“Smart” Biopolymer for a Reversible Stimuli-Responsive Platform in

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Langmuir 2008, 24, 4917-4923

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“Smart” Biopolymer for a Reversible Stimuli-Responsive Platform in Cell-Based Biochips Kyunga Na,† Jaeyeon Jung,† Okgene Kim,† Jonghwan Lee,† Tae Geol Lee,‡ Young Hwan Park,†,§ and Jinho Hyun*,†,| Department of Biosystems and Biomaterials Science and Engineering, Intelligent Textile Research Center, and Research Institute for Agriculture and Life Sciences, Seoul National UniVersity, Seoul, 151-742 Korea, and NanoBio Fusion Research Center, Korea Research Institute of Standards and Science, P.O. Box 102, Yuseong, Daejeon, 305-600 Korea ReceiVed September 21, 2007. In Final Form: January 17, 2008 The rapid response of a smart material surface to external stimuli is critical for application to cell-based biochips. The sharp and controllable phase transition of elastin-like polypeptide (ELP) enabled reversible cell adhesion on the surface by changing the temperature or salt concentration in the system. First, ELP micropatterns were prepared on a glass surface modified into aldehyde. The lysine-containing ELP (ELP-K) was genetically synthesized from E. coli for conjugation with the aldehyde on the glass surface. The phase transition of ELP was monitored in PBS and cell culture media using UV-visible spectroscopy, and a significant difference in transition temperature (Tt) was observed between the two solution systems. The micropatterning of ELP on the glass surface was performed by microcontact printing a removable polymeric template on the aldehyde-glass followed by incubation in ELP-K aqueous solution. The ELP micropatterns were imaged with atomic force microscopy and showed a monolayer thickness of ∼4 nm. Imaging from time-of-flight secondary ion mass spectroscopy confirmed that the ELP molecules were successfully immobilized on the highly resolved micropatterns. Cell attachment and detachment could be reversibly controlled on the ELP surfaces by external stimuli. The hydrophobic phase above Tt resulted in the adhesion of fibroblasts, while the detachment of cells was induced by lowering the incubation temperature below Tt. The smart properties of ELP were reliable and reproducible, demonstrating potential applications in cell-based microdevices.

Introduction Stimuli-responsive smart polymer surfaces are attractive in cell-based microdevices because they can be used to control cell attachment and detachment by external stimuli such as temperature,1 pH,2 ionic strength,3 and light.4 Derivatives of poly(Nisopropylacrylamide) (pNIPAAM), a well-known smart material, are reported to be effective for the control of cell attachment by reversible hydration/dehydration induced by temperature change.5-9 In cell-based biochips for high-throughput screening, * To whom correspondence should be addressed. E-mail: jhyun@ snu.ac.kr. † Department of Biosystems and Biomaterials Science and Engineering, Seoul National University. ‡ Korea Research Institute of Standards and Science. § Intelligent Textile Research Center, Seoul National University. | Research Institute for Agriculture and Life Sciences, Seoul National University. (1) Nath, N.; Chilkoti, A. Fabrication of a reversible protein array directly from cell lysate using a stimuli-responsive polypeptide. Anal. Chem. 2003, 75, 709-715. (2) Ju, H. K.; Kim, S. Y.; Lee, Y. M. pH/temperature-responsive behaviors of semi-IPN and comb-type graft hydrogels composed of alginate and poly (Nisopropylacrylamide). Polymer 2001, 42, 6851-6857. (3) Kumar, A.; Galaev, I. Y.; Mattiasson, B. Isolation and separation of alphaamylase inhibitors I-1 and I-2 from seeds of ragi (Indian finger millet, Eleusine coracana) by metal chelate affinity precipitation. Bioseparation 1998, 7, 129136. (4) Edahiro, J.; Sumaru, K.; Tada, Y.; Ohi, K.; Takagi, T.; Kameda, M.; Shinbo, T.; Kanamori, T.; Yoshimi, Y. In situ control of cell adhesion using photoresponsive culture surface. Biomacromolecules 2005, 6, 970-974. (5) Xu, F. J.; Zhong, S. P.; Yung, L. Y. L.; Kang, E. T.; Neoh, K. G. Surfaceactive and stimuli-responsive polymer-Si(100) hybrids from surface-initiated atom transfer radical polymerization for control of cell adhesion. Biomacromolecules 2004, 5, 2392-2403. (6) Ebara, M.; Yamato, M.; Hirose, M.; Aoyagi, T.; Kikuchi, A.; Sakai, K.; Okano, T. Copolymerization of 2-carboxyisopropylacrylamide with N-isopropylacrylamide accelerates cell detachment from grafted surfaces by reducing temperature. Biomacromolecules 2003, 4, 344-349. (7) Ito, Y. Surface micropatterning to regulate cell functions. Biomaterials 1999, 20, 2333-2342.

the toxicity of the culturing surface is critical, especially for long-term cell incubation, and limitations in controlling transition temperature further restrict its application. Elastin-like polypeptide (ELP) is a protein-based biopolymer that exhibits thermal inverse phase transition similar to pNIPAAM.10 The most distinctive features of an ELP-linked surface as a smart platform are (1) reversible control of cell adhesion, (2) rapid response to external stimuli, and (3) nontoxicity. The biocompatibility of ELP has been demonstrated in endothelial cells in Vitro11,12 and in ViVo,13,14 and an ELP surface would be a proper platform for a cell-based biochip capable of responding to external stimuli without damaging cells. Cultivated adherent cells are generally recovered using trypsin, but a surface grafted with ELP molecules could easily detach the cells by thermally induced phase transition.15 A rapid response to external stimuli is a requirement in cell-based biochips that (8) Wong, J. Y.; Leach, J. B.; Brown, X. Q. Balance of chemistry, topography, and mechanics at the cell-biomaterial interface: Issues and challenges for assessing the role of substrate mechanics on cell response. Surf. Sci. 2004, 570, 119-133. (9) Falconnet, D.; Csucs, G.; Grandin, H. M.; Textor, M. Surface engineering approaches to micropattern surfaces for cell-based assays. Biomaterials 2006, 27, 3044-3063. (10) Gil, E. S.; Hudson, S. A. Stimuli-reponsive polymers and their bioconjugates. Prog. Polym. Sci. 2004, 29, 1173-1222. (11) Nicol, A.; Gowda, D. C.; Urry, D. W. Cell-adhesion and growth synthetic elastomeric materices containing Arg-Gly-Asp-Ser-3. J. Biomed. Mater. Res. 1992, 26, 393-413. (12) Urry, D. W.; Parker, T. M.; Reid, M. C.; Gowda, D. C. Biocompatibility of the bioelastic materials, poly (GVGVP) and its gamma-irradiation cross-linked matrix - summary of generic biological test-results J. Bioact. Compat. Polym. 1991, 6, 263-282. (13) Betre, H.; Liu, W.; Zalutsky, M. R.; Chilkoti, A.; Kraus, V. B.; Setton, L. A. A thermally responsive biopolymer for intra-articular drug delivery. J. Controlled Release 2006, 115, 175-182. (14) Liu, W. E.; Dreher, M. R.; Furgeson, D. Y.; Peixoto, K. V.; Yuan, H.; Zalutsky, M. R.; Chilkoti, A. Tumor accumulation, degradation and pharmacokinetics of elastin-like polypeptides in nude mice. J. Controlled Release 2006, 116, 170-178.

10.1021/la702796y CCC: $40.75 © 2008 American Chemical Society Published on Web 03/19/2008

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Scheme 1. Strategy for Cell Patterning on the Stimuli-Responsive Substratea

a (A) Micropatterning of templating polymer on the surface, (B) immobilization of ELP on the surface, (C) removal of polymer template, (D) incubation of FN, (E) seeding of cells, and (F) decrease of temperature below Tt.

directly reflect environmental change. The thermal inverse phase transition temperature (Tt) of ELP is determined by its repetitive peptide sequence and the molecular weight of the polypeptides, and the transition occurs very rapidly and reversibly within a range of ∼2 °C.16 We describe the genetic synthesis of lysine-tagged ELP (ELPK) with a Tt of ∼30 °C for fabricating a thermoresponsive culture surface. A removable polymer template was microcontact-printed (µCP) on a glass surface derivatized with epoxide (Scheme 1) for the micropatterning of ELP-K. After conjugating ELP-K onto the glass surface and dissolving the polymer template, highly resolved ELP-K micropatterns were efficiently created on the surface. The successful micropatterning of the polypeptide was confirmed using ELP-K conjugated with fluorescent dye by confocal microscopy and atomic force microscopic (AFM) images. Time-of-flight secondary ion mass spectrometric (TOFSIMS) images of the ELP-K micropatterns also verified the highly resolved ELP microstructure on the surface. Reversible cell attachment by the control of cell incubation temperature on the ELP-modified substrate was successfully demonstrated. Materials and Methods Synthesis and Characterization of ELP. The ELP gene was oligomerized by the process of recursive directional ligation consisting of [(VPGVG)4(VPGKG)]8[VPGVG]40. ELP with a molecular weight of 82 kDa was expressed in the E. coli strain BLR(DE)3 (Novagen). The ELP was purified by the inverse transition cycling method and the concentration measured by UV-visible spectroscopy using an extinction coefficient at 280 nm. The purity and molecular weight (15) Okano, T.; Yamada, N.; Okuhara, M.; Sakai, H.; Sakurai, Y. Mechanism of cell detachment from temperature-modulated hydrophilic-hydrophobic polymer surfaces. Biomaterials 1995, 16, 297-303. (16) Chilkoti, A.; Dreher, M. R.; Meyer, D. E. Design of thermally responsive, recombinant polypeptide carriers for targeted drug delivery. AdV. Drug DeliVery ReV. 2002, 54, 1093-1111.

were determined by silver staining SDS-PAGE gels. The inverse transition temperature of ELP was characterized by the change in optical density (turbidity) at 350 nm using UV-visible spectroscopy in the temperature range from 22 to 40 °C. A 200 µM ELP solution was prepared with either PBS or cell culture media for the measurement of transition temperature. The inverse phase transition temperature was determined as the temperature at which the turbidity of the ELP solution was 50% of its maximum. Preparation of ELP-Patterned Glass. A 1 cm × 1 cm glass was cleaned with RBS 35 detergent (Fluka) and methanol/HCl (1:1 v/v). The glass was further washed with ethanol and dried overnight at 60 °C. For the silanation of a glass surface, a 1 cm × 1 cm piece of glass was immersed in 1% 3-glycidoxypropyl trimethoxysilane (Sigma) for 8 h. The silanized glass was reacted with 100 mM NaCl solution (pH4.0) and 47 mM NaIO4 overnight followed by drying with nitrogen. The micropatterning of ELP-K molecules on aldehydederivatized glass was performed by the process of soft lithography.17 First, a positive feature of an elastomeric stamp was fabricated by casting poly(dimethyl siloxane) (PDMS) against the photoresist on a silicon master with 80 µm or 100 µm features. The PDMS stamp was oxidized for 20 s using a commercially available hydrophilizer before µCP. Thin films of a random terpolymer18 were spin-cast from a 50/50 (v/v) mixture of H2O/ethanol onto the oxidized PDMS stamp at 2000 rpm for 2 min. The stamp was brought into conformal contact with the glass surface for 1 min, resulting in the transfer of the polymer micropatterns to the glass surface. Subsequently, the micropatterned glass was incubated with 0.2 mg/mL ELP-K in 50 mM sodium carbonate buffer (pH 9.2) and 5 mM NaCNBH3 for 5 h at 20 °C. After the covalent immobilization of ELP-K onto the surface, the polymeric layer was removed with the mixture of H2O/ ethanol. The ELP-patterned glasses were stored in PBS at 4 °C until further use. (17) Xia, Y. N.; Whitesides, G. M. Soft lithography. Annu. ReV. Mater. Sci. 1998, 28, 153-184. (18) Irvine, D. J.; Mayes, A. M.; Griffith, L. G. Nanoscale clustering of RGD peptides at surfaces using comb polymers. 1. Synthesis and characterization of comb thin films. Biomacromolecules 2001, 2, 85-94.

“Smart” Biopolymer Characterization of the ELP-Patterned Surface. AFM images were collected in the noncontact mode of the XE-100 model (PSIA, Korea) in air using NCHR silicon nitride cantilevers (Nanosensors, St. Louis, MO; spring constant 42 N/m; tip radius 10 nm). The typical observation of the rms noise of force was about 20 pN, which is in good agreement with the estimated thermal force fluctuations of 18 pN. TOF-SIMS images were obtained to identify the covalently immobilized ELP by a TOF-SIMS V instrument (ION-TOF GmbH) using 25 keV Bi32+ primary ions. High mass resolution exceeding M/∆M ≈ 10 000 was obtained using a high-current bunching system. The ion current in the bunching system was 0.1 pA at 5 kHz with a pulse duration of 16 ns. Positive and negative ionic mass spectra were acquired over a mass range of m/z 0-150. The raster area of the primary ions was 300 µm × 300 µm, which was chargecompensated for glass-slide samples by low-energy electron flooding. The primary ion dose was maintained at less than 1012 ions/cm2 to ensure static SIMS conditions.19 Cell Culture and Fabrication of Cell Array. The ELP-patterned surface was incubated with 1 µg/mL to 50 µg /mL of bovine fibronectin solution in PBS (FN, Sigma) at 37 °C for 30 min. NIH 3T3 fibroblasts (ATCC) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 µg/mL streptomycin, and 7.5 mM HEPES (Gibco) at 37 °C in a humidified atmosphere with 5.0% CO2. The fibroblasts were seeded on the micropatterned surface at a density of 5 × 105 cells/mL. The first group of cells was incubated for 18 h at 37 °C, and then gently rinsed with fresh culture media. Subsequently, the phase transition of the ELP-conjugated glass surface was induced by reducing the temperature to 20 °C. The second group of cells was seeded on the same micropatterned surface and incubated for another 18 h at 37 °C and then rinsed with fresh culture media. The phase transition of the ELP-conjugated glass surface was again induced by reducing the temperature to 20 °C. The three cyclic phase transitions were performed under a clean environment as mentioned above, and the cells in each step were imaged under phase-contrast microscopy.

Results and Discussion The fabrication strategy for the stimuli-responsive cell adhesion platform is shown in Scheme 1. A removable polymer template was microcontact printed on the aldehyde-derivatized glass (Scheme 1A). The substrate was subsequently incubated with a 2 µM ELP solution in 50 mM sodium carbonate buffer (pH 9.2) at 20 °C and 5 mM cyanoborohydride. ELP was covalently immobilized on the free region of the polymer template on the aldehyde-silaned glass surface through the reaction of the primary amine groups to form a Schiff base with the aldehyde groups (Scheme 1B). The glass was immersed in the mixture of H2O/ ethanol for 1 h to remove the pattern of the polymer template, and then the glass with the ELP brushes immobilized to form a smart platform was finally prepared (Scheme 1C). The ELPpatterned surface was incubated with fibronectin in PBS buffer at 37 °C for 30 min to promote cell attachment (Scheme 1D). Prior to the incubation of the 3T3 fibroblasts, the substrate was immersed in the culture media for 1 h to sustain the hydrophobic phase of the ELP molecules. The attachment and detachment of the cells on the ELP region were controlled by incubation temperature (Scheme 1E,F). We synthesized the amine-functionalized ELP for covalent immobilization onto the aldehyde-derivatized glass surface. An ELP gene library containing a lysine was constructed through the recursive directional ligation method based on the monomer [(VPGVG)4(VPGKG)]. Successful construction was confirmed by DNA agarose gel electrophoresis and DNA sequencing. The (19) Lee, T. G.; Shon, H. K.; Lee, K. B.; Kim, J.; Choi, I. S.; Moon, D. W. Time-of-flight secondary ion mass spectrometry chemical imaging analysis of micropatterns of streptavidin and cells without labeling. J. Vac. Sci. Technol., A 2006, 24, 1203-1207.

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Figure 1. Inverse phase transition of ELP in either PBS or cell culture media.

ELP molecules expressed from a gene composed of 2400 bp were purified by inverse transition cycling. The peptide sequence of the synthesized ELP was [(VPGVG)4(VPGKG)]8[VPGVG]40 and its molecular weight was 82 kDa, as measured by SDSPAGE. The inverse phase transition of the ELP solution could be observed by the change of solution turbidity detected using a UV-visible spectrometer. We obtained the inverse phase transition profiles of ELP solution in either PBS or culture media, which is the actual environment in cell culture (Figure 1). In previous reports, transition curves were obtained in standard buffer solutions such as PBS or deionized water, although the ELP molecules were intended for application in quite different environments.20,21 Since cell incubation media are complex systems containing proteins, antibiotics, growth factors, and other surface active materials, the transition behavior in cell incubation media must be different from the transitions obtained in standard buffer solutions. As shown in Figure 1, ELP molecules showed a sharp transition in both the cell incubation media and the PBS solution. Tt shifted significantly from 35 °C in PBS to 30 °C in culture media, demonstrating the effects of the solvent system. Tt can be influenced by several extrinsic factors including solvent, ELP concentration, and ionic strength. The phase transition of ELP is a change in the folded and unfolded state of the protein. The unfolding ELP remains disordered, and random coils are fully hydrated when the temperature is below Tt. In contrast, ELP molecules fold when temperatures are above Tt, driven by hydrophobic forces. This folding results in the formation of intramolecular contacts between nonpolar regions of the ELP molecules. The components in culture media prevent ELP molecules from interacting with water molecules, providing an explanation for the observed shift in Tt between solvents. Characterization of Tt in culture media will be more meaningful if the ELP is ultimately to be applied to a cell culture system. We selected a glass plate as a generic platform for a smart cell biochip because it is biocompatible, transparent and, most of all, it is easy to functionalize the surface for conjugation with biomolecules. As shown in Scheme 1, the glass surface was modified into aldehyde through the ring opening of an epoxy (20) Betre, H.; Ong, S. R.; Guilak, F.; Chilkoti, A.; Fermor, B.; Setton, L. A. Chondrocytic differentiation of human adipose-derived adult stem cells in elastinlike polypeptide. Biomaterials 2006, 27, 91-99. (21) Dreher, M. R.; Liu, W. G.; Michelich, C. R.; Dewhirst, M. W.; Chilkoti, A. Thermal cycling enhances the accumulation of a temperature-sensitive biopolymer in solid tumors. Cancer Res. 2007, 67, 4418-4424.

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Figure 2. Immobilization of ELP molecules on the glass surface. (A) Optical microscopic image of negative circle micropatterns of polymer template on the glass. (B) Noncontact mode AFM image of ELP micropatterns after the removal of a polymer template layer. (C) Line profile of B. The scan size was 22 µm × 22 µm.

layer. The modification was routinely performed as described in the literature.22 We fabricated stimuli-responsive ELP micropatterns on glass using µCP due to its flexibility and simplicity.23 The PDMS stamps, inked with a templating polymer solution, made a conformal contact to the glass surface, and the thin polymer layer with micropatterns was transferred onto the surface. The thin polymer layer is removable with an aqueous solution containing alcohol and was used as a physical template for the formation of ELP micropatterns. The phase-contrast optical microscopic image showed stable micropatterns of the polymer template on the glass surface even after harsh ultrasonic cleaning (Figure 2A). ELP conjugation with aldehyde on the glass surface could be performed by immersing a glass plate micropatterned with a removable template in the ELP-K solution in sodium carbonate buffer with a pH of 9.2. The positive feature of the immobilized ELP molecules was imaged with AFM after the removal of the template by the mixture of H2O/ethanol (Figure 2B). The AFM image showed that ELP molecules uniformly covered the micropatterning area. The height of ELP micropatterns on glass was ∼4 nm as measured in noncontact mode. This height is close to the results reported in the literature, confirming the successful immobilization of ELP on the glass.24 TOF-SIMS is a powerful analytical tool for revealing detailed information about molecular species at interfaces. Positive and negative TOF-SIMS spectra can identify characteristic ions from each species on the surface and determine the biomolecules adsorbed on the surface.25 In the experiment, the positive spectra included the fragments of specific amino acid residues such as (22) Schlapak, R.; Pammer, P.; Armitage, D.; Zhu, R.; Hinterdorfer, P.; Vaupel, M.; Fruhwirth, T.; Howorka, S. Glass surfaces grafted with high-density poly(ethylene glycol) as substrates for DNA oligonucleotide microarrays. Langmuir 2006, 22, 277-285. (23) Park, T. H.; Shuler, M. L. Integration of cell culture and microfabrication technology. Biotechnol. Prog. 2003, 19, 243-253. (24) Hyun, J.; Lee, W. K.; Nath, N.; Chilkoti, A.; Zauscher, S. Capture and release of proteins on the nanoscale by stimuli-responsive elastin-like polypeptide “switches”. J. Am. Chem. Soc. 2004, 126, 7330-7335. (25) Canavan, H. E.; Graham, D. J.; Cheng, X. H.; Ratner, B. D.; Castner, D. G. Comparison of native extracellular matrix with adsorbed protein films using secondary ion mass spectrometry. Langmuir 2007, 23, 50-56.

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CH4N+ (m/z ) 30, glycine (G)) and C4H10N+ (m/z ) 72, valine (V)). The peaks at CN- (m/z ) 26) and CNO- (m/z ) 42) in the negative spectrum were characteristic for the polyamide protein backbone. The peaks at CH2N+(m/z ) 28) and HSiO2(m/z ) 61) showed high intensity in the TOF-SIMS spectra, demonstrating the existence of a silane layer on the surface (Figure 3A,B).26 TOF-SIMS positive-ion and negative-ion images were used to chemically identify the immobilized ELP on the aldehydeglass surface (Figure 3C,D). Figure 3C,D shows positive and negative TOF-SIMS images of an ELP-patterned glass surface that were collected on the basis of specific peaks confirmed in the spectra. Nitrogen-containing ions were selected for imaging the spatial distribution of ELP, while HSiO-, C2H3O+, and CHO+ were selected for the imaging of aldehyde silane (Figure 3C,D). In the negative ion spectrum of ELP, nitrogen-containing peaks such as CN- (m/z ) 26) and CNO- (m/z ) 42) were characteristic and showed good contrast in the images (Figure 3C). CH4N+ (m/z ) 30, glycine (G)), C4H8N+ (m/z ) 70, proline (P)), and C4H10N+ (m/z ) 72, valine (V)) showed high intensity in the positive ion images corresponding with the amino acid sequence of ELP (Figure 3C). For the aldehyde silane surface, peaks at CHO+ (m/z ) 29), C2H3O+ (m/z ) 41), C2H- (m/z ) 25), C2HO(m/z ) 41), and HSiO3-(m/z ) 61) could be observed, and high contrast images were obtained (Figure 3D). The interfacial phase transition of immobilized ELP and solution-phase ELP was exploited to show proof-of-principle that the surface chemistry could be manipulated by external stimuli. In order to create a reversible surface, ELP was covalently immobilized on the aldehyde-derivatized glass surface (glass of 1 cm × 1 cm) without micropatterns. To visualize the capture of solution-phase ELP on the immobilized ELP, the solutionphase ELP was fluorescently labeled prior to use. The hydrophobic interaction between ELP molecules above their Tt enabled the capture of solution-phase ELP onto the surface-immobilized ELP (Figure 4). After washing the surface at temperatures below their Tt, the intensity of fluorescence was dramatically decreased, because reversing the environmental conditions to reverse the phase transition of the ELP resulted in desorption of captured ELP molecules from the ELP surface. The immobilized ELP successfully changed the surface property along with the ELP solution, and the binding of solution-phase ELP on ELPimmobilized glass was reversible in three cyclic characterizations, as shown in Figure 4. A number of parameters influence cell adhesion and growth, such as chemistry, topography, and mechanics, among others.27,28 Of these, hydrophilic and hydrophobic surfaces have been widely investigated, and modifying the culture surface with plasma, chemicals, irradiation, and grafting has been suggested to create more suitable surfaces for cell adhesion.29,30 An ELP-micropatterned surface provides a thermoresponsive modifier for cell adhesion in biomedical microdevices, because the phase transition (26) Vanooij, W. J.; Sabata, A. Characterization of films of organofunctional silanes by TOF-SIMS. 2. Films of gamma-APS, AEAPS and FPS on cold-rolled steel and cold-rolled zinc substrates. Surf. Interface Anal. 1993, 20, 475-484. (27) Roach, P.; Eglin, D.; Rohde, K.; Perry, C. C. Modern biomaterials: a review-bulk properties and implications of surface modifications. J. Mater. Sci.: Mater. Med. 2007, 18, 1263-1277. (28) Milner, K. R.; Siedlecki, C. A. Submicron poly(L-lactic acid) pillars affect fibroblast adhesion and proliferation. J. Biomed. Mater. Res., Part A 2007, 82A, 80-91. (29) Alvarez-Barreto, J. F.; Shreve, M. C.; Deangelis, P. L.; Sikavitsas, V. I. Preparation of a functionally flexible, three-dimensional, biomimetic poly(Llactic acid) scaffold with improved cell adhesion. Tissue Eng. 2007, 13, 12051217. (30) Hsu, S. H.; Chuang, S. C.; Chen, C. H.; Chen, D. C. Endothelial cell attachment to the gamma irradiated small diameter polyurethane vascular grafts. Biomed. Mater. Eng. 2006, 16, 397-404.

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Figure 3. TOF-SIMS images of ELP micropatterns on aldehyde glass (ELP was immobilized inside the circles). (A) The principal component (PC) 1 loadings plot from PCA of the positive ion spectra, (B) the PC 1 loadings plot from PCA of the negative ion spectra, (C) secondary ion images of ELP at CH4N+, C4H8N+, C4H10N+, CN-, and CNO- peaks and (D) secondary ion images of aldehyde silane at CHO+, C2H3O+, C2H-, C2HO-, and HSiO3- peaks.

of ELP changes the ability of cells to adhere to the surface by transitioning between hydrophilic and hydrophobic interactions at Tt. The adhesion of cells on the surface is mediated by integrin receptors, which are transmembrane receptors binding to adhesive motifs that are present in ECM proteins including FN, collagen, and laminin. In this study, FN was adsorbed on the ELP micropatterns to promote cell adhesion. The adsorption of FN (pH 7.4 in PBS) was performed at 37 °C (which is not necessary, but was selected as an optimal temperature for most mammalian cell experiments) to induce FN adsorption on the ELP molecules, which showed their phase transition at ∼35 °C. The spatial distribution of adsorbed FN was confirmed by immunostaining the surface with a rabbit anti-bovine FN antibody followed by a FITC-conjugated goat anti-rabbit IgG and observing the stained surface by confocal fluorescence microscopy (Figure 5A,B). We discovered that FN was adsorbed on the ELP micropatterns as well as the aldehyde-terminated background, which is moderately

hydrophobic (water contact angle ∼40 °C), although a relatively large amount of FN was adsorbed on the ELP region (Figure 5B). Forty-micrometer-wide negative ELP stripe features with 80 µm spacing and 80-µm-diameter negative ELP circle features with 80 µm spacing were fabricated on the aldehyde-terminated glass for cell micropatterning. Prior to cell incubation, the surfaces were immersed in cell culture media for 30 days and the stability of ELP micropatterns on glass was confirmed by imaging the surface with AFM (results not shown). To investigate the optimal concentration of FN for cell attachment on the ELP surface, aldehyde-glass surfaces with negative features of ELP micropatterns were immersed in four different concentrations of FN solution (0, 1, 10, and 50 µg/mL) at 37 °C, which was a higher temperature than the Tt of the immobilized ELP. The ELP surface showed the optimal adsorption of FN at a concentration of ∼10 µg/mL, and this concentration was used for the modification of the surface in cell culture experiments (Figure 5C).

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Figure 4. Reversible adsorption and desorption of solution-phase ELP labeled with Alexa 488 on the ELP-immobilized surface without micropatterns. Each cycle was performed by adding and rinsing solution-phase ELP molecules labeled with Alexa488 with the change of temperature. The fluorescence intensity was obtained by averaging all pixels in the imaging region (magnification: 200×). Each point in the figure shows the average value and error bar of fluorescence intensities obtained from ten separate pieces of glass.

Figure 6. Optical image of the cell micropattern. (A) The first group of cells was incubated for 18 h at 37 °C, and then gently rinsed with fresh culture media. (B) The phase transition of the ELPconjugated glass surface was induced by reducing the temperature to 20 °C. (C) FN was added to the immobilized ELP surface for the re-seeding of cells and washed with fresh media at 37 °C. Subsequently, the second group of cells was seeded on the same micropatterned surface, incubated for another 18 h at 37 °C, and then rinsed with fresh culture media. (D) Negative ELP micropattern of stripe (40 µm width with 80 µm spacing) and circle features (80 µm in diameter). The phase transition of the ELP-conjugated glass surface was induced by reducing the temperature to 20 °C. Figure 5. Immunostaining of absorbed FN on the ELP micropattern surface with a rabbit anti-bovine FN antibody, followed by a FITCconjugated goat anti-rabbit IgG. Fluorescence image of FN at concentrations of (A) 0 µg/mL and (B) 10 µg/mL. (C) Histogram of fluorescence intensity at the different FN concentrations.

3T3 fibroblast cells were incubated on the surfaces for 18 h at 37 °C to adhere cells onto the surface. Cells attached and spread over the glass without any specific features on the surface, due to the high level of FN adsorbed on the ELP surface as well as on the aldehyde-glass surface (Figure 6A). Subsequently, the cell incubation temperature was reduced to 20 °C to induce the detachment of cells from the surface due to the hydrophilic phase change of ELP molecules on the outside features (Figure 6B). The detachment of cells did not occur spontaneously; it usually took several minutes to dislodge the cells completely from the surface after rinsing with fresh culture media. We hypothesize that strong interactions between surface-immobilized ELP and ECM (FN) mediate cell attachment above Tt, but decreasing the temperature below Tt makes the surface hydrophilic and results in desorption of the ECM along with the cultured cells. In addition,

flexibility of ELP molecules is enhanced during the hydrophilic phase transition and results in the detachment of cells from the surface. Since FN is desorbed from the immobilized ELP surface below Tt, the surface density of FN on the immobilized ELP decreases below Tt. For this reason, FN was added to the ELP surface each cycle at 37 °C before cell re-seeding. The second group of cells was seeded on the same micropatterned surface and incubated for another 18 h at 37 °C. The cells spread over the regions at 37 °C, but began to detach at 20 °C on the ELP micropatterned region despite the presence of FN on the surface. The second cell culture was carried out under the same protocols as the previous cell culture to observe the reversible formation of cell micropatterns by detachment on the hydrophilic ELP surface. Cell adhesion could be controlled by the phase transition of the incubation surface, and stripe and circle features of cell micropatterns could be created as shown in Figure 6C,D. Control experiments were performed with aldehyde-modified glass and tissue culture polystyrene surfaces to see the effects of temperature on the cells. The detachment of cells on the surfaces was not observed at either 20 or 37 °C, as shown on the aldehyde region

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in Figure 6. The viability study demonstrated that over 95% of the cells were healthy, indicating that the surfaces were nontoxic to the cells during the experiments.

Conclusions This paper describes the genetic synthesis of ELP molecules containing lysine that were used for conjugation with an aldehydefunctionalized glass surface. The immobilization of ELP molecules was simply achieved using a removable polymer template created on the surface. ELP micropatterns on the surface were verified by AFM and TOF-SIMS imaging at the characteristic signal peak. The smart transition of ELP micropatterns between hydrophilic and hydrophobic surfaces at Tt enabled the control of reversible

cell adhesion by incubation temperature. To enhance cell adhesion on the surface, FN was adsorbed on the ELP platform prior to cell seeding. Fibroblasts were seeded and spread properly on the adhesive platform at temperatures exceeding Tt due to the hydrophobic phase of ELP, and they were detached from the surface at temperatures below Tt, demonstrating efficient and selective cell adhesion through the control of surface chemistry. Acknowledgment. This work was supported by a Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2005-041- D00947). LA702796Y