Ultrathin Poly(N-isopropylacrylamide) Grafted Layer on Polystyrene Surfaces for Cell Adhesion/Detachment Control Yoshikatsu Akiyama, † Akihiko Kikuchi, † Masayuki Yamato, † and Teruo Okano* ,† Institute of Advanced Biomedical Engineering and Science, COE Program for 21st Century, Tokyo Women’s Medical University, 8-1 Kawadacho, Shinjuku-ku, Tokyo 162-8666, Japan Received November 14, 2003. In Final Form: April 12, 2004 We investigated physicochemical properties of two types of poly(N-isopropylacrylamide) (PIPAAm)- grafted tissue culture polystyrene (TCPS) surfaces, to elucidate the influential factors for thermally regulated cell adhesion and detachment to PIPAAm-grafted surfaces. The two types of PIPAAm-grafted surfaces were prepared by the electron beam polymerization method. Attenuated total reflection Fourier transform infrared spectroscopy revealed that amounts of the grafted polymers were 1.4 ( 0.1 μg/cm 2 for PIPAAm-1.4 and 2.9 ( 0.1 μg/cm 2 for PIPAAm-2.9. Both PIPAAm-grafted surfaces showed hydrophobic/hydrophilic property alterations in response to temperature. However, PIPAAm-1.4 surfaces were more hydrophobic (cos θ ) 0.21 at 37 °C and cos θ ) 0.35 at 20 °C) than PIPAAm-2.9 (cos θ ) 0.42 at 37 °C and cos θ ) 0.50 at 20 °C) both above and below the PIPAAm’s transition temperature. Thicknesses of the grafted PIPAAm layers were estimated to be 15.5 ( 7.2 nm for PIPAAm-1.4 and 29.5 ( 8.4 nm for PIPAAm-2.9, by the use of UV excimer laser and atomic force microscope. Bovine carotid artery endothelial cells (ECs) adhere to the surfaces of PIPAAm-1.4 and proliferate to form confluent cell monolayers. The cell monolayers were harvested as single cell sheets by temperature decrease from 37 to 20 °C. On the contrary, ECs did not adhere to the surfaces of PIPAAm-2.9. This phenomenon was correlated with an adsorption of cell adhesion protein, fibronectin, onto surfaces of PIPAAm-1.4 and -2.9. In the case of nano-ordered thin grafted surfaces, the surface chain mobility is strongly influenced by the thickness of PIPAAm grafted layers because dehydration of PIPAAm chains should be enhanced by the hydrophobic TCPS surfaces. PIPAAm graft amounts, that is, thickness of the PIPAAm grafted layers, play a crucial role in temperature-induced hydrophilic/hydrophobic property alterations and cell adhesion/detachment behavior. Introduction We have been carrying out the preparation of thermo- responsive polymer-modified surfaces with designated molecular configuration at the interfaces. 1-3 Thermo- responsive polymers poly(N-isopropylacrylamide) (PI- PAAm) and its derivatives are used as the surface modifiers. 1-3 These surfaces are utilized to propose new chromatographic separation methods for a variety of types of bioactive compounds in a sole aqueous mobile phase. 4-6 We further applied the thermoresponsive surfaces for thermally regulated cell adhesion and detachment 7-9 and extended the idea to tissue engineering. 10-13 Confluently cultured cell monolayers on hydrophobized PIPAAm- modified surfaces at 37 °C detach as single cell sheets by lowering the culture temperature to 20 °C where the modified surfaces become hydrophilic due to PIPAAm’s hydration/dehydration transition at 32 °C. In our pre- liminary studies, graft amounts of PIPAAm on the surfaces have significant influence on cell adhesion behavior. 14,15 However, detailed mechanisms to explain why cells cannot adhere on the surfaces with high amounts of grafted PIPAAm chains are unclear. Such phenomenon was also found for PIPAAm dip-coated surfaces or PPAAm hydro- gels. In the present paper, we focused on the correlation of the thickness of PIPAAm covalently grafted layers on tissue culture polystyrene (TCPS) surfaces and cell adhesion/detachment behavior. For this purpose, we utilized limited excimer laser ablation and atomic force microscopic methods to determine the thickness of PI- PAAm grafted layers. Preliminary ellipsometry measure- ment did not work to determine the grafted layer thickness, since the refractive indices are similar for polystyrene and PIPAAm. Then, we investigated thermoresponsive * Corresponding author. Phone: +81-3-3353-8111 ext. 30233. Fax: +81-3-3359-6046. E-mail: tokano@abmes.twmu.ac.jp. † Core Researches for Evolutional Science and Technology (CREST), Japan Science and Technology Agency. (1) Takei, Y. G.; Aoki, T.; Sanui, K.; Ogata, N.; Sakurai, Y.; Okano, T. Macromolecules 1994, 27, 6163-6166. (2) Yakushiji, T.; Sakai, K.; Kikuchi, A.; Aoyagi, T.; Sakurai, Y.; Okano, T. Langmuir 1998, 14, 4657-4662. (3) Kikuchi, A.; Okano, T. Prog. Polym. Sci. 2002, 27, 1165-1193. (4) Kanazawa, H.; Yamamoto, K.; Matsushima, Y.; Takai, N.; Kikuchi, A.; Sakurai, Y.; Okano, T. Anal. Chem. 1996, 68, 100-105. (5) Kanazawa, H.; Kashiwase, Y.; Yamamoto, K.; Matsushima, Y.; Kikuchi, A.; Sakurai, Y.; Okano, T. Anal. Chem. 1997, 69, 823-830. (6) Kanazawa, H.; Sunamoto, T.; Matsushima, Y.; Kikuchi, A.; Sakurai, Y.; Okano, T. Anal. Chem. 2000, 72, 5961-5966. (7) Yamada, N.; Okano, T.; Sakai, H.; Karikusa, F.; Sakurai, Y. Makromol. Chem. Rapid. Commun. 1990, 11, 571-576. (8) Okano, T.; Yamada, N.; Sakai, H.; Sakurai, Y. J. Biomed. Mater. Res. 1993, 27, 1243-1251. (9) von Recum, H. A.; Kim, S. W.; Kikuchi, A.; Okuhara, M.; Sakurai, Y.; Okano, T. J. Biomater. Sci., Polym. Ed. 1998, 9, 1241-1254. (10) Kushida, A.; Yamato, M.; Konno, C.; Kikuchi, A.; Sakurai, Y.; Okano, T. J. Biomed. Mater. Res. 2000, 51, 216-223. (11) Shimizu, T.; Yamato, M.; Kikuchi, A.; Okano, T. Tissue Eng. 2001, 7, 141-151. (12) Yamato, M.; Shimizu, T.; Harimoto, M.; Hirose, M.; Kushida, A.; Kwon O. H.; Kikuchi, A.; Okano, T. In Tissue Engineering for Therapeutic Use 5; Ikada, Y., Ohshima, N., Eds.; Elsevier Science: Amsterdam, 2001; pp 93-100. (13) Yamato, M.; Okano, T. Macromol. Chem. Symp. 2001, 14 (2), 21-29. (14) Sakai, H.; Doi, Y.; Okano, T.; Yamada, N.; Sakurai, Y. In Advanced Biomaterials in Biomedical Engineering and Drug Delivery Systems; Ogata, N., Feijen, J., Kim, S. W., Okano, T., Eds.; Springer: Tokyo, 1996; pp 229-230. (15) Yamato, M.; Konno, C.; Koike, S.; Isoi, Y.; Shimizu, T.; Kikuchi, A.; Makino, K.; Okano, T. J. Biomed. Mater. Res. 2003, 67A, 1065- 1071. 5506 Langmuir 2004, 20, 5506-5511 10.1021/la036139f CCC: $27.50 © 2004 American Chemical Society Published on Web 05/26/2004