JP7232442B2 - Substrate for cell culture that reversibly changes surface phase separation structure in response to light - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Law 1. Name of publisher: The Society of Polymer Science, Japan Publication name: “The Society of Polymer Science and Technology Proceedings, Vol. 67, No. 1 [2018]” Publication date: May 8, 2018 2. Meeting name: The 67th (2018) SPSJ Annual Meeting Announcement date: May 24, 2018 3. Publisher name: The Chemical Society of Japan Publication name: “The Chemical Society of Japan Autumn Program 8th CSJ Chemistry Festa 2018 Program Collection” Publication date: September 26, 2018 4. Name of Meeting: The 8th CSJ Chemistry Festa 2018 Autumn Event of the Chemical Society of Japan Presentation Date: October 24, 2018

TECHNICAL FIELD The present invention relates to a block copolymer that reversibly changes its surface phase separation structure in response to light, a method for producing the same, a cell culture substrate using the block copolymer, and the like.

Humoral signaling factors such as growth factors and differentiation-inducing factors have been used to control cell behavior in in vitro cell culture. On the other hand, it was revealed that the microstructure, wettability, and material properties such as elastic modulus of the cell culture substrate surface lead to changes in the microenvironment of the cell scaffold structure, affecting cell behavior (non-patent literature). 1, 2). In recent years, research that attempts to control cell behavior using only scaffolding materials has attracted a great deal of attention. allows control of cell behavior through control of material properties by external stimuli. Furthermore, since the in vivo environment changes from moment to moment, it is possible to apply it to new culture forms by imitating it.

Among external stimuli, light-based external stimuli can provide local, immediate, and non-contact stimuli. Photoresponsive substrates that control cell attachment and detachment using this property have also been reported, but since they induce surface state changes due to photodegradation reactions, their decomposition products are toxic to cells. and unexpected side effects (Non-Patent Document 3).

Among them, a technology has already been developed to control the adhesiveness of cells by changing the wettability and molecular mobility of the culture substrate surface using an external stimulus such as a change in temperature. There is a famous material as a material. Poly(N-isopropylacrylamide) (PIPAAm), a well-known thermoresponsive polymer, has a lower critical solution temperature (LCST) of approximately 32°C in water. On the other hand, at temperatures below the LCST, it becomes hydrophilic and the polymer chains hydrate. Utilizing the dynamic wettability change at this time and the change in molecular mobility due to hydration of polymer chains, a cell attachment/detachment and cell separation control system was constructed (Non-Patent Document 4).

However, fine manipulations such as patterning are difficult for an external stimulus such as a temperature change due to the range selectivity of the change and the time required for a continuous parameter change such as temperature.

Nagase K., et al., J. Mater. Chem. B, 2017, 5, 5924-5930 Engler, A. J. et al., Cell 2006, 126, 677-689. Nakanishi, J. et al., J. Am. Chem. Soc. 2004, 126, 16314-16315. Nagase K., et al., J. Mater. Chem., 2012, 22, 19514-19522.

An object of the present invention is to prepare a polymer material having a substituent group that responds to light and causes reversible photoisomerization, and to provide a photoresponsive cell culture material and a method for producing the same. .

The present inventors have conducted intensive research and synthesized a diblock copolymer composed of two types of methacrylates as a photoresponsive cell culture material. A monomer based on spirobenzopyran, one of the photochromic molecules that responds to photochromic reactions, was introduced into the diblock copolymer. Then, for the purpose of creating a fine structure on the surface of the material, we used the phenomenon of phase separation, which spontaneously forms a fine structure including micro- and nano-scales by self-aggregation due to the difference in physical properties of each block of the diblock copolymer. . For this purpose, a culture substrate having a microstructure on the surface was prepared by coating the photoresponsive diblock copolymer on a substrate that was hydrophobically modified by laminating an organic layer on the surface, and the present invention was completed. let me

ただし、
環Aが、C₅～C₈の芳香環であり、
R¹が、C₁～C₁₈の置換されていてもよい直鎖又は分枝状のアルキル基であり、
R²が、H又はC₁～C₁₇の置換されていてもよい直鎖又は分枝状のアルキル基であり、
R¹の炭素数 ＞ R²の炭素数であり、
R³、R⁴及びR⁵が、それぞれ独立して、H又は-CH₃から選択され、
Xaが、O又はSから選択され、
Yaが、-NO₂、-CN、-COOH、-F、-Cl、-Br、-I、-OH、-NH₂、-CH₃から選択され、及び、
nが、5～200であり、
xが、5～200であり、
yが、5～50である。 Specifically, the present invention provides a block copolymer characterized by being represented by the following formula (I):

however,
Ring A is a C ₅ to C ₈ aromatic ring,
R ¹ is a C ₁ to C ₁₈ optionally substituted linear or branched alkyl group,
R ² is H or an optionally substituted linear or branched C ₁ to C ₁₇ alkyl group;
The number of carbon atoms in R ¹ > the number of carbon atoms in R ² and
^R3 , ^R4 and ^R5 are each independently selected from H or _-CH3 ;
Xa is selected from O or S;
Ya is selected from _-NO2 , -CN, -COOH, -F, -Cl, -Br, -I, -OH, _-NH2 , _-CH3 , and
n is 5 to 200,
x is between 5 and 200;
y is 5-50.

In the formula (I) of the block copolymer of the present invention,
n is from 100 to 180,
x is between 60 and 140;
y may be 9-30.

ただし、
nが、133～146であり、
xが、74～115であり、
yが、11～25である。 In the block copolymer of the present invention, the compound of formula (I) may be represented by the following formula (II):

however,
n is from 133 to 146,
x is between 74 and 115;
y is 11-25.

The present invention also provides a phase-separated structure system in which the compound of formula (I) or (II) is coated on an organic layer surface-fixed to the surface of a substrate made of glass or silicon.

In the phase-separated structure system of the present invention, the surface-immobilized organic layer may be an organic layer having an organic compound immobilized on the surface by a silicon compound reagent or an organic layer containing a random copolymer.

In the phase separation structure system of the present invention, the silicon compound reagent is trimethoxy(methyl)silane or 1,1,1,3,3,3-hexamethyldisilazane, 1,3-dibutyl-1,1,3 ,3-tetramethyldisilazane or 1,3-diethyl-1,1,3,3-tetramethyldisilazane. .

ただし、
R⁶が、-COOH又は-CH₂OHであり、
R⁷が、-C₆H₅又は-S-(CH₂)₁₁-CH₃であり、
pが、20～200であり、
qが、20～200である。 In the phase-separated structure system of the present invention, the random copolymer may have the structure of formula (III) below:

however,
^R6 is -COOH or _-CH2OH ,
^R7 is _-C6H5 or _-S- ( _CH2 ) ₁₁ - _CH3 ;
p is between 20 and 200;
q is between 20 and 200;

In the formula (III) of the phase separation structure system of the present invention,
p is between 55 and 60;
q is between 54 and 59;
Sometimes.

In the phase-separated structure system of the present invention, the phase-separated structure system may have a phase-separated structure in which the reversible surface phase-separated structure changes in response to light.

In the phase-separated structure system of the present invention, the change in the surface phase-separated structure may be accompanied by a change in the fine uneven structure of the surface, the wettability of the film surface, or the hardness.

In the phase-separated structure system of the present invention, the phase-separated structure system may be a substrate for cell culture.

In the phase separation structure system of the present invention, the substrate for cell culture reversibly adheres and detaches cultured cells or detaches and re-adheres adhered cultured cells in response to light. or regulate the differentiation, spreading, migration, or changes in gene and protein expression of cultured cells.

ただし、nが、5～200であり；

次に、
下記反応式２で表されるように、4,4′-アゾビス(4-シアノペンタン酸)を用いて、前記PMMAと、SpMA （1'-(2-Methacryloyloxyethyl)-3',3'-dimethyl-6-nitrospiro(2H-1-benzopyran-2,2'-indoline)）と、n－ブチルメタクリレート（n-Butyl methacrylate） とを反応させる工程、
を含む製造方法を提供する；

ただし、
nが、5～200であり、
xが、5～200であり、
yが、5～50である。
Furthermore, the present invention provides a method for producing the block copolymer: PMMA-bP (nBMA-stat-SpMA),
As shown in Reaction Formula 1 below, 4,4′-azobis(4-cyanopentanoic acid) is used as a polymerization initiator, and 4,4′-azobis(4-cyanopentanoic acid) is used as a RAFT polymerization chain transfer agent. A step of reacting methylmethacrylate with 4-cyanopentanoic acid dithiobenzoate to prepare PMMA (poly-methylmethacrylate);

provided that n is 5 to 200;

next,
As represented by the following Reaction Scheme 2, 4,4′-azobis(4-cyanopentanoic acid) was used to react the PMMA with SpMA (1′-(2-Methacryloyloxyethyl)-3′,3′-dimethyl). -6-nitrospiro(2H-1-benzopyran-2,2'-indoline)) and n-butyl methacrylate reacting,
to provide a manufacturing method comprising;

however,
n is 5 to 200,
x is between 5 and 200;
y is 5-50.

The invention also provides the use of said block copolymers for the production of phase-separated structural systems.

Furthermore, the present invention reversibly changes the surface phase separation structure in response to light, and the change causes the cultured cells to adhere and detach, or the adhered cultured cells to detach and re-adhere. Alternatively, the use of said separate structure system for controlling differentiation, spreading, migration of cultured cells or changes in gene and protein expression is provided.

INDUSTRIAL APPLICABILITY According to the present invention, a polymer material having a substituent that responds to light and undergoes reversible photoisomerization can be produced, and a photoresponsive cell culture material and a method for producing the same can be provided.

¹ H-NMR spectrum of block copolymer: PMMA-bP (nBMA-stat-SpMA). ¹ H-NMR spectrum of random copolymer: P(MMA-r-nBMA). The figure which shows the image of the phase-separated surface structure by an atomic force microscope, and the change of the phase-separated structure by UV light. Scale bar represents 5 μm. Fig. 3 is an atomic force microscope image of the phase-separated surface structure in reversible switching of the phase-separated structure by repeated irradiation of UV light and visible light. Scale bar represents 5 μm. Investigation of changes in the surface relief structure of the phase-separated surface structure of MMA : nBMA : SpMA = 146 : 115 : 21 and 133 : 115 : 25 polymer-modified substrates by irradiation with UV light or visible light. FIG. 2 shows a surface and a cross-section of a phase-separated structure system; Surface and cross section of a phase-separated structure system on a polymer-modified substrate of MMA : nBMA : SpMA = 127 : 118 : 28 when UV light is irradiated to cause a change in the surface relief structure of the phase-separated surface structure The figure showing the change of the height difference of uneven|corrugated structure. Micrographs of cells cultured on a cell culture substrate having a phase-separated structure surface capable of photoswitching. Scale bars represent 200 μm on top and 100 μm on bottom, respectively. Micrographs showing reversible changes in cell adhesion when cells cultured on a cell culture substrate with a photoswitchable phase-separated structure surface are repeatedly irradiated with UV light and visible light, and changes in surface structure and cell adhesion The figure which represented typically. Micrographs of cell adhesion and detachment observed when cells cultured on a cell culture substrate irradiated with UV light or visible light are irradiated with light different from the light initially irradiated to change the surface layer separation. . The scale bar of the micrograph of the cells represents 200 μm, and the scale bar of the image showing the phase-separated structure shown in the middle represents 5 μm.

1. Block Copolymers One of the embodiments of the present invention is block copolymers.

The present invention prepares a material comprising a block copolymer having spirobenzopyran that responds to light and undergoes reversible photoisomerization to produce a photoresponsive cell culture material.

A phenomenon called phase separation, in which fine structures including micro- and nano-scales are spontaneously formed by self-aggregation due to differences in the physical properties of blocks of block polymers, is used to fabricate fine structures on the surface of materials. In order to induce this phenomenon, for example, a diblock copolymer of methyl methacrylate and n-butyl methacrylate is synthesized. Furthermore, in order to dynamically change this fine structure and its physical properties, for example, spirobenzopyran-based monomers, which are photochromic molecules that respond reversibly to light, are attached to n-butyl methacrylate blocks. Introduce. This photoresponsive diblock copolymer is modified by spin coating on a substrate such as a glass substrate that has been hydrophobically modified by laminating an organic layer such as an alkyl chain on the surface, and heat treatment is applied to the surface. A culture substrate having a microstructure is produced. Spirobenzopyran undergoes photoisomerization from the ring-closed, low-polarity spiropyran form to the ring-opened, zwitterionic merocyanine form upon irradiation with UV light. The aggregation morphology of the copolymer can be varied.

ただし、
環Aが、C₅～C₈の芳香環であり、
R¹が、C₁～C₁₈の置換されていてもよい直鎖又は分枝状のアルキル基であり、
R²が、H又はC₁～C₁₇の置換されていてもよい直鎖又は分枝状のアルキル基であり、
R¹の炭素数 ＞ R²の炭素数であり、
R³、R⁴及びR⁵が、それぞれ独立して、H又は-CH₃から選択され、
Xaが、O又はSから選択され、
Yaが、-NO₂、-CN、-COOH、-F、-Cl、-Br、-I、-OH、-NH₂、-CH₃から選択され、及び、
nが、5～200であり、
xが、5～200であり、
yが、5～50である。 Specifically, the block copolymer of the present embodiment provides a block copolymer characterized by being represented by the following formula (I):

however,
Ring A is a C ₅ to C ₈ aromatic ring,
R ¹ is a C ₁ to C ₁₈ optionally substituted linear or branched alkyl group,
R ² is H or an optionally substituted linear or branched C ₁ to C ₁₇ alkyl group;
The number of carbon atoms in R ¹ > the number of carbon atoms in R ² and
^R3 , ^R4 and ^R5 are each independently selected from H or _-CH3 ;
Xa is selected from O or S;
Ya is selected from _-NO2 , -CN, -COOH, -F, -Cl, -Br, -I, -OH, _-NH2 , _-CH3 , and
n is 5 to 200,
x is between 5 and 200;
y is 5-50.

In the above formula (I),
n is from 100 to 180,
x is between 60 and 140;
It can be a block copolymer in which y is 9-30.

ただし、
nは133～146であり、
xは74～115であり、
yは、11～25である。 In the block copolymer of the present invention, the compound of formula (I) can be a block copolymer represented by the following formula (II):

however,
n is between 133 and 146;
x is between 74 and 115;
y is 11-25.

In the present invention, spirobenzopyran can be used as an example of the photoresponsive unit. Spirobenzopyran is a molecule that exhibits a greater polar change and also causes a steric structural change by photoisomerization compared to azobenzene. Spirobenzopyran can provide a driving force sufficient for phase separation structural conversion by light alone, except under conditions of high molecular mobility such as liquid surface and high temperature conditions.

More specifically, spirobenzopyrans are photochromic molecules. Photochromic molecules are a general term for molecules whose physical properties can be reversibly photoisomerized by irradiation with light.

Structural changes due to photoisomerization of spirobenzopyran, which is a photochromic molecule used in the present invention, are shown below. Spirobenzopyran photoisomerizes from the spiropyran type, which is a closed ring structure with low polarity, to the merocyanine type, which is an open ring structure having a zwitterion, by irradiation with UV light at a wavelength of about 365 nm (see formula A below). At this time, the merocyanine type shows absorption at 580 nm, which the spiropyran type does not, and exhibits its complementary color, violet. Spirobenzopyran has a thermodynamically stable structure of the spiropyran type.

The physical properties of each block and the ratio of each block are important for the formation of a phase-separated structure by self-aggregation of a diblock copolymer. Therefore, in synthesizing diblock copolymers, a technique for accurately controlling the molecular chain length is required. Accordingly, in the present invention, as a specific example thereof, a polymer is polymerized using reversible addition-fragmentation chain transfer polymerization (RAFT polymerization), which is one of living radical polymerizations.

RAFT polymerization is a polymerization method that can produce a polymer with a narrow molecular weight distribution by selecting an appropriate chain transfer agent (RAFT agent) and using it during synthesis. In the reaction process of RAFT polymerization, in addition to initiation reaction, propagation reaction, and termination reaction, which are typical reaction processes of living radical polymerization, there are equilibrium reactions mediated by RAFT agents (see Scheme 1). A single RAFT agent can store radicals between two polymer chains while rapidly transferring them, allowing the overall polymerization to proceed slowly. As a result, it becomes difficult for a difference in degree of polymerization to occur between the polymer chains, and it is possible to polymerize a polymer having a narrow molecular weight distribution.

In the present invention, by using this RAFT polymerization, a block copolymer having a narrow range of degree of polymerization, i.e., having high homogeneity, is produced, thereby producing a phase-separated structure that reversibly reacts to light as described below. system can be manufactured.

The present block copolymer can be produced according to the methods described in the embodiments and examples of "3. Production method of block copolymer" below. In addition, it can be used according to the embodiment described in "4. Use of Block Copolymer" below and the methods described in Examples.

2. Phase Separated Structure System Another embodiment of the present invention is a phase separated structure system. As described above, the present invention produces a polymer material having spirobenzopyran that responds to light and undergoes reversible photoisomerization, develops a photoresponsive cell culture material, and obtains a phase separation structure. As a body system, for the preparation of the fine structure of the material surface, the phase separation phenomenon that spontaneously forms fine structures including micro and nano scales by self-aggregation due to the difference in physical properties of each block of the block polymer is used.

In order to induce this phenomenon, for example, a diblock copolymer of methyl methacrylate and n-butyl methacrylate is synthesized. Furthermore, in order to dynamically change this fine structure and its physical properties, for example, spirobenzopyran-based monomers, which are photochromic molecules that respond reversibly to light, are attached to n-butyl methacrylate blocks. Introduce.

Modification by spin coating on a substrate such as a glass substrate which has been hydrophobically modified by laminating the photoresponsive diblock copolymer on the surface, for example, on an organic layer comprising an alkyl group or an organic layer containing a random copolymer. Then, a culture substrate having a microstructure on its surface is produced by heat-treating. That is, in this case, a phase-separated structure system in which an organic layer and a photoresponsive diblock copolymer are laminated on a glass substrate is formed and used.

Spirobenzopyran in this phase-separated structural system undergoes photoisomerization from the ring-closed, low-polar spiropyran form to the ring-opened, zwitterionic merocyanine form upon irradiation with UV light. The aggregation morphology of the introduced diblock copolymer can be changed by structural change (see formula (III) above).

Specifically, as an example of the present embodiment, a phase-separated structure in which the compound of formula (I) is coated on an organic layer surface-fixed to the surface of a substrate made of glass or silicon system.

In the phase-separated structure system of the present invention, the surface-immobilized organic layer may be an organic layer having an organic compound immobilized on the surface by a silicon compound reagent.

In the phase separation structure system of the present invention, the silicon compound reagent is trimethoxy(methyl)silane or 1,1,1,3,3,3-hexamethyldisilazane, 1,3-dibutyl-1,1,3 ,3-tetramethyldisilazane or 1,3-diethyl-1,1,3,3-tetramethyldisilazane.

ただし、
R⁶が、-COOH又は-CH₂OHであり、
R⁷が、-C₆H₅又は-S-(CH₂)₁₁-CH₃であり、
pが、20～200であり、
qが、20～200である。 Further, when a random copolymer is used as the organic layer, the random copolymer prepares and uses a phase-separated structure system having a structure of the following formula (III):

however,
^R6 is -COOH or _-CH2OH ,
^R7 is _-C6H5 or _-S- ( _CH2 ) ₁₁ - _CH3 ;
p is between 20 and 200;
q is between 20 and 200;

The separation structure system of the present invention can reversibly change the surface phase separation structure in response to light.

In the phase-separated structure system of the present invention, the change in the surface phase-separated structure can be accompanied by a change in the fine uneven structure of the film surface, a change in wettability and/or a change in hardness.

In this specification, the term "change in fine uneven structure" means that the height difference of the surface in the range of 10 to 30 nm, preferably in the range of 15 to 25 nm, becomes a height difference below this range, or the height difference disappears, or a height difference within the above numerical range is generated from a state in which the height difference is smaller than or not present in the above range.

The phase-separated structure system of the present invention can be used as a substrate for cell culture.

The phase separation structure can be confirmed by, for example, surface analysis of the polymer-modified substrate using an atomic force microscope. The structure of phase separation differs depending on the composition of the polymer and the concentration at which the polymer is spin-coated. For example, by irradiating this substrate with UV light, this structure can change due to the photoresponse and can exhibit a large change such that it completely disappears under certain conditions. Furthermore, by irradiating the substrate from which the phase separation structure has disappeared with visible light, the fine structure derived from the phase separation can be revived.

The phase separation structure system of the present invention reversibly adheres and detaches cultured cells in response to light, or causes differentiation, spreading, migration, or changes in gene and protein expression of cultured cells. It can be used as a controllable substrate for cell culture.

For example, using the phase separation structure system of the present invention as a substrate for cell culture, adhesion of cells to the substrate, detachment of cultured cells adhered to the substrate, or detachment of cultured cells Re-adherence of cultured cells to the coated substrate can be brought about by dynamic, reversible changes in the microstructure of the membrane surface upon irradiation with visible or UV light.

That is, by irradiating the surface of the phase-separated structure system of the present invention with visible light, the fine uneven structure on the surface becomes smaller or disappears, and by irradiating with UV light, the surface of the phase-separated structure system becomes finer. It is a phase-separated structure system having a characteristic that the uneven structure reversibly becomes relatively large. In this phase-separated structure system, the cultured cells adhered when the fine uneven structure on the surface is small or disappears are exposed to the fine uneven structure by irradiating the surface of the phase-separated structure system with UV light. Detachment of the cultured cells is triggered when the cells grow significantly larger. On the other hand, the cultured cells adhered to the surface of the phase-separated structure system when the fine uneven structure on the surface was relatively large due to the irradiation of UV light, the fine uneven structure on the surface became relatively large due to the irradiation of visible light. or disappears from the surface of the phase-separated structure system.

Therefore, it is possible to control the adhesion, detachment, re-adhesion, etc. of cultured cells to the surface of the phase-separated structure system of the present invention by irradiating visible light or UV light.

3. Method for Making Block Copolymers Another embodiment of the present invention is a method for making block copolymers used in the phase-separated structure system.

下記反応式２に表されるように、前記PMMA、4,4′-アゾビス(4-シアノペンタン酸) （4,4’-azobis(4-cyanopentanoic acid)）、SpMA （1'-(2-Methacryloyloxyethyl)-,3',3'-dimethyl-6-nitrospiro(2H-1-benzopyran-2,2'-indoline)）と、n－ブチルメタクリレート（n-Butyl methacrylate） とを反応させる工程、
を含む製造方法である。

A specific example of the method for producing the block copolymer is a method for producing the block copolymer: PMMA-bP (nBMA-stat-SpMA),
4-Cyanopentanoic acid dithiobenzoate and 4,4'-azobis(4-cyanopentanoic acid) (4,4'-azobis(4 -cyanopentanoic acid)) and methylmethacrylate to prepare PMMA (Poly-Methylmethacrylate);

As shown in Reaction Scheme 2 below, the PMMA, 4,4′-azobis(4-cyanopentanoic acid) (4,4′-azobis(4-cyanopentanoic acid)), SpMA (1′-(2- Methacryloyloxyethyl)-,3',3'-dimethyl-6-nitrospiro(2H-1-benzopyran-2,2'-indoline)) and n-butyl methacrylate,
It is a manufacturing method including

A more specific method for producing a block copolymer will be described in detail in the examples below.

4. Use of Block Copolymers Another embodiment of the present invention is the use of said block copolymers for the manufacture of phase separated structural systems.

Already, block copolymers and methods for preparing phase-separated structural systems have been presented above. According to the methods described above, the block copolymers can be used to prepare phase-separated structural systems. The phase-separated structure system reversibly adheres and detaches cultured cells in response to light, or causes differentiation, spreading, and migration of cultured cells, or changes in gene and protein expression. It can be used as a substrate for cell culture that can be controlled.

All documents mentioned herein are hereby incorporated by reference in their entirety. The examples described herein are illustrative of embodiments of the invention and should not be construed as limiting the scope of the invention.

Synthetic materials and methods of spirobenzopyran (SpOH)

Reagents In this example, the following reagents were used.
2,3,3-Trimethyl-3H-indole (97 %, Wako Pure Chemical Industries, Ltd., Osaka, Japan)
2-Bromoethanol (97 %, Wako Pure Chemical Industries, Ltd.)
Acetonitrile (99.5 %, Wako Pure Chemical Industries, Ltd.)
Potassium hydroxide (85.0 %, Wako Pure Chemical Industries, Ltd.)
2-Hydroxy-5-nitrobenzaldehyde (Wako Pure Chemical Industries, Ltd.)
Diethyl ether (98.0 %, Wako Pure Chemical Industries, Ltd.)
Hexane (96.0 %, Wako Pure Chemical Industries, Ltd.)
Ethanol (99.5 %, Wako Pure Chemical Industries, Ltd.)

Apparatus In this example, the following apparatus was used.
Nuclear magnetic resonance (NMR) spectrometer (1H-NMR; UNITY INOVA 400 MHz spectrometer, Varian, Palo Alto, CA, USA)

1-(2-Hydroxyethyl)-2,3,3-trimethyl-3H-indolium bromide (1-(2-Hydroxyethyl)-2,3,3-trimethyl-3H-indolium bromide; hereinafter referred to as TMIBR) Synthesis TMIBR was synthesized with the amount shown in Table 1 as shown in Reaction Formula 3 below. Here, the molar ratio of 2,3,3-Trimethyl-3H-indole to 2,3,3-Trimethyl-3H-indole is 1:1.50 so that there is sufficient 2-bromoethanol. I made it so that

Acetonitrile (100 mL) was added as a solvent to a two-neck eggplant flask (300 mL), and nitrogen bubbling was performed. 2,3,3-Trimethyl-3H-indole (13.0 g, 81.6 mmol) and 2-bromoethanol (15.2 g, 122.0 mmol) were mixed with acetonitrile in a two-necked flask and heated at 90°C under a nitrogen atmosphere. The reaction was allowed to reflux for an hour. After completion of the reaction, acetonitrile was removed at 45°C using an evaporator. The remaining purple oily liquid was ultrasonically washed three times with hexane (25 mL) to obtain TMIBR.

1H-NMR spectrum of TMIBR (400 MHz CDCl ₃ )
1H-NMR ( _CDCl3 ): δ = 7.79-7.54 (m, aromatic, 4H), 4.89 (t, CH2 _- OH, 2H), 4.19 (t, N- _CH2 - _CH2 , 2H), 3.12 ( s, C- _CH3 , 3H), 1.65 (s, C-( _CH3 ) ₂ , 6H).

9,9,9a-Trimethyl-2,3,9a-tetrahydro-oxazolo[3,2-a]indole Synthesis of a]indole; TMIO) TMIO was synthesized in the amounts shown in Table 2 as represented by Reaction Formula 4 below. Here, potassium hydroxide was prepared at a molar ratio of 1:1.18 with respect to 2,3,3-trimethyl-3H-indole used in the synthesis of TMIBR.

An aqueous potassium hydroxide solution was prepared by dissolving potassium hydroxide (5.40 g, 96.2 mmol) in H ₂ O (300 mL) in an ice bath. An aqueous potassium hydroxide solution was added to TMIBR (whole amount), and the mixture was stirred at room temperature for 10 minutes to react. Diethyl ether (300 mL) was used to separate three times, and the diethyl ether layer was extracted. After liquid separation, diethyl ether was removed using an evaporator to obtain TMIO [yield: 15.6 g (76.7 mmol), yield: 94.0%].

1H-NMR spectrum of TMIO (400 MHz CDCl ₃ )
1H-NMR ( _CDCl3 ): δ = 7.13-6.72 (m, aromatic, 4H), 3.82-3.66 (m, O- _CH2- , 2H), 3.57-3.43 (m, N- _CH2- ,2H). , 1.42 and 1.37 (s, C-(CH ₃ ) ₂ , 6H), 1.16 (s, C-CH ₃ , 3H).

2-(3',3'-dimethyl-6-nitro-3'H-spiro[chromen-2,2'-indol]-1'-yl)-ethanol (2-(3',3'-dimethyl- Synthesis of 6-nitro-3'H-spiro[chromene-2,2'-indol]-1'-yl)-ethanol; SpOH was synthesized at the charge. Here, the molar ratio of 2-hydroxy-5-nitrobenzaldehyde to TMIO was adjusted to 1:1.34 so that a sufficient amount of 2-hydroxy-5-nitrobenzaldehyde was present.

Ethanol (40 mL) was added as a solvent to a two-neck eggplant flask (300 mL). TMIO (15.6 g, 66.9 mmol) and 2-hydroxy-5-nitrobenzaldehyde (17.2 g, 103.0 mmol) were mixed with ethanol in a two-neck flask and nitrogen bubbling was performed. Under nitrogen atmosphere, reflux reaction was carried out at 90° C. for 24 hours. After completion of the reaction, ethanol was removed using a suction filtration device, and the obtained solid was further washed with ethanol. SpOH was obtained by drying in a vacuum dryer (FVO-30, Fine, Tokyo, Japan) at 50°C for 6 hours [yield: 23.6 g (67.0 mmol), yield: 87.4%].

1H-NMR spectrum of SpOH (400 MHz CDCl ₃ )
1H-NMR ( _CDCl3 ): δ = 8.03 and 8.00 (d, ortho-aromatic to _NO2 , 2H), 7.21-6.66 (m, aromatic, 6H), 5.88 (d, -CH=CH-CO-, 1H ), 5.87 (d, -CH=CH-CO-, 1H), 3.84-3.69 (m, CH ₂ -OH, 3H), 3.50-3.30 (m, N-CH ₂ -, 2H), 1.29 and 1.20 ( s, C-( _CH3 ) ₂ , 6H).

Materials and Methods for Synthesis of Spirobenzopyran Monomer (SpMA) In this example, the following reagents were used.
2-(3',3'-dimethyl-6-nitro-3'H-spiro[chromen-2,2'-indol]-1'-yl) - ethanol (SpOH)
Methacrylic chloride (97.0 %, Wako Pure Chemical Industries, Ltd.)
Triethylamine (99.0 %, Kanto Chemical Co., Ltd.)
N,N-Dimethyl-4-aminopyridine (99.0 %, Wako Pure Chemical Industries, Ltd.)
Hydrochloric acid (36 %, Wako Pure Chemical Industries, Ltd.)
Sodium bicarbonate (99.5 %, Nacalai Tesque Co., Ltd., Kyoto, Japan)
Magnesium Sulfate (98.0 %, Wako Pure Chemical Industries, Ltd.)
Methylene chloride (99.5 %, Wako Pure Chemical Industries, Ltd.)
Hexane (96.0 %, Wako Pure Chemical Industries, Ltd.)

In this example, the following apparatus was used.
Nuclear magnetic resonance (NMR) spectrometer (1H-NMR; UNITY INOVA 400 MHz spectrometer, Varian, Palo Alto, CA, USA)

1'-(2-Methacryloyloxyethyl)-,3',3'-dimethyl-6-nitrospiro(2H-1-benzopyran-2,2'-indoline) (1'-(2-Methacryloyloxyethyl)-,3' Synthesis of ,3'-dimethyl-6-nitrospiro (2H-1-benzopyran-2,2'-indoline; hereinafter referred to as SpMA) The molar ratio of methacryloyl chloride to SpOH was adjusted to 1:1.29 so that a sufficient amount of methacryloyl chloride was present.

Methylene chloride (100 mL) previously dehydrated with magnesium sulfate was added as a solvent to a two-neck eggplant flask (300 mL). SpOH (5.00 g, 14.2 mmol) and N,N-dimethyl-4-aminopyridine (0.237 g, 1.94 mmol) were mixed with methylene chloride in a two-necked eggplant flask and nitrogen bubbling was performed. Triethylamine (1.82 g, 18.0 mmol) and methacrylic acid chloride (1.91 g, 18.3 mmol) were mixed with the remaining methylene chloride (50 mL), added dropwise in an ice bath under a nitrogen atmosphere for 1 hour, and allowed to react for 24 hours. After completion of the reaction, it was washed with hydrochloric acid (100 mM, 500 mL), saturated sodium hydrogen carbonate (1.14 M, 500 mL) and H ₂ O (100 mL) in that order, and the recovered methylene chloride layer was dehydrated with magnesium sulfate. Methylene chloride was removed using an evaporator, hexane (1 L) heated to 50° C. was added, and the mixture was stirred for 2 hours. The solution was cooled in a freezer to precipitate SpMA, which was collected by suction filtration [yield 2.39 g (5.69 mmol) yield 40.1%].

1H-NMR spectrum of SpMA (400 MHz CDCl ₃ )
1H-NMR ( _CDCl3 ): δ = 8.03 and 8.00 (d, ortho-aromatic to _NO2 , 2H), 7.23-6.67 (m, aromatic, 6H), 6.07 and 5.56 (s, _CH2 =C, 2H) , 5.87 (d, -CH=CH-CO-, 1H), 4.30 (t, N- _CH2 - _CH2- , 2H), 3.58-3.40 (m,N- _CH2- , 2H), 1.92 (s , CH ₃ -C=CH ₂ , 3H), 1.28 and 1.17 (s, C-(CH ₃ ) ₂ , 6H).

Materials and Methods for Synthesizing Dithiobenzoic Acid 4-Cyanopentanoate (CPADB), a RAFT Polymerization Chain Transfer Agent In this example, the following reagents were used.
4,4'-azobis(4-cyanopentanoic acid) (98.0 %, Wako Pure Chemical Industries, Ltd.)
Benzyl chloride (99.0 %, Wako Pure Chemical Industries, Ltd.)
Sodium Methoxide (96.0 %, Tokyo Chemical Industry Co., Ltd.)
Sulfur (98.0 %, Wako Pure Chemical Industries, Ltd.)
Methanol (99.8 %, Wako Pure Chemical Industries, Ltd.)
Diethyl ether (98.0 %, Wako Pure Chemical Industries, Ltd.)
Hydrochloric acid (36.0 %, Wako Pure Chemical Industries, Ltd.)
Sodium hydroxide (97.0 %, Wako Pure Chemical Industries, Ltd.)
Potassium ferricyanide(III) (99.0 %, Wako Pure Chemical Industries, Ltd.)
Ethyl acetate (99.5 %, Wako Pure Chemical Industries, Ltd.)
Hexane (96.0 %, Wako Pure Chemical Industries, Ltd.)
Silica gel (Wakogel ^R C-200; Wako Pure Chemical Industries, Ltd.)

In this example, the following apparatus was used.
Nuclear magnetic resonance (NMR) spectrometer (1H-NMR; UNITY INOVA 400 MHz spectrometer, Varian, Palo Alto, CA, USA)
Thin layer chromatography: Aluminum TLC plate (TLC Silica gel 60 F254, silica gel coated with flourescent indicator F254, EMD Millipore, MA, USA)

Synthesis of dithiobenzoic acid (hereinafter referred to as DTBA) DTBA was synthesized in the amounts shown in Table 5 as represented by Reaction Formula 7 below. Here, the molar ratio of sodium methoxide and sulfur to benzyl chloride was adjusted to 1:1.98.

Sodium methoxide (27.0 g, 0.500 mol) was added to a two-necked eggplant flask (500 mL), and methanol (250 mL) previously dehydrated with magnesium sulfate was added as a solvent while cooling with ice. As soon as the sodium methoxide dissolved and the heat generation subsided, sulfur (16.0 g, 0.499 mol) was added, and the mixture was stirred while bubbling nitrogen. Benzyl chloride (31.9 g, 0.252 mmol) was added dropwise with ice cooling under a nitrogen atmosphere. After completion of the dropwise addition, reflux reaction was carried out at 68° C. for 18 hours. After completion of the reaction, the reaction solution was ice-cooled, and the precipitated solid was removed by suction filtration. Methanol was removed with an evaporator, ice-cooled H ₂ O (150 mL) was added, and suction filtration was performed again. It was washed with diethyl ether (100 mL) three times and the _H2O layer was collected. 1.0 M Hydrochloric acid (250 mL) was added to the recovered H ₂ O layer, and liquid separation operation was performed three times with diethyl ether (100 mL) to extract the produced DTBA into the diethyl ether layer. Separation operation was performed three times with 1.0 M sodium hydroxide (750 mL), and the generated DTBA was extracted into the H ₂ O layer to obtain a DTBA solution.

Synthesis of di(thiobenzoyl) disulfide (hereinafter referred to as DTD) DTD was synthesized as shown in Reaction Formula 8 below with the amount shown in Table 6. Here, the molar ratio of potassium ferricyanide (III) to benzyl chloride used in the synthesis of DTD was 1:1.24.

Potassium ferricyanide (III) (102.5 g, 0.311 mol) was dissolved in H ₂ O (1000 mL), and the DTBA solution was added dropwise to react with stirring. The resulting reddish purple solid was collected by suction filtration. After washing with H ₂ O until the washings were colorless, the DTD was obtained by drying under reduced pressure [Yield 19.9 g (64.9 mmol) Yield 51.5 %].

1H-NMR spectrum of DTD (400 MHz CDCl ₃ )
1H-NMR ( _CDCl3 ): δ = 8.09 (m, ortho-aromatic, 2H), 7.61 (m, para-aromatic, 1H), 7.45 (m, meta-aromatic, 2H).

Synthesis of 4 -Cyanopentanoic acid dithiobenzoate (hereafter referred to as CPADB) CPADB was synthesized as shown in Reaction Formula 9 below with the amount shown in Table 7. Here, the molar ratio of 4,4'-azobis(4-cyanopentanoic acid) to DTD is 1:1.49 so that a sufficient amount of 4,4'-azobis(4-cyanopentanoic acid) is present. I made it so that

DTD (12.3 g, 40.1 mmol) and 4,4'-azobis(4-cyanopentanoic acid) (16.8 g, 59.9 mmol) were added to a two-necked eggplant flask (500 mL) and dissolved in ethyl acetate (230 mL). and nitrogen bubbling was applied while stirring. A reflux reaction was carried out at 85° C. for 24 hours under a nitrogen atmosphere. Ethyl acetate was removed using an evaporator and purified by column chromatography (stationary phase; silica gel, developing solvent; hexane:ethyl acetate = 3:2) (Rf value: 0.32) to obtain CPADB [yield 3.9 g ( 14.0 mmol) Yield 34.9 %].

1H-NMR spectrum of CPADB (400 MHz CDCl ₃ )
1H-NMR (CDCl ₃ ): δ = 7.91 (m, ortho-aromatic, 2H), 7.58 (m, para-aromatic, 1H), 7.40 (m, meta-aromatic, 2H), 2.77-2.42 (m, - _CH2 - _CH2- , 4H), 1.95 (d, _-CH3 , 3H).

Materials and Methods for Polymerizing PMMA-bP (nBMA-stat-SpMA) In this example, the following reagents were used.
1'-(2-methacryloyloxyethyl)-,3',3'-dimethyl-6-nitrospiro(2H-1-benzopyran-2,2'-indoline) (SpMA)
4- Cyanopentanoic acid dithiobenzoate (hereinafter referred to as CPADB)
Methyl methacrylate (98.0%, Wako Pure Chemical Industries, Ltd.) was obtained and used after purification.
n-butyl methacrylate (98.0%, Wako Pure Chemical Industries, Ltd.) was obtained and used after purification.
4,4'-Azobis(4-cyanopentanoic acid) (98.0 %, Wako Pure Chemical Industries, Ltd.)
1,4-dioxane (99.5 % Wako Pure Chemical Industries; distilled at 120°C)

In this example, the following apparatus was used.
Nuclear magnetic resonance (NMR) spectrometer (1H-NMR; UNITY INOVA 400 MHz spectrometer, Varian, Palo Alto, CA, USA)
High Performance Liquid Chromatography (GPC; EXTREMA, JASCO Corporation, Tokyo, Japan)
UV measurement device : UV-4075 (JASCO Corporation)
Autosampler: AS-4050 (JASCO Corporation)
Column Oven : CO-4060 (JASCO Corporation)
Column : KF-G4A (Shodex, Showa Denko K.K., Tokyo, Japan), KF-806L (Shodex, Showa Denko K.K.), KF-806L (Shodex, Showa Denko K.K.)

Polymerization of poly-methylmethacrylate (hereinafter referred to as PMMA) As represented by Reaction Formula 10 below, PMMA was synthesized with the amounts shown in Tables 8 and 9. Here, the charging amount was set so that the amount of methyl methacrylate was about 130 units. In addition, since a polymer larger than the target unit was obtained with the amount charged in Table 8, synthesis was performed again with the amount charged in Table 9 with a reduced proportion of methyl methacrylate. The polymers obtained here were named PMMA#1 and PMMA#2, respectively.

Dithiobenzoic acid 4-cyanopentanoate (PMMA#1; 0.0626 g, 0.22 mmol, PMMA#2; 0.0714 g, 0.26 mmol) and 4,4'-azobis(4- cyanopentanoic acid) (144 units; 0.0133 g, 0.0464 mmol) was added. After purging with nitrogen three times, 1,4-dioxane (35 mL) was added dropwise with a syringe, and nitrogen bubbling was performed. Methyl methacrylate (3.8 mL) was added dropwise with a syringe under a nitrogen atmosphere, and the mixture was reacted at 70°C for 21 hours. After completion of the reaction, PMMA was purified by a reprecipitation method (good solvent; tetrahydrofuran, poor solvent; methanol) [PMMA-1; yield 2.76 g, yield 75.2%] [PMMA#2; yield 2.10 g, yield 57.1%].

1H-NMR spectrum of PMMA_1 (400 MHz CDCl ₃ )
1H-NMR (CDCl ₃ ): δ = 7.89 (broad, ortho-aromatic H), 7.52 (broad, para-aromatic H), 7.36 (broad, meta-aromatic H), 3.78-3.42 (broad, -O-CH ₃ ), 2.56 (broad, -CH ₂ -CH ₂ -), 2.07-1.74 (broad, -CH ₂ -), 1.02-0.84 (broad, methacrylic methylene).

1H-NMR spectrum of PMMA_2 (400 MHz CDCl ₃ )
1H-NMR (CDCl ₃ ): δ = 7.89 (broad, ortho-aromatic H), 7.52 (broad, para-aromatic H), 7.36 (broad, meta-aromatic H), 3.71-3.55 (broad, -O-CH ₃ ), 2.53 (broad, -CH ₂ -CH ₂ -), 2.07-1.76 (broad, -CH ₂ -), 1.02-0.84 (broad, methacrylic methylene).

Synthesis of PMMA-bP (nBMA-stat-SpMA) PMMA-bP (nBMA-stat-SpMA) was synthesized in the amounts shown in Tables 10 to 13 as shown in Reaction Formula 11 below. Here, we designed four types of diblock copolymers with different numbers of units, and two types of polymers with a ratio of MMA: nBMA: SpMA of about 146:120: about 28, 146: about 75: about 15, and 133: About 120: I made it about 28. PMMA-bP(nBMA-stat-SpMA)_1, PMMA-bP(nBMA-stat-SpMA)_2, PMMA-bP(nBMA-stat-SpMA)_3, PMMA-bP( nBMA-stat-SpMA)_4.

PMMA, 4,4'-azobis(4-cyanopentanoic acid) and SpMA were added to an eggplant flask (100 mL). After purging with nitrogen three times, 1,4-dioxane (6 mL) was added dropwise with a syringe, and freeze degassing was performed five times. In a nitrogen atmosphere, n-butyl methacrylate was added dropwise with a syringe, and reacted at 70°C for 21 hours. After completion of the reaction, PMMA-bP (nBMA-stat-SpMA) was purified by a reprecipitation method (good solvent; tetrahydrofuran, poor solvent; methanol). The amount and yield of PMMA-bP (nBMA-stat-SpMA) obtained are shown below.
[PMMA-bP(nBMA-stat-SpMA)_1; Yield 100 mg Yield 50.0 % ]
[PMMA-bP(nBMA-stat-SpMA)_2; Yield 1.51 g Yield 75.4 % ]
[PMMA-bP(nBMA-stat-SpMA)_3; Yield 0.93 g Yield 66.5 % ]
[PMMA-bP(nBMA-stat-SpMA)_4; Yield 1.59 g Yield 75.5 % ]

1H-NMR spectrum of PMMA-bP(nBMA-stat-SpMA)_1 (400 MHz CDCl ₃ )
1H-NMR (CDCl ₃ ): δ = 8.03 (broad, ortho -aromatic to NO ₂ ), 7.21-6.74 (broad, aromatic), 5.88 (broad, -CH=CH-CO-), 3.94 (broad, N- CH2 _{- CH2-} , _-CH2 - _CH2 -O-), 3.60-3.55 (broad, -O- _{CH2- CH2-} ,O=CO- _CH3 ), 1.94-1.81 (broad, -CH ₂ -), 1.40-0.85 (broad, C-(-CH ₃ ) ₂ ), 1.02-0.85 (broad, methacrylic methyl).

Figure 1 shows the 1H-NMR spectrum (400 MHz CDCl ₃ ) of PMMA-bP(nBMA-stat-SpMA)_2.
1H-NMR (CDCl ₃ ): δ = 8.02 (broad, ortho -aromatic to NO ₂ ), 7.21-6.69 (broad, aromatic), 5.89 (broad, -CH=CH-CO-), 3.93 (broad, N- CH2 _{- CH2-} , _-CH2 - _CH2 -O-), 3.60-3.55 (broad, -O- _{CH2- CH2-} ,O=CO- _CH3 ), 1.94-1.81 (broad, -CH ₂ -), 1.45-0.85 (broad, C-(-CH ₃ ) ₂ ), 1.02-0.85 (broad, methacrylic methyl).

1H-NMR spectrum of PMMA-bP(nBMA-stat-SpMA)_3 (400 MHz CDCl ₃ )
1H-NMR (CDCl ₃ ): δ = 8.01 (broad, ortho -aromatic to NO ₂ ), 7.21-6.69 (broad, aromatic), 5.89 (broad, -CH=CH-CO-), 3.94 (broad, N- CH2 _{- CH2-} , _-CH2 - _CH2 -O-), 3.60 (broad, -O- _CH2 - _CH2- , O=CO- _CH3 ), 1.95-1.82 (broad, _-CH2- ), 1.39-0.85 (broad, C-(-CH ₃ ) ₂ ), 1.03-0.85 (broad, methacrylic methyl).

1H-NMR spectrum of PMMA-bP(nBMA-stat-SpMA)_4 (400 MHz CDCl ₃ )
1H-NMR (CDCl ₃ ): δ = 8.00 (broad, ortho -aromatic to NO ₂ ), 7.21-6.74 (broad, aromatic), 5.87 (broad, -CH=CH-CO-), 3.94 (broad, N- CH2 _{- CH2-} , _-CH2 - _CH2 -O-), 3.60 (broad, -O- _CH2 - _CH2- ,O=CO- _CH3 ), 1.94-1.82 (broad, _-CH2- ), 1.40-0.85 (broad, C-(-CH ₃ ) ₂ ), 1.02-0.85 (broad, methacrylic methyl).

The molecular weight Mn and the molecular weight distribution Mw/Mn of the synthesized four types of PMMA-bP (nBMA-stat-SpMA) were measured by 1H-NMR and GPC (developing solvent: THF). The results are shown in Table 14. Based on the composition ratio of the obtained polymers, the names of the polymers were expressed as MMA:nBMA:SpMA=146:155:25, 146:115:21, 146:74:11, 133:115:25.

Material and Method for Decomposing Impurities on Substrate Surface with Piranha Solution In this example, the following reagents were used.
Sulfuric acid (95.0 %, Wako Pure Chemical Industries, Ltd.)
Hydrogen peroxide (30.0%, Wako Pure Chemical Industries, Ltd.)

The substrate was treated with a piranha solution for cleaning the glass substrate used for substrate preparation and exposing the silanol groups. The piranha solution was prepared with ice cooling so that the volume fraction of sulfuric acid and hydrogen peroxide was 7:3. When the heat generation disappeared, the glass substrate was immersed in the piranha solution and treated for 24 hours. The piranha solution has a strong oxidizing power, and by oxidizing the organic matter adhering to the substrate surface, removes the organic matter from the substrate surface and exposes silanol groups on the substrate surface.
After the piranha solution treatment was completed, the piranha solution was thoroughly washed with Milli-Q water. The piranha-cleaned substrate was immersed in Milli-Q water and preserved to prevent new organic soiling. When used for the silane coupling reaction or measurement, the substrate that had been stored in Milli-Q water was dried under reduced pressure for 3 hours, and the water on the surface of the glass substrate was sufficiently dried before use.

Material and Method for Alkylating Substrate Surface by Silane Coupling Reaction In this example, the following reagents were used.
Toluene (99.0 %, Wako Pure Chemical Industries, Ltd.)
Trimethoxysilane (C1; 98.0 %, Tokyo Chemical Industry Co., Ltd.)
1,1,1,3,3,3-Hexamethyldisilazane (HMDS; 96.0 %, Wako Pure Chemical Industries, Ltd.)
Acetone (99.5 %, Wako Pure Chemical Industries, Ltd.)

Trimethoxy(methyl)silane (C1) Modification A silane coupling solution was prepared to fix the polymer thin film to be modified. Trimethoxy(methyl)silane (0.83 mL) was mixed in toluene (100 mL) and stirred for 15 minutes with nitrogen bubbling. Thereafter, the glass substrate treated with the piranha solution in Example 5 was immersed in the silane coupling solution and reacted at 50° C. for 24 hours. After completion of the reaction, the silane coupling solution was thoroughly washed with Milli-Q water and dried under reduced pressure for 3 hours to fabricate a C1-modified substrate.

1,1,1,3,3,3-Hexamethyldisilazane modification 1,1,1,3,3,3-Hexamethyldisilazane (200 μL) was cast and dried at 140° C. for 15 minutes using a dryer. After that, it was immersed in acetone, subjected to ultrasonic cleaning for 15 minutes, and dried under reduced pressure for 1 hour to fabricate the HMDS-modified substrate.

ただし、
nが、20～200であり、
mが、20～200である。 Synthesis of random copolymer of MMA and nBMA: P(MMA-r-nBMA)
A random copolymer of MMA and nBMA: P(MMA-r-nBMA) was synthesized according to Reaction Formula 12 below.

however,
n is 20 to 200,
m is 20-200.

(1) Materials The following reagents were used.
・4-Cyano-4-[(dodecylsulfanyl-thiocarbonyl)sulfanyl]pentanol (4-Cyano-4-[(dodecylsulfanyl-thiocarbonyl)sulfanyl]pentanol; 99.0%, Sigma-Aldrich)
- Methyl methacrylate (98.0%, Wako Pure Chemical Industries, Ltd.) was obtained and used after purification.
- n-butyl methacrylate (98.0%, Wako Pure Chemical Industries, Ltd.) was obtained and used after purification.
・4,4'-Azobis(4-cyanopentanoic acid; 98.0 %, Wako Pure Chemical Industries, Ltd.)
- 1,4-dioxane (99.5% Wako Pure Chemical Industries, Ltd.) was obtained and used after distillation.
・Tetrahydrofuran (99.5 %, Wako Pure Chemical Industries, Ltd.)
・Methanol (99.8 %, Wako Pure Chemical Industries, Ltd.)

(2) Synthesis conditions Table 15 shows the synthesis conditions.

(3) Reaction 4,4'-Azobis(4-cyanopentanoic acid) (15.2mg, 0.054mmol) and (4-Cyano-4-[(dodecylsulfanyl-thiocarbonyl)sulfanyl]pentanoic acid) were added to a two-neck eggplant flask (100 mL). (120.6 mg, 0.3 mmol) was added.After purging with nitrogen, 1,4-dioxane (8 mL) was added dropwise and nitrogen bubbling was performed.Methyl methacrylate (1.31 mL) and butyl methacrylate (1.31 mL) were added under a nitrogen atmosphere. (1.79 mL) was added dropwise and reacted at 70° C. for 21 hours.
After completion of the reaction, reprecipitation was performed using tetrahydrofuran as a good solvent and methanol as a poor solvent to obtain a rubbery solid.
Further, the resulting solid was dissolved in tetrahydrofuran, which is a good solvent, and the solvent was removed under reduced pressure to obtain P(MMA-r-nBMA). (Yield 1.257 g, Yield 44.1% )
¹ H-NMR spectrum of P(MMA-r-nBMA) is shown in FIG.

Material and Method for Modification of Photoresponsive Polymer on Glass Substrate Surface by Spin Coater In this example, the following materials were used.
PMMA-bP (nBMA-stat-SpMA)
Toluene (99.0 %, Wako Pure Chemical Industries, Ltd.)
Micro cover glass 24 mm x 24 mm x 0.13-0.17 mm (modified with C1 or HMDS above)

In this example, the following apparatus was used.
Spin coater (ACT-300DH, Active Co., Ltd., Saitama, Japan)

In order to fabricate a polymer thin film with uniform thickness, modification by spin coating was performed. The spin coating method is sometimes used in the manufacturing process of semiconductors, and is a modification method that can stably produce a thin film. The synthesized photoresponsive diblock copolymer was dissolved in toluene (360 μL, 10 mg/mL) and spin-coated on the silane-modified glass substrate in Example 6 under the rotation conditions shown in Table 15 under a nitrogen atmosphere. After that, it was subjected to thermal annealing at 140°C for 12 hours under reduced pressure.

Materials and Methods for Observation of Phase Separation Surface Structure by Atomic Force Microscope In the present example, the following materials were used.
Micro cover glass 24 mm x 24 mm x 0.13-0.17 mm (modified with polymer)
Cantilever (OMCL-AC240TS-W2, Olympus Corporation, Tokyo, Japan) (spring constant 2.0 N/m, resonant frequency 70 kHz)

In this example, the following apparatus was used.
Atomic force microscope (Dimension 3100 Nanoscope IV, Veeco, Plainview, New York, USA)
Atomic force microscope (MultiMode Nanoscope IIIa, Bruker AXS KK , Billerica, Massachusetts, USA)
UV lamp (365 nm wavelength handheld; UVGL-58, 1.2 mW/cm2, UVP, CA, USA).

In order to investigate the effect of the type of alkyl layer and the composition of the polymer on the phase-separated structure of the substrate surface, and the effect of the photoisomerization of spirobenzopyran on the phase-separated structure of the substrate surface, C1 and HMDS silanes were used. Four different polymers (MMA:nBMA:SpMA = 146:155:25, 146:115:21, 146:74:11, 133:115:25) were added to the modified substrate at a concentration of 5 mg/mL, respectively. Spin-coated substrates were made (7 mg/mL was also made only for MMA:nBMA:SpMA=146:155:25 and 133:115:25). The surface microstructure was then observed using an atomic force microscope. The substrate was measured at room temperature in the air under the conditions of tapping mode, measurement range of 20 μm, and scan speed of 1 Hz. UV light irradiation was performed for 90 seconds from a distance of 1 cm from the substrate using a UV lamp (365 nm wavelength).

Results and Discussion FIG. 3 shows an image of the phase-separated surface structure by an atomic force microscope. First, we focused on the substrate before UV light irradiation. MMA : nBMA : SpMA = 146 : 74 : 11, 5 mg/mL modified substrate has some structure on the surface, but it was considered that phase separation did not occur from the height and the shape of the outline of the structure. . Regarding other polymer-modified substrates, it was confirmed that the same polymer formed a similar surface structure derived from phase separation, regardless of the type of silane agent. However, the shape of phase separation varied depending on the composition of the polymer. MMA : nBMA : SpMA = 146 : 155 : 25 The substrate modified with 5 mg/mL showed a white dot-like sea-island structure, and the substrate modified with 7 mg/mL showed white domains. showed a lamellar structure with a series of It was considered that this was caused by the change in the amount of polymer molecules existing on the substrate surface due to the difference in concentration when the substrate was modified with the polymer. The white dot domain portion that appeared on the substrate modified at 5 mg/mL was increased by the polymer molecule, which increased the area of the domain and fused with different white dot domains in the vicinity, resulting in the above lamellar structure. was showing. The 146 : 115 : 21 polymer showed a lamellar structure with continuous white domains, while the 133 : 115 : 25 polymer showed a sea-island structure with white dots. It was considered that these structural differences depend on the composition ratio of the polymer.
Next, we will focus on the substrate after UV light irradiation. MMA : nBMA : SpMA = 146 : 74 : 11 polymer-modified substrate showed no change regardless of UV light irradiation. Furthermore, in the MMA : nBMA : SpMA = 146 : 155 : 25 polymer, the substrate modified at 5 mg/mL on the two types of alkyl layers became white dot-like, resulting in the disappearance of the phase separation structure from the sea-island structure. , Of the substrates modified at 7 mg/mL, the HMDS-grafted alkyl chains showed a lamellar structure with a series of white domains and a phase-separated structure that disappeared. Among them, the substrate with C1-grafted alkyl chains showed only a transformation from a lamellar structure with a series of white domains to a sea-island structure with white dots, and the change was not as great as the disappearance of the phase-separated structure. The reason for this difference was thought to be related to the formation of a complex polymer network with multiple silane molecules by C1. When this C1 polymer network is close to a monolayer as in the HMDS modification, the photoisomerization of spirobenzopyran is sufficient as the driving force for phase-separated structural transformation without the polymer being trapped in the network. As the C1 polymer network becomes more complex, the modified polymer is trapped in the network, restricting the mobility of the molecule, and the photoisomerization of spirobenzopyran provides the driving force to eliminate the phase-separated structure. thought it wasn't. It could be considered that this was caused by the instability of the C1 modification. On the other hand, with respect to other polymers, similar conversion was observed with the same polymer regardless of the type of silane agent. Focusing on the 146 : 115 : 21 polymer-modified substrate, the lamellar structure with a series of white domains was transformed into a sea-island structure with white dots. However, due to the difference in the type of alkyl layer, the size of each dot in the sea-island structure, which had no difference before UV light irradiation, became different after UV light irradiation. There was unevenness in the size of the white dots, and in the case of the HMDS-modified substrate, there were many dots that looked as if adjacent dots were fused together.

Observation Material and Method for Reversible Switching of Phase Separation Structure by Visible Light In this example, the following materials were used.
Micro cover glass 24 mm x 24 mm (modified with polymer)
Cantilever (OMCL-AC240TS-W2, Olympus Corporation) (spring constant 2.0 N/m, resonance frequency 70 kHz)

In this example, the following apparatus was used.
Atomic force microscope (Dimension 3100 Nanoscope IV, Veeco, Plainview, New York , USA)
Atomic force microscope (MultiMode Nanoscope IIIa, Bruker AXS KK , Billerica, Massachusetts, USA)
UV lamp (365 nm wavelength handheld; UVGL-58, 1.2 mW/cm2, UVP, CA, USA).
LED (530 nm wavelength; LAJ4C, 8500 cd, Iwasaki Electric Co., Ltd., Tokyo, Japan).

In order to investigate the effect of spirobenzopyran on the phase-separated structure of the substrate surface by photoisomerization by UV light irradiation and visible light irradiation, respectively, three kinds of polymer MMA : nBMA : SpMA = 146 were applied to the HMDS-modified substrate. : 115 : 21, 133 : 115 : 25 and 127 : 118 : 28 were spin-coated at concentrations of 5, 7 and 10 mg/mL. The surface microstructure was then observed using an atomic force microscope. The substrate was at room temperature in the atmosphere, and the measurement mode was the tapping mode, the measurement range was 20 μm, and the scan speed was 1 Hz. UV light irradiation was performed for 90 seconds from a distance of 1 cm from the substrate. Visible light irradiation was performed for 1800 seconds from a distance of 20 cm from the substrate.

Results FIG. 4 shows an atomic force microscope image of the phase-separated surface structure in the reversible switching of the phase-separated structure by visible light. 5 and 6 show cross-sectional views of phase-separated surface structures. First, focus on the substrate before UV light irradiation. MMA : nBMA : SpMA = 146 : 115 : 21, 5 mg/mL modified substrate could not confirm the phase separation structure on the surface unlike previous results. It was confirmed that the other polymer-modified substrates formed a similar sea-island structure derived from phase separation, although there was a difference in the size of the white dots.
We pay attention to the difference in the phase separation structure due to the difference in the modification concentration during spin coating for each polymer. At MMA : nBMA : SpMA = 146 : 115 : 21, the area per dot was larger at 10 mg/mL than at 7 mg/mL. In addition, on the MMA : nBMA : SpMA = 133 : 115 : 25 polymer-modified substrate, the area per dot increased as the modification concentration during spin coating increased.
Next, we will focus on the substrate after UV light irradiation. On all modified substrates, the surface structure due to phase separation disappeared by UV light irradiation. After further irradiation with visible light, observation revealed that the polymer-modified substrate with MMA : nBMA : SpMA = 146 : 115 : 21 recovered the surface structure similar to that before UV light irradiation, regardless of the modification concentration. rice field. On the MMA : nBMA : SpMA = 133 : 115 : 25 polymer-modified substrate, the surface structure derived from phase separation was recovered by visible light irradiation, but the shape of the recovered surface structure differed depending on the modification concentration. The substrate modified with 5 mg/mL had a shape intermediate between a lamellar structure with white dots and a sea-island structure. The substrate modified at 7 mg/mL showed brown dots, and the surface structure was as if the white and brown domains were reversed. At 10 mg/mL, there was a tendency for a slight series of white dots to appear, but the shape was generally similar to that of the surface structure before UV light irradiation.
After further irradiation with UV light, observation revealed that the surface structure of all the modified substrates had disappeared, as was the case with the initial UV light irradiation. From the above, it can be concluded that a surface structure derived from phase separation is formed on a substrate modified with polymers of two types of composition regardless of the modification concentration, and that structure can be destroyed by UV light irradiation and restored by visible light irradiation. It was shown to have reversible properties.
In addition, the fine uneven structure on the surface when irradiated with visible light had a height difference of 19.23±1.35 nm, and this height difference almost disappeared when irradiated with UV light (Fig. 6).

Application of photoswitchable phase separation structure surface to cell culture substrate (1)
Materials and Methods In this example, the following materials were used.
Bovine aortic endothelial cells (BAECs; B304-05, used for passage numbers 16, Cell Applications, San Diego, USA)
Micro cover glass 24 mm x 24 mm x 0.13-0.17 mm (modified with polymer)
Dulbecco's Modified Eagle Medium (DMEM; Lot# L7N3114, Wako Pure Chemicals, Osaka, Japan)
Fetal bovine serum (FBS; Lot# 12868, Biowest, Nuaille, France)
Penicillin and streptomycin (P/S; Lot# 1864855, Gibco Life Technologies, Grand Island, NY, USA)
Dulbecco's phosphate buffered saline (PBS; Lot# L7K2513, Sigma-Aldrich, St. Louis, MO, USA
Trypsin/EDTA (Trypsin/ethylenediaminetetraacetic acid; T/E; Lot# TWP7024, Sigma-Aldrich, St. Louis, MO, USA)

In this example, the following apparatus was used.
Phase contrast microscope (Eclipse Ti-E/B, Nikon Corporation, Tokyo, Japan)

The medium used for cell culture was DMEM with FBS at a final concentration of 10% and PS at a final concentration of 1%. 1×PBS was prepared by diluting 10×PBS with sterilized water. 1xT/E was prepared by diluting 10xT/E with 1xPBS. These reagents were stored at 4°C and warmed to 37°C before use. Cells were subcultured by inoculating a cell culture dish (φ100 mm) to 10% confluency and culturing in a 37°C, 5% CO ₂ incubator. Here, 2.0×10 ⁵ cells/cm ² is defined as BAEC confluency. Passaging was performed once every 3 days before reaching 100% confluence. Regarding the subculture operation, after removing the medium with an aspirator, the cells were washed twice with 1×PBS. After that, 1×T/E was added and reacted in a 37° C., 5% CO ₂ incubator for about 3 minutes to detach the cells from the dish. After stopping the T/E reaction by adding medium, the cell pellet was collected by centrifugation at 1200 rpm for 3 minutes. The supernatant was removed and fresh medium was added to prepare a cell suspension, which was used to inoculate a new TCPS dish.

In order to evaluate whether the cells seeded on the prepared polymer-modified substrate adhere and spread, the cell morphology was observed using a phase-contrast microscope on days 1 to 4 and day 7 after seeding. As a control, cells seeded on a TCPS φ100 mm dish were used. A small amount of instant adhesive was applied to the four corners of the prepared polymer-modified substrate, and the substrate was pasted on a Petri dish of φ100 mm. For comparison, a portion of the Petri dish where no substrate was adhered was also observed. FIG. 7 is a photomicrograph of cells cultured on a cell culture substrate having a photoswitchable phase-separated structure surface. No radiography was performed on the magnified images on day 1 of TCPS and on day 7 of TCPS (they reached confluence at day 4). Focusing on the results of day 1 and day 2, compared with the behavior of the cells seeded on TCPS, many of the cells on the polymer-modified substrate had a round morphology and did not develop sufficiently due to insufficient adhesion. This was also true for Petri dishes, where we found that polymer-modified substrates adhered slowly. As the number of culture days increased, TCPS reached confluence on day 3-day 4, whereas cells seeded on polymer-modified substrates and petri dishes reached 30% and 50-60% confluence on day 7, respectively. only reached On the other hand, on day 7, most of the adherent cells stretched out their arms and could also be observed to be stretched.

By using the cell culture substrate having a phase-separated structure surface of the present invention, cell behavior such as cell detachment can be controlled.

Application of photoswitchable phase separation structure surface to cell culture substrate (2)
In the same manner as in Example 10, in order to investigate the effect of spirobenzopyran on the phase separation structure of the substrate surface and cultured cells due to photoisomerization by UV light irradiation and visible light irradiation, A polymer (MMA:nBMA:SpMA = 133:115:25) was spin-coated at a concentration of 10 mg/mL on the HMDS-laminated modified substrate to fabricate a phase-separated structural system.
Thereafter, in the same manner as in Example 11, C2C12 (P10-15) was seeded on the surface of the phase separation structure system at 2.0×10 ⁴ cells/cm ² , cultured for 2 days, and cell adhesion and detachment were observed. Observation was made using a phase-contrast microscope.
In addition, when UV light irradiation was performed at a distance of 1 cm from the substrate for 90 seconds, detachment of the cells was induced and removed. The surface of the phase separation structure system was washed with 1×PBS. After cleaning, visible light irradiation was performed for 1800 seconds from a distance of 20 cm from the substrate. In addition, cell seeding, UV light irradiation, washing and visible light irradiation were repeated four times.

Results Figure 8 shows the adhesion, detachment, re-adhesion, and re-detachment of cultured cells when cells cultured on a cell culture substrate having a phase-separated structure surface capable of photoswitching were repeatedly irradiated with UV light and visible light. It is the figure which represented typically the observed micrograph, surface structure, and the change of adhesiveness of a cell. By using the cell culture substrate having the phase-separated structure surface of the present invention as a phase-separated structure system, cell detachment could be reversibly controlled.

Application of photoswitchable phase separation structure surface to cell culture substrate (3)
In the same manner as in Example 10, in order to examine the effect of spirobenzopyran on the phase-separated structure of the substrate surface and cultured cells due to photoisomerization by UV light irradiation and visible light irradiation, respectively, a polymer was applied to the HMDS-modified substrate. MMA : nBMA : SpMA = 133 : 115 : 25 was spin-coated at a concentration of 10 mg/mL. After that, three types of substrates were prepared: a substrate not irradiated with UV light and visible light, a substrate irradiated with UV light, and a substrate irradiated with UV light and visible light. C2C12 (P10-15) was seeded on each of these three substrates at 2.0×10 ⁴ cells/cm ² in the same manner as in Example 11, cultured for 2 days, and the cells were observed using a phase-contrast microscope. bottom.

Results FIG. 9 shows micrographs of cells cultured on cell culture substrates irradiated with different lights and irradiated with UV light or visible light.
In three types of substrates (initial state): a substrate not irradiated with UV light and visible light, a substrate irradiated with UV light, and a substrate irradiated with UV light and visible light, regardless of the presence or absence of a phase separation structure, Cell spreading, adhesion and proliferation were observed.
Three types of substrates: substrates irradiated with UV light or visible light, substrates not irradiated with UV light and visible light, substrates irradiated with UV light, and substrates irradiated with UV light and visible light (after light irradiation) state), induction of cell detachment was observed upon irradiation with visible or UV light, which induces a transition in phase separation.
That is, it was clarified that the cells do not recognize the phase separation structure itself, but the dynamic conversion of the phase separation structure.

Claims (15)

A block copolymer characterized by being represented by the following formula (I):

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however,
Ring A is a C ₅ to C ₈ aromatic ring,
R ¹ is a C ₁ to C ₁₈ optionally substituted linear or branched alkyl group,
R ² is H or an optionally substituted linear or branched C ₁ to C ₁₇ alkyl group;
The number of carbon atoms in R ¹ > the number of carbon atoms in R ² and
^R3 , ^R4 and ^R5 are each independently selected from H or _-CH3 ;
Xa is selected from O or S;
Ya is selected from _-NO2 , -CN, -COOH, -F, -Cl, -Br, -I, -OH, _-NH2 , _-CH3 , and
n is 5 to 200,
x is between 5 and 200;
y is 5-50. In the above formula (I),
n is from 100 to 180,
x is between 60 and 140;
The block copolymer according to claim 1, characterized in that y is 9-30. The block copolymer according to claim 1, wherein the compound of formula (I) is represented by formula (II):

______________________________________________________________________
however,
n is from 133 to 146,
x is between 74 and 115;
y is 11-25. Phase separation characterized in that the compound of formula (I) according to any one of claims 1 to 3 is coated on an organic layer surface-fixed to the surface of a substrate made of glass or silicon. structure system. 5. The phase separation according to claim 4, wherein the surface-immobilized organic layer is an organic layer having an organic compound immobilized on the surface by a silicon compound reagent or an organic layer containing a random copolymer. structure system. The silicon compound reagent is trimethoxy(methyl)silane or 1,1,1,3,3,3-hexamethyldisilazane, 1,3-dibutyl-1,1,3,3-tetramethyldisilazane or 1, 6. A phase-separated structure system according to claim 5, characterized in that it contains a compound selected from 3-diethyl-1,1,3,3-tetramethyldisilazane. The phase-separated structure system according to claim 5, wherein the random copolymer has a structure of formula (III):

however,
^R6 is -COOH or _-CH2OH ,
^R7 is _-C6H5 or _-S- ( _CH2 ) ₁₁ - _CH3 ;
p is between 20 and 200;
q is between 20 and 200; In the formula (III),
p is between 55 and 60;
q is between 54 and 59;
The phase-separated structure system according to claim 7, characterized in that: The phase separation structure according to any one of claims 4 to 8, wherein the phase separation structure system has a phase separation structure in which a reversible surface phase separation structure changes in response to light. body system. 10. The phase-separated structure system according to claim 9, wherein the change in the surface phase-separated structure accompanies a change in the fine uneven structure of the surface, the wettability of the film surface, or the hardness. The phase separation structure system according to any one of claims 4 to 10, characterized in that said phase separation structure system is a substrate for cell culture. The cell culture substrate reversibly adheres and detaches cultured cells or detaches and re-adheres adhered cultured cells in response to light, or differentiates and spreads cultured cells. 12. The phase-separated structure system according to claim 11 , which regulates , migration, or changes in gene and protein expression. Block copolymer according to any one of claims 1 to 3: A method for producing PMMA-bP (nBMA-stat-SpMA),
As shown in Reaction Formula 1 below, 4,4′-azobis(4-cyanopentanoic acid) is used as a polymerization initiator, and 4,4′-azobis(4-cyanopentanoic acid) is used as a RAFT polymerization chain transfer agent. A step of reacting methylmethacrylate with 4-cyanopentanoic acid dithiobenzoate to prepare PMMA (poly-methylmethacrylate);

provided that n is 5 to 200;

next,
As represented by the following Reaction Scheme 2, 4,4′-azobis(4-cyanopentanoic acid) was used to react the PMMA with SpMA (1′-(2-Methacryloyloxyethyl)-3′,3′-dimethyl). -6-nitrospiro(2H-1-benzopyran-2,2'-indoline)) and n-butyl methacrylate reacting,
A manufacturing method, characterized in that it comprises;

however,
n is 5 to 200,
x is between 5 and 200;
y is 5-50. Use of block copolymers according to any one of claims 1 to 3 for the production of phase-separated structural systems. Reversibly changing the surface phase separation structure in response to light, and by the change, adhesion and detachment of cultured cells, detachment and re-adhesion of adhered cultured cells, or differentiation of cultured cells , spreading, migration or changes in gene and protein expression.

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