Detachment Behavior on Dual

Feb 19, 2010 - E-mail: [email protected]., § .... Thermal-Responsive Block Copolymers for Surface with Reversible Switchable Wettability. Jin-Jin ...
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Protein Adsorption and Cell Adhesion/Detachment Behavior on Dual-Responsive Silicon Surfaces Modified with Poly(N-isopropylacrylamide)-block-polystyrene Copolymer Qian Yu,† Yanxia Zhang,† Hong Chen,*,†,§ Feng Zhou,† Zhaoqiang Wu,† He Huang,†,§ and John L. Brash‡ †

State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, Wuhan 430070, PR China, and ‡School of Biomedical Engineering and Department of Chemical Engineering, McMaster University, Hamilton, Ontario, Canada. §Current address: Collage of Chemistry, Chemical Engineering and Materials Science, Soochow University, Soochow, Jiangsu, PR China. Received December 10, 2009. Revised Manuscript Received February 3, 2010

Diblock copolymer grafts covalently attached to surfaces have attracted considerable attention because of their special structure and novel properties. In this work, poly(N-isopropylacrylamide)-block-polystyrene (PNIPAAm-b-PS) brushes were prepared via surface-initiated consecutive atom-transfer radical polymerization on initiator-immobilized silicon. Because of the inherent thermosensitivity of PNIPAAm and the hydrophobicity difference between the two blocks, the modified surfaces were responsive to both temperature and solvent. Moreover, the diblock copolymer brushes exhibited both resistance to nonspecific protein adsorption and unique cell interaction properties. They showed strong protein resistance in both phosphate-buffered saline and blood plasma. In particular, fibrinogen adsorption from plasma at either room temperature or body temperature was less than 8 ng/cm2, suggesting that the surfaces might possess good blood compatibility. In addition, the adhesion and detachment of L929 cells could be “tuned”, and the ability to control the detachment of cells thermally was restored by block polymerization of hydrophobic, cell-adhesive PS onto a thicker PNIPAAm layer. In addition to providing a simple and effective design for advanced cell-culture surfaces, these results suggest new biomedical applications for PNIPAAm.

1. Introduction Diblock copolymers have received considerable attention because of their many interesting properties and applications, including nanopatterning on solid surfaces.1-4 When diblock copolymers are physically adsorbed or spin-coated from solution onto a solid surface, they generally undergo microphase separation and form nanometer-sized domains via a self-assembly process.5 These unique nanostructures provide surfaces with potential biomedical applications. For example, Kumar et al. have investigated protein adsorption behavior on amphiphilic diblock copolymer ultrathin films.6-8 They found that protein molecules selectively self-segregate onto hydrophobic domains, suggesting that such surfaces might be used as highly suitable functional protein sensor substrates. The formation of protein patterns could be interpreted mainly in terms of the differences in hydrophilicity of the two blocks, though other factors may also influence the adsorption behavior.9 Liu et al. obtained protein nanopatterns using a highly organized diblock copolymer template consisting of two hydrophobic components of different flexibility.10 In addition to forming *Corresponding author. Tel:þ86-27-87168305. Fax:þ86-27-87168305. E-mail: [email protected]. (1) Lazzari, M.; Lopez-Quintela, M. A. Adv. Mater. 2003, 15, 1583–1594. (2) Krishnamoorthy, S.; Hinderling, C.; Heinzelmann, H. Mater. Today 2006, 9, 40–47. (3) Hamley, I. W. Nanotechnology 2003, 14, R39–R54. (4) Park, C.; Yoon, J.; Thomas, E. L. Polymer 2003, 44, 6725–6760. (5) Fasolka, M. J.; Mayes, A. M. Annu. Rev. Mater. Res. 2001, 31, 323–355. (6) Kumar, N.; Parajuli, O.; Hahm, J.-i. J. Phys. Chem. B 2007, 111, 4581–4587. (7) Kumar, N.; Hahm, J.-i. Langmuir 2005, 21, 6652–6655. (8) Kumar, N.; Parajuli, O.; Gupta, A.; Hahm, J.-i. Langmuir 2008, 24, 2688– 2694. (9) Lau, K. H. A.; Bang, J.; Kim, D. H.; Knoll, W. Adv. Funct. Mater. 2008, 18, 3148–3157. (10) Liu, D.; Wang, T.; Keddie, J. L. Langmuir 2009, 25, 4526–4534.

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protein patterns, microphase-separated structures are expected to produce surfaces that are protein-resistant11 and cell-adhesionresistant12 and therefore have improved blood and tissue compatibility.13 It should be noted that most of these surfaces were prepared using diblock copolymers synthesized by bulk polymerization methods and were placed on the surface via spin casting. The covalent grafting approach reported in the present work should give surfaces that are more stable and more versatile (tunable) with respect to surface structure. In a typical diblock copolymer brush structure, one of the blocks is tethered to the surface and the other block stretches away from the surface to interact with the contacting medium.14,15 Such chemically grafted polymer layers are inherently more stable than layers formed by physical adsorption or spin coating. The behavior and properties of tethered diblock copolymer brushes are interesting in that because of the different properties of the two blocks (hydrophobicity, flexibility, etc.) the brushes may undergo a structural rearrangement and property change in response to external stimuli.16-19 For example, Brittain’s group has reported (11) Feng, L.; Gu, G.; Chen, M.; Wu, L. Macromol. Mater. Eng. 2007, 292, 754– 761. (12) George, P. A.; Donose, B. C.; Cooper-White, J. J. Biomaterials 2009, 30, 2449–2456. (13) Nojiri, C.; Okano, T.; Koyanagi, H.; Nakahama, S.; Park, K. D.; Kim, S. W. J. Biomater. Sci., Polym. Ed. 1992, 4, 75–88. (14) Zhao, B.; Brittain, W. J. Prog. Polym. Sci. 2000, 25, 677–710. (15) Boyes, S. G.; Granville, A. M.; Baum, M.; Akgun, B.; Mirous, B. K.; Brittain, W. J. Surf. Sci. 2004, 570, 1–12. (16) Zhao, B.; Brittain, W. J. J. Am. Chem. Soc. 1999, 121, 3557–3558. (17) Zhao, B.; Brittain, W. J. Macromolecules 2000, 33, 8813–8820. (18) Granville, A. M.; Boyes, S. G.; Akgun, B.; Foster, M. D.; Brittain, W. J. Macromolecules 2005, 38, 3263–3270. (19) Rowe, M. A.; Hammer, B. A. G.; Boyes, S. G. Macromolecules 2008, 41, 4147–4157.

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on a wide variety of diblock copolymer brushes; they found that these brushes underwent self-reorganization and exhibited different surface properties in response to block-selective solvents.20-24 In principle, it should also be possible to obtain surfaces having dual functionality by combining the respective properties of the two blocks in a given system.25-27 Properties may be varied by regulating the composition of the blocks, the relative block length, and the block sequence.28,29 Therefore, diblock copolymer brushes have attracted increasing attention because of their unique structure and novel properties, and extensive research has been carried out on the design of new diblock copolymer brushes not only for theoretical studies but also for their potential applications.30-34 However, most of the previous reports on diblock copolymer brushes have been focused on surface wettability or topography, and their biointeractions have received less attention. To the best of our knowledge, there are few reports on the control of protein adsorption and cell adhesion by diblock copolymer brushes. In the work reported here, poly(N-isopropylacrylamide)-blockpolystyrene (PNIPAAm-b-PS) brushes were prepared via surfaceinitiated consecutive atom-transfer radical polymerization (ATRP) on initiator-immobilized silicon surfaces. PNIPAAm is the best known of the thermoresponsive polymers, displaying reversible solubility changes in response to temperature in the vicinity of 32 °C (the lower critical solution temperature (LCST)) in aqueous solution.35 PNIPAAm-modified surfaces exhibit good protein resistance at room temperature.36,37 They have also been used as cell-culture substrates with unique behavior that allows the release of cell layers by cycling through the LCST.38-41 However, it has been suggested that this thermoresponsive cell adhesion/detachment behavior will disappear when the grafted PNIPAAm layer is beyond a “critical thickness range”.41,42 Therefore, there is a need to develop systems that extend the application areas of PNIPAAm-based cell-culture substrates.43 (20) Ayres, N.; Boyes, S. G.; Brittain, W. J. Langmuir 2007, 23, 182–189. (21) Ayres, N.; Cyrus, C. D.; Brittain, W. J. Langmuir 2007, 23, 3744–3749. (22) Boyes, S. G.; Akgun, B.; Brittain, W. J.; Foster, M. D. Macromolecules 2003, 36, 9539–9548. (23) Granville, A. M.; Boyes, S. G.; Akgun, B.; Foster, M. D.; Brittain, W. J. Macromolecules 2004, 37, 2790–2796. (24) Granville, A. M.; Brittain, W. J. Macromol. Rapid Commun. 2004, 25, 1298– 1302. (25) Lindqvist, J.; Nystrom, D.; Ostmark, E.; Antoni, P.; Carlmark, A.; Johansson, M.; Hult, A.; Malmstrom, E. Biomacromolecules 2008, 9, 2139–2145. (26) Rahane, S. B.; Floyd, J. A.; Metters, A. T.; Kilbey, S. M. Adv. Funct. Mater. 2008, 18, 1232–1240. (27) Wang, X.; Xiao, X.; Wang, X. H.; Zhou, J. J.; Li, L.; Xu, J. Macromol. Rapid Commun. 2007, 28, 828–833. (28) Yu, K.; Han, Y. C. Soft Matter 2009, 5, 759–768. (29) Xu, C.; Wu, T.; Drain, C. M.; Batteas, J. D.; Fasolka, M. J.; Beers, K. L. Macromolecules 2006, 39, 3359–3364. (30) Brittain, W. J.; Boyes, S. G.; Granville, A. M.; Baum, M.; Mirous, B. K.; Akgun, B.; Zhao, B.; Blickle, C.; Foster, M. D. Adv. Polym. Sci. 2006, 198, 125– 147. (31) Tsukagoshi, T.; Kondo, Y.; Yoshino, N. Colloids Surf., B 2007, 55, 19–25. (32) Gao, X.; Feng, W.; Zhu, S.; Sheardown, H.; Brash, J. L. Langmuir 2008, 24, 8303–8308. (33) Xu, F. J.; Zhong, S. P.; Yung, L. Y. L.; Tong, Y. W.; Kang, E.-T.; Neoh, K. G. Biomaterials 2006, 27, 1236–1245. (34) Osborne, V. L.; Jones, D. M.; Huck, W. T. S. Chem. Commun. 2002, 1838– 1839. (35) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163–249. (36) Cheng, X.; Canavan, H. E.; Graham, D. J.; Castner, D. G.; Ratner, B. D. Biointerphases 2006, 1, 61–72. (37) Huber, D. L.; Manginell, R. P.; Samara, M. A.; Kim, B.-I.; Bunker, B. C. Science 2003, 301, 352–354. (38) da Silva, R. M. P.; Mano, J. F.; Reis, R. L. Trends Biotechnol. 2007, 25, 577– 583. (39) Kikuchi, A.; Okano, T. J. Controlled Release 2005, 101, 69–84. (40) Yoshida, M.; Langer, R.; Lendlein, A.; Lahann, J. Polym. Rev. 2006, 46, 347–375. (41) Li, L.; Zhu, Y.; Li, B.; Gao, C. Langmuir 2008, 24, 13632–13639. (42) Akiyama, Y.; Kikuchi, A.; Yamato, M.; Okano, T. Langmuir 2004, 20, 5506–5511. (43) Kong, B.; Choi, J. S.; Jeon, S.; Choi, I. S. Biomaterials 2009, 30, 5514–5522.

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With PNIPAAm as one block, PS was chosen as the second block because it is a typical hydrophobic polymer and is widely used in cell-culture applications. In the present work, we investigated the stimuli-responsive properties of PNIPAAm-b-PSmodified surfaces with respect to temperature and solvent. We also studied the interactions of proteins and cells with the modified surfaces, including the influence of temperature on cell adhesion and detachment. Interestingly, our results indicated that the PNIPAAm-b-PS-modified surfaces can both facilitate cell adhesion and resist nonspecific protein adsorption; these properties are of great importance in biomedical applications, especially tissue engineering.44

2. Experiments 2.1. Materials. N-Isopropyl acrylamide (NIPAAm, Acros, 99%) was recrystallized from a toluene/hexane solution (50% v/v) and dried under vacuum prior to use. Styrene (Shanghai Chemical Reagent Co.) was washed three times with a 5 wt % sodium hydroxide solution and twice with distilled water. After drying with anhydrous magnesium sulfate, the monomer was purified by distillation under reduced pressure and stored in a refrigerator immediately after distillation. Copper(I) bromide (CuBr, Fluka, 98%) was purified by stirring in acetic acid, washing with methanol, and drying under vacuum. 3-Aminopropyltriethoxysilane (APTES, Aldrich), bromoisobutyryl bromide (BIBB, Fluka), ethyl 2-bromoisobutyrate (EBIB, Aldrich), and 1,1,4,7,7-pentamethyldiethylenetriamine (PMDETA, Aldrich, 99%) were used as received. All other solvents were purchased from Shanghai Chemical Reagent Co. and purified according to standard methods before use. Silicon wafers [n-doped, (100)-oriented, 0.56 mm thick, 100 mm diameter] were purchased from the laboratory of Guangzhou Semiconductor Materials (Guangzhou, China). The as-received silicon wafers were cut into square chips of about 0.5 cm  0.5 cm. Deionized water purified by a Millipore waterpurification system to give a minimum resistivity of 18.2 MΩ 3 cm was used in all experiments. Nitrogen gas was of high-purity grade. Fibrinogen (MW = 341 kDa, plasminogen-free) was purchased from Calbiochem (La Jolla, CA). Citrated normal human blood plasma was purchased from the Wuhan blood center. L929 cells were supplied by the China Center for Type Culture Collection (CCTCC). 2.2. Preparation of PNIPAAm-b-PS-Grafted Silicon. The pretreatment of silicon wafers for the immobilization of initiator followed the procedures reported in our previous work.45 Surface-initiated ATRP grafting of NIPAAm was carried out in a glovebox purged with nitrogen. NIPAAm (6.25 g, 55.23 mmol), PMDETA (0.7 mL, 3.35 mmol), and CuBr (0.16 g, 1.12 mmol) were dissolved in a 1:1 mixture of methanol and water (25 mL). The reaction solution was sonicated for 2 min and then was added to a glass vessel in which the initiatorfunctionalized wafers were placed. Polymerization was carried out at room temperature under a nitrogen atmosphere for 2 h. After the desired period of time, the polymerization was stopped by adding a methanol solution of CuBr2/PMDETA. The obtained PNIPAAm-grafted silicon wafers were removed from the solution, rinsed with abundant deionized water to remove unreacted NIPAAm monomer and PNIPAAm that was not grafted, and then dried under a flow of nitrogen. The SiPNIPAAm surfaces were used as macroinitiators for the subsequent surface-initiated ATRP of PS to produce linear diblock copolymer brushes. ATRP of styrene was carried out according to a previously reported method22,23 with some modifications. Briefly, CuBr (55 mg, 0.38 mmol) was dissolved in a mixture of styrene (13.5 mL, 117.8 mmol) and anisole (16.5 mL). After the (44) Bhat, R. R.; Chaney, B. N.; Rowley, J.; Liebmann-Vinson, A.; Genzer, J. Adv. Mater. 2005, 17, 2802–2807. (45) Wu, Z.; Chen, H.; Liu, X.; Zhang, Y.; Li, D.; Huang, H. Langmuir 2009, 25, 2900–2906.

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Yu et al. Scheme 1. Process for Grafting PNIPAAm-b-PS from a Silicon Surfacea

a

(1) Aminosilanization of surface. (2) Immobilization of initiator. (3) SI-ATRP of NIPAAm. (4) ATRP of styrene initiated from NIPAAm grafts.

mixture was degassed (two freeze-pump-thaw cycles), PMDETA (157 μL, 0.75 mmol) was added and degassing was continued for two cycles. The solution was then transferred to a 100 mL Schlenk flask containing initiator-functionalized silicon wafers, and EBIB (46 μL, 0.31 mmol) was added by syringe. Polymerization was allowed to proceed at 110 °C for 8 h. Afterwards, the wafers were taken out of the polymerization solution and rinsed thoroughly with toluene. To remove physically adsorbed polymer, the wafers were Soxhlet extracted with dichloromethane for 24 h, cleaned ultrasonically in dichloromethane, and finally dried under a flow of nitrogen.

2.3. Treatment of Si-PNIPAAm-b-PS Surfaces with Different Solvents. Surfaces were treated with different solvents to investigate possible surface rearrangement. Water was chosen as a good solvent for the PNIPAAm segment, and cyclohexane was chosen as a good solvent for the PS segment. The PNIPAAmb-PS surfaces were immersed in 20 mL of deionized water at room temperature or cyclohexane at 40 °C for 1 h. The samples were removed from the solvent and dried in a flow of nitrogen, followed by water contact angle measurement. 2.4. Surface Characterization. The chemical composition of the modified silicon surfaces was determined with an ESCALAB MK II X-ray photoelectron spectrometer (XPS) (VG Scientific Ltd.). All XPS data were analyzed using XPS Peak 4.1 software. The topology of the modified silicon surfaces was studied with a DI Nanoscope V atomic force microscope (AFM) (Vecco). The rms surface roughness was calculated from the roughness profiles. To investigate the surface morphologies in water, the samples were immersed in deionized water for 3 h and then lyophilized prior to AFM examination. The thickness of the polymer grafts on the silicon substrate was measured with an M88 spectroscopic ellipsometer (J. A. Woollam Co., Inc.). The static water contact angles of the pristine and functionalized silicon surfaces were measured using the sessile drop method on a C201 optical contact angle meter (Solon Information Technology Co., Ltd.). 2.5. Protein Adsorption. Fibrinogen (Fg) was dissolved in phosphate-buffered saline (PBS) (pH 7.4) and radiolabeled with 125 I (Isotope Company of China, Beijing, PR China) using the iodine monochloride (ICl) technique. Unbound radioactive iodide was removed by ion-exchange chromatography on AG-1-X4 resin (Bio-Rad Laboratories, Hercules, CA). For studies of Fg adsorption from buffer, labeled and unlabeled proteins were mixed (1/9 labeled/unlabeled) to a total concentration of 1 mg/ mL. For studies of Fg adsorption from blood plasma, labeled Fg was added to the plasma in an amount corresponding to 10% of the endogenous Fg level. Unmodified and modified silicon wafers were first immersed in PBS for 12 h to achieve complete hydration and then transferred to 96-well microtiter plates, with each well containing 250 μL of protein solution. 8584 DOI: 10.1021/la904663m

Adsorption was allowed to proceed for 3 h under static conditions at either room temperature (23 °C) or body temperature (37 °C). Following adsorption, the surfaces were immediately immersed in fresh protein-free PBS for 10 min (three times) to remove loosely adsorbed protein. The samples were then wicked onto filter paper and transferred to clean counting vials for radioactivity determination using a Wizard 300 1480 Automatic Gamma Counter (Perkin-Elmer Life Sciences). 2.6. Cell Culture. All samples were placed in the wells of a 48well tissue culture plate. L929 cells, derived from mouse connective tissue fibroblasts, were cultured in RPMI medium 1640 (Gibco) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/mL penicillin, and 10 mg/mL streptomycin. L929 cells were seeded at a density of 3.0  104 cells/cm2 and incubated at 37 °C in a humidified atmosphere containing 5% CO2 for 2 days. The culture medium was replaced every other day. After a predetermined incubation time, the samples were washed twice with PBS at 37 °C to remove unattached cells and then fixed with 2.5% glutaraldehyde and dehydrated in a series of ethanol solutions (30-100%). For the study of cell detachment, the cellattached samples were incubated at 25 °C for 1 day. The same washing and fixation procedures were conducted as described above. The surfaces were then examined by scanning electron microscopy (SEM, JSM-5610LV) after coating a thin platinum layer.

3. Results and Discussion 3.1. Formation of PNIPAAm-b-PS Diblock Brushes on Flat Silicon Substrates via Consecutive Surface-Initiated ATRP. The general process for the formation of PNIPAAm-bPS block copolymer brushes on a silicon wafer surface by ATRP is illustrated in Scheme 1. First, a homogeneous, dense monolayer of initiator was immobilized on the silicon surface. Then SIATRP of NIPAAm was carried out by immersing the initiatormodified silicon in the reaction medium for 2 h at room temperature. To stop the polymerization and to convert reactive sites into the corresponding dormant species, the polymerization solution was replaced by a degassed methanol solution of CuBr2/PMDETA. The resulting PNIPAAm-grafted layer had a thickness of ∼42 nm as determined by ellipsometry, consistent with the value reported elsewhere.46 Prior to the grafting of the PS block, the PNIPAAm-grafted surfaces were washed thoroughly with deionized water to remove any unreacted NIPAAm monomer and ungrafted PNIPAAm. The ATRP of styrene was then carried out in anisole as the solvent (46) Sun, T. L.; Wang, G. J.; Feng, L.; Liu, B. Q.; Ma, Y. M.; Jiang, L.; Zhu, D. B. Angew. Chem., Int. Ed. 2004, 43, 357–360.

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Figure 1. XPS survey spectra and high-resolution C 1s spectra (inset) of (a) PNIPAAm- and (b) PNIPAAm-b-PS-grafted surfaces. The peak assignments are (I) C-C/H, (II) C-N, and (III) CdO.

at 110 °C for 8 h with the grafted PNIPAAm brushes as the macroinitiator. The PNIPAAm chain ends were reactivated to initiate PS grafting. Free EBIB initiator was added to the polymerization medium to generate a sufficient concentration of the deactivator copper(II) species; this is necessary for the formation of homogeneous films with grafts of controlled length, especially in the beginning stages of the ATRP process. Because of the possible “contamination” of immobilized polymer with polymer in solution, the resulting PNIPAAm-b-PS-modified surfaces were Soxhlet extracted with dichloromethane for 24 h and ultrasonicated in dichloromethane to remove loosely adsorbed polymer. Chain growth results in an increase in thickness and can be detected by ellipsometry.32 In this work, an increase in thickness of ∼2.6 nm compared to the initial PNIPAAm layer indicated the formation of PS blocks. Changes in the chemical composition of silicon surfaces after grafting were determined from XPS data. Figure 1 shows survey spectra of the PNIPAAm- and PNIPAAm-b-PS-grafted surfaces. For the PNIPAAm surface, the C/N/O atomic ratio was determined to be 73.6:12.3:13.0, close to the theoretical values for PNIPAAm, suggesting that the surface was completely covered with a PNIPAAm layer. Because the thickness of the grafted PS layer, ∼2.6 nm, was less than the sampling depth of the XPS measurement (∼10 nm), the N 1s and O 1s components are still discernible after the addition of the PS block. The high-resolution C 1s spectra are also shown in Figure 1 (inset). These were curve fitted using peaks with binding energies of 284.6, 286.2, and 287.4 eV, attributable to chemical bonding environments C-C/H, C-N, and CdO, respectively. Because PS is composed mainly of carbon and hydrogen, it is expected that the carbon content should increase after the addition of the PS block; indeed, the C/N ratio increased from 5.97 to 6.73 and the C-C/H peak increased from 52.9 to 55.9%. Static contact angle measurements indicated the wettability of the functionalized silicon surfaces at various stages. The pristine silicon substrate was strongly hydrophilic (contact angle ∼2°). It became more hydrophobic after modification by PNIPAAm, with angles of 57.3° at room temperature and 77.4° at 37 °C. This strong temperature dependence of surface wettability further demonstrated the formation of a PNIPAAm layer. After the copolymerization of PS, the contact angle increased to 62.1° at room temperature. This compares to the much greater value (>90°) for silicon modified with PS alone19,23 and suggests that the relatively short PS segments of the block copolymers did not cover the surface completely. Langmuir 2010, 26(11), 8582–8588

Figure 2. AFM phase images of Si-PNIPAAm and Si-PNIPAAm-b-PS surfaces before (a, b) and after water treatment (c, d), respectively. The scale bar in all images is 200 nm.

3.2. Surface Topography. The surface morphology of the PNIPAAm surfaces before and after block copolymerization of PS was investigated by AFM, and phase images of these two surfaces are shown in Figure 2. The PNIPAAm-grafted layer was very smooth with an rms roughness value of ∼0.624 nm and with no evidence of heterogeneity (Figure 2a), indicating uniform coverage of PNIPAAm. On the Si-PNIPAAm-b-PS surface (Figure 2b), however, two distinct domains were apparent, presumably indicating phase segregation of the PNIPAAm and PS blocks. Correspondingly, the rms roughness value increased to 1.69 nm. This obvious change might be rationalized on the basis that the outer PS segments were not long enough to cover the underlying layer completely whereas being just self-assembled and dispersed in the PNIPAAm layer. Because protein adsorption experiments were carried out in buffer solution, the morphology was also investigated in the wet state. Because the glass-transition temperatures of PS and PNIPAAm are above room temperature, the chains are immobile and the morphology of the outermost layer of the block brush is not expected to be greatly changed if the samples are exposed only briefly to the aqueous solution. Thus AFM images after treatment with water and then freeze drying could reflect the surface topography in water as suggested by others.28,46 Upon immersion in water, it is assumed that the grafted layers will undergo reorganization. However, the phase image and roughness of the PNIPAAm surface in the wet state showed little change compared to those in the dry state (Figure 2c). For the Si-PNIPAAm-g-PS DOI: 10.1021/la904663m

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Figure 3. Reversible change in the static water contact angle (room temperature) for the PNIPAAm-b-PS brushes in response to alternating contact with water and cyclohexane, respectively. Data consist of the mean ( standard error (n = 3).

surface (Figure 2d), the domain structure seen in the dry state disappeared, the surface became essentially homogeneous, and the roughness decreased. Under wet conditions, it is expected that the hydrophilic PNIPAAm chains will extend to the aqueous interface and form a shield around the PS segments. The thinner PS layer would facilitate PNIPAAm segment mobility during the rearrangement process. As a result, the aggregates started to merge and the outermost surface should be a more or less homogeneous PNIPAAm layer. 3.3. Determination of the Stimuli-Responsive Nature of the Diblock Copolymer Brushes. 3.3.1. Treatment of Surfaces with Block-Selective Solvents. Previous studies suggested that surfaces grafted with diblock copolymer brushes may undergo reorganization and exhibit different wettability in response to different solvents.16,17,32 Therefore, experiments were performed on the Si-PNIPAAm-b-PS surfaces to determine whether segment rearrangement occurred upon block-selective solvent treatment. Exposure to water, which is a good solvent for the inner PNIPAAm blocks but a poor solvent for the outer PS blocks, would be expected to cause the PS segments to retreat inward from the interface and mix with the PNIPAAm chains, resulting in a surface with PNIPAAm-like wettability. The contact angle value observed (∼58°, Figure 3) is in agreement with this expectation. Following treatment with cyclohexane, a good solvent for PS, the contact angle increased to ∼66°, presumably because of exposure of the more hydrophobic PS block at the outermost surface. However, because the water contact angles on pure PS surfaces are on the order of 90°,19,23 it is clear that there is still some mixing of blocks at the interface. The intermediate value of the water contact angle along with the indication mentioned previously that the short PS segments could not cover the surface completely suggests that both PNIPAAm and PS blocks were present at the interface. This solvent-sensitive wettability of the PNIPAAm-b-PS brushes was reversible for repeated cycles of alternating treatment by water and cyclohexane (Figure 3). 3.3.2. Temperature Dependence of Wettability. It has been reported that the wettability of PNIPAAm-grafted surfaces undergoes a temperature-dependent transition near the LCST (∼32 °C) of PNIPAAm.47,48 Below the LCST, the PNIPAAm (47) Balamurugan, S.; Mendez, S.; Balamurugan, S. S.; O’Brien, M. J.; Lopez, G. P. Langmuir 2003, 19, 2545–2549. (48) Liang, L.; Rieke, P. C.; Fryxell, G. E.; Liu, J.; Engehard, M. H.; Alford, K. L. J. Phys. Chem. B 2000, 104, 11667–11673.

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Figure 4. Water contact angles of Si-PNIPAAm and Si-PNIPAAm-b-PS surfaces at 23 and 37 °C. Data consist of the mean ( standard error (n = 3).

chains are strongly hydrated with an extended conformation, but when heated to above the LCST, the polymer undergoes a phase transition to a collapsed morphology due to dehydration. This response will be modified when NIPAAm is copolymerized with other monomers that are either hydrophilic or hydrophobic.49-51 In this work, water contact angles of Si-PNIPAAm and SiPNIPAAm-b-PS surfaces below and above the LCST were measured. It can be seen in Figure 4. that the Si-PNIPAAm surfaces exhibited higher contact angles at 37 than at 23 °C, consistent with other reports;41,46 the difference may be explained by the competition between intermolecular and intramolecular hydrogen bonding during the phase transition.46 After block copolymerization with PS, the contact angle values increased at both 23 and 37 °C because of the addition of more hydrophobic PS segments; the thermoresponsive wettability was still present. 3.4. Protein Adsorption. Fibrinogen (Fg) was used as a model protein to investigate protein adsorption on the modified surfaces because it is abundant in plasma and plays a major role in blood coagulation.52,53 Adsorption from 1 mg/mL Fg in phosphate-buffered saline (PBS) was measured at both room temperature and body temperature. It can be seen in Figure 5 that at room temperature adsorption on the PNIPAAm-grafted surface was significantly less than on the pristine silicon surface. PNIPAAm has been shown by others to have good protein resistance.37,54 Water bound to the PNIPAAm chains via intermolecular hydrogen bonds46 may be responsible for protein resistance by preventing intimate molecular contact between protein and the surface, similar to the situation for poly(2methacryloyloxyethyl phosphorylcholine)(PMPC) and OEG (oligo(ethylene glycol)).55-57 (49) Gil, E. S.; Hudson, S. A. Prog. Polym. Sci. 2004, 29, 1173–1222. (50) Barker, I. C.; Cowie, J. M. G.; Huckerby, T. N.; Shaw, D. A.; Soutar, I.; Swanson, L. Macromolecules 2003, 36, 7765–7770. (51) Feil, H.; Bae, Y. H.; Feijen, J.; Kim, S. W. Macromolecules 1993, 26, 2496– 2500. (52) Kwak, D.; Wu, Y. G.; Horbett, T. A. J. Biomed. Mater. Res. 2005, 74A, 69– 83. (53) Shen, M. C.; Wagner, M. S.; Castner, D. G.; Ratner, B. D.; Horbett, T. A. Langmuir 2003, 19, 1692–1699. (54) Cho, E. C.; Kim, Y. D.; Cho, K. J. Colloid Interface Sci. 2005, 286, 479–486. (55) Chen, S.; Zheng, J.; Li, L.; Jiang, S. J. Am. Chem. Soc. 2005, 127, 14473– 14478. (56) Li, L.; Chen, S.; Zheng, J.; Ratner, B. D.; Jiang, S. J. Phys. Chem. B 2005, 109, 2934–2941. (57) Ostuni, E.; Chapman, R. G.; Holmlin, R. E.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 5605–5620.

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Figure 5. Adsorption of Fg from PBS buffer and blood plasma (inset) over a 3 h period on pristine and modified silicon surfaces at 23 and 37 °C. Data consist of the mean ( standard error (n = 3).

Adsorption on the Si-PNIPAAm-b-PS surface was only slightly greater than on the Si-PNIPAAm, indicating that the introduction of the PS block did not affect the protein resistance. This result is consistent with the observed surface morphology of the Si-PNIPAAm-b-PS surface after water treatment, suggesting that most of the PS segments were buried in the PNIPAAm layer. At 37 °C, Fg adsorption increased on both the Si-PNIPAAm and Si-PNIPAAm-b-PS surfaces, in agreement with the observed wettability changes indicating that hydrophobic interactions are the main driving force for protein adsorption.58 To investigate the protein resistance of the surfaces under conditions closer to physiological, Fg adsorption from blood plasma was studied (inset, Figure 5). It is seen that adsorption levels were much less than from buffer and that the effect of temperature was no longer significant. Fg adsorption is known to be involved in the early stages of blood-material interactions, and the amount of adsorbed Fg may be related to blood compatibility. Horbett et al. have suggested that the adhesion and activation of platelets should become insignificant at Fg adsorption levels below 10 ng/cm2.53 Adsorption was highest on the Si-PNIPAAm-b-PS surface but was less than 8 ng/cm2 Fg whether at room temperature or 37 °C. These block-copolymergrafted surfaces may therefore be useful in blood-contacting applications, for example, as a bioinert material for biosensors. Further research on coagulant activity and platelet interactions will be required to establish blood compatibility fully. 3.5. Cell Adhesion and Detachment. Because Okano and co-workers pioneered the chemical grafting of thermoresponsive PNIPAAm onto tissue culture polystyrene (TCPS) as the basis for cell sheet tissue engineering,59 PNIPAAm-coated surfaces have been applied as cell-culture substrates that enable the reversible adhesion and detachment of cells by temperature control.38-41 Previous research suggested that thermoresponsive cell adhesion/ detachment behavior is dependent on the thickness of the PNIPAAm film41,42,60,61 and that the response weakens or even disappears when the PNIPAAm layer is beyond a “critical thickness range”.41,42 For thicker PNIPAAm layers, cells could (58) Sigal, G. B.; Mrksich, M.; Whitesides, G. M. J. Am. Chem. Soc. 1998, 120, 3464–3473. (59) Okano, T.; Yamada, N.; Okuhara, M.; Sakai, H.; Sakurai, Y. Biomaterials 1995, 16, 297–303. (60) Mizutani, A.; Kikuchi, A.; Yamato, M.; Kanazawa, H.; Okano, T. Biomaterials 2008, 29, 2073–2081. (61) Xu, F. J.; Zhong, S. P.; Yung, L. Y. L.; Kang, E. T.; Neoh, K. G. Biomacromolecules 2004, 5, 2392–2403.

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Figure 6. SEM images of L929-cells on (a) an unmodified Si surface at 37 °C, (b) a Si-PNIPAAm surface at 37 °C, (c) a SiPNIPAAm-b-PS surface at 37 °C, and (d) a Si-PNIPAAm-b-PS surface at 23 °C. The scale bar in all images is 100 μm.

not adhere even at 37 °C because more polymer chains in the outermost surface regions remain hydrated and the surfaces remain hydrophilic and cell-resistant.41,42,60 It seemed possible that this limitation could be resolved by incorporating hydrophobic elements such as polystyrene blocks into the PNIPAAm chains as in the present work. Figure 6 shows SEM images of L929 fibroblasts cultured on pristine and modified silicon surfaces. It is seen that cells readily attached and proliferated on the bare silicon surface (Figure 6a). Most of the cells were elongated and spindle-shaped whereas some were spherical, probably because of the high cell density. After grafting PNIPAAm, somewhat surprisingly, cell attachment and growth were prevented (Figure 6b) presumably because the thickness of the PNIPAAm grafted layer was beyond the “critical thickness”.41,42,60 Cell adhesion and growth were significantly greater on the Si-PNIPAAm-b-PS surface (Figure 6c). The adherent cells spread uniformly and again adopted a spindlelike shape; the overall appearance was similar to that of the bare silicon surface except that the cell density was smaller. The appearance of pseudopods or cell processes indicates that the Si-PNIPAAm-b-PS surface promoted cell growth. Therefore, with the addition of the PS block, the surface transitioned from being cell-repellent to cell-adhesive. Temperature-dependent celldetachment experiments were performed to investigate the role of the PS block. It was found that the Si-PNIPAAm-b-PS surface regained the property of cell detachment in response to temperature: many of the adherent cells became detached when the temperature decreased from 37 °C to room temperature (Figure 6d). This behavior may be related to a conformational transition of the PNIPAAm block through the LCST: at 37 °C, the inner PNIPAAm segments are dehydrated and adopt a collapsed conformation; more PS segments are located in the outermost layer, which enhances the adsorption of cell-adhesive proteins (such as vitronectin) and thus promotes cell spreading and proliferation62 (Supporting Information). At lower temperature, the PNIPAAm chains are strongly hydrated with an extended conformation. As mentioned previously, in culture medium most PS segments will be buried in the PNIPAAm layer and the surface will be cell-repellent. Thus, the data presented here indicate that thermoresponsive PNIPAAm graft-based cell-culture substrates can be fine tuned by modifying the grafts with (62) Steele, J. G.; Dalton, B. A.; Johnson, G.; Underwood, P. A. J. Biomed. Mater. Res. 1993, 27, 927–940.

DOI: 10.1021/la904663m

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hydrophobic polymer blocks. For block copolymer grafts in general, the relative block length plays a critical role in determining the surface properties28,32 and is expected to influence the density and morphology of adherent cells. Investigations of this parameter in the PNIPAAm-PS system are in progress.

4. Conclusions In this work, diblock copolymer PNIPAAm-b-PS-grafted surfaces were prepared via surface-initiated consecutive atomtransfer radical polymerization on initiator-immobilized silicon. AFM showed that the surfaces were microphase-separated with hydrophobic PS nanodomains dispersed in a continuous PNIPAAm phase. Upon exposure to water, the domain structure disappeared and the surface became more homogeneous with decreased roughness. These surfaces were shown to be responsive to both temperature and solvent. Exposure to different solvents selective for one block or another caused wettability and corresponding structural changes. The changes were reversible and could be cycled repeatedly. Surface wettability was also temperature-sensitive as expected because of the transition of the PNIPAAm block through its lower critical solution temperature. The significant feature of these new diblock copolymer graft surfaces is that they resist nonspecific protein adsorption and facilitate cell adhesion at the appropriate temperature. The modified surface showed protein resistance at room temperature similar to that of analogous homoPNIPAAm even though the

8588 DOI: 10.1021/la904663m

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outer PS block is hydrophobic and protein-adsorptive. The adsorption of fibrinogen from plasma over a 3 h period was less than 8 ng/cm2 at either room temperature or 37 °C. Therefore, it appears that this block-copolymer-grafted surface may possess good blood compatibility. Experiments with L929 fibroblasts showed very little adhesion on the Si-PNIPAAm surface with a PNIPAAm layer thickness of ∼42 nm even at 37 °C. In contrast, the PNIPAAm-b-PS-grafted surface supported significant adhesion and promoted cell growth. It was shown that the adherent cells could be detached by decreasing the temperature below the LCST, suggesting that surfaces grafted with block copolymers of PNIPAAm may provide the basis for novel cell-culture systems. Acknowledgment. We are grateful to Professor Hongwei Ma’s group for assistance with ellipsometry measurements. This work was supported by the National Natural Science Foundation (20634030, 20974086, and 20920102035), the Ministry of Education (NCET0606055), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. Supporting Information Available: Western blot analysis of vitronectin adsorption on the modified surfaces. This material is available free of charge via the Internet at http:// pubs.acs.org.

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