a result of SEC measurement, Mn was 80,000, Mw was 152,500, and PD was 1.91. From the integrated values in the ¹ H-NMR spectrum, x/y was 68/32(%), and m/n was 33/67(%).

Example 14
Synthesis of poly(butyl methacrylate-ran-benzyl methacrylate)-block-poly[(1-acryloyl-3-isopropylimidazolidin-2-one)-ran-(1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one)]-1 (Block copolymer (14))

1.53 g (8.40 mmol) of 1-acryloyl-3-isopropylimidazolidin-2-one synthesized by the method of Reference Example-4, 715 mg (3.61 mmol) of 1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one synthesized by the method of Reference Example-5, 636 mg (4.00 mmol in terms of monomer units) of P(BMA-ran-BnMA)(1) synthesized in Reference Example-14, and 4.51 mg (14.6 μmol) of V-70 were dissolved in 16 mL of DMF. This DMF solution was freeze-degassed, sealed, and stirred at 40° C. for 18 hours. The reaction solution was diluted with DMF, and precipitation purification was performed in an excess amount of a mixed solvent of methanol/water (=1/1). The precipitate was collected and dried, and then washed with diethyl ether to obtain 2.13 g (yield: 73.8%) of a light pink block copolymer (14). As a result of SEC measurement, Mn was 70,500, Mw was 190,100, and PD was 2.70. The integral values in the ¹ H-NMR spectrum showed x/y=68/32(%), p/q=51/49(%), and m/n=33/67(%).

Example 15
Synthesis of polybenzyl acrylate-block-poly[(1-acryloyl-3-isopropylimidazolidin-2-one)-ran-(1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one)] (block copolymer (15))

1.53 g (8.40 mmol) of 1-acryloyl-3-isopropylimidazolidin-2-one synthesized by the method of Reference Example-4, 715 mg (3.60 mmol) of 1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one synthesized by the method of Reference Example-5, 704 mg (4.00 mmol in terms of monomer units) of PBnA (1) synthesized in Reference Example-15, and 5.37 mg (17.4 μmol) of V-70 were dissolved in 16 mL of 2-methoxyethanol. This 2-methoxyethanol solution was freeze-degassed, sealed, and stirred at 40° C. for 18 hours. The reaction solution was poured into an excess amount of water, and the resulting precipitate was collected and dried, and then washed with diethyl ether to obtain 2.03 g (yield: 70.0%) of a pale pink block copolymer (15). As a result of SEC measurement, Mn was 67,400, Mw was 127,900, and PD was 1.90. From the integrated values in the ¹ H-NMR spectrum, x/y was 68/32(%), and m/n was 31/69(%).

Reference Example 16
Synthesis of poly(butyl methacrylate-ran-benzyl methacrylate (P(BMA-ran-BnMA)(2)) by RAFT polymerization-2

3.20 mL (20.1 mmol) of butyl methacrylate, 3.40 mL (20.0 mmol) of benzyl methacrylate, 28.0 mg (100 μmol) of 4-cyano-4-[(thiobenzoyl)sulfanyl]pentanoic acid, and 6.52 mg (40.2 μmol) of AIBN were dissolved in 4 mL of DMF. This DMF solution was freeze-degassed, sealed, and heated and stirred at 70° C. for 18 hours. The reaction solution was diluted with THF and precipitated and purified in an excess amount of methanol to obtain 6.02 g (yield: 94.2%) of pink solid poly(butyl methacrylate-ran-benzyl methacrylate) (P(BMA-ran-BnMA)(2)). As a result of SEC measurement, Mn was 37,400, Mw was 48,400, and PD was 1.29. The integral value in the ¹ H-NMR spectrum indicated that p/q was 50/50 (%).

Example 16
Synthesis of poly(butyl methacrylate-ran-benzyl methacrylate)-block-poly[(1-acryloyl-3-isopropylimidazolidin-2-one)-ran-(1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one)]-2 (Block copolymer (16))

267 g (1.47 mmol) of 1-acryloyl-3-isopropylimidazolidin-2-one, 125 mg (0.631 mmol) of 1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one, 1.62 g (10.2 mmol in terms of monomer units) of P(BMA-ran-BnMA) (2) synthesized in Reference Example-16, and 7.44 mg (24.1 μmol) of V-70 were dissolved in 12 mL of 2-methoxyethanol. This 2-methoxyethanol solution was freeze-degassed, sealed, and stirred at 40° C. for 18 hours. The reaction solution was added to an excess amount of water, and precipitation purification was performed. The precipitate was collected and dried, and then washed with diethyl ether to obtain 1.77 g (yield: 88.2%) of a light pink block copolymer (16). As a result of SEC measurement, Mn was 44,600, Mw was 62,500, and PD was 1.40. From the integrated values in the ¹ H-NMR spectrum, x/y=75/25(%), p/q=50/50(%), and m/n=84/14(%).

Reference Example 17
Synthesis of poly(butyl methacrylate-ran-benzyl methacrylate (P(BMA-ran-BnMA)(3)) by RAFT polymerization-3

3.20 mL (20.1 mmol) of butyl methacrylate, 3.40 mL (20.0 mmol) of benzyl methacrylate, 13.9 mg (49.6 μmol) of 4-cyano-4-[(thiobenzoyl)sulfanyl]pentanoic acid, and 3.49 mg (21.5 μmol) of AIBN were dissolved in 4 mL of DMF. This DMF solution was freeze-degassed, sealed, and heated and stirred at 70° C. for 18 hours. The reaction solution was diluted with THF and precipitated and purified in an excess amount of methanol to obtain 6.25 g (yield: 97.9%) of pink solid poly(butyl methacrylate-ran-benzyl methacrylate) (P(BMA-ran-BnMA) (3)). As a result of SEC measurement, Mn was 66,100, Mw was 95,200, and PD was 1.44. The integral value in the ¹ H-NMR spectrum indicated that p/q was 50/50 (%).

Example 17
Synthesis of poly(butyl methacrylate-ran-benzyl methacrylate)-block-poly[(1-acryloyl-3-isopropylimidazolidin-2-one)-ran-(1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one)]-3 (Block copolymer (17))

267 g (1.47 mmol) of 1-acryloyl-3-isopropylimidazolidin-2-one, 125 mg (0.631 mmol) of 1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one, 1.62 g (10.2 mmol in terms of monomer units) of P(BMA-ran-BnMA) (3) synthesized in Reference Example-17, and 3.74 mg (12.1 μmol) of V-70 were dissolved in 12 mL of 2-methoxyethanol. This 2-methoxyethanol solution was freeze-degassed, sealed, and stirred at 40° C. for 18 hours. The reaction solution was added to an excess amount of water, and precipitation purification was performed. The precipitate was collected and dried, and then washed with diethyl ether to obtain 1.45 g (yield: 71.9%) of a light pink block copolymer (17). As a result of SEC measurement, Mn was 75,400, Mw was 116,400, and PD was 1.54. From the integral values in the ¹ H-NMR spectrum, x/y=77/23(%), p/q=50/50(%), and m/n=87/13(%).

Reference Example 18
Synthesis of polyethyl methacrylate (PEtMA) by RAFT polymerization 4.57 g (40.0 mmol) of ethyl methacrylate, 44.7 mg (160 μmol) of 4-cyano-4-[(thiobenzoyl)sulfanyl]pentanoic acid, and 10.4 mg (63.1 μmol) of AIBN were dissolved in 2 mL of DMF. This DMF solution was freeze-degassed, sealed, and stirred at 70 ° C for 18 hours. The reaction solution was diluted with THF and precipitated and purified in an excess amount of methanol to obtain 3.69 g (yield: 65.6%) of pink solid polybenzyl methacrylate (PEtMA). As a result of SEC measurement, Mn was 17,300, Mw was 20,600, and PD was 1.19.

Example 18
Synthesis of polyethyl methacrylate-block-poly[(1-acryloyl-3-isopropylimidazolidin-2-one)-ran-(1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one)]-1 (Block copolymer (18))

1.53 g (8.40 mmol) of 1-acryloyl-3-isopropylimidazolidin-2-one, 714 mg (3.60 mmol) of 1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one, 571 mg of PEtMA (5.00 mmol in terms of monomer units) synthesized in Reference Example-18, and 5.72 mg (18.5 μmol) of V-70 were dissolved in 16 mL of 2-methoxyethanol. This solution was freeze-degassed, sealed, and stirred at 40° C. for 18 hours. The reaction solution was added to an excess amount of water, and precipitation purification was performed. The precipitate was collected, dried, and washed with diethyl ether to obtain 1.85 g (yield: 65.6%) of a pale pink block copolymer (18). As a result of SEC measurement, Mn was 65,700, Mw was 109,600, and PD was 1.67. The integral values in the ¹ H-NMR spectrum gave x/y=67/33(%) and m/n=41/59(%).

Reference Example 19
Synthesis of poly(ethyl methacrylate) (P ⁱ BuMA) by RAFT polymerization-1
5.70 g (40.1 mmol) of isobutyl methacrylate, 44.7 mg (160 μmol) of 4-cyano-4-(thiobenzoylthio)pentanoic acid, and 10.4 mg (63.0 μmol) of AIBN were dissolved in 2 mL of DMF. This DMF solution was freeze-degassed, sealed, and stirred at 70° C. for 18 hours. The reaction solution was diluted with THF and purified by precipitation in an excess amount of methanol to obtain 4.46 g (yield: 78.3%) of pink solid polyisobutyl methacrylate (P ⁱ BuMA). As a result of SEC measurement, Mn was 20,100, Mw was 23,400, and PD was 1.16.

Example 19
Synthesis of polyisobutyl methacrylate-block-poly[(1-acryloyl-3-isopropylimidazolidin-2-one)-ran-(1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one)]-1 (Block copolymer (19))

1.53 g (8.40 mmol) of 1-acryloyl-3-isopropylimidazolidin-2-one, 714 mg (3.60 mmol) of 1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one, 570 mg of P ⁱ BuMA (4.01 mmol in terms of monomer units) synthesized in Reference Example-19, and 4.99 mg (16.2 μmol) of V-70 were dissolved in 16 mL of 2-methoxyethanol. This solution was freeze-degassed, sealed, and stirred at 40° C. for 18 hours. The reaction solution was added to an excess amount of water, and precipitation purification was performed. The precipitate was collected, dried, and then washed with diethyl ether to obtain 1.45 g (yield: 51.3%) of a pale pink block copolymer (19). As a result of SEC measurement, Mn was 77,700, Mw was 132,700, and PD was 1.71. The integral values in the ¹ H-NMR spectrum gave x/y=67/33(%) and m/n=33/67(%).

Reference Example 20
Synthesis of polybenzyl methacrylate (PBnMA (5)) by RAFT polymerization-5
6.90 mL (40.7 mmol) of benzyl methacrylate, 56.7 mg (203 μmol) of 4-cyano-4-(thiobenzoylthio)pentanoic acid, and 13.2 mg (80.9 μmol) of AIBN were dissolved in 2 mL of DMF. This DMF solution was freeze-degassed, sealed, and stirred at 70° C. for 18 hours. The reaction solution was diluted with THF and purified by precipitation in an excess amount of methanol to obtain 7.15 g (yield: 99.8%) of pink solid polybenzyl methacrylate (PBnMA). As a result of SEC measurement, Mn was 21,300, Mw was 24,900, and PD was 1.17.

Example 20
Synthesis of polybenzyl methacrylate-block-poly(1-acryloyl-3-isopropylimidazolidin-2-one) (block copolymer (20))-1

2.19 g (12.0 mmol) of 1-acryloyl-3-isopropylimidazolidin-2-one, 706 mg (4.01 mmol in terms of monomer units) of PBnMA (5) synthesized in Reference Example-20, and 4.79 mg (15.5 μmol) of V-70 were dissolved in 16 mL of 2-methoxyethanol. This solution was freeze-degassed, sealed, and stirred at 40° C. for 18 hours. The reaction solution was added to an excess amount of water and subjected to precipitation purification. The precipitate was collected, dried, and then washed with diethyl ether to obtain 2.73 g (yield: 95.8%) of a light pink block copolymer (20). As a result of SEC measurement, Mn was 46,800, Mw was 85,900, and PD was 1.84. From the integrated value in the ¹ H-NMR spectrum, m/n=25/75(%).

Example 21
Synthesis of polybenzyl methacrylate-block-poly[(1-acryloyl-3-isopropylimidazolidin-2-one)-ran-(1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one)]-6 (Block copolymer (21))

1.97 g (10.8 mmol) of 1-acryloyl-3-isopropylimidazolidin-2-one, 240 mg (1.21 mmol) of 1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one, 705 mg (4.00 mmol in terms of monomer units) of PBnMA (5) synthesized in Reference Example-20, and 4.81 mg (15.6 μmol) of V-70 were dissolved in 16 mL of 2-methoxyethanol. This solution was freeze-degassed, sealed, and stirred at 40° C. for 18 hours. The reaction solution was added to an excess amount of water, and precipitation purification was performed. The precipitate was collected, dried, and then washed with diethyl ether to obtain 2.54 g (yield: 87.1%) of a pale pink block copolymer (21). As a result of SEC measurement, Mn was 52,500, Mw was 92,600, and PD was 1.76. The integral values in the ¹ H-NMR spectrum gave x/y=88/12(%) and m/n=26/74(%).

Example 22
(2-4) Synthesis of polybenzyl methacrylate-block-poly[(1-acryloyl-3-isopropylimidazolidin-2-one)-ran-(1-acryloylimidazolidin-2-one)]-1 (Block copolymer (22))

1.53 g (8.41 mmol) of 1-acryloyl-3-isopropylimidazolidin-2-one, 505 mg (3.60 mmol) of 1-acryloyl imidazolidin-2-one, 706 mg of PBnMA (5) (4.01 mmol in terms of monomer units) synthesized in Reference Example-20, and 4.94 mg (16.0 μmol) of V-70 were dissolved in 16 mL of 2-methoxyethanol. This solution was freeze-degassed, sealed, and stirred at 40° C. for 18 hours. The reaction solution was added to an excess amount of water, and precipitation purification was performed. The precipitate was collected, dried, and washed with diethyl ether to obtain 2.33 g (yield: 84.9%) of a pale pink block copolymer (JST-18806). As a result of SEC measurement, Mn was 53,700, Mw was 93,900, and PD was 1.75. The integral values in the ¹ H-NMR spectrum gave x/y=70/30(%) and m/n=28/72(%).

Example 23
(2-5) Synthesis of polybenzyl methacrylate-block-poly[(1-acryloyl-3-isopropylimidazolidin-2-one)-ran-(1-acryloyl-3-methylimidazolidin-2-one)]-1 (Block copolymer (23))

1.97 g (10.8 mmol) of 1-acryloyl-3-isopropylimidazolidin-2-one, 185 mg (1.20 mmol) of 1-acryloyl-3-methylimidazolidin-2-one, 706 mg (4.01 mmol in terms of monomer units) of PBnMA (5) synthesized in Reference Example-20, and 4.95 mg (16.0 μmol) of V-70 were dissolved in 16 mL of 2-methoxyethanol. This solution was freeze-degassed, sealed, and stirred at 40° C. for 18 hours. The reaction solution was added to an excess amount of water, and precipitation purification was performed. The precipitate was collected, dried, and then washed with diethyl ether to obtain 2.62 g (yield: 91.6%) of a pale pink block copolymer (23). As a result of SEC measurement, Mn was 48,500, Mw was 88,600, and PD was 1.83. The integral values in the ¹ H-NMR spectrum gave x/y=89/11(%) and m/n=26/74(%).

Example 24
Synthesis of polybenzyl methacrylate-block-poly[(1-acryloyl-3-ethylimidazolidin-2-one)-ran-(1-acryloyl-3-benzylimidazolidin-2-one)]-1 (Block copolymer (24))

1.92 g (11.4 mmol) of 1-acryloyl-3-ethylimidazolidin-2-one, 138 mg (0.599 mmol) of 1-acryloyl-3-benzylimidazolidin-2-one, 705 mg (4.00 mmol in terms of monomer units) of PBnMA (5) synthesized in Reference Example-20, and 4.92 mg (16.0 μmol) of V-70 were dissolved in 16 mL of 2-methoxyethanol. This solution was freeze-degassed, sealed, and stirred at 40° C. for 18 hours. The reaction solution was added to an excess amount of water, and precipitation purification was performed. The precipitate was collected, dried, and then washed with diethyl ether to obtain 2.15 g (yield: 77.8%) of a light pink block copolymer (24). As a result of SEC measurement, Mn was 56,000, Mw was 100,800, and PD was 1.80. The integral values in the ¹ H-NMR spectrum gave x/y=94/6(%) and m/n=28/72(%).

Reference Example 21
Synthesis of polybutyl methacrylate (PBMA(4)) by RAFT polymerization-4
Butyl methacrylate (6.40 mL, 40.2 mmol), 4-cyano-4-(thiobenzoylthio)pentanoic acid (13.9 mg, 50.0 μmol), and AIBN (3.67 mg, 22.3 μmol) were dissolved in 2 mL of DMF. This DMF solution was freeze-degassed, sealed, and stirred at 70° C. for 18 hours. The reaction solution was diluted with THF and precipitated and purified in an excess amount of methanol to obtain 5.61 g (yield: 98.1%) of pink solid polybutyl methacrylate (PBMA). As a result of SEC measurement, Mn was 64,100, Mw was 86,900, and PD was 1.35.

Example 25
Synthesis of polybutyl methacrylate-block-poly[(1-acryloyl-3-isopropylimidazolidin-2-one)-ran-(1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one)]-6 (Block copolymer (25))

762 mg (4.18 mmol) of 1-acryloyl-3-isopropylimidazolidin-2-one, 92.0 mg (0.464 mmol) of 1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one, 1.71 mg (12.0 mmol in terms of monomer units) of PBMA (4) synthesized in Reference Example-21, and 5.52 mg (17.9 μmol) of V-70 were dissolved in 14 mL of DMF. This solution was freeze-degassed, sealed, and stirred at 40° C. for 15 hours. The reaction solution was added to an excess amount of a mixed solvent of methanol/water (=1/3) and subjected to precipitation purification. The precipitate was collected, dried, and then washed with diethyl ether to obtain 1.78 g (yield: 69.6%) of a light pink block copolymer (25). As a result of SEC measurement, Mn was 76,500, Mw was 131,600, and PD was 1.72. From the integrated values in the ¹ H-NMR spectrum, x/y=89/11(%) and m/n=84/16(%).

Example 26
Synthesis of polybutyl methacrylate-block-poly[(1-acryloyl-3-isopropylimidazolidin-2-one)-ran-(1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one)]-7 (Block copolymer (26))

1.97 g (10.8 mmol) of 1-acryloyl-3-isopropylimidazolidin-2-one, 238 mg (1.20 mmol) of 1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one, 569 mg (4.00 mmol in terms of monomer units) of PBMA (4) synthesized in Reference Example-21, and 1.89 mg (6.13 μmol) of V-70 were dissolved in 20 mL of DMF. This solution was freeze-degassed, sealed, and stirred at 40° C. for 15 hours. The reaction solution was added to an excess amount of a mixed solvent of methanol/water (=1/3), and precipitation purification was performed. The precipitate was collected, dried, and then washed with diethyl ether to obtain 1.12 g (yield: 40.4%) of a pale pink block copolymer (26). As a result of SEC measurement, Mn was 151,200, Mw was 352,600, and PD was 2.33. The integral values in the ¹ H-NMR spectrum gave x/y=88/12(%) and m/n=47/53(%).

Example 27
Synthesis of polybutyl methacrylate-block-poly[(1-acryloyl-3-isopropylimidazolidin-2-one)-ran-(1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one)]-8 (Block copolymer (27))

2.95 g (16.2 mmol) of 1-acryloyl-3-isopropylimidazolidin-2-one, 357 mg (1.80 mmol) of 1-acryloyl-3-(2-methoxyethyl)imidazolidin-2-one, 567 mg (3.99 mmol in terms of monomer units) of PBMA (4) synthesized in Reference Example-21, and 1.99 mg (6.45 μmol) of V-70 were dissolved in 25 mL of DMF. This solution was freeze-degassed, sealed, and stirred at 40° C. for 15 hours. The reaction solution was added to an excess amount of a mixed solvent of methanol/water (=1/7), and precipitation purification was performed. The precipitate was collected, dried, and washed with diethyl ether to obtain 1.37 g (yield: 35.3%) of a pale pink block copolymer (27). As a result of SEC measurement, Mn was 253,900, Mw was 735,300, and PD was 2.90. The integral values in the ¹ H-NMR spectrum gave x/y=88/12(%) and m/n=39/61(%).
(Evaluation of cultureware coated with block copolymer)
Cultureware was prepared using block copolymers (1) to (11), (13) to (15), and (25) to (27) according to the following procedure, and was evaluated. Evaluation of block copolymer (12) was omitted because its structure is similar to that of block copolymer (11). For comparison, a dish not coated with a block copolymer was also prepared as a cultureware.
I. Preparation of cultureware [Preparation of surface treatment agent]
Isopropanol or 2-methoxyethanol was added to the block copolymer, and the mixture was stirred to completely dissolve it, and then filtered through a regenerated cellulose filter (manufactured by Sartorius) with a pore size of 0.2 μm to prepare a surface treatment agent. Note that the concentration of the block polymer was changed depending on the block polymer in order to keep the coating thickness approximately constant.
[Coat to dish 1]
1.0 mL of the surface treatment agent was added to a tissue culture dish (AGC Technoglass, diameter 6 cm) and allowed to stand for 1 minute. Excess surface treatment agent was collected with a Pasteur pipette, and the surface of the dish was then air-dried and thermally annealed at 60° C. for 60 minutes to prepare a culture vessel coated with the block copolymer.
[Coat to dish 2]
100 μL of the surface treatment agent was added to the center of a tissue culture dish (AGC Technoglass, diameter 6 cm), and spin-coated using a spin coater at a rotation speed of 2,000 rpm for a rotation time of 60 seconds to prepare a culture vessel coated with the block copolymer.

In either case, the thickness of the coating was measured at five points from the center to the edge of the culture vessel using a microspectrophotometer (OPTM-A1, manufactured by Otsuka Electronics).
II. Cell culture and detachment treatment [Culture process]
Human bone marrow-derived mesenchymal stem cells (manufactured by Lonza Japan Co., Ltd., Product Code: PT-2501) were seeded on the culture equipment at a cell density of 0.75 x ¹⁰ to 1.0 x ¹⁰ cells/dish and cultured at 37°C and 5% _CO2 concentration. The culture medium used was Dulbecco-Voigt modified Eagle's minimum essential medium (DMEM: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) containing 10 vol% fetal bovine serum (manufactured by BioWest, Colombia), and the culture period was 5 to 8 days.
[Cooling treatment process]
After the incubation step was completed, the culture supernatant was gently removed, and fresh medium cooled to 4° C. was added, followed by cooling at room temperature for 10 minutes or more.
[Cell detachment process]
After the cooling process, the culture medium was pipetted 10 times with a 1 mL pipetter so that the culture medium was applied to the entire culture surface of the culture substrate, and the culture medium was recovered together with the cells. The recovered culture medium was centrifuged at 160 rcf, 25°C, and 5 minutes, after which the supernatant was removed and 500 μL of new medium was added and suspended. 10 μL of the obtained cell suspension was added to a slide for measuring cell count (manufactured by Thermo Fisher Scientific, product name: Countess Cell Counting Chamber Slide), and the cell number was measured using an automatic cell counter (manufactured by Thermo Fisher Scientific, product name: Countess II) (number of cells recovered after cooling). After the culture medium was recovered, the cells remaining in the culture equipment were recovered by treating them with trypsin at 37° C. for 10 minutes, and the number of cells was counted and added to the number of cells recovered after cooling to obtain the total number of cells.

The results are shown in Table 1.

Claims (4)

The following general formula (1)

(wherein R ¹ is a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a benzyl group, or a group represented by the following general formula (2):

(wherein p represents an integer of 2 to 4, and R ⁴ represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a 2-methoxyethyl group, or an acetyl group.) R ² represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. R ³ represents a hydrogen atom or a methyl group.) and a hydrophobic block chain having a repeating unit represented by the following general formula (3):

(wherein R ⁵ represents an alkyl group having 1 to 6 carbon atoms, a cyclohexyl group, a benzyl group, or a phenylethyl group; R ⁶ represents a hydrogen atom or a methyl group; provided that when R ⁶ is a hydrogen atom, R ⁵ is a methyl group, an ethyl group, a cyclohexyl group, or a benzyl group) ,
the copolymer, wherein a ratio of the block chain (m) having a repeating unit represented by the general formula (1) to the hydrophobic block chain (n) having a repeating unit represented by the general formula (3) is m/n=21/79 to m/n=87/13. A coating agent comprising the copolymer according to claim 1 . A cell culture material comprising a substrate coated with the coating agent according to claim 2 . A cell culture method, comprising using the cell culture material according to claim 3 .