Langmuir 2005, 21, 2317-2322
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In Situ Study of the Thermoresponsive Behavior of Micropatterned Hydrogel Films by Imaging Ellipsometry Dirk Schmaljohann,*,† Mirko Nitschke,‡,§ Roland Schulze,‡ Andreas Eing,| Carsten Werner,‡,§ and Klaus-Jochen Eichhorn‡ Welsh School of Pharmacy, Cardiff University & Cardiff Institute of Tissue Engineering and Repair (CITER), Redwood Building, King Edward VII Avenue, Cardiff CF10 3XF, Wales, U.K., and Institute of Polymer Research Dresden, Hohe Strasse 6, 01069 Dresden, The Max Bergmann Center of Biomaterials Dresden, Hohe Str. 27, 01069 Dresden, Germany, and Nanofilm Technologie GmbH, Anna-Vandenhoeck-Ring 5, 37081 Go¨ ttingen, Germany Received September 26, 2004. In Final Form: December 21, 2004 A patterned hydrogel was immobilized on a polymer substrate by low-pressure argon plasma treatment using a masking technique. The polymer sample showed a thermoresponsive aggregation behavior in the region of 35-37 °C. The micropatterned, thermoresponsive hydrogel film has been characterized with imaging ellipsometry. The characterization was carried out on the dry film as well as on a swollen sample in water. The thermoresponsive behavior was studied in deionized water by temperature-dependent measurements in a solid-liquid cell. Through imaging ellipsometry, it was possible to distinguish the different regions of interest on a micrometer scale and to follow the swelling of the hydrogel part as a function of the temperature. It was possible to visualize the swelling as 3D profiles of ∆ at various temperatures. Long-term changes of the sample could also be detected, which cannot be picked up by conventional ellipsometry.
Introduction The field of tissue engineering opens a variety of applications in regenerative medicine.1-3 These methods are strongly dependent on the versatility of a used scaffold. Biocompatibility is necessary for in vivo as well as in vitro application. Hydrogels have been widely used in tissue engineering due to their biocompatibility both as an implantable device and as an in vitro cultivation carrier.4 The hydrogels serve as an excellent scaffold due their ability to simulate the properties of living tissue, such as swelling in water and softness to allow local rearrangement combined with a certain mechanical strength for cell adhesion. Furthermore, diffusivity of nutrients and metabolites is favorable for applications in cell cultivation. The preparation of flat homogeneous surfaces allows the study of cell action under standardized conditions, whereas heterogeneous substrates give the potential to create more complex structures and devices. The ultimate goal is the preparation of a three-dimensional implantable scaffold, which supports the implanted cells and provides the right biological, chemical, and physicomechanical stimuli for the desired response as functional tissue. In vivo studies are already under way, but there is still a need to study biological systems under standardized conditions to relate the properties of the scaffold to the biological response.5-7 Stimuli-responsive hydrogels can expose or hide their surface functionalities, and they can change integral * To whom correspondence should be addressed. E-mail:
[email protected]. † Cardiff University & CITER. ‡ Institute of Polymer Research Dresden. § The Max Bergmann Center of Biomaterials. | Nanofilm Technologie GmbH. (1) Langer, R.; Tirrell, D. A. Nature 2004, 428, 487-492. (2) Okano, T., Ed.; Biorelated Polymers and Gels; Academic Press: San Diego, 1998. (3) Kwon, O. H.; Kikuchi, A.; Yamato, M.; Okano, T. Biomaterials 2003, 24, 1223-1232. (4) Lee, K. Y.; Mooney, D. J. Chem. Rev. 2001, 101, 1869-1879.
characteristics (hydrophilicity, charge) in relation to varied degrees of swelling.8 The hydrogels and the corresponding soluble polymers exhibit a volume phase transition upon a change in, e.g., the environmental pH, temperature, or illumination due to an altered balance of competing interactions (i.e., electrostatic forces, hydrophobic dehydration). For use in cell culture technologies, the variation of the environmental conditions has to occur within a physiological range of settings. Poly(N-isopropylacrylamide) (PNiPAAm) represents a very successful and widespread example of a thermoresponsive hydrogel or polymer, respectively, showing a transition between 20 and 40 °C.9 To enhance thermoresponsive cell culture carriers by implementation of matrix biopolymers and growth factors,3,10-12 we recently prepared a series of graft copolymers consisting of PNiPAAm as the polymer backbone and poly(ethyleneglycol) (PEG) as side chains.13 These PNiPAAm-containing polymers exhibit a lower critical solution temperature (LCST), where the volume phase transition of PNiPAAm goes along with a transition from hydrophilic to hydrophobic. And that finally converts an all-hydrophilic graft copolymer, PNiPAAm-g-PEG, at (5) Lutolf, M. P.; Weber, F. E.; Schmoekel, H. G.; Schense, J. C.; Kohler, T.; Mu¨ller, R.; Hubbell, J. A. Nat. Biotechnol. 2003, 21, 513518. (6) Hubbell, J. A. Curr. Opin. Biotechnol. 2003, 14, 551-558. (7) Hou, Q.; De Bank, P. A.; Shakesheff, K. M. J. Mater. Chem. 2004, 14, 1915-1923. (8) McCormick, C. L., Ed.; Stimuli-responsive Water-soluble and Amphiphilic Polymers; ACS Symposium Series No. 780; American Chemical Society: Washington, DC, 1999. (9) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163-249. (10) Van Recum, H.; Okano, T.; Wan Kim, S. J. Controlled Release 1998, 55, 121-130. (11) Shimizu, T.; Yamato, M.; Akutsu, T.; Shibata, T.; Isoi, Y.; Kikuchi, A.; Umezu, M.; Okano, T. J. Biomed. Mater. Res. 2002, 60, 110-117. (12) Harimoto, M.; Yamato, M.; Hirose, M.; Takahashi, C.; Isoi, Y.; Kikuchi, A.; Okano, T. J. Biomed. Mater. Res. 2002, 62, 464-470. (13) Schmaljohann, D.; Gramm, S. Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem.) 2002, 43, 758-759.
10.1021/la0476128 CCC: $30.25 © 2005 American Chemical Society Published on Web 02/08/2005
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low temperatures into an amphiphilic polymer above the LCST. The corresponding hydrogels have a so-called lower gel transition temperature (LGTT) in the same region as the LCST. Following the studies on surface-immobilized, biocompatible hydrogels,14,15 we were able to demonstrate the immobilization and cross-linking of thermoresponsive water-soluble polymers by low-pressure argon plasma exposure.16,17 The resulting surface-immobilized, thermoresponsive hydrogels exhibit a temperature-dependent swelling and collapsing behavior with different regimes of swelling. Swelling and collapsing were monitored quantitatively by following the change of the thickness and refractive index of the layers, which could be calculated from spectroscopic ellipsometry data. The volume phase transition was in the range of 32-37 °C, which allowed the modulation of surface properties within the setting of cell cultivation experiments. Furthermore, we were able to show that these changes in the surface properties allow the control of cell attachment and detachment of mouse fibroblasts.18 The potential of this method covers various applications in regenerative medicine such as the preparation of transferable sheets of cells.19-22 The microstructuring of these hydrogels will broaden these applications even more. Several groups have accomplished a patterning of hydrogels, especially surface-immobilized hydrogels in bioanalytical systems and in tissue engineering. Pishko et al. fabricated arrays based on poly(ethylene glycol) (PEG) using photolithographic and soft lithographic techniques.23-25 Toner et al. patterned PEG by photolithography to create cell arrays.26 Langer et al. introduced a soft lithographic approach also for PEG patterning.27 And we were able to pattern poly(HEMA) hydrogels using a soft lithographic approach.28,29 Thermoresponsive PNiPAAm hydrogels have also been patterned for sensor and actuator application.30,31 And Ober et al. microstructured 3-D scaffolds via a two-photon lithography approach.32,33 (14) Nitschke, M.; Menning, A.; Werner, C. J. Biomed. Mater. Res. 2000, 50, 340-343. (15) Nitschke, M.; Zschoche, S.; Baier, A.; Simon, F.; Werner, C. Surf. Coat. Technol. 2004, 185, 120-125. (16) Schmaljohann, D.; Nitschke, M.; Beyerlein, D.; Werner, C. Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem.) 2003, 44, 196-197. (17) Schmaljohann, D.; Beyerlein, D.; Nitschke, M.; Werner, C. Langmuir 2004, 20, 10107-10114. (18) Schmaljohann, D.; Oswald, J.; Jørgensen, B.; Nitschke, M.; Beyerlein, D.; Werner, C. Biomacromolecules 2003, 4, 1733-1739. (19) Ebara, M.; Yamato, M.; Aoyugi, T.; Kikuchi, A.; Sakai, K.; Okano, T. Biomacromolecules 2004, 5, 505-510. (20) Akiyama, Y.; Kikuchi, A.; Yamato, M.; Okano, T. Langmuir 2004, 20, 5506-5511. (21) Tsuda, Y.; Kikuchi, A.; Yamato, M.; Sakurai, Y.; Umezu, M.; Okano, T. J. Biomed. Mater. Res., Part A 2004, 69A, 70-78. (22) Cheng, X. H.; Wang, Y. B.; Hanein, Y.; Bohringer, K. F.; Ratner, B. D. J. Biomed. Mater. Res., Part A 2004, 70A, 159-168. (23) Sirkar, K.; Pishko, M. V. Anal. Chem. 1998, 70, 2888-2894. (24) Koh, W.-G.; Itle, L. J.; Pishko, M. V. Anal. Chem. 2003, 75, 57835789. (25) Koh, W.-G.; Pishko, M. Langmuir 2003, 19, 10310-10316. (26) Revzin, A.; Tompkins, R. G.; Toner, M. Langmuir 2003, 19, 98559862. (27) Suh, K. Y.; Seong, J.; Khademhosseini, A.; Laibnis, P. E.; Langer, R. Biomaterials 2004, 25, 557-563. (28) Chiellini, F.; Bizarri, R.; Ober, C. K.; Schmaljohann, D.; Solaro, R.; Chiellini, E. Macromol. Rapid Commun. 2001, 22, 1284-1287. (29) Chiellini, F.; Bizarri, R.; Ober, C. K.; Schmaljohann, D.; Yu, T.; Saltzman, W. M.; Solaro, R.; Chiellini, E. Macromol. Symp. 2003, 197, 369-379. (30) Kuckling, D.; Hoffmann, J.; Plo¨tner, M.; Ferse, D.; Kretschmer K.; Adler, H.-J. P.; Arndt, K.-F.; Reichelt, R. Polymer 2003, 44, 44554462. (31) Harmon, M. E.; Tang, M.; Frank, C. W. Polymer 2003, 44, 45474556. (32) Yu, T.; Ober, C. K. Biomacromolecules 2003, 4, 1126-1131.
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Photolithography is an effective tool to create microstructured substrates.34 Here, the change in the solubility characteristics of a polymer-coated substrate after exposure to radiation allows the selective removal of one part of the polymer coating. If the irradiated part becomes soluble in the developer, it is called a positive-tone resist. When it becomes insoluble, it is a negative-tone resist. The latter one provides a tool for the preparation of patterned hydrogels, when water-soluble polymers are subjected to the cross-linking step.35 Low-pressure argon plasma generates high-intensity UV light, which leads to cross-linking of polymers by random chain scission and recombination. Thus, plasma can be used to micropattern a polymer-coated substrate in the contact mode, when the substrate is covered by a mask. Even though this does not allow high-resolution lithography, it has the potential to pattern the substrate in the micrometer range, which is in the size range of cells, and it is therefore suitable for modulation of cell-substrate interaction. Surface characterization is a critical point within this project. It is well-known that ellipsometry is a fast and noninvasive method for characterizing the properties of polymers at interfaces and in thin films. The wellestablished ellipsometry techniques are null ellipsometry in the polarizer-compensator sample analyzer configuration (PCSA)36 and variable-angle spectroscopic ellipsometry (VASE).37 These techniques usually probe a spot of ca. 1 mm diameter on the substrate (null ellipsometry) or of several square millimeters (VASE). However, it does not give any spatial resolution. For small spot measurements (ca. 30-50 µm beam diameter) additional focusing optics are necessary. Over the past decade a technique called imaging ellipsometry emerged,38-41 which combines a typical ellipsometry experiment with an objective and a CCD camera as a detector. Now, each pixel on the CCD camera allows a separate ellipsometry experiment. So, the best spatial resolution can be in the range of 1-2 µm. Imaging ellipsometry is gaining increasing interest in particular for visualization of biomolecules and biological interaction on a substrate,42 e.g., an antigen-antibody interaction as a model of a biosensor43 or surface-grafted polypeptides.44 In this paper we describe the micropatterning of a thermoresponsive hydrogel and the characterization of its temperature-dependent swelling and collapsing behavior, analyzed by imaging ellipsometry. The experiments are carried out as proof-of-principle for the lithographic method. The thermoresponsive behavior was investigated, and also the film properties were studied as a function of the spatial position. (33) Zhou, W.; Kuebler, S. M.; Braun, K. L.; Yu, T.; Cammack, J. K.; Ober, C. K.; Perry, J. W.; Marder, S. R. Science 2002, 296, 1106-1109. (34) Rai-Choudhury, P., Ed. Handbook of Microlithography, Micromachining, and Microfabrication Volume 1: Microlithography; SPIE Optical Engineering Press: Bellingham, WA, 1997. (35) Thompson, L. F., Willson, C. G., Bowden, M. J., Eds.; Introduction to Microlithography; American Chemical Society: Washington, DC, 1994. (36) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and Polarized Light; North-Holland: Amsterdam, 1977. (37) Debe, M. K.; Field, D. R. J. Vac. Sci. Technol. 1991, A9, 1265. (38) Liu, A.; Wayner, P. C., Jr.; Plawsky, J. L. Appl. Opt. 1994, 33, 1223. (39) Harke, M.; Teppner, R.; Schulz, O.; Motschmann, H.; Orendi, H. Rev. Sci. Instrum. 1997, 68, 3130-3134. (40) Ho¨nig, D.; Mo¨bius, D. J. Phys. Chem. 1991, 95, 4590. (41) Goodall, D. G.; Stevens, C. W.; Beaglehole, D.; Gee, M. L. Langmuir 1999, 15, 4579-4583. (42) Arwin, H. Thin Solid Films 1998, 313-314, 764-774. (43) Jin, G.; Tengvall, P.; Lundstro¨m, I.; Arwin, H. Anal. Biochem. 1995, 232, 69-72. (44) Beyerlein, D.; Kratzmu¨ller, T.; Eichhorn, K.-J. Vib. Spectrosc. 2002, 29, 223-227.
Thermoresponsive Behavior of Hydrogel Films
Experimental Section Materials. The graft copolymer P1 (Figure 1) was synthesized from N-isopropylacrylamide (NiPAAm) and poly(ethylene glycol) monomethyl ether monomethacrylate via free radical copolymerization. Experimental details are given elsewhere.13 Low-Pressure Plasma Immobilization. Nonbranched fluorocarbon films with a structure close to that of PTFE were prepared on silicon substrates with an oxide layer of 50 nm, which allows the ellipsometric investigation of the hydrogel preparation. The fluorocarbon films, kindly provided by the Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences (Chernogolovka, Russia), were deposited by plasma polymerization. Tetrafluoroethylene (C2F4) was introduced downstream into a low-pressure argon discharge. Silicon wafers were placed further downstream of the discharge. The thickness of the obtained fluorocarbon films was about 80 nm.45 The fluorocarbon surfaces were treated in argon plasma as described below for 120 s to obtain an appropriate wetting behavior for spin coating. Thin films of the polymer were prepared on the fluorocarbon surfaces by spin coating from a 0.5% w/w solution in CHCl3. A spin coater, RC5 by Karl Suss, France, was operated at a velocity of 5000 rpm and an acceleration rate of 5000 rpm/s () 83.3 s-2). The polymer films were immobilized on the fluorocarbon surface using low-pressure argon plasma. The plasma treatment was carried out in a computer-controlled MicroSys apparatus by Roth&Rau, Germany. The cylindrical vacuum chamber, made of stainless steel, has a diameter of 350 mm and a height of 350 mm. The base pressure obtained with a turbomolecular pump is 7 at 25 °C is also comparable to that of the nonpatterned samples. Topographic Analysis of Thermoresponsive Hydrogel Collapsing. Ellipsometric Imaging Analysis of Hydrogel Collapsing. The major advantage of imaging ellipsometry, compared to conventional ellipsometry techniques, is the potential to display a two-dimensional area plot with an ellipsometric parameter in the third dimension, giving a surface map which results in a 3D profile. This can illustrate the effectiveness of the patterning process, and it allows the tracking of surface heterogeneities. In comparison to atomic force microscopy (AFM) as another surface analysis method, imaging ellipsometry is much faster, because it circumvents the slow scanning process. Relatively fast processes, which cannot be picked up by AFM, will be determined. Figure 5 demonstrates this with an example of a 3D profile of ∆ at three selected temperatures. The constant decrease of ∆, which goes along with a decrease in the film thickness d (see Figure 4), is illustrated as a change in the “height” at the hydrogel sections. The imaging 3D profile shows also that the hydrogel only changes its film thickness with the temperature increase; there is no change in the resolution of the pattern. Thus, swelling and collapsing of the hydrogel only occur in the z-direction as opposed to nonimmobilized hydrogel samples, where swelling occurs in the x-, y-, and z-directions. This observation indicates the effectiveness of the surfaceimmobilization process, which prevents swelling in the xand y-directions. Small heterogeneities at the surface are also visible; this will be discussed in the following section. Time-Dependent Effects. Surface heterogeneities on the sample were observed after storage of the sample under
Figure 4. ∆, Ψ, d, and n of the hydrogel (ROI (a), compare Figure 2) as functions of the temperature during the second heating in water, T equilibrium: (a) ∆, Ψ vs temperature, (b) d, n vs temperature.
Figure 5. 3D profile of ∆ during collapsing of the hydrogel upon heating: (a) T ) 25 °C, (b) T ) 30 °C, (c) T ) 35 °C.
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Conclusions
Figure 6. Ellipsometric contrast image of the hydrogel pattern: (a) dry in the cell, (b) in water after 20 min, and (c) after 1 day of equilibration.
water for 1 day. This is presented by an ellipsometric contrast image in Figure 6. The origin of these spots on the surface is not quite clear yet. A dewetting process of the underlying PTFElike layer might be associated with this observation. Another possible explanation is a surface contamination; however, further studies need to be carried out to identify the physicochemical processes inducing this behavior. If surface contamination is the origin of these spots, this would probably interfere with the volume phase transition in this particular region, because small changes in the microenvironment influence the transition temperature.51 This again shows the advantage of imaging ellipsometry compared to conventional ellipsometric techniques. (51) Mao, H.; Li, C.; Zhang, Y.; Furyk, S.; Cremer, P. S.; Bergbreiter, D. E. Macromolecules 2004, 37, 1031-1036.
A thermoresponsive hydrogel has been surfaceimmobilized and micropatterned using a photolithographic process. UV irradiation by low-pressure plasma exposure through a mask was utilized for the patterning in the contact-printing mode. The lithographic process was demonstrated as proof-of-principle without consideration of a high resolution. With this process hydrogel patches separated by 60 µm wide grooves were prepared and subsequently analyzed by imaging ellipsometry. First, the success of the patterning was confirmed by imaging ellipsometry in the dry state. The temperaturedependent swelling and collapsing in deionized water were then studied using the localized nulling mode, and 3D profiles of the thickness-sensitive ellipsometric angle ∆ were measured as a function of temperature. The imaging mode of the measurement allowed the determination of collapsing of the hydrogel through the temperature stimulus, and it was possible to visualize surface heterogeneities upon storage of the sample in water. The patterning of thermoresponsive hydrogels has a great impact on the preparation of next-generation cell culture carriers, and the hydrogels serve as model implantable devices in the field of tissue engineering. Further studies are necessary both on fundamental physicochemical characterization and on biological studies such as cell response, proliferation, and differentiation. Acknowledgment. We thank Prof. B. Voit for valuable discussion and support of this work. LA0476128
