JP6779577B2 - Block copolymer of N-vinylimidazolidine-2-one compound and vinyl esters and cell culture material using it - Google Patents

Description

The present invention relates to a block copolymer of an N-vinylimidazolidine-2-one compound and vinyl esters, which is expected to be applied as a material for cell culture or a temperature-responsive polymer material.

In recent years, there has been active development of temperature-responsive materials synthesized using stimulus-responsive materials, particularly N-isopropylacrylamide (NIPAAM). In the case of NIPAAM homopolymers, the lower critical solution temperature (LCST) is 32 ° C. near body temperature, and at lower temperatures the NIPAAM homopolymers are soluble in water, but at higher temperatures they are insoluble in water. It has been known. Copolymers of NIPAAM and various comonomer have been synthesized, and their application to drug release control materials, chromatographic separation gels, cell culture beds, and the like is being investigated. In addition, there are reports that LCST is regulated and the affinity for a specific protein is enhanced by controlling the type and composition of comonomer (Non-Patent Documents 1 to 4).

On the other hand, Patent Documents 1 and 2 describe N-vinylimidazolidine-2-one derivatives and polymers thereof. Specifically, poly (1-vinylimidazolidine-2-one), poly (1-methyl-3-vinylimidazolidine-2-one), poly (1-ethyl-).
Synthesis of 3-vinylimidazolidine-2-one) and poly (1-butyl-3-vinylimidazolidine-2-one), or poly (1-methyl-3-vinylimidazolidine-2-one)
Although a bactericidal composition using (on) is shown, synthesis of a block copolymer of the N-vinylimidazolidine-2-one compound according to the present invention and vinyl esters, and cell culture using the block copolymer. There is no description about materials for materials and temperature responsive materials.

Japanese Unexamined Patent Publication No. 2001-172262.

Japanese Unexamined Patent Publication No. 2001-172333.

Langmuir, Vol. 4, pp. 4657-4662 (1998).

Macromolecules, Vol. 33, pp. 8312-8316 (2000).

Biomacromolecules, Vol. 5, pp. 505-510 (2004).

Langmuir, Vol. 28, pp. 16623-16637 (2012).

Provided is a block copolymer of N-vinylimidazolidine-2-one that can be applied as a material for cell culture.

As a result of diligent studies to solve the above problems, the present inventors have made a poly (N) having a specific structure.
-By using a block copolymer consisting of a (vinyl imidazolidine-2-one compound) chain and a poly (vinyl ester) chain as a coating agent component, the surface of the carrier can be easily treated and treated with a coating agent. The present invention has been completed by discovering that the obtained carrier can be used as a material for cell culture.

That is, the present invention has the following general formula (1).

(In the formula, R ¹ represents an alkyl group or an acyl group having 2 or 3 carbon atoms, and x is 5 to 5000.
Represents an integer of. ) Is the main component of the block, and the following general formula (2)

(In the formula, R ² represents an acyl group having 2 to 18 carbon atoms, and y represents an integer of 5 to 10000.)
It relates to a block copolymer having a block whose main component is a chain represented by (hereinafter, referred to as the block copolymer of the present invention). More reversible additional cleavage chain transfer (RA)
FT) Using the polymerization method, the following general formula (3)

(In the formula, R ¹ represents an alkyl group or an acyl group having 2 or 3 carbon atoms.) N-
Vinyl imidazolidine-2-one compound or the following general formula (4)

(In the formula, R ² represents an acyl group having 2 to 18 carbon atoms.) The step of polymerizing one of the vinyl esters represented by the number of carbon atoms and the step of polymerizing the other are performed alternately or once. The present invention relates to a method for producing a block copolymer characterized by the above. The present invention also relates to a coating agent containing the block copolymer of the present invention and a cell culture material obtained by treating with the coating agent.

The present invention will be described in more detail below. In the N-vinylimidazolidine-2-one compounds of the general formula (1) and the general formula (3), R ¹ represents an alkyl group or an acyl group having 2 to 3 carbon atoms, and an ethyl group, a propyl group, an isopropyl group, Cyclopropyl group, acetyl group,
Examples thereof include a propionyl group. In the vinyl esters of the general formula (2) and the general formula (4), R ² represents an acyl group having 2 to 18 carbon atoms, and an acetyl group, a propionyl group, a butyryl group, an isobutyryl group, a pentanoyl group, a 3-methylbutyryl group, 2,2-Dimethylpropanoyl group, hexanoyl group, heptanoyyl group, cyclohexanecarbonyl group, octanoyl group, nonanoyl group, decanoyyl group, dodecanoyl group, tetradecanoyl group, hexadecanoyl group, octadecanoyl group, benzoyl group, 4 Examples thereof include the -tert-butylbenzoyl group. A propionyl group, a butyryl group, an isobutylyl group, a pentanoyl group, a 3-methylbutyryl group, a 2,2-dimethylpropanoyl, a hexanoyl group, a cyclohexanecarbonyl group, or a benzoyl group is preferable from the viewpoint of excellent coating property. x is 5
An integer of 5000 and y represent an integer of 5 to 10000, but x is excellent in coating property.
Is preferably in the range of 20 to 1000, and y is preferably in the range of 10 to 1000.

The block copolymer of the present invention can be produced by using the RAFT polymerization method. The manufacturing process can be roughly divided into two processes. Monomer A is N-vinylimidazolidine-
It is either one of the 2-one compound (3) or the vinyl esters (4), and the other is monomer B.

In the first step, RAFT polymerization is carried out in the coexistence of monomer A, a radical polymerization initiator and a RAFT agent, and a polymer in which a RAFT agent is bonded to the end of polymer A obtained by polymerizing monomer A (this is macro RAFT of polymer A). It is a process of manufacturing (called an agent).

In the second step, RAFT polymerization is performed in the coexistence of the macro RAFT agent, monomer B, and radical polymerization initiator of the polymer A produced earlier, and the polymer B obtained by polymerizing the monomer B is bonded to one end of the polymer A. This is a step of producing a block copolymer (diblock copolymer).

A RAFT agent is bound to the end of this block copolymer, which acts as a macro RAFT agent, and by using this macro RAFT agent instead of the RAFT agent in the first step, polymer A-polymer B-polymer A and each of them. It is possible to produce a triblock copolymer in which the chains of the above are linked. By repeating the process in the same manner, a multi-block copolymer in which arbitrary chains are connected can be produced.

The molar amount of the monomer [M] and the molar amount of the radical species generated from the radical polymerization initiator [I.
] Ratio [M] / [I ·], the degree of polymerization can be controlled, and it is sufficient to charge more than the amount of monomers satisfying the x and y values of the block copolymer of the present invention, and the conversion rate of the monomers can be changed to x and y. It may be controlled so as to satisfy the value of. The amount [RAFT] used of the RAFT agent may be the same as or more than [I ·], and generally [RAFT] / [I ·] is 1-10, preferably 1.
Adjust so that it is 5 to 5. The conversion rate of the monomer is the type of RAFT agent, [RAFT] / [I
-] Value, the presence or absence or type of solvent, the initial concentration of the monomer, the viscosity of the solution, the polymerization temperature, the polymerization time, etc. can be arbitrarily controlled.

As the radical polymerization initiator used in the first step and the second step, potassium persulfate,
Inorganic peroxides such as ammonium persulfate can be exemplified, and N, N, N'called polymerization accelerators
, N'-Tetramethylethylenediamine, N, N-dimethylparatoluidine and other amine compounds can be used in combination to enable rapid polymerization at low temperatures. In addition, as radical initiators, dilauroyl peroxide, benzoyl peroxide, di-tert-
Organic peroxides such as butyl peroxide, tert-butyl hydroperoxide, cumene hydroperoxide, and 2,2'-azobisisobutyronitrile (AIBN), 2,2.
Azo compounds such as'-azobis (4-methoxy-2,4-dimethylvaleronitrile) (V-70) and azobiscyclohexanecarbonitrile can be exemplified. Above all, an azo compound is preferably used from the viewpoint of suppressing side reactions.

The RAFT agent used in the first step is not particularly limited as long as the monomer A can be controlledly polymerized and the monomer B can be controlledly polymerized using the macro-RAFT agent of the polymer A, and a known RAFT agent used in the RAFT polymerization of non-conjugated monomers. Can be used. Generally, dithiocarbamic acid compounds and dithiocarbonate ester compounds are known, and more specifically, N-methyl-N-phenyldithiocarbamate cyanomethyl, N, N-diphenyldithiocarbamate cyanomethyl, N-methyl-N-4- pyridyl dithiocarbamate cyanomethyl, N, N-diphenyl dithiocarbamate ethoxycarbonylmethyl, can be exemplified such as ethoxy dithiocarboxylate 1-cyanoethyl, the types and combinations of R ¹ and R ^2, selection of the RAFT agent by order of addition polymerization May be different. R
Regarding the selection of AFT agents, for example, Macromolecules, Vol. 45, 5321
It is advisable to refer to publicly known documents such as page -5342 (2012).

A solvent can be used in the first step and the second step to allow the polymerization reaction to proceed smoothly. Available solvents include, for example, water, methanol, ethanol, propanol, isopropyl alcohol, butanol, isobutanol, hexanol, pentane, etc.
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, ethyl acetate , Acetonitrile, dimethyl sulfoxide, N, N-dimethylformamide (DMF), N, N-dimethylacetamide (DMAc), ammonia, trimethylamine, diethylamine, triethylamine,
Diisopropylamine, butylamine, dibutylamine, tributylamine, pyridine,
Picoline and the like can be used, but the present invention is not limited to these. In addition, two or more of these solvents can be mixed and used. In the polymerization reaction, the polymerization can be easily controlled by lowering the oxygen concentration in the reaction vessel to the extent that the radical reaction is not hindered, such as under a vacuum or an inert gas atmosphere. The temperature of the polymerization reaction usually proceeds smoothly within the range of 0 ° C. to 150 ° C.

When the monomer A or the monomer B is RAFT-polymerized in either or both of the first step and the second step, the other copolymerizable with these monomers as long as the properties as a material for cell culture are not impaired. Monomer C can be added, i.e., with monomer A or monomer B as the main component and a small amount of monomer C added to make the chain of one or both blocks a random copolymer block. When the block containing the general formula (1) and / or the general formula (3) as a main component becomes a random copolymerization block, x and / or y shall represent the degree of polymerization of the corresponding random copolymerization block. .. As monomer C, RAF
There is no particular limitation as long as T copolymerization is possible, but the following general formula (2a)

(In the formula, R ^2a represents an acyl group having 2 to 18 carbon atoms, and an acetyl group, a propionyl group, a butyryl group, an isobutyryl group, a pentanoyl group, a 3-methylbutyryl group, a 2,2-dimethylpropanoyl group, a hexanoyl group, Examples thereof include a heptanoid group, a cyclohexanecarbonyl group, an octanoyl group, a nonanoyl group, a decanoyyl group, a dodecanoyl group, a tetradecanoyl group, a hexadecanoyl group, an octadecanoyl group, a benzoyl group, and a 4-tert-butylbenzoyl group.) In addition to the vinyl esters represented by (2a), the following general formula (5)

(In the formula, R ³ represents a hydrogen atom, an alkyl group having 1 to 12 carbon atoms or an acyl group having 1 to 12 carbon atoms. Even if the alkyl group having 1 to 12 carbon atoms has a branched and / or cyclic structure. Often, methyl group, ethyl group, propyl group, isopropyl group, cyclopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group, cyclobutyl group, pentyl group, neopentyl group, isopentyl group, tert-pentyl group. , 1-Methylbutyl group, 1-ethylpropyl group, cyclobutylmethyl group, cyclopentyl group, hexyl group, 1-methylpentyl group, 4-methylpentyl group, 1-ethylbutyl group, 2-ethylbutyl group, cyclohexyl group, heptyl group , 1-Methylhexyl group, 1-ethylpentyl group, cyclohexylmethyl group, cycloheptyl group, octyl group, 1-methylheptyl group, 2-ethylhexyl group, nonyl group, decyl group, 3,7-dimethyloctyl group, undecyl Examples include a group, a dodecyl group, and the like. The acyl group having 1 to 12 carbon atoms may have a branched and / or cyclic structure, and a formyl group, an acetyl group, a propionyl group, a butyryl group, an isobutyryl group, a valeryl group, and an isovaleryl group. N-vinylimidazolidine-2- represented by a group, 2,2-dimethylpropanoyl group, hexanoyl group, heptanoyle group, cyclohexanecarbonyl group, octanoyl group, nonanoyl group, decanoyl group, dodecanoyl group, etc.) On compound, the following general formula (6)

(In the formula, R ⁴ represents an acyl group having 2 to 18 carbon atoms, and an acetyl group, a propionyl group, a butyryl group, an isobutyryl group, a pentanoyl group, a 3-methylbutyryl group, a 2,2-dimethylpropanoyl group, a hexanoyl group, Examples thereof include a heptanoid group, a cyclohexanecarbonyl group, an octanoyl group, a nonanoyl group, a decanoyyl group, a dodecanoyl group, a tetradecanoyl group, a hexadecanoyl group, an octadecanoyl group, a benzoyl group, and a 4-tert-butylbenzoyl group.) Vinylamides represented by, 1-vinylpyrrolidin-2-one, 3-
Examples include vinyl-1,3-oxazoline-2-one, 1-vinylimidazoline and the like.

A coating agent can be prepared by dissolving the block copolymer of the present invention in a solvent. The solvent used for the coating agent is not particularly limited as long as the block copolymer of the present invention can be dissolved, applied to the surface of the carrier, and then the solvent can be removed, but water, methanol, ethanol, propanol, isopropyl alcohol, butanol, etc. 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, 2-butoxyethanol and the like can be used, but the present invention is not limited thereto. In addition, two or more of these solvents can be mixed and used.

The concentration of the coating agent is not particularly limited as long as it does not interfere with the coating, but it is generally preferable to adjust the concentration of the block copolymer of the present invention to 0.01 to 20% by weight.

The surface of a carrier having various shapes such as a petri dish made of various materials such as glass and resin, a bag, a sponge-like porous base material, a granular porous base material, and a fiber base material such as a non-woven fabric or a woven fabric of the present invention A material for cell culture can be prepared by treating with a coating agent containing a block copolymer. The method for treating the coating agent is not particularly limited, and a known method can be used. For example, a dip method, a spray method, a casting method, a spin casting method, an inkjet printing method, or the like can be used.

By treating the surface of carriers of various materials and shapes with a coating agent containing the block copolymer of the present invention, the influence of the surface of these carriers on cells can be reduced, and cell culture, preservation of cultured cells,
Since it can improve the convenience of handling such as collection and transfer of cultured cells, it is expected to be applied as a biocompatible material and a material for cell culture.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto. The molecular weight of the obtained polymer was determined from the GPC results. The GPC system is GL-7400 manufactured by GL Science (detector: GL-7456, column (4): TSK.
Gel Super H5000, H4000 × 2, H2000, column temperature: 40 ° C., developing solvent: 0.01 M LiCl DMF solution, standard polystyrene equivalent) was used. The structure and copolymerization composition ratio of the polymer are AVANCE III-4 manufactured by Bruker-Biospin.
It was determined from the result of ¹ H-NMR measurement using 00.

Reference example 1

10.3 g (120 mmol) of imidazolidine-2-one and 100 mL of propionic anhydride were added to a 200 mL eggplant flask, and the mixture was stirred at 150 ° C. for 1 hour. By allowing the solid to cool to room temperature, recovering the precipitated solid, and washing with a small amount of cold ethanol, the colorless solid 1-
Propionyl imidazolidine-2-one (13.0 g) (yield: 76.2%) was obtained. ¹ H-
NMR (400MHz, CDCl ₃ , ppm), δ: 1.66 (3H, t, J = 7.4H)
z), 2.93 (2H, q, J = 7.4Hz), 3.50 (2H, t, J = 8.1Hz)
, 3.96 (2H, t, J = 8.1Hz), 5.68 (1H, bs). EI-MS, m /
z: 142 (M) ⁺ , 127 (M-CH ₃ ) ⁺ , 113 (MC ₂ H ₅ ) ⁺ , 100. I
R (neat, cm ^-1 ), ν: 3230 (w), 3129 (w), 2983 (w), 2
898 (w), 1734 (m), 1670 (m), 1377 (m), 1265 (m), 1
159 (m), 1062 (m), 1026 (m), 941 (m).

1-Propionyl imidazolidine-2 in a 100 mL Schlenk tube under an argon atmosphere
-On 2.86 g (20.1 mmol), copper (I) iodide 0.19 g (1.0 mmol)
, N, N'-dimethylethylenediamine 0.21 mL (2.0 mmol), potassium carbonate 5.53 g (40.0 mmol) and 1 M vinyl bromide THF solution 40.0 mL (4)
0.0 mmol) was added, and the mixture was stirred at 70 ° C. for 7 hours. The reaction mixture was filtered through Celite to concentrate the filtrate. By purification by alumina column chromatography (developing solution: chloroform), 2.83 g of 1-propionyl-3-vinylimidazolidine-2-one (VIm (C = O) Et) (yield: 83.) as a colorless solid. 6%) was obtained. ^{1 1} H-NMR (400 MHz, CDCl ₃ , ppm), δ: 1.17 (3H, t, J = 7.4 Hz), 2.96 (2H, q, J = 7.4 Hz), 3.57 (2H) , T, J = 8.2Hz), 3.93 (2H, t, J = 8.2Hz), 4.33 (1H, dd, J = 1.1, 15.9Hz), 4.46 (1H, 1H, dd, J = 1.1, 9.0Hz), 6.99 (1H, dd, J = 9.0, 15.9Hz). EI-MS, m / z: 168 (M) ⁺ , 139 (MC ₂ H ₅ ) ⁺ , 111 (M-COC ₂ H ₅ ) ⁺ . IR (neat, cm ^-1 ), ν: 3111 (w), 2979 (w), 1716 (m), 1691 (m), 1633 (m), 1402 (m), 1360 (m), 1360 (m) , 1230 (m), 1049 (m).

Example 1

VIm (C = O) Et (1.68 g; 10.0 mmol), V-70 (12.3 mg; 40.0 μmol) and cyanomethyl N, N-diphenyldithiocarbamate (22.8 mg;) obtained in Reference Example 1. 80.0 μmol) was weighed in a 10 mL eggplant flask equipped with a two-way cock and dissolved in 2.0 mL of DMF. Argon gas was introduced into the DMF solution with a needle, and bubbling was performed for 30 minutes under ice-cooling. The needle was pulled out, sealed, and stirred at 25 ° C. for 24 hours. When the polymerization solution was added dropwise to 200 mL of diethyl ether, 0.906 g of poly (1-propionyl-3-vinylimidazolidine-2-one) (PVIm (C = O) Et-RAFT) was recovered as a colorless powder. Mw: 29100, PD: 1.58, yield: 53.9%.

PVIm (C = O) Et-RAFT (0.408 g; 22.1 μmol), vinyl benzoate (VB, 1.42 g; 9.60 mmol) and AIBN (1.64 mg; 10.0 μmol), two-sided cock. It was taken in the attached 10 mL eggplant flask and dissolved in 1.4 mL of DMAc. After freezing and degassing using an oil diffusion pump, the tube was sealed and stirred at 60 ° C. for 24 hours. When the polymerization solution was added dropwise to 50 mL of methanol, 0.717 g of the target block copolymer PVIm (C = O) Etx-block-PVBy was recovered as a pale yellow powder. Mw: 64000, PD: 2.40, Yield: 39.2%. ^{1 The 1} H-NMR spectrum is shown in FIG. 1 and the GPC elution curve is shown in FIG. Composition from the ratio of the integrated value of the peak around 2.8 ppm attributed to the polymer derived from PVIm (C = O) Et in FIG. 1 and the integrated value of the peak around 5.2 ppm attributed to the polymer derived from VB. When the ratio was calculated, it was x / y = 34/66.

Example 2

Vinyl benzoate (VB, 1.682 g; 11.4 mmol), AIBN (6.24 mg; 38.0 μmol) and cyanomethyl N, N-diphenyldithiocarbamate (54.0 mg; 190 μmol), 10 mL with a two-sided cock. It was weighed in an eggplant flask and dissolved in 3.8 mL of ethyl acetate. After freezing and degassing using an oil diffusion pump, the tube was sealed and stirred at 60 ° C. for 18 hours. When the polymerization solution was added dropwise to 200 mL of hexane, 0.896 g of poly (vinyl benzoate) (PVB-RAFT) was recovered as a colorless powder. Mw: 5400, PD: 1.21, Yield: 51.6%.

PVB-RAFT (67.1 mg; 14.9 μmol), VIm (C = O) Et (0.336 g; 2.00 mmol) and AIBN (66 mg; 40 μmol) were placed in a 5 mL eggplant flask with a two-sided cock. 0.2 mL of ethyl acetate and 0.2 mL of DMF were added and dissolved. After freezing and degassing using an oil diffusion pump, the tube was sealed and stirred at 60 ° C. for 24 hours. When the polymerization solution was added dropwise to 50 mL of diethyl ether, 0.189 g of the target block copolymer PVBy-block-PVIm (C = O) Etx was recovered as a colorless powder. Mw: 48700, PD: 2.01, Yield: 46.9%. ^{1 The 1} H-NMR spectrum is shown in FIG. 3, and the GPC elution curve is shown in FIG. Composition from the ratio of the integrated value of the peak around 2.8 ppm attributed to the polymer derived from VIm (C = O) Et in FIG. 3 and the integrated value of the peak around 5.2 ppm attributed to the polymer derived from VB. When the ratio was calculated, it was x / y = 84/16.

Example 3
(Cover glass coating and temperature responsiveness evaluation)
0.02 g of the block copolymer obtained in Example 1 was dissolved in 2.0 mL of DMF to prepare a 1 wt% DMF solution, which was used as a coating agent. A coating agent was applied to a round cover glass having a diameter of 15 mm manufactured by Matsunami Glass Industry Co., Ltd. by a spin coating method, and the cover glass was coated with a block copolymer. The bubble (3 μL) contact angle (θ) at 37 ° C. and 22 ° C. in water on the surface of the cover glass was measured using a contact angle meter DM300 manufactured by Kyowa Interface Science Co., Ltd. The water contact angles (180 ° −θ) at 37 ° C and 22 ° C were 36.8 ° and 25.8 °, respectively, which effectively acted as a temperature-responsive surface.

(Cell culture evaluation and exfoliation evaluation)
The cover glass was fitted into a 24-well plate manufactured by Corning Inc. and used as a base material for cell culture evaluation. Mouse connective tissue-derived cells (L929 cells) were seeded at a density of 1.9 × 10 ⁴ cells / well. As the medium, Dulbecco Vogt's modified Eagle's minimum essential medium (DMEM medium) containing 10 vol% fetal bovine serum was used, and 1.0 mL was added per well.
Inverted phase-contrast microscope (10 × 10 times) at 37 ° C. and 5% CO ₂ concentration at predetermined time intervals.
It was observed at Olympus IX73). The cell adhesion density reached 480 cells / mm ² in 3 days with respect to the initial cell density of 100 cells / mm ² , and cell proliferation was confirmed. Cultured cells are the base material 1
After culturing until it covered 00%, the substrate was cooled to 15 ° C. and the cells were observed again after 30 minutes. After cooling for 30 minutes, 100% of the cells were detached.

Example 4
(Cover glass coating and temperature responsiveness evaluation)
0.02 g of the block copolymer obtained in Example 2 was dissolved in 2.0 mL of DMF to prepare a 1 wt% DMF solution, which was used as a coating agent. A coating agent was applied to a round cover glass having a diameter of 15 mm manufactured by Matsunami Glass Industry Co., Ltd. by a spin coating method, and coated with a random copolymer. The bubble (3 μL) contact angle (θ) at 37 ° C. and 22 ° C. in water on the surface of the cover glass was measured using a contact angle meter DM300 manufactured by Kyowa Interface Science Co., Ltd. The water contact angles (180 ° -θ) at 37 ° C and 22 ° C were 40.4 ° and 32.8 °, respectively, which effectively acted as a temperature-responsive surface.

(Cell culture evaluation and exfoliation evaluation)
The cover glass was fitted into a 24-well plate manufactured by Corning Inc. and used as a base material for cell culture evaluation. Mouse connective tissue-derived cells (L929 cells) were seeded at a density of 1.9 × 10 ⁴ cells / well. As the medium, Dulbecco Vogt's modified Eagle's minimum essential medium (DMEM medium) containing 10 vol% fetal bovine serum was used, and 1.0 mL was added per well.
Inverted phase-contrast microscope (10 × 10 times) at 37 ° C. and 5% CO ₂ concentration at predetermined time intervals.
It was observed at Olympus IX73). The cell adhesion density reached 550 cells / mm ² in 3 days with respect to the initial cell density of 100 cells / mm ² , and cell proliferation was confirmed. Cultured cells are the base material 1
After culturing to cover 00%, the substrate was cooled to 15 ° C. and the cells were observed again after 30 minutes and 60 minutes. Cooling for 30 minutes gives 60% of cells, cooling for 60 minutes gives cells 8
0% peeled off.

Example 5

Vinyl benzoate (VB, 1.78 g; 12.0 mmol), AIBN (3.3 mg; 20 μmol) and cyanomethyl N, N-diphenyldithiocarbamate (11.4 mg; 40.0 μmol), 10 mL with a two-sided cock. It was weighed in an eggplant flask and dissolved in 4.0 mL of ethyl acetate. Argon gas was introduced into the solution with a needle and bubbled at room temperature for 30 minutes. The needle was pulled out, sealed, and stirred at 80 ° C. for 23 hours. When the polymerization solution was added dropwise to 400 mL of hexane, 0.676 g of poly (vinyl benzoate) (PVB-RAFT) was recovered as a colorless powder. Mw: 25700, PD: 1.52, Yield: 38.0%.

PVB-RAFT (0.396 g; 23.3 μmol), VIm (C = O) Et (0.235 g; 1.40 mmol) and AIBN (0.96 mg; 5.8 μmol) in a 5 mL eggplant with a two-sided cock. It was placed in a flask and 0.35 mL of DMAc was added to dissolve it. After freezing and degassing using an oil diffusion pump, the tube was sealed and stirred at 60 ° C. for 64 hours. When the polymerization solution was added dropwise to 50 mL of diethyl ether, 0.351 g of the target block copolymer PVBy-block-PVIm (C = O) Etx was recovered as a colorless powder. Mw: 40200, PD: 1.71, Yield: 55.6%. ^{1 The 1} H-NMR spectrum is shown in FIG. 5, and the GPC elution curve is shown in FIG. Composition from the ratio of the integrated value of the peak around 1.1 ppm attributed to the polymer derived from VIm (C = O) Et in FIG. 5 and the integrated value of the peak around 5.2 ppm attributed to the polymer derived from VB. When the ratio was calculated, it was x / y = 24/76.

Example 6
(Dish coating and temperature responsiveness evaluation)
0.15 g of the block copolymer obtained in Example 5 was dissolved in 30 g of 2-methoxyethanol to prepare a 0.5 wt% 2-methoxyethanol solution, which was used as a coating agent. 1 mL of the obtained coating agent was added to a dish for tissue culture with a diameter of 6 cm, the lid was closed, and the dish was allowed to stand for 5 minutes, and then the coating agent on the dish was sucked up and removed with a pastur pipette. After allowing to stand in an oven whose internal temperature was set to 70 ° C. for 1 hour, the surface of the dish was washed with distilled water and dried under reduced pressure to coat the surface of the dish with a block copolymer. The bubble (3 μL) contact angle (θ) at 37 ° C. and 22 ° C. in water on the surface of the dish was measured using a contact angle meter DM300 manufactured by Kyowa Interface Science Co., Ltd. The water contact angles (180 ° -θ) at 37 ° C and 15 ° C were 50 ° and 43 °, respectively, which effectively acted as a temperature-responsive surface.

(Cell culture evaluation and exfoliation evaluation)
DS Pharma mouse fibroblasts (3T3-E1) were seeded on the above dish at a cell density of 100 cells / mm ² . As the medium, Eagle's minimum essential medium α-modified type (α-MEM medium) containing 10 vol% fetal bovine serum was used. The cells were cultured at 37 ° C. and a CO ₂ concentration of 5%, and observed with an inverted phase-contrast microscope (Olympus IX73) at 10 × 10 times every predetermined time. The cell adhesion density reached 500 cells / mm ² in 3 days, and cell proliferation was confirmed. After culturing until the cultured cells covered 100% of the substrate, the substrate was cooled to 15 ° C., and the cells were observed again after 30 minutes and 60 minutes. The cells were detached by 40% by cooling for 30 minutes and by 60% by cooling for 60 minutes.

Example 7
(Cell culture evaluation and exfoliation evaluation)
The dish obtained in Example 6 was seeded with mesenchymal stem cells derived from human bone marrow of LONZA at a cell density of 100 cells / mm ² . As the medium, a special medium (MSCGM Bullet Kit) manufactured by LONZA was used. The cells were cultured at 37 ° C. and a CO ₂ concentration of 5%, and observed with an inverted phase-contrast microscope at 10 × 10 times every predetermined time. The cell adhesion density reached 230 cells / mm ² in 3 days, and cell proliferation was confirmed. After culturing until the cultured cells covered 100% of the substrate, the substrate was cooled to 15 ° C., and the cells were observed again after 30 minutes and 60 minutes. The cells were detached by 40% by cooling for 30 minutes and by 60% by cooling for 60 minutes.

Example 8
(Cell culture evaluation and exfoliation evaluation)
The dishes obtained in Example 6 were seeded with 100 human precursor adipocytes (No. CA802s05a) from Toyobo Co., Ltd. at a cell density of 100 cells / mm ² . As the medium, a human precursor adipocyte proliferation medium (No. CA811K500) manufactured by Toyobo Co., Ltd. was used. The cells were cultured at 37 ° C. and a CO ₂ concentration of 5%, and observed with an inverted phase-contrast microscope of 10 × 10 times at predetermined time intervals. The cell adhesion density reached 270 cells / mm ² in 3 days, and cell proliferation was confirmed. After culturing until the cultured cells covered 100% of the substrate, the substrate was cooled to 15 ° C., and the cells were observed again after 30 minutes and 60 minutes. After cooling for 30 minutes, 70% of the cells were detached, and after cooling for 60 minutes, 90% of the cells were detached.

Example 9

VIm (C = O) Et (2.52 g; 15.0 mmol), V-70 (18.5 mg; 60.0 μmol) and cyanomethyl N, N-diphenyldithiocarbamate (34.) obtained by the method of Reference Example 1. 1 mg; 120 μmol) was weighed into a 15 mL eggplant flask with a two-sided cock and dissolved in 3.0 mL of DMF. Argon gas was introduced into the DMF solution with a needle, and bubbling was performed for 30 minutes under ice-cooling. The needle was pulled out, sealed, and stirred at 25 ° C. for 24 hours. When the polymerization solution was added dropwise to 300 mL of diethyl ether, 2.253 g of poly (1-propionyl-3-vinylimidazolidine-2-one) (PVIm (C = O) Et-RAFT) was recovered as a colorless powder. Mw: 26100, PD: 1.36, yield: 89.4%.

PVIm (C = O) Et-RAFT (0.280 g; 14.6 μmol), vinyl hexanoate (HB, 0.498 g; 3.50 mmol) and V-40 (1.8 mg; 7.3 μmol) in two directions. It was taken in a 10 mL eggplant flask with a cock and dissolved in 0.5 mL of DMAc. After freezing and degassing using an oil diffusion pump, the tube was sealed and stirred at 100 ° C. for 24 hours. When the polymerization solution was added dropwise to 50 mL of diethyl ether, 0.282 g of the target block copolymer PVIm (C = O) Etx-block-PHBy was recovered as a pale yellow powder. Mw: 40500, PD: 1.38, yield: 36.2%. ^{1 The 1} H-NMR spectrum is shown in FIG. 7, and the GPC elution curve is shown in FIG. Composition from the ratio of the integrated value of the peak around 1.1 ppm attributed to the polymer derived from VIm (C = O) Et in FIG. 7 and the integrated value of the peak around 0.9 ppm attributed to the polymer derived from HB. When the ratio was calculated, it was x / y = 62/38.

Example 10
(Dish coating and temperature responsiveness evaluation)
The dish was coated in the same manner as in Example 6 (coating on the dish) except that the block copolymer obtained in Example 9 was used, and the surface of the dish was coated with the block copolymer. The bubble (3 μL) contact angle (θ) at 37 ° C. and 22 ° C. in water on the surface of the dish was measured using a contact angle meter DM300 manufactured by Kyowa Interface Science Co., Ltd. The water contact angles (180 ° -θ) at 37 ° C and 22 ° C were 54 ° and 46 °, respectively, which effectively acted as a temperature-responsive surface.

(Cell culture evaluation and exfoliation evaluation)
Evaluation was carried out in the same manner as in Example 6 (cell culture evaluation and exfoliation evaluation) except that the above dish was used. The cell adhesion density reached 490 cells / mm ² in 3 days, and cell proliferation was confirmed.

Example 11
(Cell culture evaluation and exfoliation evaluation)
Evaluation was carried out in the same manner as in Example 7 (cell culture evaluation and exfoliation evaluation) except that the dish prepared in Example 10 was used. The cell adhesion density reached 220 cells / mm ² in 3 days, and cell proliferation was confirmed. After culturing until the cultured cells covered 100% of the substrate, the substrate was cooled to 15 ° C., and the cells were observed again after 30 minutes and 60 minutes. After cooling for 30 minutes, 70% of the cells were detached, and after cooling for 60 minutes, 90% of the cells were detached.
Example 12
(Cell culture evaluation and exfoliation evaluation)
Evaluation was carried out in the same manner as in Example 8 (cell culture evaluation and exfoliation evaluation) except that the dish prepared in Example 10 was used. The cell adhesion density reached 280 cells / mm ² in 3 days, and cell proliferation was confirmed. After culturing until the cultured cells covered 100% of the substrate, the substrate was cooled to 15 ° C., and the cells were observed again after 30 minutes and 60 minutes. After cooling for 30 minutes, 70% of the cells were detached, and after cooling for 60 minutes, 90% of the cells were detached.

Reference example 2

5.18 g (60.2 mmol) of imidazolidine-2-one and 50 mL of acetic anhydride were added to a 100 mL eggplant flask, and the mixture was stirred at an oil bath temperature of 150 ° C. for 1 hour. After allowing to cool to room temperature, the precipitated solid was collected by filtration and washed with a small amount of cold ethanol to obtain 4.94 g (yield: 64.1%) of 1-acetylimidazolidine-2-one as a colorless solid. It was. ^{1 1} H-NMR (400 MHz, CDCl ₃ , ppm), δ: 2.50 (3H, s), 3.50 (2H, t, J = 8.1 Hz), 3.95 (2H, t, J = 8) .1Hz), 5.63 (1H, bs). EI-MS, m / z: 128 (M) ⁺ , 113 (M-CH ₃ ) ⁺ , 100 (M-CO) ⁺ . IR (neat, cm ^-1 ), ν: 3249 (w), 3149 (w), 1741 (m), 1643 (m), 1387 (m), 1340 (m), 1259 (m), 1167 (m) , 1072 (m), 1038 (m), 949 (m).

1.29 g (10.1 mmol) of 1-acetylimidazolidine-2-one, 98 mg (0.52 mmol) of copper (I) iodide, 0.11 mL of N, N'-dimethylethylenediamine in a 50 mL Schlenk tube under an argon atmosphere. (1.0 mmol), 2.76 g (20.0 mmol) of potassium carbonate and 20.0 mL (20.0 mmol) of 1 M solution of vinyl bromide in THF were added, and the mixture was stirred at 80 ° C. for 9 hours. The reaction mixture was filtered through Celite to concentrate the filtrate. Purification by alumina column chromatography (developing solution: chloroform) gave 1.46 g (yield: 94.4%) of 1-acetyl-3-vinylimidazolidine-2-one (VImAc) as a colorless liquid. .. ^{1 1} H-NMR (400 MHz, CDCl ₃ , ppm), δ: 2.54 (3H, s), 3.57 (2H, t, J = 8.2 Hz), 3.93 (2H, t, J = 8) .2Hz), 4.34 (1H, dd, J = 1.2, 15.8Hz), 4.48 (1H, dd, J = 1.2, 9.0Hz), 6.99 (1H, dd, J = 9.0, 15.8 Hz). EI-MS, m / z: 154 (M) ⁺ , 111 (M-CHO) ⁺ . IR (naet, cm ^-1 ), ν: 2966 (w), 2910 (w), 1722 (m), 1678 (m), 1630 (m), 1475 (m), 1429 (m), 1360 (m) , 1302 (m), 1257 (m), 1101 (m), 1036 (m).
Example 13

VImAc (308 mg; 2.00 mmol), AIBN (1.3 mg; 7.9 μmol) and cyanomethyl N, N-diphenyldithiocarbamate (4.6 mg; 16 μmol) obtained by the method of Reference Example 2 were added to a two-sided flask. It was weighed in a attached 10 mL eggplant flask and dissolved in 0.4 mL of DMAc. After freezing and degassing using an oil diffusion pump, the tube was sealed and stirred at 60 ° C. for 24 hours. When the polymerization solution was added dropwise to 40 mL of diethyl ether, 0.165 g of poly (1-acetyl-3-vinylimidazolidine-2-one) (PVImAc-RAFT) was recovered as a colorless powder. Mw: 19600, PD: 1.32, Yield: 53.6%.

PVImAc-RAFT (0.155 g; 11.7 μmol), vinyl benzoate (VB, 0.830 g; 5.60 mmol) and AIBN (1.0 mg; 5.8 μmol) in a 10 mL eggplant flask with a two-sided cock. It was taken and dissolved in 0.8 mL of DMAc. After freezing and degassing using an oil diffusion pump, the tube was sealed and stirred at 60 ° C. for 36 hours. When the polymerization solution was added dropwise to 80 mL of diethyl ether, 0.357 g of the target block copolymer PVImAcx-block-PVBy was recovered as a colorless powder. Mw: 40300, PD: 1.63, Yield: 36.2%. ^{1 The 1} H-NMR spectrum is shown in FIG. 9, and the GPC elution curve is shown in FIG. The composition ratio was calculated from the ratio of the integrated value of the peak around 3.6 ppm attributed to the polymer derived from VImAc in FIG. 9 and the integrated value of the peak around 5.2 ppm attributed to the polymer derived from VB. / Y = 34/66.

Example 14
(Dish coating and temperature responsiveness evaluation)
The dish was coated in the same manner as in Example 6 (coating on the dish) except that the block copolymer obtained in Example 9 was used, and the surface of the dish was coated with the block copolymer. The bubble (3 μL) contact angle (θ) at 37 ° C. and 22 ° C. in water on the surface of the dish was measured using a contact angle meter DM300 manufactured by Kyowa Interface Science Co., Ltd. The water contact angles (180 ° -θ) at 37 ° C and 15 ° C were 34 ° and 23 °, respectively, which effectively acted as a temperature-responsive surface.

Example 15

VImAc (0.770 g; 5.00 mmol), V-70 (6.2 mg; 20.0 μmol) and cyanomethyl N, N-diphenyldithiocarbamate (11.4 mg; 40.0 μmol) obtained by the method of Reference Example 2 Was weighed in a 10 mL eggplant flask equipped with a two-way cock and dissolved in 1.0 mL of DMF. After freezing and degassing using an oil diffusion pump, the tube was sealed and stirred at 25 ° C. for 22 hours. When the polymerization solution was added dropwise to 100 mL of diethyl ether, 0.558 g of poly (1-acetyl-3-vinylimidazolidine-2-one) (PVImAc-RAFT) was recovered as a colorless powder. Mw: 17800, PD: 1.19, yield: 72.5%.

PVImAc-RAFT (0.300 g; 20.0 μmol), vinyl benzoate (VB, 0.356 g; 2.40 mmol) and AIBN (1.6 mg; 10 μmol) were placed in a 10 mL eggplant flask with a two-sided cock. It was dissolved in 0.48 mL of DMAc. After freezing and degassing using an oil diffusion pump, the tube was sealed and stirred at 60 ° C. for 24 hours. When the polymerization solution was added dropwise to 50 mL of diethyl ether, 0.344 g of the target block copolymer PVImAcx-block-PVBy was recovered as a colorless powder. Mw: 26600, PD: 1.39, Yield: 52.4%. ^{1 The 1} H-NMR spectrum is shown in FIG. 11 and the GPC elution curve is shown in FIG. The composition ratio was calculated from the ratio of the integrated value of the peak around 3.6 ppm attributed to the polymer derived from VImAc in FIG. 11 and the integrated value of the peak around 5.2 ppm attributed to the polymer derived from VB. / Y = 67/33.

Example 16
(Dish coating and temperature responsiveness evaluation)
The dish was coated in the same manner as in Example 6 (coating on the dish) except that the block copolymer obtained in Example 11 was used, and the surface of the dish was coated with the block copolymer. The bubble (3 μL) contact angle (θ) at 37 ° C. and 22 ° C. in water on the surface of the dish was measured using a contact angle meter DM300 manufactured by Kyowa Interface Science Co., Ltd. The water contact angles (180 ° -θ) at 37 ° C and 15 ° C were 36 ° and 29 °, respectively, which effectively acted as a temperature-responsive surface.

The block copolymer of the present invention reduces the influence of these carrier surfaces on cells by treating the surface of carriers of various materials and shapes as a coating agent, and cultures cells, preserves cultured cells, and cultured cells. Biocompatible materials, because they can improve the convenience of handling such as collection and transfer of cells.
It is expected to be applied as a material for cell culture.

^{1 1} H-NMR spectrum chart (CDCl ₃ solution) of PVIm (C = O) Etx-block-PVBy obtained in Example 1. GPC chart of PVIm (C = O) Etx-block-PVBy (solid line) and PVIm (C = O) Et-RAFT (dashed line) obtained in Example 1. ¹ H-NMR spectrum chart (CDCl ₃ solution) of PVBy-block-PVIm (C = O) Etx obtained in Example 2. GPC chart of PVBy-block-PVIm (C = O) Etx (solid line) and PVB-RAFT (dashed line) obtained in Example 2. ¹ H-NMR spectrum chart (CDCl ₃ solution) of PVBy-block-PVIm (C = O) Etx obtained in Example 5. GPC chart of PVBy-block-PVIm (C = O) Etx (solid line) and PVB-RAFT (dashed line) obtained in Example 5. ¹ H-NMR spectrum chart (CDCl ₃ solution) of PVIm (C = O) Etx-block-PHBy obtained in Example 9. GPC chart of PVIm (C = O) Etx-block-PHBy (solid line) and PVIm (C = O) Et-RAFT (dashed line) obtained in Example 9. ^{1 1} H-NMR spectrum chart of PVImAcx-block-PVBy obtained in Example 13 (CDCl ₃ solution). GPC chart of PVImAcx-block-PVBy (solid line) and PPVImAc-RAFT (dashed line) obtained in Example 13. ^{1 1} H-NMR spectrum chart of PVImAcx-block-PVBy obtained in Example 15 (CDCl ₃ solution). GPC chart of PVImAcx-block-PVBy (solid line) and PPVImAc-RAFT (dashed line) obtained in Example 15.

Claims (7)

The following general formula (1)

(In the formula, R ¹ represents an alkyl group or an acyl group having 2 or 3 carbon atoms, and x represents an integer of 10 to 5000. ) A block whose main component is a chain represented by a chain, and the following general formula (2). )

(In the formula, R ² represents an acyl group having 3 to 7 carbon atoms, and y represents an integer of 10 to 1000. )
A block copolymer having a block whose main component is a chain represented by. The block copolymer according to claim 1, wherein the block copolymer comprises a chain represented by one general formula (1) and a chain represented by one general formula (2). Polymer. Using the reversible addition cleavage chain transfer polymerization method, the following general formula (3)

(In the formula, R ¹ represents an alkyl group or an acyl group having 2 or 3 carbon atoms.) An N-vinylimidazolidine-2-one compound represented by or the following general formula (4).

(In the formula, R ² represents an acyl group having 3 to 7 carbon atoms. ) It is characterized in that the step of polymerizing one of the vinyl esters represented by () and the step of polymerizing the other are alternately performed. The method for producing a block copolymer according to claim 1. Using the reversible addition cleavage chain transfer polymerization method, the following general formula (3)

(In the formula, R ¹ represents an alkyl group or an acyl group having 2 or 3 carbon atoms.) An N-vinylimidazolidine-2-one compound represented by or the following general formula (4).

(In the formula, R ² represents an acyl group having 3 to 7 carbon atoms. ) It is characterized in that the step of polymerizing one of the vinyl esters represented by () and the step of polymerizing the other are performed once. The method for producing a block copolymer according to claim 2. Claims characterized by the use of cyanomethyl N-methyl-N-phenyldithiocarbamate, cyanomethyl N, N-diphenyldithiocarbamate or cyanomethyl N-methyl-N- (4-pyridyl) dithiocarbamate as the reversible addition cleavage chain transfer agent. Item 3. The method for producing a block copolymer according to Item 3. A coating agent containing the block copolymer according to claim 1 or 2. A cell culture material obtained by treating with the coating agent according to claim 6.

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