Star-Shaped Thermoresponsive Polymers with Various Functional

Dec 19, 2017 - This study demonstrates the facile preparation of poly(N-isopropylacrylamide) (PNIPAM)-immobilized Petri dishes by drop-casting a star-...
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Article Cite This: Langmuir 2018, 34, 653−662

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Star-Shaped Thermoresponsive Polymers with Various Functional Groups for Cell Sheet Engineering Yu Sudo,† Ryuki Kawai,† Hideaki Sakai,‡ Ryohei Kikuchi,§ Yuta Nabae,*,† Teruaki Hayakawa,† and Masa-aki Kakimoto† †

Department of Materials Science and Engineering, School of Materials and Chemical Technology, Tokyo Institute of Technology, 2-12-1 S8-26, Ookayama, Meguro-ku, Tokyo 152-8552, Japan ‡ Zellech Inc., Studio3 10F, KFC-Bldg., 1-6-1, Yokoami, Sumida-ku, Tokyo 130-0015, Japan § Ookayama Materials Analysis Division, Technical Department, Tokyo Institute of Technology, 2-12-1 S7-26, Ookayama, Meguro-ku, Tokyo 152-8552, Japan S Supporting Information *

ABSTRACT: This study demonstrates the facile preparation of poly(N-isopropylacrylamide) (PNIPAM)-immobilized Petri dishes by drop-casting a star-shaped copolymer of hyperbranched polystyrene (HBPS) possessing PNIPAM arms (HBPS-g-PNIPAM) functionalized with polar groups. HBPS was synthesized via reversible addition− fragmentation chain transfer (RAFT) self-condensing vinyl polymerization (SCVP), and HBPS polymers with different terminal structures were prepared by changing the monomer structure. HBPS-g-PNIPAM was synthesized by the grafting of PNIPAM from each terminal of HBPS. To tune the cell adhesion and detachment properties, polar functional groups such as carboxylic acid and dimethylamino groups were introduced to HBPS-g-PNIPAM. Based on surface characterization using scanning transmission electron microscopy (STEM), X-ray photoelectron spectroscopy (XPS), and contact angle measurements, the advantage of the hyperbranched structure for the PNIPAM immobilization was evident in terms of the uniformity, stability, and thermoresponsiveness. Successful cell sheet harvesting was demonstrated on dishes coated with HBPSg-PNIPAM. In addition, the cell adhesion and detachment properties could be tuned by the introduction of polar functional groups.

1. INTRODUCTION Recently, tissue engineering, an innovative medical technology, has developed rapidly. Cell sheet engineering is a key technological requirement for tissue engineering, in which cell sheets can be prepared by cultivating cells collected from patients, which are subsequently implanted back to regenerate damaged tissues. Okano and co-workers1−3 have reported many examples of successful cell sheet recovery from thermoresponsive cell culture dishes modified with poly(Nisopropylacrylamide) (PNIPAM), which is known to have a lower critical solution temperature (LCST) at 32 °C in aqueous solution.4 Such PNIPAM-immobilized polystyrene (PS) Petri dishes show good affinity for cells above the LCST but poor affinity below the LSCT. Commercial dishes are currently manufactured by electron beam polymerization,5,6 which requires complicated equipment and is expensive; moreover, it is difficult to modify the PNIPAM chemical structure. To modify Petri dishes with PNIPAM easily, plasma polymerization,7,8 the grafting-onto method,9 the grafting-from method,10,11 and the casting method12,13 have been developed. Our research group has been developing promising polymer materials for cell sheet engineering.13−16 We have focused on © 2017 American Chemical Society

copolymers that can generate thermoresponsive cell culture dishes by simple drop-casting onto ordinary PS Petri dishes. Copolymers of hyperbranched polystyrene and PNIPAM (HBPS-g-PNIPAM) have been demonstrated to be good modifiers for PS dishes, exhibiting excellent temperatureresponsive behavior. In the next stage, tailor-made thermoresponsive surfaces are required to target a wider range of cells, and the introduction of various functional groups to HBPS-gPNIPAM will enable the fine-tuning of the resulting surface. Self-condensing vinyl polymerization (SCVP) was proposed and developed by Fréchet,17,18 Matyjaszewski,19,20 and their co-workers to synthesize hyperbranched polymers using an AB* monomer that contains an initiation site for atom transfer radical polymerization (ATRP). The applicability of SCVP can be expanded to other controlled radical polymerizations, such as (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO)-mediated polymerization18,21,22 and reversible addition−fragmentation chain transfer (RAFT)23 polymerization. Because of the high Received: December 12, 2017 Revised: December 19, 2017 Published: December 19, 2017 653

DOI: 10.1021/acs.langmuir.7b04213 Langmuir 2018, 34, 653−662

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Figure 1. Synthesis of various HBPS-g-PNIPAM samples.

2. EXPERIMENTAL SECTION

compatibility with various monomers and the mild reaction conditions of RAFT polymerization, RAFT-SCVP is expected to be a promising methodology to synthesize hyperbranched polymers with flexible designs.24−27 In this study, RAFT-SCVP was utilized to synthesize HBPSs using different styrene monomers to prepare different terminal structures. Because such HBPSs have a chain transfer agent (CTA) at the termini, they were used as macro CTAs25,27−30 for subsequent RAFT polymerization of NIPAM to obtain star-shaped copolymers (HBPS-g-PNIPAM). Furthermore, this type of HBPS-gPNIPAM is easily functionalized with polar groups because it can be copolymerized with various functional monomers. In this study, carboxyl groups and amino groups were introduced into the copolymers to vary their affinity for cells, enabling effective cell cultivation.10,31,32 This paper describes the synthesis of the polymers summarized in Figure 1 and their application to cell sheet engineering. Various thermoresponsive surfaces were prepared by simply drop-casting these copolymers onto ordinary PS Petri dishes. The role of the hyperbranched structure on the polymer immobilization is investigated by surface analyses using scanning tunneling electron microscopy (STEM), X-ray photoelectron spectroscopy (XPS), and contact angle measurements. In addition, the effect of polar functional groups introduced to star-shaped copolymer is also determined by examining the cell adhesion and detachment properties.

2.1. Materials. 2,2′-Azobis(isobutyronitrile) (AIBN, 98%) was purchased from Sigma-Aldrich Japan and recrystallized from methanol. N-(Isopropylacrylamide) (NIPAM, > 98.0%) was purchased from Tokyo Chemical Industry Co., Ltd. (TCI) and recrystallized three times from hexane. t-Butyl acrylate (TCI, > 98.0%) and 2-(dimethylamino)ethyl acrylate (TCI, > 98.0%) were purified by passage through an alumina column before use. Silica gel (average pore size 6 nm) was purchased from Fuji Silysia Chemical Ltd. 2-(Dodecylthiocarbonothioylthio)-2-methylpropanoic acid was prepared by a literature method.33 The other reagents were purchased from TCI, Sigma-Aldrich Japan, or Wako Pure Chemical Industries, Ltd. and used without further purification. 2.2. Methods. NMR (1H, 400 MHz) spectra were obtained using a JNM-ECS 400 NMR spectrometer (JEOL). The molecular weights of the prepared HBPS-C3, -C6, and -C12 were determined by gel permeation chromatography (GPC) in tetrahydrofuran eluent relative to PS standards using a Shodex GPC-101 system equipped with a refractive index (RI) detector and an LF-804 column (Showa Denko). The molecular weights of the copolymers were determined by GPC in N,N-dimethylformamide (DMF) containing 0.05 M lithium bromide relative to PS standards using a Viscotek TDA 302 system with a refractive index detector and a TSKgel α-M column (Tosoh Corporation). Contact angle measurements were performed at 50 and 20 °C with 2.0 μL of deionized water using Kyowa DM-501YH equipped with a homemade thermostatic chamber. XPS measurements were conducted using a JSP-9010MC (JEOL) spectrometer equipped with a monochromatic Al Kα source. A cross section of the polymer-coated substrate was obtained using an Ultracut UCT ultramicrotome (Leica) and observed using an SU9000 (Hitachi 654

DOI: 10.1021/acs.langmuir.7b04213 Langmuir 2018, 34, 653−662

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70 °C for 24 h, and reprecipitated into a water/methanol (v/v = 1/4) solution. The residue was collected and dried in vacuo overnight at 40 °C to obtain HBPS-tBA1-C3 (yield 81%). Other samples (x = 5, 10, and 20) were synthesized by changing the stoichiometric ratio of tbutyl acrylate to the terminal groups of 2a. For deprotection, HBPS-tBA1-C3 (0.5 g), trifluoroacetic acid (1.53 g, 13.4 mmol), and dichloromethane (5.0 mL) were placed in a 10 mL round-bottomed flask. The solution was stirred at room temperature for 48 h. After removing the trifluoroacetic acid and dichloromethane by rotary evaporation, a dichloromethane/methanol (v/v = 10/1) solution was added, and the mixture was concentrated again by rotary evaporation. The obtained brown solid was dried in vacuo at 45 °C overnight, yielding HBPS-AA1-C3. 2.3.5. Synthesis of HBPS-g-AA-b-PNIPAM (HBPS-AAx-PNIPAMC3). HBPS-AA1-C3 (12.7 mg), NIPAM (0.350 g, 3.08 mmol), AIBN (0.612 mg), THF (0.67 mL), and methanol (0.33 mL) were added to a 5 mL ampule. After degassing the solution and sealing the vial in a similar manner as described above, the mixture was stirred at 70 °C for 24 h and subsequently reprecipitated into a water/methanol (v/v = 1/4) solution. The precipitate was dried in vacuo at 40 °C overnight to obtain HBPS-AA1-PNIPAM-C3 (yield 76%). 2.3.6. Synthesis of HBPS-g-DMA (HBPS-DMAx-C3). 2a (0.400 g, 1.49 mmol of termini), 2-(dimethylamino)ethyl acrylate (DMA, 0.320 g, 2.24 mmol), AIBN (24.5 mg, 0.149 mmol), toluene (1.24 mL), and THF (1.24 mL) were added into a 5 mL ampule. After degassing the solution and sealing the vial in a similar manner as described above, the mixture was stirred at 70 °C for 24 h and reprecipitated into cold hexane. The precipitate was dried in vacuo at 40 °C overnight to obtain the HBPS-DMA1-C3 polymer (yield 76%). 2.3.7. Synthesis of HBPS-g-DMA-b-PNIPAM (HBPS-DMAx-PNIPAM-C3). HBPS-DMA1-PNIPAM-C3 was prepared by the RAFT polymerization of NIPAM from polymer HBPS-DMA1-C3 in a similar manner to the synthesis of HBPS-AA1-PNIPAM-C3 (yield 80%). 2.3.8. Preparation of Linear Polystyrene and Its Block Copolymer with PNIPAM (LPS-PNIPAM). LPS-PNIPAM was synthesized by a previously reported method.15 Styrene (1.71 g, 16.4 mmol), 2(dodecylthiocarbonothioylthio)-2-methylpropanoic acid (17.1 mg, 47 mmol), AIBN (0.772 mg, 4.7 mmol), and dry THF (2 mL) were added to a 5 mL ampule and degassed by three freeze−pump−thaw cycles. After the ampule had been sealed, the mixture was stirred at 70 °C for 24 h. After quenching the reaction by dipping the ampule into ice water, the reaction mixture was diluted with THF and poured into hexane. After filtration, the collected white solid was dried at 40 °C in vacuo overnight to obtain polymer 3. The obtained polymer 3 (13 mg) was further reacted with NIPAM (0.5 g, 4.42 mmol) and AIBN (0.032 mg, 0.19 mmol) in a 5 mL ampule for RAFT polymerization in the manner described above. 2.3.9. Preparation of Polymer-Coated Dishes (HBPS-PNIPAM/ dish, HBPS-AAx-PNIPAM-C3/dish, HBPS-DMAx-PNIPAM-C3/dish, and LPS-PNIPAM/dish). HBPS-PNIPAM-C3 was dissolved in THF/methanol (v/v = 1/4) (concentration of PNIPAM segment: 6.66 mg in 10 mL), drop-cast onto a commercial PS Petri dish (Becton, Dickinson and Company; Falcon3001, 9.6 cm2), and dried in air at room temperature for 90 min to obtain the product, which is denoted HBPS-PNIPAM-C3/dish. The other samples, HBPSPNIPAM-C6/dish, HBPS-PNIPAM-C12/dish, HBPS-AA-PNIPAMC3/dish, HBPS-DMA-PNIPAM-C3/dish, and LPS-PNIPAM/dish, were prepared in the same manner using the corresponding copolymers. 2.3.10. Cell-Culture Experiments. Mouse 3T3 fibroblasts were seeded on the polymer-coated dishes as follows. Two milliliters of medium (Dulbecco’s modified Eagle medium - high glucose; DMEM) with 10% fetal calf serum were placed into a polymer-coated dish, and a medium containing 1 × 105 mouse 3T3 fibroblasts was added. The cells were cultivated in a CO2 incubator (37 °C, 5% CO2). After 4 days, the surface of the culture dish was observed with an optical microscope at 37 °C. Subsequently, the sample was placed in an incubator (20 °C, 5% CO2) for 15 min, and the surface was observed again.

High-Technologies) equipped with a Genesis APEX energy dispersive X-ray (EDX) spectrometer (EDAX). 2.3. Experimental Procedure. 2.3.1. Preparation of Styrene Monomers with Various CTAs (1a−c). Chain transfer monomer 1a was synthesized as previously reported.28 Sodium methoxide (TCI, 5 mol/L in methanol) (4.04 mL, 0.0202 mol) and dry methanol (36 mL) were placed in a 100 mL round-bottomed flask, and 1propanethiol (TCI, > 98.0%, 0.02 mol) was slowly added. The mixture was stirred in a nitrogen atmosphere at room temperature for 15 min. Carbon disulfide (TCI, > 98.0%, 1.9 g, 0.025 mol) was added dropwise, and the mixture was stirred for an additional 15 min. 4Vinylbenzyl chloride (TCI, > 90%, 3.72 g, 0.022 mol) was then added, and the mixture was stirred for 3 h. After adding water to the reaction solution, the product was extracted with dichloromethane. The organic layer was washed with pure water three times, dried over magnesium sulfate, and the solvent removed by rotary evaporation. The crude product was purified by silica gel chromatography using hexane as an eluent. The target compound 1a was obtained as a bright yellow viscous oil. The other monomers, 1b and 1c, were prepared in a similar manner using 1-hexanethiol and 1-dodecanethiol, respectively. 1a: yield 87%; 1H NMR (CDCl3, δ in ppm): 7.36 (d, 2H, aromatic), 7.30 (d, 2H, aromatic), 6.69 (dd, 1H, CH-), 5.73 and 5.25 (d, 2H, CH2), 4.60 (s, 2H, Ph−CH2-S-), 3.36 (t, 2H, −CH2−C2H5), 1.74 (m, 2H, −CH2−CH3), 1.02 (q, 3H, −CH3). 1b: yield 86%; 1H NMR (CDCl3, δ in ppm): 7.36 (d, 2H, aromatic), 7.29 (d, 2H, aromatic), 6.69 (dd, 1H, CH-), 5.73 and 5.25 (d, 2H,  CH2), 4.60 (s, 2H, Ph−CH2−S-), 3.36 (t, 2H, −CH2−C5H11), 1.70 (m, 2H, −CH2−C4H9), 1.45−1.35 (m, 2H, −CH2−C3H7), 1.31−1.27 [m, 4H, −(CH2)2−CH3], 0.89 (t, 3H, −CH3). 1c: yield 85%; 1H NMR (CDCl3, δ in ppm): 7.36 (d, 2H, aromatic), 7.30 (d, 2H, aromatic), 6.67 (dd, 1H, CH-), 5.74 and 5.25 (d, 2H, CH2), 4.60 (s, 2H, Ph−CH2−S-), 3.37 (t, 2H, −CH2−C11H23), 1.70 (m, 2H, −CH2−C10H21), 1.43−1.36 (m, 2H, −CH2−C9H19), 1.36−1.26 [m, 16H, −(CH2)8−CH3], 0.88 (t, 3H, −CH3). 2.3.2. Synthesis of Hyperbranched Polystyrene (HBPS, 2a−c). CTA styrene monomer 1a (0.500 g, 1.86 mmol), AIBN (30.6 mg, 0.186 mmol), and toluene (0.34 mL) were added to a 5 mL ampule and degassed by three freeze−pump−thaw cycles. The ampule was sealed, heated with stirring at 65 °C for 24 h, and then dipped into ice water to quench the reaction. The reaction mixture was diluted with THF and poured into cold hexane. The viscous polymer (2a) was collected by decantation and dried under vacuum at 45 °C. 2b and 2c were synthesized in a similar manner to 1b and 1c, respectively. 2a: yield 90%; 1H NMR (CDCl3, δ in ppm): 7.3−6.4 (aromatic), 4.7−4.4 (Ph−CH2-S, S−CH), 3.4−3.3 [−CH−SC(S)S−CH2-], 3.3−3.1 [−CH2−SC(S)S−CH2-], 1.8−1.6 (−CH2−), 1.1−0.8 (−CH3). 2b: yield 87%; 1H NMR (CDCl3, δ in ppm): 7.4−6.4 (aromatic), 4.7−4.4 (Ph−CH2−S, S−CH), 3.4−3.3 [−CH−SC(S)S−CH2-], 3.3−3.2 [−CH2−SC(S)S−CH2-], 1.8−1.2 (−CH2−), 1.0−0.8 (−CH3). 2c: yield 85%; 1H NMR (CDCl3, δ in ppm): 7.4−6.3 (aromatic), 4.8−4.4 (Ph−CH2−S, S−CH), 3.5−3.3 [−CH−SC(S)S−CH2-], 3.3−3.2 [−CH2−SC(S)S−CH2-], 1.8−1.2 (−CH2−), 1.1−0.8 (−CH3). 2.3.3. Synthesis of HBPS-g-PNIPAM (Grafting of NIPAM from HBPS) (HBPS-PNIPAM-C3, C6, C12). 2a (0.020 g, 0.075 mmol of terminal), NIPAM (0.70 g, 6.2 mmol), AIBN (1.22 mg, 0.0075 mmol), THF (1.0 mL), and toluene (1.0 mL) were added to a 5 mL ampule and degassed by three freeze−pump−thaw cycles. The ampule was sealed, heated under stirring at 65 °C for 24 h, and then dipped into ice water to quench the reaction. The reaction mixture was diluted with THF and poured into a hexane/acetone (v/v = 7/3) solution. After filtration, the residue was dried in vacuo at 40 °C overnight to obtain HBPS-PNIPAM-C3 as a whitish powder (yield 88%). HBPS-PNIPAM-C6 and HBPS-PNIPAM-C12 were synthesized in a similar manner to 2b and 2c, respectively. 2.3.4. Synthesis of HBPS-g-tBA and HBPS-g-AA (Modification of HBPS Termini with Carboxylic Acids) (HBPS-AAx-C3). 2a (0.4 g, 1.49 mmol of terminal), t-butyl acrylate (0.287 g, 2.24 mmol), AIBN (24.5 mg, 0.149 mmol), toluene (1.24 mL), and THF (1.24 mL) were added to a 5 mL ampule. After degassing the solution and sealing the vial in a similar manner as described above, the mixture was stirred at 655

DOI: 10.1021/acs.langmuir.7b04213 Langmuir 2018, 34, 653−662

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Figure 2. 1H NMR spectra of the polymers obtained from 2a. Solvent: DMSO-d6 for HBPS-AA20-C3 and chloroform-d for the others.

Table 1. Characterization of Hyperbranched Polystyrene and Copolymers functional group sample

Mna (PDI)

DB

CNIPAMb/ wt %

LPS-PNIPAM HBPS (2a) HBPS (2b) HBPS (2c) HBPS-PNIPAM-C3 HBPS-PNIPAM-C6 HBPS-PNIPAM-C12 HBPS-AA1-PNIPAM-C3 HBPS-AA5-PNIPAM-C3 HBPS-AA10-PNIPAM-C3 HBPS-AA20-PNIPAM-C3 HBPS-DMA1-PNIPAM-C3 HBPS-DMA5-PNIPAM-C3 HBPS-DMA10-PNIPAM-C3 HBPS-DMA20-PNIPAM-C3

113600 (1.2) 4900 (3.4) 5400 (2.0) 7100 (1.7) 142000 (4.6) 125000 (3.9) 101000 (2.7) N.A. N.A. N.A. N.A. 151000 (5.1) 198000 (3.4) 39000 (6.7) 34300 (4.0)

0.40 0.41 0.38 -

94.0 -

-

95.7 91.5 93.6 91.5 89.4 85.4 80.8 92.1 83.8 78.5 73.4

-

-

-

density/mmol g−1

type

-COOHc

-N(CH3)2d

0.29 0.86 1.65 2.28 0.20 0.82 1.26 1.70

terminal type

density/mmol g−1

-C12H25 -C3H7 -C6H13 -C12H25 -C3H7 -C6H13 -C12H25 -C3H7

0.0088 3.7 3.2 2.5 0.16 0.28 0.24 0.31 0.39 0.54 0.71 0.29 0.60 0.80 0.98

-C3H7

a

Determined by GPC using PS calibration. bPNIPAM content calculated from eq 5 for HBPS-DMAx-PNIPAM-C3 samples, and eq 4 for the other samples. cQuantified by back-titration with NaOH (aq) and HCl (aq). dQuantified by back-titration with a p-toluenesulfonic acid aqueous solution and NaOH (aq).

3. RESULTS AND DISCUSSION

r=

3.1. Preparation of HBPS, HBPS-g-PNIPAM, HBPS-gPAA-b-PNIPAM, and HBPS-g-PDMA-b-PNIPAM. The molecular architectures and synthetic scheme for the polymers in this study are summarized in Figure 1. HBPSs with propyl (2a), hexyl (2b), and dodecyl (2c) terminal groups were synthesized by the RAFT-SCVP of the corresponding monomers (1a−c). The number-average degree of polymerization of the hyperbranched polystyrenes was estimated to be 18 (for 2a), 17 (for 2b), and 18 (for 2c) from their molecular weights. To evaluate the degree of branching (DB) of the hyperbranched polystyrene, the purified polymer was analyzed by 1H NMR spectroscopy (Figures 2A and S1). DB was determined using eqs 1 and 2 based on the SCVP kinetic theory according to the previous literature,34,35 where r is the reactivity ratio, B* is the ratio of unreacted chain transfer agent group, and z is a parameter introduced for the variable transformation of reaction time (details in the Supporting Information, Figure S1). The DB values are summarized in Table 1.

e −z

e −z +z−1

(1)

DB = 2e−z(1 − e−z)

(2)

B* B* − ln B* − 1

(3)

r=

HBPS-AAx-C3 and HBPS-DMAx-C3 (x = 1, 5, 10, 20) were synthesized by modifying HBPS (2a) with acrylic acid and dimethylaminoethyl acrylate, respectively, where x represents the stoichiometric number of the units introduced to the terminal. The actual quantity of polar functional groups in HBPS-AAx-C3 and HBPS-DMAx-C3 was determined by backtitration. The HBPS-AAx-C3 samples were dispersed in NaOH aqueous solutions. Subsequently, the residual NaOH was titrated with an HCl aqueous solution. The HBPS-DMAx-C3 samples were also tested in a similar manner using aqueous solutions of p-toluenesulfonic acid and NaOH. As shown by the results summarized in Table 1, the polar functional groups were successfully introduced to the termini of the hyperbranched polystyrene. 656

DOI: 10.1021/acs.langmuir.7b04213 Langmuir 2018, 34, 653−662

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Figure 3. GPC-RI traces of (A) HBPS-C3, -C6, and -C12 in THF and (B) HBPS-PNIPAM-C3, -C6, and -C12 in DMF containing lithium bromide.

Figure 4. STEM images of (A) HBPS-PNIPAM-C6/dish and (B) LPS-PNIPAM/dish.

Figure 5. XPS spectra at C 1s and N 1s regions of (A, B) HBPS-PNIPAM-C6/dish before the rinse, (C, D) HBPS-PNIPAM-C6/dish after the rinse, (E, F) LPS-PNIPAM/dish before the rinse, and (G, H) LPS-PNIPAM/dish after the rinse.

the nitrogen content obtained by the elemental analysis, and 0.1237 is the theoretical nitrogen content in the homo PNIPAM. The CNIPAM value of the sample containing dimethylamino ethyl groups (HBPS-DMAx-PNIPAM) was calculated by combining the elemental analysis and 1H NMR spectra based on eq 5. In eq 5, ANIPAM and ADMA are the integrated 1H NMR peaks (Figure 2F) corresponding to NIPAM (4.01 ppm) and DMA (2.55 ppm) units, respectively. The calculated values are summarized in Table 1.

PNIPAM was grafted from 2a, 2b, and 2c to obtain the starshaped copolymers HBPS-PNIPAM-C3, HBPS-PNIPAM-C6, and HBPS-PNIPAM-C12, respectively. In the preparation of 2a, 2b, and 2c, the number of chain transfer agents in a single molecule corresponds to the number of the component units, i.e., the degree of polymerization. Therefore, the resulting starshaped copolymers have approximately 18 (for 2a), 17 (for 2b), and 18 (for 2c) arms per molecule. Similarly, PNIPAM was grafted from HBPS-AAx-C3 and HBPS-DMAx-C3 to obtain HBPS-AAx-PNIPAM-C3 and HBPS-DMAx-PNIPAMC3, respectively. The PNIPAM contents (CNIPAM) in HBPSPNIPAM-C3, -C6, -C12, and HBPS-AAx-PNIPAM-C3 were determined by elemental analysis using eq 4, where Cnitrogen is

C NIPAM = 657

Cnitrogen 0.1237

× 100

(4) DOI: 10.1021/acs.langmuir.7b04213 Langmuir 2018, 34, 653−662

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Langmuir C NIPAM =

Cnitrogen 0.1237

×

2ANIPAM × 100 ADMA + 2ANIPAM

(5)

All polymers, except for HBPS-AA20-PNIPAM-C3 and HBPSDMAx-PNIPAM-C3 (x = 5, 10, and 20), had high CNIPAM values of over 85%, suggesting the successful formation of starshaped copolymers with a polystyrene core and long PNIPAM arms. It was difficult to graft long PNIPAM chains to HBPSAA20-PNIPAM-C3 and HBPS-DMAx-PNIPAM-C3 (x = 5, 10, 20), probably because of the deactivation of the CTA. 1 H NMR spectra and GPC-RI traces of the polymers are shown in Figures 2, 3, and S2−4, indicating the successful syntheses of these polymers. As shown in Figure 2, similar spectra were obtained from HBPS-AAx-PNIPAM-C3 and HBPS-DMAx-PNIPAM-C3 to HBPS-PNIPAM-C3, suggesting similarly high CNIPAM. 3.2. Surface Characterization of Polymer-Coated Dishes. The STEM cross-sectional images of the polymercoated dishes (HBPS-PNIPAM-C6/dish and LPS-PNIPAM/ dish) are shown in Figure 4. The PS segment was stained with ruthenium(VIII) tetroxide before slicing for both samples. On the HBPS-PNIPAM-C6/dish, a uniform polymer layer covering the PS dish can be clearly seen. The presence of PNIPAM was confirmed by the detection of nitrogen in this layer using EDX (Figure 4A inset). The thickness of the copolymer layer is approximately 25 nm. In contrast, the image of the LPSPNIPAM/dish shows a slightly thinner polymer layer (15 nm) and several particles of 50 nm diameter. In this comparison, HBPS-PNIPAM-C6 seems to have the advantage against LPSPNIPAM in terms of the uniformity of the immobilized polymer on the PS dishes. The stability of the polymer coating in water was evaluated by XPS analysis. Both HBPS-PNIPAM-C6/dish and LPSPNIPAM/dish were immersed in Milli-Q water at room temperature for 24 h and dried overnight. The XPS spectra of the C 1s and N 1s regions before and after the treatment are shown in Figure 5. Peak fitting of the C 1s peak resolved the spectrum into four major peaks36−39 representing (i) aromatic carbon (284.2 eV), (ii) C*H2−C*H (285.1 eV), (iii) C*H−N (286.3 eV), and (iv) C*O (288.0 eV) and confirming the presence of PNIPAM and PS. In the N 1s region, a single peak corresponding to N*H−C(O) was observed at 400.0 eV.40 These XPS results support the presence of PNIPAM in the polymer coating the surfaces. The atomic ratios of nitrogen to carbon (N/C) of the samples before and after the rinse were evaluated from the peak intensities in Figure 5 and are summarized in Figure 6. The decrease in the N/C ratio after the rinse with water was smaller in the HBPS-PNIPAM-C6/ dish. This indicates that the HBPS-PNIPAM-C6 copolymer was immobilized onto the PS dish more strongly than the LPSPNIPAM copolymer owing to its characteristic structure that exposes PS segments to the PS dish efficiently.15 On the LPSPNIPAM/dish, a considerable amount of the copolymer seemed to be washed away because the PS segment was not well immobilized on the PS dish. To discuss the coating stability in a situation that is closer to that of the actual culture medium (DMEM), a similar test was also conducted by immersing HBPS-PNIPAM-C6/dish and LPS-PNIPAM/dish in DMEM (2 mL) at room temperature for 24 h. The surface was rinsed with Milli-Q water thoroughly and dried overnight. The XPS wide scan spectra shown in Figures S5−6 suggest the contamination by substances contained in the DMEM. Fortunately, the atomic ratio N/C could be evaluated using

Figure 6. Atomic ratios of N to C on the prepared dishes.

the peak area of C 1s and N 1s regions, as shown in Figure 6. Comparing the atomic ratio N/C before and after treatment with DMEM, the reduction in the amount of polymer showed similar behavior to the case of the pure water rinse. The thermoresponsive behavior of the immobilized copolymers was investigated using contact angle measurements with water. Figure 7 shows the time dependence of the static water contact angles on the polymer-coated surfaces at 20 and 50 °C for 41 s. The pristine PS dish did not show any thermoresponsiveness (Figure 7a). As shown in Figure 7b, the contact angles of HBPS-PNIPAM-C3, -C6, and -C12 measured at 50 °C showed relatively steady values throughout the measurement time. In contrast, the contact angles at 20 °C decreased after the addition of water, resulting in lower contact angles than those at 50 °C. These results indicate that the surface was hydrophobic at 50 °C and hydrophilic at 20 °C, thus exhibiting the expected thermoresponsiveness. The effect of the alkyl chain length was demonstrated by a slight change in the contact angles at 41 s. The higher contact angles with longer alkyl chains probably arise because of the hydrophobicity derived from the alkyl chain (Figure 5b). In the case of LPS-PNIPAM/dish, the contact angle decreased in both cases at 50 and 20 °C.15 This is probably because the unstable coating of LPS-PNIPAM, as indicated by XPS, was removed, dissolving into the water drop and affecting the contact angle. The samples with carboxylic acid groups, HBPS-AAxPNIPAM-C3/dish, showed a similar trend to that of HBPSPNIPAM-C3/dish, and the contact values did not change even as the number of carboxylic acid groups increased. In contrast, HBPS-DMAx-PNIPAM-C3/dish showed very low contact angles at 20 °C because of the presence of the amino groups. Based on these characterizations of the polymer-coated surfaces, the star-shaped copolymers seem to be advantageous for surface modification compared to the linear copolymers. The star-shaped copolymers are suitable for immobilizing PNIPAM onto PS dishes in terms of uniform coating, high stability against water rinsing, and excellent thermoresponsiveness. These differences between the star-shaped and linear copolymers probably originate from the characteristics in the segregation behaviors of the hyperbranched and linear PS segments, as reported previously.15 The hyperbranched polymer bundles of many polymer chains may be well exposed to the PS dish, resulting in enhanced interactions between the PS segments and the dish. 3.3. Cell Cultivation Experiments. The polymer-coated dishes were tested for cell cultivation to evaluate the cell 658

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Figure 7. Contact angles of (A) pristine PS dish, (B) HBPS-PNIPAM-C3, C6, C12/dish and LPS-PNIPAM/dish, (C) HBPS-AAx-PNIPAM-C3/ dish, and (D) HBPS-DMAx-PNIPAM-C3/dish.

Figure 8. Microscopy images (40×) of the cultivated cells with a density of 1 × 105 cells/dish on (A−C) HBPS-PNIPAM-C3/dish, (E−G) HBPSAA1-PNIPAM-C3/dish, and (I, K) HBPS-DMA1-PNIPAM-C3/dish, and 1 × 104 cells/dish on (D) HBPS-PNIPAM-C3/dish, (H) HBPS-AA1PNIPAM-C3/dish, and (L) HBPS-DMA1-PNIPAM-C3/dish.

20 °C. In contrast, the cell sheets did not detach from the LPSPNIPAM/dish (not shown).15 In the case of HBPS-AAx-PNIPAM-C3/dish, which was modified with carboxylic acid groups, the cells adhered as well as with HBPS-PNIPAM-C3 at 37 °C; however, part of the cell sheet was still attached to the dish even after cooling to 20 °C. This might be because of the low molecular weight of PNIPAM14 or increased electrostatic interactions between the cells and the copolymer owing to the carboxylic acid groups.

adhesion and detachment properties using mouse 3T3 fibroblasts. Figure 8 shows typical microscopic images of the cultivated cells on the polymer-coated dishes. The results of the cell adhesion and detachment tests are summarized in Table 2. HBPS-PNIPAM-C3, -C6, and -C12/dish allowed the cells to adhere and proliferate at 37 °C, achieving confluence after incubation for 4 days. The cell sheets detached spontaneously from the substrates when they were cooled to 659

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4. CONCLUSIONS Star-shaped copolymers consisting of polystyrene and PNIPAM were synthesized and drop-cast onto polystyrenebased dishes to fabricate thermoresponsive cell culture dishes. Hyperbranched polystyrenes (HBPSs) with various terminal structures were synthesized via RAFT-SCVP from styrene monomers with various CTAs. PNIPAM were grafted from the termini of the HBPS via RAFT polymerization to obtain starshaped copolymers: HBPS-g-PNIPAM. Furthermore, polar functional groups such as carboxylic acid and dimethylamino groups were introduced to the termini of the HBPS, and then PNIPAM was sequentially grafted to obtain HBPS-g-PAA-bPNIPAM and HBPS-g-PDMA-b-PNIPAM. STEM, XPS, and contact angle measurements have revealed that the star-shaped copolymers could be coated uniformly and immobilized strongly onto PS dishes. Furthermore, the polymers showed excellent thermoresponsive behavior compared with the linear copolymer (LPS-PNIPAM). The introduction of a carboxylic acid group did not have a clear effect on the surface properties, whereas the dimethylamino group seemed to enhance the cell adhesion property. This paper demonstrates the versatile syntheses of starshaped copolymers of hyperbranched polystyrene and PNIPAM with polar functional groups, and a facile preparation of PNIPAM-immobilized cell culture dishes by simple dropcasting. Such technology enables the fabrication of highly compatible thermoresponsive dishes suitable for the culture of various types of cells that cannot currently be used in cell sheet engineering, such as single sheets of keratinocytes. We believe that this technique will contribute to the exciting future of tissue engineering.

Table 2. Cell Adhesion and Detachment Properties cell seed density: 1 × 10 per dish sample LPS-PNIPAM/dish HBPS-PNIPAM-C3/ dish HBPS-PNIPAM-C6/ dish HBPS-PNIPAM-C12/ dish HBPS-AA1-PNIPAMC3/dish HBPS-AA5-PNIPAMC3/dish HBPS-AA10-PNIPAMC3/dish HBPS-AA20-PNIPAMC3/dish HBPS-DMA1PNIPAM-C3/dish HBPS-DMA5PNIPAM-C3/dish HBPS-DMA10PNIPAM-C3/dish HBPS-DMA20PNIPAM-C3/dish

5

cell cell adhesiona detachmentb

cell seed density: 1 × 104 per dish cell adhesiona

++ ++

++

+

++

++

-

++

++

+

+

+

++

+

-

++

+

++

+

+

++

++

-

N.A.

-

-

N.A.

-

N.A.

a Cell adhesion + + Proliferated to confluence after 4 days cultivation; + Adhered to dishes but did not achieve confluence after cultivation for 4 days. bCell detachment + + Spontaneously detached from the surfaces by cooling; + Partially detached from the surface; - Did not detach from the surface.

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ASSOCIATED CONTENT

S Supporting Information *

This is consistent with earlier studies that have reported that cells adhere strongly to surfaces modified with carboxylic acid groups because of their effects on cell adhesive proteins.10,41,42 In the cases of HBPS-DMA5-PNIPAM-C3/dish, HBPSDMA10-PNIPAM-C3/dish, and HBPS-DMA20-PNIPAMC3/dish, which were modified with amino groups, the cells did not adhere well, and no cell sheets could be obtained from these dishes. In contrast, the dish with HBPS-DMA1PNIPAM-C3 exhibited good cell adhesion, and the cells achieved confluence at 37 °C. This sample also exhibited good cell detachment properties, providing a cell sheet spontaneously at 20 °C. To highlight the effect of the polar functional groups introduced to the copolymer, the cell adhesion experiments were conducted under severe cultivation conditions. The cells were seeded at a low density (1 × 104 per dish) and incubated for 120 h at 37 °C on three different dishes modified with HBPS-PNIPAM-C3, HBPS-AA1-PNIPAM-C3, and HBPSDMA1-PNIPAM-C3. As a result, the number of cells on HBPS-DMA1-PNIPAM-C3 increased remarkably compared to the other two samples, indicating better cell adhesion on HBPS-DMA1-PNIPAM than the others. These results indicate that the insertion of a few dimethylamino groups in HBPS-gPNIPAM enhances cell adhesion. These experimental results suggest that HBPS-g-PNIPAM synthesized via RAFT-SCVP is promising for cell sheet engineering, exhibiting a clear advantage over its linear analog. Furthermore, the cell adhesion can be easily tuned by inserting polar functional groups. Importantly, this tuning can be achieved by simply casting the copolymers.

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b04213. Elemental components of the polymers; Calculation of DB; NMR and XPS spectra; proposed mechanism; and microscopy imaging (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]. Tel. +81-3-5734-2429. Fax. +81-3-5734-2429. ORCID

Yuta Nabae: 0000-0002-9845-382X Teruaki Hayakawa: 0000-0002-1704-5841 Notes

The authors declare no competing financial interest.

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REFERENCES

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