Star-Shaped Thermoresponsive Polymers with Various Functional

Subscriber access provided by The Library | The University of Auckland

Article

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 Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b04213 • Publication Date (Web): 19 Dec 2017 Downloaded from http://pubs.acs.org on December 26, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Title Star-shaped thermoresponsive polymers with various functional groups for cell sheet engineering

Authors Yu Sudo1, Ryuki Kawai1, Hideaki Sakai2, Ryohei Kikuchi3, Yuta Nabae1*, Teruaki Hayakawa1, and Masa-aki Kakimoto1

Affiliations 1

Department of Materials Science and Engineering, School of Materials and Chemical Technology, Tokyo Institute of

Technology, 2-12-1 S8-26, Ookayama, Meguro-ku 2

3

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

Keywords

hyperbranched polymer, cell sheet engineering, thermoresponsive polymer, block copolymer

[Corresponding author]

1

ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 33

Yuta Nabae

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

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 HBPS-g-PNIPAM. In addition, the cell adhesion and detachment properties could

be tuned by the introduction of polar functional groups.

1. Introduction

2


Page 3 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Recently, tissue engineering, an innovative medical technology, has developed rapidly. Cell sheet engineering is a

key technological requirement in tissue engineering, in which cell sheets can be prepared by cultivating cells collected from the patients, which are subsequently implanted back to regenerate damaged tissues. Okano and coworkers1–3 have

reported many examples of successful cell sheet recovery from thermoresponsive cell culture dishes modified with

poly(N-isopropylacrylamide) (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 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 temperature-responsive 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-g-PNIPAM 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 coworkers to synthesize hyperbranched polymers using an AB* monomer that contains an initiation site for atom

3


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 33

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 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-g-PNIPAM 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 Fig. 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.

4


Page 5 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

2. Experimental section 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

5


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 33

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 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

1-propanethiol (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. 4-Vinylbenzyl 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 was 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-),

6


Page 7 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

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).

7


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 33

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 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 t-butyl 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

8


Page 9 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

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-PNIPAM-C3)

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)

9


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 33

HBPS-DMA1-PNIPAM-C3 was prepared by the RAFT polymerization of NIPAM from polymer HBPS-DMA1-C3

in a similar manner as 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,

10


Page 11 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

HBPS-PNIPAM-C6/dish, HBPS-PNIPAM-C12/dish, HBPS-AA-PNIPAM-C3/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 four 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.

11


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 33

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

3. Results and Discussion 3.1. Preparation of HBPS, HBPS-g-PNIPAM, HBPS-g-PAA-b-PNIPAM, and HBPS-g-PDMA-b-PNIPAM

The molecular architectures and synthetic scheme for the polymers in this study are summarized in Fig. 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

12


Page 13 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

branching (DB) of the hyperbranched polystyrene, the purified polymer was analyzed by 1H-NMR spectroscopy (Figs.

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, Fig. S1). The DB

values are summarized in Table 1.

‫=ݎ‬

௘ ష೥ ௘ ష೥ ା௭ିଵ

DB = 2݁ ି௭ (1 − ݁ ି௭ )

‫=ݎ‬

஻∗ ஻ ∗ ି୪୬ ஻ ∗ ିଵ

(1) (2)

(3)

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

back titration. The HBPS-AAx-C3 samples were dispersed in NaOH aqueous solutions. Subsequently, the residual

NaOH was titrated with a 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.

PNIPAM was grafted from 2a, 2b, and 2c to obtain the star-shaped 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

13


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 33

polymerization. Therefore, the resulting star-shaped 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-PNIPAM-C3, respectively. The PNIPAM contents (CNIPAM) in HBPS-PNIPAM-C3, -C6, -C12, and HBPS-AAx-PNIPAM-C3 were determined by elemental analysis using Eq. 4,

where Cnitrogen is 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 (Fig. 2F) corresponding to NIPAM (4.01 ppm) and DMA (2.55 ppm) units, respectively. The calculated values are summarized in Table 1. ‫୔୍୒ܥ‬୅୑ =

஼౤౟౪౨౥ౝ౛౤ ଴.ଵଶଷ଻ ஼

× 100

‫୔୍୒ܥ‬୅୑ = ×஺ ଴.ଵଶଷ଻

ଶ஺ಿ಺ುಲಾ ವಾಲ ାଶ஺ಿ಺ುಲಾ

(4) × 100

(5)

All polymers, except for HBPS-AA20-PNIPAM-C3 and HBPS-DMAx-PNIPAM-C3 (x = 5, 10, and 20), had high

CNIPAM values of over 85%, suggesting the successful formation of star-shaped copolymers with a polystyrene core and long PNIPAM arms. It was difficult to graft long PNIPAM chains to HBPS-AA20-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 Figs. 2, 3, and S2–4, indicating the successful

syntheses of these polymers. As shown in Fig. 2, similar spectra were obtained from HBPS-AAx-PNIPAM-C3 and HBPS-DMAx-PNIPAM-C3 to HBPS-PNIPAM-C3, suggesting similarly high CNIPAM.

14


Page 15 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

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

Fig. 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.

15


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 33

Table 1. Characterization of hyperbranched polystyrene and copolymers.

Sample

Mna (PDI)

DB

CNIPAMb/ wt.%

Functional group Density/

Type

94.0

Terminal

mmol g

-1

-

Type

Density/ mmol g-1

LPS-PNIPAM

113600 (1.2)

-

HBPS (2a)

4900 (3.4)

0.40

HBPS (2b)

5400 (2.0)

0.41

HBPS (2c)

7100 (1.7)

0.38

HBPS-PNIPAM-C3

142000 (4.6)

HBPS-PNIPAM-C6

125000 (3.9)

HBPS-PNIPAM-C12

101000 (2.7)

93.6

HBPS-AA1-PNIPAM-C3

N.A.

91.5

HBPS-AA5-PNIPAM-C3

N.A.

HBPS-AA10-PNIPAM-C3

N.A.

HBPS-AA20-PNIPAM-C3

N.A.

80.8

2.28

0.71

HBPS-DMA1-PNIPAM-C3

151000 (5.1)

92.1

0.20

0.29

HBPS-DMA5-PNIPAM-C3

198000 (3.4)

HBPS-DMA10-PNIPAM-C3

39000 (6.7)

HBPS-DMA20-PNIPAM-C3

34300 (4.0)

-

-

95.7 -

-

-

-

91.5

89.4 85.4

83.8 78.5

-C12H25

0.0088

-C3H7

3.7

-C6H13

3.2

-C12H25

2.5

-C3H7

0.16

-C6H13

0.28

-C12H25

0.24

0.29

-COOHc

-N(CH3)2d

73.4

0.86 1.65

0.82 1.26

1.70

0.31

-C3H7

-C3H7

0.39 0.54

0.60 0.80

0.98

a

Determined by GPC using PS calibration.

b

PNIPAM content calculated from Eq. 5 for HBPS-DMAx-PNIPAM-C3 samples, and Eq. 4 for the other samples.

c

Quantified by back titration with NaOH (aq) and HCl (aq).

d

Quantified by back titration with a p-toluenesulfonic acid aqueous solution and NaOH (aq).

3.2. Surface characterization of polymer-coated dishes

The STEM cross-sectional images of the polymer-coated dishes (HBPS-PNIPAM-C6/dish and LPS-PNIPAM/dish)

are shown in Fig. 4. The PS segment was stained with ruthenium(VIII) tetroxide before slicing for both samples. On the

16


Page 17 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

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 (Fig. 4A inset). The thickness of the copolymer layer is approximately 25 nm. In contrast, the image of the LPS-PNIPAM/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 LPS-PNIPAM 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

LPS-PNIPAM/dish were immersed in Milli-Q water at room temperature for 24 h and dried overnight. The XPS spectra

of the C1s and N1s regions before and after the treatment are shown in Fig. 5. Peak fitting of the C1s 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 N1s 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 Fig. 5 and are summarized in Fig. 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 LPS-PNIPAM copolymer owing to its characteristic structure that exposes PS segments to the PS dish efficiently.15 On the LPS-PNIPAM/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

17


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 33

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 Figs. S5–6 suggest the contamination by substances contained in the DMEM. Fortunately, the atomic

ratio N/C could be evaluated using the peak area of C1s and N1s regions, as shown in Fig. 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. Fig. 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 (Fig. 7a). As shown in Fig. 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 (Fig. 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-AAx-PNIPAM-C3/dish, showed a similar trend to that of HBPS-PNIPAM-C3/dish, and the contact

18


Page 19 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

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.

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

19


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 33

Fig. 5. XPS spectra at C1s and N1s 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.

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

20


Page 21 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Fig. 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.

3.3. Cell cultivation experiments

The polymer-coated dishes were tested for cell cultivation to evaluate the cell adhesion and detachment properties

using mouse 3T3 fibroblasts. Fig. 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 four days.

21


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 33

The cell sheets detached spontaneously from the substrates when they were cooled to 20 °C. In contrast, the cell sheets did not detach from the LPS-PNIPAM/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. 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,

HBPS-DMA10-PNIPAM-C3/dish, and HBPS-DMA20-PNIPAM-C3/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-DMA1-PNIPAM-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 HBPS-DMA1-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-g-PNIPAM enhances cell adhesion.

22


Page 23 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

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.

Fig. 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) HBPS-AA1-PNIPAM-C3/dish, and (I, K) HBPS-DMA1-PNIPAM-C3/dish, and 1 × 104

cells/dish

on

(D)

HBPS-PNIPAM-C3/dish,

(H)

HBPS-AA1-PNIPAM-C3/dish,

HBPS-DMA1-PNIPAM-C3/dish.

23


and

(L)

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 33

Table 2. Cell adhesion and detachment properties. Cell seed density: 1 × 105 per dish

Sample

Cell seed density: 1 × 104 per dish

Cell adhesiona

Cell detachmentb

Cell adhesiona

LPS-PNIPAM/dish

++

-

-

HBPS-PNIPAM-C3/dish

++

++

+

HBPS-PNIPAM-C6/dish

++

++

HBPS-PNIPAM-C12/dish

++

++

HBPS-AA1-PNIPAM-C3/dish

+

+


++

+


++

+


++

+

HBPS-DMA1-PNIPAM-C3/dish

+

++


-

N.A.


-

N.A.


-

N.A.

a

b

Cell adhesion

+

-

++

-

++

Proliferated to confluence after four days cultivation;

+

Adhered to dishes but did not achieve confluence after cultivation for four days;

Cell detachment ++

Spontaneously detached from the surfaces by cooling;

+

Partially detached from the surface;

-

Did not detach from the surface.

4. Conclusions Star-shaped copolymers consisting of polystyrene and PNIPAM were synthesized and drop-cast onto

polystyrene-based dishes to fabricate thermoresponsive cell culture dishes. Hyperbranched polystyrenes (HBPSs) with

24


Page 25 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

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 star-shaped 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-b-PNIPAM

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 star-shaped copolymers of hyperbranched polystyrene and

PNIPAM with polar functional groups, and a facile preparation of PNIPAM-immobilized cell culture dishes by simple

drop-casting. 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.

Supporting Information Table S1, details of DB calculation, and Figure S1-7.

25


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 33

References (1)

Okano, T.; Yamada, N.; Sakai, H.; Sakurai, Y. A Novel Recovery System for Cultured Cells Using Plasma-Treated

Polystyrene Dishes Grafted with poly(N-Isopropylacrylamide). J. Biomed. Mater. Res. 1993, 27 (10), 1243–1251.

(2)

Owaki, T.; Shimizu, T.; Yamato, M.; Okano, T. Cell Sheet Engineering for Regenerative Medicine: Current

Challenges and Strategies. Biotechnol. J. 2014, 9 (7), 904–914.

(3)

Matsuura, K.; Utoh, R.; Nagase, K.; Okano, T. Cell Sheet Approach for Tissue Engineering and Regenerative

Medicine. J. Control. Release 2014, 190, 228–239.

(4)

Schild, H. G. Poly(N-Isopropylacrylamide): Experiment, Theory and Application. Prog. Polym. Sci. 1992, 17 (2),

163–249.

(5)

Fukumori, K.; Akiyama, Y.; Kumashiro, Y.; Kobayashi, J.; Yamato, M.; Sakai, K.; Okano, T. Characterization of

Ultra-Thin Temperature-Responsive Polymer Layer and Its Polymer Thickness Dependency on Cell

Attachment/detachment Properties. Macromol. Biosci. 2010, 10 (10), 1117–1129.

(6)

Tsuda, Y.; Kikuchi, A.; Yamato, M.; Nakao, A.; Sakurai, Y.; Umezu, M.; Okano, T. The Use of Patterned Dual

Thermoresponsive Surfaces for the Collective Recovery as Co-Cultured Cell Sheets. Biomaterials 2005, 26 (14),

1885–1893.

(7)

Vickie Pan, Y.; Wesley, R. A.; Luginbuhl, R.; Denton, D. D.; Ratner, B. D. Plasma Polymerized

N-Isopropylacrylamide: Synthesis and Characterization of a Smart Thermally Responsive Coating.

Biomacromolecules 2001, 2 (1), 32–36.

26


Page 27 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(8)

Langmuir

Canavan, H. E.; Cheng, X.; Graham, D. J.; Ratner, B. D.; Castner, D. G. Cell Sheet Detachment Affects the

Extracellular Matrix: A Surface Science Study Comparing Thermal Liftoff, Enzymatic, and Mechanical Methods. J.

Biomed. Mater. Res. Part A 2005, 75A (1), 1–13.

(9)

Kim, S. J.; Kim, W. Il; Yamato, M.; Okano, T.; Kikuchi, A.; Kwon, O. H. Successive Grafting of PHEMA and

PIPAAm onto Cell Culture Surface Enables Rapid Cell Sheet Recovery. Tissue Eng. Regen. Med. 2013, 10 (3), 139–

145.

(10)

Takahashi, H.; Matsuzaka, N.; Nakayama, M.; Kikuchi, A.; Yamato, M.; Okano, T. Terminally Functionalized

Thermoresponsive Polymer Brushes for Simultaneously Promoting Cell Adhesion and Cell Sheet Harvest.

Biomacromolecules 2012, 13 (1), 253–260.

(11)

Stenzel, M. H.; Zhang, L.; Huck, W. T. S. Temperature-Responsive Glycopolymer Brushes Synthesized via RAFT

Polymerization Using the Z-Group Approach. Macromol. Rapid Commun. 2006, 27 (14), 1121–1126.

(12)

Nakayama, M.; Yamada, N.; Kumashiro, Y.; Kanazawa, H.; Yamato, M.; Okano, T. Thermoresponsive

poly(N-Isopropylacrylamide)-Based Block Copolymer Coating for Optimizing Cell Sheet Fabrication. Macromol.

Biosci. 2012, 12 (6), 751–760.

(13)

Gillet, R.; Sakai, H.; Nabae, Y.; Hayakawa, T.; Kakimoto, M. Synthesis of Hyperbranched-Linear

poly(N-Isopropylacrylamide) Polymers with a Poly(siloxysilane) Hyperbranched Macroinitiator, and Their

Application to Cell Culture on Glass Substrates. Polym. J. 2016, 48 (10), 1007–1012.

(14)

Sudo, Y.; Sakai, H.; Nabae, Y.; Hayakawa, T.; Kakimoto, M. Preparation of Hyperbranched

27


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 33

Polystyrene-G-poly(N-Isopropylacrylamide) Copolymers and Its Application to Novel Thermo-Responsive Cell

Culture Dishes. Polymer 2015, 70, 307–314.

(15)

Sudo, Y.; Sakai, H.; Nabae, Y.; Hayakawa, T.; Kakimoto, M. Role of Hyperbranched Polystyrene on

Thermo-Responsive Cell Culture Dishes Prepared by Hyperbranched Polystyrene-g-poly(N-Isopropylacrylamide).

Polymer 2016, 100, 77–85.

(16)

Park, B. R.; Nabae, Y.; Surapati, M.; Hayakawa, T.; Kakimoto, M. Poly(N-Isopropylacrylamide)-Modified Silica

Beads with Hyperbranched Polysiloxysilane for Three-Dimensional Cell Cultivation. Polym. J. 2013, 45 (2), 210–

215.

(17)

Fréchet, J. M. J.; Henmi, M.; Gitsov, I.; Aoshima, S.; Leduc, M. R.; Grubbs, R. B. Self-Condensing Vinyl

Polymerization: An Approach to Dendritic Materials. Science 1995, 269 (5227), 1080–1083.

(18)

Hawker, C. J.; Fréchet, J. M. J.; Grubbs, R. B.; Dao, J. Preparation of Hyperbranched and Star Polymers by A

“living”, Self-Condensing Free Radical Polymerization. J. Am. Chem. Soc. 1995, 117 (43), 10763–10764.

(19)

Wang, J. S.; Matyjaszewski, K. Controlled/“living” radical Polymerization. Halogen Atom Transfer Radical

Polymerization Promoted by a Cu(I)/Cu(II) Redox Process. Macromolecules 1995, 28 (23), 7901–7910.

(20)

Wang, J. S.; Matyjaszewski, K. Controlled“living” radical Polymerization. Atom Transfer Radical Polymerization in

the Presence of Transition-Metal Complexes. J. Am. Chem. Soc. 1995, 117 (6), 5614–5615.

(21)

Leduc, M. R.; Hawker, C. J.; Dao, J.; Fréchet, J. M. J. Dendritic Initiators For “living” radical Polymerizations: A

Versatile Approach to the Synthesis of Dendritic-Linear Block Copolymers. J. Am. Chem. Soc 1996, 118 (45),

28


Page 29 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

11111–11118.

(22)

Grubbs, R. B.; Hawker, C. J.; Dao, J.; Fréchet, J. M. J. A Tandem Approach to Graft and Dendritic Graft Copolymers

Based on“Living” Free Radical Polymerizations. Angew. Chemie Int. Ed. English 1997, 36 (3), 270–272.

(23)

Wang, Z.; He, J.; Tao, Y.; Yang, L.; Jiang, H.; Yang, Y. Controlled Chain Branching by RAFT-Based Radical

Polymerization. Macromolecules 2003, 36 (20), 7446–7452.

(24)

Rikkou-Kalourkoti, M.; Elladiou, M.; Patrickios, C. S. Synthesis and Characterization of Hyperbranched

Amphiphilic Block Copolymers Prepared via Self-Condensing RAFT Polymerization. J. Polym. Sci. Part A Polym.

Chem. 2015, 53 (11), 1310–1319.

(25)

Ghosh Roy, S.; De, P. Facile RAFT Synthesis of Side-Chain Amino Acids Containing pH-Responsive

Hyperbranched and Star Architectures. Polym. Chem. 2014, 5 (21), 6365–6378.

(26)

Alfurhood, J. A.; Bachler, P. R.; Sumerlin, B. S. Hyperbranched Polymers via RAFT Self-Condensing Vinyl

Polymerization. Polym. Chem. 2016, 7 (20), 3361–3369.

(27)

Haldar, U.; Roy, S. G.; De, P. POSS Tethered Hybrid “inimer” Derived Hyperbranched and Star-Shaped Polymers

via SCVP-RAFT Technique. Polymer (Guildf). 2016, 97, 113–121.

(28)

Zhang, C.; Zhou, Y.; Liu, Q.; Li, S.; Perrier, S. S.; Zhao, Y. Facile Synthesis of Hyperbranched and Star-Shaped

Polymers by RAFT Polymerization Based on a Polymerizable Trithiocarbonate. Macromolecules 2011, 44 (7), 2034–

2049.

(29)

Zhang, M.; Liu, H.; Shao, W.; Ye, C.; Zhao, Y. Versatile Synthesis of Multiarm and Miktoarm Star Polymers with a

29


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 33

Branched Core by Combination of Menschutkin Reaction and Controlled Polymerization. Macromolecules 2012, 45

(23), 9312–9325.

(30)

Li, C.; Liu, H.; Tang, D.; Zhao, Y. Synthesis, Postmodification and Fluorescence Properties of Reduction-Cleavable

Core-Couplable Miktoarm Stars with a Branched Core. Polym. Chem. 2015, 6, 1474–1486.

(31)

Nagase, K.; Kobayashi, J.; Kikuchi, A.; Akiyama, Y.; Kanazawa, H.; Okano, T. Thermally-Modulated

On/off-Adsorption Materials for Pharmaceutical Protein Purification. Biomaterials 2011, 32 (2), 619–627.

(32)

Nagase, K.; Hatakeyama, Y.; Shimizu, T.; Matsuura, K.; Yamato, M.; Takeda, N.; Okano, T. Thermoresponsive

Cationic Copolymer Brushes for Mesenchymal Stem Cell Separation. Biomacromolecules 2015, 16 (2), 532–540.

(33)

Skey, J.; O’Reilly, R. K. Facile One Pot Synthesis of a Range of Reversible Addition-Fragmentation Chain Transfer

(RAFT) Agents. Chem. Commun. (Camb). 2008, No. 35, 4183–4185.

(34)

Yan, D.; Müller, A. H. E.; Matyjaszewski, K. Molecular Parameters of Hyperbranched Polymers Made by

Self-Condensing Vinyl Polymerization. 2. Degree of Branching. Macromolecules 1997, 30, 7024–7033.

(35)

Matyjaszewski, K.; Gaynor, S. G.; Müller, A. H. E. Preparation of Hyperbranched Polyacrylates by Atom Transfer

Radical Polymerization. 2. Kinetics and Mechanism of Chain Growth for the Self-Condensing Vinyl Polymerization

of 2-((2-Bromopropionyl)oxy)ethyl Acrylate. Macromolecules 1997, 30 (23), 7034–7041.

(36)

Barth, G.; Linder, R.; Bryson, C. Advances in Charge Neutralization for XPS Measurements of Nonconducting

Materials. Surf. Interface Anal. 1988, 11 (6–7), 307–311.

(37)

Canavan, H. E.; Graham, D. J.; Cheng, X.; Ratner, B. D.; Castner, D. G. Comparison of Native Extracellular Matrix

30


Page 31 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

with Adsorbed Protein Films Using Secondary Ion Mass Spectrometry. Langmuir 2007, 23 (1), 50–56.

(38)

Zhang, Y.; Teo, B. M.; Postma, A.; Ercole, F.; Ogaki, R.; Zhu, M.; Städler, B. Highly-Branched

poly(N-Isopropylacrylamide) as a Component in Poly(dopamine) Films. J. Phys. Chem. B 2013, 117 (36), 10504–

10512.

(39)

Walo, M.; Przybytniak, G.; Barsbay, M.; Güven, O. Functionalization of Poly(ester-Urethane) Surface by

Radiation-Induced Grafting of N-Isopropylacrylamide Using Conventional and Reversible Addition-Fragmentation

Chain Transfer-Mediated Methods. Polym. Int. 2016, 65 (2), 192–199.

(40)

Leonard, D.; Chevolot, Y.; Bucher, O.; Sigrist, H.; Mathieu, H. J. ToF-SIMS and XPS Study of Photoactivatable

Reagents Designed for Surface Glycoengineering - Part I.

N-(m-(3-(Trifluoromethyl)diazirine-3-yl)phenyl)-4-Maleimido-Butyramide (MAD) on Silicon, Silicon Nitride and

Diamond. Surf. Interface Anal. 1998, 26 (11), 783–792.

(41)

Tidwell, C. D.; Ertel, S. I.; Ratner, B. D.; Tarasevich, B. J.; Atre, S.; ALLARA, D. L. Endothelial Cell Growth and

Protein Adsorption on Terminally Functionalized, Self-Assembled Monolayers of Alkanethiolates on Gold.

Langmuir 1997, 13 (13), 3404–3413.

(42)

Michael, K. E.; Vernekar, V. N.; Keselowsky, B. G.; Meredith, J. C.; Latour, R. A.; García, A. J. Adsorption-Induced

Conformational Changes in Fibronectin due to Interactions with Well-Defined Surface Chemistries. Langmuir 2003,

19 (19), 8033–8040.

31


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TOC graphic

32


Page 32 of 33

Page 33 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

TOC graphic 85x47mm (300 x 300 DPI)


Star-Shaped Thermoresponsive Polymers with Various Functional

Recommend Documents