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Cell Adhesion Properties of Patterned Poly(acrylic acid)/ Poly(allylamine hydrochloride) Multilayer Films Created by Room-Temperature Imprinting Technique Yingxi Lu, Junqi Sun,* and Jiacong Shen State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin UniVersity, Changchun 130012, People’s Republic of China ReceiVed March 31, 2008. ReVised Manuscript ReceiVed May 15, 2008 Patterned poly(acrylic acid) (PAA)/poly(allylamine hydrochloride) (PAH) multilayer films with line structures of different lateral size and vertical height were fabricated by a room-temperature imprinting technique, and their cell adhesion properties were investigated. The nonimprinted PAA/PAH multilayer films are cytophilic toward NIH/3T3 fibroblasts and HeLa cells whether PAA or PAH is the outer most layer. In contrast, the PAA/PAH multilayer films with a 6.5-µm-line/3.5-µm-space pattern structure are cytophobic toward NIH/3T3 fibroblasts and HeLa cells when the height of the lines is 1.29 µm. By either increasing the lateral size of the patters to 69-µm-line/43-µm-space or decreasing the height of the imprinted lines to ∼107 nm, imprinted PAA/PAH multilayer films become cytophilic. This kind of transition of cell adhesion behavior derives from the change of the physical pattern size of the PAA/PAH multilayer films and is independent of the chemical composition of the films. The easy patterning of layer-by-layer assembled polymeric multilayer films with the room-temperature imprinting technique provides a facile way to tailor the cellular behavior of the layered polymeric films by simply changing the pattern dimensions.
Introduction The layer-by-layer (LbL) assembly technique, as developed by Decher and co-workers in the early 1990s, has become a powerful way to fabricate functional composite film materials with precise control of film composition and structures.1,2 In the past decade, more and more efforts have been expended to use LbL assembled polyelectrolyte multilayer films for biorelated applications including drug delivery, biosensors, biomimetics, and tissue engineering.3 The LbL assembled multilayer films, characterized by the easily tailored film composition, topography, thickness, and abundance of building blocks for film construction and their capability of depositing on various kinds of substrates with any geometry, provide a platform to investigate the adhesion of cells on these film surfaces. Promotion and resistance of cell adhesion by LbL assembled multilayer films are equally important. For example, endovascular stents4 and damaged blood vessels5 need a cytophobic multilayer coating to prevent adhesion and proliferation of vascular smooth muscles which can cause occlusion of the blood flow. Coatings which can improve bone cell adhesion are required for bone implants.6 Rubner and coworkers demonstrated that LbL assembled synthetic polyelec* To whom correspondence should be addressed. Phone: 0086-43185168723. Fax: 0086-431-85193421. E-mail:
[email protected]. (1) Decher G.; Schlenoff, J. B. Multilayer Thin Films-Sequential Assembly of Nanocomposite Materials; Wiley-VCH: Weinheim, Germany, 2002. (2) (a) Decher, G. Science 1997, 277, 1232. (b) Zhang, X.; Shen, J. C. AdV. Mater. 1999, 11, 1139. (c) Hammond, P. T. AdV. Mater. 2004, 16, 1271. (d) Bertrand, P.; Jonas, A.; Laschewsky, A.; Legras, R. Macromol. Rapid Commun. 2000, 21, 319. (e) Caruso, F. Chem. Eur. J. 2000, 6, 413. (f) Zhang, X.; Chen, H.; Zhang, H. Y. Chem. Commun. 2007, 1395. (3) Tang, Z. Y.; Wang, Y.; Podsiadlo, P.; Kotov, N. A. AdV. Mater. 2006, 18, 3203. (4) (a) Tan, Q. G.; Ji, J.; Barbosa, M. A.; Fonseca, C.; Shen, J. C. Biomaterials 2003, 24, 4699. (b) Fu, J. H.; Ji, J.; Yuan, W. Y.; Shen, J. C. Biomaterials 2005, 26, 6684. (c) Salloum, D. S.; Olenych, S. G.; Keller, T. C. S.; Schlenoff, J. B. Biomacromolecules 2005, 6, 161. (5) Thierry, B.; Winnik, F. M.; Merhi, Y.; Tabrizian, M. J. Am. Chem. Soc. 2003, 125, 7494. (6) (a) Tryoen-To´th, P.; Vautier, D.; Haikel, Y.; Voegel, J.-C.; Schaaf, P.; Chluba, J.; Ogier, J. J. Biomed. Mater. Res., Part A 2002, 60, 657. (b) Zhu, H.; Ji, J.; Barbosa, M. A.; Shen, J. C. J. Biomed. Mater. Res., Part B 2004, 71, 159.
trolyte multilayer films can be either cytophobic or cytophilic depending on the swelling capacities of the films, where highly swellable films prohibit cell adhesion and highly ionically crosslinked films support cell adhesion.7 The swelling capacities of the films can be tuned by changing the deposition pH of the films as well as the choice of materials. Meanwhile, the mechanical properties of polyelectrolyte multilayer films play a determinant role in cell adhesion where stiff films are more favorable to cell adhesion and spreading than soft films.8 Additionally, LbL assembled multilayer films can cover cytotoxic substrates to render them biocompatible.9 Adhesion of cells is a complicated process. Different types of cells exhibit different adhesion behavior even on the same LbL assembled films. In generally, many factors such as surface charges, wettability, chemical composition, surface roughness, film thickness, mechanical strength, etc., can have an influence on the cellular behavior of LbL assembled polyelectrolyte multilayer films.10 Besides the above-mentioned factors, topography provides another choice to control the cellular behavior of LbL assembled multilayer films. Cellular response on chemically patterned LbL assembled films fabricated by microcontact printing,11 photo(7) Mendelsohn, J. D.; Yang, S. Y.; Hiller, J.; Hochbaum, A. I.; Rubner, M. F. Biomacromolecules 2003, 4, 96. (8) (a) Thompson, M. T.; Berg, M. C.; Tobias, I. S.; Rubner, M. F.; Van Vliet, K. J. Biomaterials 2005, 26, 6836. (b) Schneider, A.; Francius, G.; Obeid, R.; Schwinte´, P.; Hemmerle´, J.; Frisch, B.; Schaaf, P.; Voegel, J.-C.; Senger, B.; Picart, C. Langmuir 2006, 22, 1193. (9) Sinani, V. A.; Koktysh, D. S.; Yun, B.-G.; Matts, R. L.; Pappas, T. C.; Motamedi, M.; Thomas, S. N.; Kotov, N. A. Nano. Lett. 2003, 3, 1177. (10) (a) Serizawa, T.; Yamaguchi, M.; Matsuyama, T.; Akashi, M. Biomacromolecules 2000, 1, 306. (b) Thompson, M. T.; Berg, M. C.; Tobias, I. S.; Lichter, J. A.; Rubner, M. F.; Van Vliet, K. J. Biomacromolecules 2006, 7, 1990. (c) Richert, L.; Lavalle, P.; Payan, E.; Shu, X.-Z.; Prestwich, G. D.; Stoltz, J.-F.; Schaaf, P.; Voegel, J.-C.; Picart, C. Langmuir 2004, 20, 448. (d) Richert, L.; Lavalle, Ph.; Vautier, D.; Senger, B.; Stoltz, J.-F.; Schaaf, P.; Voegel, J.-C.; Picart, C. Biomacromolecules 2002, 3, 1170. (e) Richert, L.; Boulmedais, F.; Lavalle, P.; Mutterer, J.; Ferreux, E.; Decher, G.; Schaaf, P.; Voegel, J.-C.; Picart, C. Biomacromolecules 2004, 5, 284. (11) (a) Berg, M. C.; Yang, S. Y.; Hammond, P. T.; Rubner, M. F. Langmuir 2004, 20, 1362. (b) Kidambi, S.; Lee, I.; Chan, C. J. Am. Chem. Soc. 2004, 126, 16286. (c) Kidambi, S.; Sheng, L.; Yarmush, M. L.; Toner, M.; Lee, I.; Chan, C. Macromol. Biosci. 2007, 7, 344.
10.1021/la800998n CCC: $40.75 2008 American Chemical Society Published on Web 06/24/2008
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lithography,12 and microfluidic templating13 has been extensively studied. These patterned LbL assembled multilayer films can be used not only as cell resistant/promotion surfaces but also to control the spatial adhesion of different cells, which are essential to the application of these patterned films in tissue engineering and fabricating biosensor arrays. The cellular response on these chemically patterned films originates mainly from their difference of lateral chemical compositions, where cell promotion and/or cell-resistant films were laterally patterned on solid substrates. The direct study of the influence of topography on cellular response of LbL assembled multilayer films is rare. Very recently, Chan, Lee, and co-workers deposited LbL assembled poly(diallyldimethylammonium chloride)/poly(styrene sulfonate) films on polydimethylsioxane (PDMS) surfaces with various micrometer patterns and systematically investigated the cell adhesion and proliferation on these PDMS surfaces.14 Recently, we developed a room-temperature imprinting technique to pattern LbL assembled polymeric films based on either electrostatic interaction or hydrogen bonding as a driving force.15 A rigid Ni mold15a or Norland Optical Adhesives (NOA 63) polymer mold15b can be used to imprint LbL assembled polymeric films. In particular, the use of polymeric NOA 63 mold renders the imprinting technique a facile and cost-effective method to fabricate layered polymeric films with various kinds of pattern structures. The imprinting technique enables the fabrication of patterned films with large areas.16 Advantages of this patterning method lie in the easy control of the lateral dimension and vertical height of the patterns. We believe that imprinted LbL assembled multilayer films will provide not only a model to study the topography-dependent cell adhesion properties of LbL assembled multilayer films but also useful scaffolds for tissue engineering in the near future. In this work, we investigate the adhesion properties of room-temperature imprinted poly(acrylic acid) (PAA)/poly(allylamine hydrochloride) (PAH) multilayer films toward NIH/3T3 fibroblasts and HeLa cells by changing the lateral dimension and vertical height of the patterned line structures. The present study is meaningful for potential applications of room-temperature imprinted polymeric multilayer films in areas such as cell culture, tissue engineering, biosensors, etc.
Experimental Section Materials. Poly(acrylic acid) (PAA, Mw ca. 2000), poly(allylamine hydrochloride) (PAH, Mw ca. 70 000), and poly(diallyldimethylammonium chloride) (PDDA, 20 wt %, Mw ca. 100 000-200 000) were purchased from Sigma-Aldrich. Poly(dimethylsiloxane) (PDMS, Sylgard 184) was purchased from Dow Corning. Norland Optical Adhesives 63 (NOA 63) was purchased from Norland Products Inc. NIH/3T3 fibroblasts and HeLa cell lines were purchased from American type Culture Collection (ATCC). Dubelco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS), and steapsin were obtained from Gibco. Penicillin and streptomycin were purchased from Sigma-Aldrich. Six-well tissue culture polystyrene (12) Yang, S. Y.; Mendelsohn, J. D.; Rubner, M. F. Biomacromolecules 2003, 4, 987. (13) Reyes, D. R.; Perruccio, E. M.; Becerra, S. P.; Locascio, L. E.; Gaitan, M. Langmuir 2004, 20, 8805. (14) Kidambi, S.; Udpa, N.; Schroeder, S. A.; Findlan, R.; Lee, I.; Chan, C. Tissue Eng. 2007, 13, 2105. (15) (a) Lu, Y. X.; Hu, W.; Ma, Y.; Zhang, L. B.; Sun, J. Q.; Lu, N.; Shen, J. C. Macromol. Rapid Commun. 2006, 27, 505. (b) Lu, Y. X.; Chen, X. L.; Hu, W.; Lu, N.; Sun, J. Q.; Shen, J. C. Langmuir 2007, 23, 3254. (16) (a) Chou, S. Y.; Krauss, P. R.; Renstrom, P. J. Appl. Phys. Lett. 1995, 67, 3114. (b) Sotomayor Torres, C. M.; Zankovych, S.; Seekamp, J.; Kam, A. P.; Ceden˜o, C. C.; Hoffmann, T.; Ahopelto, J.; Reuther, F.; Pfeiffer, K.; Bleidiessel, G.; Gruetzner, G.; Maximov, M. V.; Heidari, B. Mater. Sci. Eng., C 2003, 23, 23. (c) Kim, Y. S.; Lee, H. H.; Hammond, P. T. Nanotechnology 2003, 14, 1140. (d) Guo, L. J. AdV. Mater. 2007, 19, 495.
Langmuir, Vol. 24, No. 15, 2008 8051 (TCPS) was purchased from Corning Inc. All chemicals were used without further purification. The NOA 63 molds were fabricated by replication of PDMS masters with line pattern structures, as described in our previous publication.15b Fabrication of Patterned PAA/PAH Multilayer Films. The LbL deposition of PAA/PAH multilayer films was conducted automatically by a programmable dipping machine (Dipping Robot DR-3, Riegler & Kirstein GmbH) at room temperature. Glass slides were immersed in slightly boiled piranha solution (3:1 mixture of 98% H2SO4 and 30% H2O2) for 20 min and rinsed with copious amounts of water. Caution: Piranha solution reacts Violently with organic material and should be handled carefully. A newly cleaned glass slide (25.4 × 76.2 mm, thickness ≈ 1.2 mm) was immersed in PDDA aqueous solution for 20 min to obtain a cationic ammoniumterminated surface and ready for PAA/PAH multilayer deposition. Subsequently, the substrate was immersed into aqueous PAA solution (pH 3.5) for 15 min to obtain a layer of PAA film. The substrate was then rinsed in two water baths for 1 min each before the next layer deposition. Next, the substrate was immersed into aqueous PAH solution (pH 7.5) for 15 min to obtain a layer of PAH film. No drying step was used in the film deposition procedure. The adsorption and rinsing steps were repeated until the desired number of bilayers was obtained. PAA/PAH films with n cycle deposition are noted as (PAA/PAH)*n. The thickness of the PAA/PAH multilayer films exhibit an exponential growth behavior with increasing number of film deposition cycles, possibly because of the serious interpenetration of the polyelectrolytes in the neighboring layers caused by the nondrying deposition film preparative process.15b PAA/PAH multilayer films with deposition cycles of 7, 15, and 20 were fabricated. Multilayers of PAA/PAH were immediately used to imprint or otherwise stored in a water bath. PAA/PAH multilayer films were imprinted using a homemade imprinting device that consists of two pieces of 30-mm diameter magnet, as described in our previous work.15b The imprinted PAA/PAH multilayer films was thermally cross-linked by heating the films at 130 °C for 12 h to stabilize the imprinted pattern structures.17 Seeding of Cells on Patterned PAA/PAH Multilayer Films. NIH/3T3 fibroblasts and HeLa cell line were cultured in DMEM supplemented with 10% heated-inactivated FCS and antibiotics (100 units/mL penicillin and 100 µg/mL streptomycin). Cells were grown at 37 °C in humidified air containing 5% CO2 and harvested at passages every 2-3 days prior to experimentation to ensure an appropriate number of viable cells. Prior to cell seeding, the imprinted PAA/PAH multilayer films were thermally cross-linked by heating at 130 °C for 12 h and then sterilized with UV light for about 10 min. Then cell suspension was added to six-well TCPS-containing glass slides coated with the imprinted PAA/PAH multilayer films. Cells were cultured in a humid, 37 °C and 5% CO2 incubator in pH 6.8-7.2 media composed of DMEM medium supplemented with 10% heated-inactivated FCS and antibiotics. NIH/3T3 and HeLa cells were seeded at a cell density of 1 × 105 and 2 × 105 cells/well, respectively. Glass slides coated with thermally cross-linked PAA/ PAH multilayer films without imprinting were used as controls in these studies. Characterization. Atomic force microscope (AFM) images were taken with a Nanoscope IIIa AFM Multimode (Digital Instruments, Santa Barbara, CA) under ambient conditions to obtain topographical information about the patterned PAA/PAH multilayer films. AFM was operated in the tapping mode using silicon cantilevers with a force constant of 40 N/m. Heights of the patterned lines were measured in at least five different cross-sectional areas for each sample. Pictures documenting cell adhesion and spreading were taken using an Olympus inverted phase microscope. The multilayer film-thickness determination was carried out on a JEOL JSM-6700F scanning electron microscope. (17) (a) Harris, J. J.; DeRose, P. M.; Bruening, M. L. J. Am. Chem. Soc. 1999, 121, 1978. (b) Balachandra, A. M.; Dai, J. H.; Bruening, M. L. Macromolecules 2002, 35, 3171. (c) Ma, Y.; Sun, J. Q.; Shen, J. C. Chem. Mater. 2007, 19, 5058.
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Results and Discussion Fabrication of Patterned PAA/PAH Multilayer Films. To evaluate the effect of pattern physical properties (i.e., lateral size and vertical height of the pattered lines here) on cell attachment and spreading, PAA/PAH multilayer films with five different line structures were separately prepared and grouped according to horizontal size and vertical height of the lines as follows: (i) fixing the vertical line height, three (PAA/PAH)*20 films with line/space structures of (a) 6.5/3.5, (b) 13.6/18.7, and (c) 69/43 µm were imprinted with different NOA 63 molds; (ii) PAA/ PAH multilayer films containing patterned structures of 6.5µm-line/3.5-µm-space with varied vertical line height were fabricated by imprinting PAA/PAH multilayer films with the number of deposition cycles being 20, 15, and 7. Previous study shows that for the (PAA/PAH)*20 multilayer films, the broader the width of the imprinted lines is, the longer imprinting time is needed.15b Therefore, a different length of imprinting time was used to obtain patterned (PAA/PAH)*20 films with varied lateral line structures. Here the imprinting was conducted at room temperature with a pressure of about 100 bar. Three-dimensional AFM images of patterned line structures with different lateral sizes after thermal cross-linking are shown in Figure 1a-c. After imprinting the (PAA/PAH)*20 multilayer films for 2 h, one can obtain a large area line structure with 6.5-µm-line/3.5-µm-space. The height of the lines is determined to be about 1.29 µm from their corresponding cross-sectional analysis (as shown in Figure 1a). With further increasing the lateral pattern size to 13.6-µm-line/18.7-µm-width, it took 6 h to obtain a patterned (PAA/PAH)*20 film where the lines have a flat top surface. The height of the lines is about 1.37 µm. This result indicates that the lateral movement of the PAA/PAH film under the same pressure becomes difficult when increasing the size of the patterns. To fabricate (PAA/PAH)*20 films with a lateral pattern of 69-µm-line/43-µm-space, the length of imprinting time increased to 12 h. As shown in Figure 1c, the crosssectional AFM analysis shows that the lines have a height of about 1.19 µm and the top of the lines is not as flat as those in Figure 1a and 1b. The as-fabricated (PAA/PAH)*20 film has a thickness of 1.09 µm, as determined from its cross-sectional SEM image.15b The height of the lines in these three cases is larger than the film thickness, indicating that pattern formation originates from the vertical compression and concomitant lateral movement of the film toward the noncontact region of the mold and films under high pressure, as already demonstrated in our previous study.15 The height difference between these three kinds of patterned films is ∼180 nm, which can be negligible when compared with the cultured cell diameter of several tens of micrometers.18 Therefore, we can regard the patterned lines in these three kinds of imprinted (PAA/PAH)*20 films as having almost the same height. By fixing the lateral pattern size to 6.5-µm-line/3.5-µm-space, the height of the imprinted lines was systematically varied to investigate the influence of vertical height of the lines on cell attachment. Multilayer films of (PAH/PAA)*15 and (PAH/ PAA)*7 were fabricated, which have a film thickness of ∼778.5 and ∼102.4 nm, respectively, as measured from their corresponding SEM images. Multilayer films of (PAH/PAA)*15 and (PAH/PAA)*7 were imprinted under a pressure of about 30 bar for 5 and 8 min, respectively, using a NOA 63 polymer mold containing 6.5-µm-space/3.5-µm-line pattern structures. As shown in Figure 1d and 1e, successful imprinting of (PAH/PAA)*15 and (PAH/PAA)*7 films was achieved. For the imprinted (PAA/ PAH)*15 film, the cross-sectional AFM analysis shown in Figure (18) Hui, E. E.; Bhatia, S. N. Langmuir 2007, 23, 4103.
Figure 1. AFM images of the imprinted PAA/PAH multilayer films. Imprinted (PAA/PAH)*20 films with line structures of 6.5-µm-line/ 3.5-µm-space (a), 13.6-µm-line/18.7-µm-space (b), and 69-µm-line/43µm-space. Imprinted (PAA/PAH)*15 (d) and (PAA/PAH)*7 (e) films with line structures of 6.5-µm-line/3.5-µm-space. Their corresponding cross-sectional analysis is also given.
1d indicates that the pattern height is 687 ( 157 nm on average. The line structure shows a slight saddle-like profile. For the (PAA/PAH)*7 multilayer films, regular line structures with a height of 107 ( 3 nm were obtained after imprinting (Figure 1e). Patterned (PAA/PAH)*20 multilayers with a height of 1.29 µm, shown in Figure 1a, were also used to investigate cell adhesion on imprinted PAA/PAH films with different vertical height. Influence of Lateral Pattern Size on Cell Adhesion. The as-prepared (PAA/PAH)*20 film constructed from 1 mg/mL PAA (pH 3.5) and 1 mg/mL PAH (pH 7.5) is cytophilic toward NIH/ 3T3 fibroblasts and HeLa cells whether PAA or PAH is the outermost layer. Because of the interpenetration film structure,
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Figure 2. Phase contrast microscope images of NIH/3T3 fibroblasts after 1 day in culture on (PAA/PAH)*20 films. (a) Nonimprinted (PAA/PAH)*20 films. Imprinted (PAA/PAH)*20 films with line structure of 6.5-µm-line/3.5-µm-space (b), 13.6-µm-line/18.7-µm-space (c), and 69-µm-line/43µm-space (d). (c) An image having both imprinted (right) and nonimprinted (left) multilayer films on the same substrate. The arrows in c serve to discern the fibroblasts. Scale bar is 100 µm.
the influence of the outermost layers to cell adhesion is minimal, as the cell shapes appear nearly identical on both surfaces. This result is consistent with that of Rubner and co-workers.7 Even after thermally cross-linking the (PAA/PAH)*20 multilayer films at 130 °C for 12 h, NIH/3T3 fibroblasts and HeLa cells readily attach to these surfaces (Figures 2a and 3a). Nevertheless, on the imprinted (PAA/PAH)*20 films, the adhesion behavior of NIH/ 3T3 fibroblasts and HeLa cells is quite different. After seeding 1 day, the NIH/3T3 fibroblasts exhibited substantial attachment and good spreading into their characteristic elongated morphologies on the nonimprinted (PAA/PAH)*20 film over the entire coated area, as shown in Figure 2a. In contrast to nonimprinted (PAA/PAH)*20 films, the imprinted (PAA/PAH)*20 films with 6.5-µm-line/3.5-µm-space completely resisted the attachment and spreading of highly adhesive NIH/3T3 fibroblasts (Figure 2b). Even after being cultured for 3 days, no attachment and spreading of fibroblast were observed. From the microscope image shown in Figure 2b, a few rounded (unattached) fibroblasts without spreading were observed. The (PAA/PAH)*20 films containing 6.5-µm-line/3.5-µm-space with the height of the lines being 1.29 µm exhibit strong cytophobic response toward NIH/3T3 fibroblasts. When fibroblasts were seeded on (PAA/PAH)*20 films with imprinted structures of 13.6-µm-line/18.7-µm-space, a few attached fibroblasts were observable on the surface of the lines (Figure 2c) but the number is very limited. The size of the attached fibroblasts is much smaller than that in Figure 2a, indicating an inefficient spreading. The attached cells could not spread as well as in Figure 2a and have small sizes. The cells show a tendency to align along the imprinted lines. When the feature size of the imprinted (PAA/PAH)*20 film increases to 69-µm-line/43-µmspace, the imprinted films become cytophilic with multiple fibroblasts nonselectively attached on the surface of imprinted
lines and spaces (Figure 2d). The individual cells spread well and are connected to each other by temporary projections (i.e., pseudopods) of the cytoplasm. Aligned growth of NIH/3T3 fibroblasts was observed on this kind of patterned surface. Therefore, the imprinted (PAA/PAH)*20 films with large feature sizes of 69-µm-line/43-µm-space are cytophilic and capable of controlling epitaxial spreading of the fibroblasts. HeLa cells are another kind of typical adherent cell line and human epithelial cells from a fatal cervical carcinoma. The adhesion behavior of HeLa cells on imprinted (PAA/PAH)*20 films was also examined. As shown in Figure 3, different adhesion behaviors of HeLa cells on imprinted and nonimprinted PAA/ PAH films were observed after the cells were cultured for 1 day. HeLa cells showed clear signs of attachment and spreading on the cross-linked nonimprinted (PAA/PAH)*20 films (Figure 3a). No attachment and spreading of HeLa cells on imprinted (PAA/ PAH)*20 films with 6.5-µm-line/3.5-µm-space (Figure 3b) were observed, indicating the cytophobic property of this kind of imprinted films toward HeLa cells. While increasing the feature size to 13.6-µm-line/18.7-µm-space, HeLa cells attached in small quantities on the top of the lines, as shown in Figure 3c. Although the cells attached to the imprinted surface, inefficient spreading was observed and the size of the cells was much smaller than those on the nonimprinted (PAA/PAH)*20 films. When HeLa cells were cultured on imprinted (PAA/PAH)*20 films with 69µm-line/43-µm-space pattern structures they attached in large quantities and spread over the whole patterned film surface (Figure 3d), indicating that imprinted (PAA/PAH)*20 films with a large feature size of 69-µm-line/43-µm-space have a similar cell adhesion behavior to the nonimprinted (PAA/PAH)*20 films. From the above results we arrive at the following conclusions: the lateral feature size of the imprinted (PAA/PAH)*20 films
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Figure 3. Phase contrast microscope images of HeLa cells after 1 day in culture on (PAA/PAH)*20 films. (a) Nonimprinted (PAA/PAH)*20 films. Imprinted (PAA/PAH)*20 films with a line structure of 6.5-µm-line/3.5-µm-space (b), 13.6-µm-line/18.7-µm-space (c), and 69-µm-line/43-µm-space (d). Scale bar is 100 µm.
has a dramatic influence on the attachment and spreading of cells. When the imprinted feature size (e.g., 6.5-µm-line/3.5µm-space) is much smaller than the dimensions of the cells, the imprinted (PAA/PAH)*20 films become completely cytophobic. The imprinted films become gradually cytophilic with very limited number of cells attached when the imprinted feature size is about similar or comparable to the cells (e.g., 13.6-µm-line/18.7-µmspace). When the imprinted feature size (e.g., 69-µm-line/43µm-space) is larger than the cells, the imprinted (PAA/PAH)*20 films are cytophilic to the cultured cells. These results further demonstrated that the cell adhesion behavior of the LbL assembled PAA/PAH films can be well tailored by simply changing the lateral feature size of the imprinted structures. Influence of Vertical Height of the Imprinted Lines on Cell Adhesion. Besides the lateral size of the imprinted PAA/ PAH films, the vertical height of the patterned structures on imprinted PAA/PAH films can also have an influence on the cell adhesion behavior. NIH/3T3 fibroblasts were cultured on imprinted PAA/PAH films containing patterns of 6.5-µm-line/ 3.5-µm space with different line heights. After seeding for 5 days, different cell adhesion behavior was observed as shown in Figure 4. As previously observed in Figure 2b, there is no cell attachment and spreading on the (PAA/PAH)*20 films with a line height of 1.29 µm (Figure 4a). Decreasing the line height to ∼687 nm on (PAA/PAH)*15 films, a small amount of fibroblasts attached on the surface of the imprinted films (Figure 4b). A single cell spread on several neighboring lines, adopting an elongated morphology. By decreasing the height of the imprinted lines from 1.29 µm to ∼687 nm, the imprinted PAA/ PAH films gradually turned from cytophobic to cytophilic. It is assumed that this kind of transformation originates mainly from the decreased line height but not the saddle-like line structure because the dimensions of the saddle-like structure are too small
compared with the cell size. As confirmed, when further decreasing the height of the line to ∼107 nm on (PAA/PAH)*7 films, a more cytophilic surface was obtained as more fibroblasts attached and spread on the imprinted films (Figure 4c). HeLa cells were also examined on these three kinds of imprinted PAA/ PAH films. The cells adhesion behavior is similar to those of NIH/3T3 fibroblasts (data not shown here), i.e., PAA/PAH films with a line height of ∼107 nm are cytophilic to HeLa cells, while PAA/PAH films with a line height of 1.29 µm are cytophobic. The PAA/PAH films with a decreased line height of ∼687 nm exhibit a transition from cytophobic to cytophilic. Therefore, by simply adjusting the vertical height of the imprinted line structures on PAA/PAH multilayer films, the cell adhesion behavior of the films can also be well tuned.
Conclusions and Outlook In the present study, we demonstrate that the cell adhesion behavior of LbL assembled PAA/PAH films depends largely on their surface topography. While the nonimprinted PAA/PAH multilayer films are always cytophilic toward NIH/3T3 fibroblasts and HeLa cells, the PAA/PAH multilayer films with a 6.5-µmline/3.5-µm-space pattern structure created by the room-temperature imprinting technique are cytophobic toward NIH/3T3 fibroblasts and HeLa cells when the height of the lines is 1.29 µm. By either increasing the lateral size of the patters to 69µm-line/43-µm-space or decreasing the height of the imprinted lines to ∼107 nm, the imprinted PAA/PAH multilayer films become cytophilic. Because there is no change of the surface chemical composition occurring after imprinting of the films, the transition of cell adhesion behavior between cytophobic and cytophilic derives from the change of the physical pattern dimension of the imprinted PAA/PAH multilayer films. The room-
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Langmuir, Vol. 24, No. 15, 2008 8055
Figure 4. Phase contrast microscope images of NIH/3T3 fibroblasts after 5 days in culture on various imprinted PAA/PAH films with line structures of 6.5-µm-line/3.5-µm-space. (a) Imprinted (PAA/PAH)*20 films with a line height of 1.29 µm. (b) Imprinted (PAA/PAH)*15 multilayers with a line height of ∼687 nm. (c) Imprinted (PAA/PAH)*7 multilayers with a line height of ∼107 nm. Scale bar is 100 µm.
temperature imprinting technique is flexible and cost effective in fabricating patterned polymeric films with designed lateral pattern size and vertical height. Meanwhile, the room-temperature imprinting technique is generally suitable to prepare patterned structures for LbL assembled polymeric multilayer films based on electrostatic interaction and other kinds of driving forces, for example, hydrogen bonding and so forth. Therefore, we have a wide range of choices for imprinted film materials for cell culture. The easy control of dimensions of imprinted patterned structures and the abundance in film materials will therefore enable the fabrication of various kinds of imprinted films with precise control of cell adhesion behavior satisfying for different requirements. Additionally, an anticipated advantage is that the imprinted layered polymeric films are continuous and capable of peeling off and can be transferred conveniently onto other kinds of useful scaffolds for cell culture purposes. Although more experiments are needed to exhaustively study the cell adhesion behavior on the patterned
PAA/PAH multilayer films with various physical topographies, we firmly believe that the room-temperature imprinting technique will become a powerful technique to pattern various polymeric multilayer films and further widen the applications of the resultant patterned films in cell culture, tissue engineering, etc. Acknowledgment. This work was supported by the National Natural Science Foundation of China (NSFC grant no. 20574029), National Basic Research Program (2007CB808000), Foundation for the Author of National Excellent Doctoral Dissertation of P. R. China (FANEDD grant no. 200323), Program for New Century Excellent Talents in University (NCET), and Jilin Provincial Science and Technology Bureau of Jilin Province (20070104). We are grateful to Dr. Xianfeng Zhou in the same key laboratory for assistance with cell adhesion experiments. LA800998N
