pubs.acs.org/Langmuir © 2009 American Chemical Society
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Thermoresponsive PEG-Based Polymer Layers: Surface Characterization with AFM Force Measurements Stefanie Kessel,†,‡ Stephan Schmidt,‡ Renate M€uller,†,‡ Erik Wischerhoff,§ Andre Laschewsky,§, Jean-Franc-ois Lutz,*,§ Katja Uhlig,^ Andreas Lankenau,^ Claus Duschl,*,^ and Andreas Fery*,‡
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† Max Planck Institute of Colloids and Interfaces, Interfaces Department, Am M€ uhlenberg 1, D-14476 Potsdam-Golm, Germany, ‡Physikalische Chemie II, Universit€ at Bayreuth, Universit€ atsstrasse 30, D-95447 Bayreuth, Germany, §Fraunhofer Institute for Applied Polymer Research, Geiselbergstrasse 69, D-14476 Potsdam-Golm, Germany, Institute of Chemistry, University of Potsdam, Karl-Liebknecht-Str. 24-25, D-14476 Potsdam-Golm, Germany, and ^Fraunhofer Institute for Biomedical Engineering IBMT, Am M€ uhlenberg 13, D-14476 Potsdam-Golm, Germany
Received August 13, 2009. Revised Manuscript Received September 28, 2009 Thermoresponsive polymer-coated surfaces based on poly(2-(2-methoxyethoxy)ethyl methacrylate-co-oligo(ethylene glycol) methacrylate) [P(MEO2MA-co-OEGMA)] allow switching between cell attachment and detachment. Here, we investigate the temperature-dependent surface interactions between the polymer coating and a colloidal probe in an aqueous medium by means of atomic force microscopy (AFM) force-distance measurements. The analysis of the adhesion forces from AFM retraction curves indentifies two kinds of regimes for the copolymer at temperatures below and above the lower critical solution temperature (LCST). Whereas at 25 C the surface interactions with the polymer in the swollen state are dominated by repulsive forces, at 37 C the surface interactions switch to attractive forces and a stronger adhesion is detected by AFM. Running several heating/cooling cycles repeatedly shows that switching the surface properties provides reproducible adhesion force values. Time-dependent measurements give insight into the switching kinetics, demonstrating that the cell response is coupled to the polymer kinetics but probably limited by the cellular rearrangements.
1. Introduction The control over cell-surface interactions is a highly important issue for biomedical and biotechnology applications.1 Although cell adhesion on 2D surfaces or in 3D scaffolds is a highly complex and still unresolved phenomenon, it can be controlled to some degree by adjusting the interaction between extracellular matrix proteins with the substrate. Smart adjustment of the cell adhesion becomes increasingly important, in particular for advanced cell assays. For example, on standard cell culture substrates (i.e., commercial polystyrene Petri dishes or well-plates), the cell adhesion to the polystyrene surface is so intense that cell detachment requires treatment with potentially harmful enzymes such as trypsin. This treatment affects the viability of the cells, as it often damages the cell surface proteins, complicating the investigation of cell interactions and signal transduction pathways. As an alternative, biocompatible materials such as poly(ethylene glycol) *To whom correspondence should be addressed. For surface modification: Jean-Franc-ois Lutz. Address: Fraunhofer-Institut f€ur Angewandte Polymerforschung, Geiselbergstrasse 69, 14476 Potsdam (Germany). Fax: (þ49) 331568-3000. Telephone: (þ49) 331-568-1127. E-mail: jean-francois.lutz@ iap.fraunhofer.de. Website: http://www.bioactive-surfaces.com. For cell assay: Claus Duschl. Address: Fraunhofer-Institut f€ur Biomedizinische Technik, Am M€uhlenberg 13, 14476 Potsdam (Germany). Fax: (þ49) 33158187-399. Telephone: (þ49) 331-58187-300. E-mail: claus.duschl@ibmt. fhg.de. For AFM force measurements: Andreas Fery. Address: Universit€at Bayreuth, Universit€atsstrasse 30, 95440 Bayreuth (Germany). Fax: þ49 (0)921 55-2059. Telephone: þ49 (0)921 55-2751. E-mail: andreas.fery@ uni-bayreuth.de. (1) Wischerhoff, E.; Badi, N.; Lutz, J.-F.; Laschewsky, A. Soft Matter, submitted. (2) Prime, K. L.; Whitesides, G. M. J. Am. Chem. soc. 1993, 115(23), 10714– 10721. (3) Kingshott, P.; McArthur, S.; Thissen, H.; Castner, D. G.; Griesser, H. J. Biomaterials 2002, 23(24), 4775–4785.
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(PEG) emerged, which are characterized by excellent protein- and cell-repellent properties.2,3 Yet, although very useful, biorepellent PEG surfaces remain passive materials, preventing noninvasive control over cell adhesion. Recently, some efforts have been made to achieve an effective control over cellular interactions with synthetic surfaces. Research interests have been directed toward the development of new smart polymer systems that can be reversibly switched between interacting and noninteracting states.4 An advantage of such stimuli-responsive polymers is that cell adhesion and detachment is reversible and can be controlled upon an external signal.5 The switchable surfaces react on external stimuli such as light,6 temperature,7-9 electric potential, pH value,10 or ionic strength.11 Thermoresponsive polymers exhibit a temperature-dependent solution and surface behavior. Slight changes in the environment, for example, heating above the lower critical solution temperature (LCST), cause dramatic changes in the polymer hydration state and bioadhesion. The most frequently studied thermoresponsive polymer is poly(N-isopropylacrylamide) (PNIPAM). This polymer exhibits an LCST in water with a sharp (4) Cole, M. A.; Voelcker, N. H.; Thissen, H.; Griesser, H. J. Biomaterials 2009, 30(9), 1827–1850. (5) Mano, J. F. Adv. Eng. Mater. 2008, 10(6), 515–527. (6) Tada, Y.; Sumaru, K.; Kameda, M.; Ohi, K.; Takagi, T.; Kanamori, T.; Yoshimi, Y. J. Appl. Polym. Sci. 2006, 100(1), 495–499. (7) Yamato, M.; Utsumi, M.; Kushida, A.; Konno, C.; Kikuchi, A.; Okano, T. Tissue Eng. 2001, 7(4), 473–480. (8) Cunliffe, D.; Alarcon, C. D.; Peters, V.; Smith, J. R.; Alexander, C. Langmuir 2003, 19(7), 2888–2899. (9) Jonas, A. M.; Glinel, K.; Oren, R.; Nysten, B.; Huck, W. T. S. Macromolecules 2007, 40(13), 4403–4405. (10) Zhu, X.; De Graaf, J.; Winnik, F. M.; Leckband, D. Langmuir 2004, 20(4), 1459–1465. (11) Spruijt, E.; Choi, E. Y.; Huck, W. T. S. Langmuir 2008, 24(19), 11253– 11260.
Published on Web 11/05/2009
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phase transition point in the physiologically relevant range (i.e., at ∼32 C in pure water),12 thus allowing control over various surface properties.13-15 However, PNIPAM can undergo hydrogen-bridging with the amide groups of proteins, rendering PNIPAM likely not truly bioinert and thus prone to distort the results of certain cell assays. Recent studies described the control of protein- and cell-adhesion with a new class of thermoresponsive polymers based on oligo(ethylene glycol)methacrylates.16-18 Indeed, it was recently demonstrated that random copolymers of 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA) and oligo(ethylene glycol) methacrylate (OEGMA, Mn ∼ 475 g mol-1) exhibit a LCST in water, which can be precisely adjusted by the copolymer composition.19-22 These polymers can be synthesized by atom transfer radical polymerization (ATRP), and using initiators with appropriate functional groups they can be easily immobilized on a wide variety of surfaces (e.g., gold or glass) using grafting-from or grafting-onto strategies.23,24 Jonas et al. reported that model surfaces modified with P(MEO2MA-coOEGMA) brushes exhibit switchable properties in the nearphysiological temperature range.9 Shortly after, Laschewsky, Lutz, and co-workers demonstrated that P(MEO2MA-coOEGMA)-modified surfaces can be exploited to control fibroblast adhesion and detachment in a very convenient temperature range (i.e., 25-37 C).16,18 However, the physicochemical understanding of this process is still limited. For the investigation of the surface transitions and structural changes triggered by the temperature shift, a sensitive analytic method is required. Atomic force microscopy (AFM) forcedistance-measurements have emerged as a versatile tool to study all kinds of surface forces.25 Applications address fundamental questions in contact mechanics as well as surface forces. In particular, the colloidal probe technique26 proved useful for the quantification of surface forces, for example, adhesion forces between a polymer-coated surface and a colloidal particle.27,28 For example, the colloidal probe configuration was used to characterize the switch from repulsive to attractive forces on PNIPAM-coated surfaces when raising the temperature above the LCST, indicating a transition in the hydration state of PNIPAM chains.29,30
(12) Heskins, M.; Guillet, J. E. J. Macromol. Sci., Part A: Pure Appl. Chem. 1968, 2(8), 1441–1455. (13) Balamurugan, S.; Mendez, S.; Balamurugan, S. S.; , M. J.; Lopez, G. P. Langmuir 2003, 19(7), 2545–2549. (14) Kidoaki, S.; Ohya, S.; Nakayama, Y.; Matsuda, T. Langmuir 2001, 17(8), 2402–2407. (15) Schmidt, S.; Motschmann, H.; Hellweg, T.; von Klitzing, R. Polymer 2008, 49(3), 749–756. (16) Wischerhoff, E.; Uhlig, K.; Lankenau, A.; Borner, H. G.; Laschewsky, A.; Duschl, C.; Lutz, J.-F. Angew. Chem., Int. Ed. 2008, 47(30), 5666–5668. (17) Gao, X.; Kucerka, N.; Nieh, M.-P.; Katsaras, J.; Zhu, S.; Brash, J. L.; Sheardown, H. Langmuir 2009, 25(17), 10271–10278. (18) Wischerhoff, E.; Glatzel, S.; Uhlig, K.; Lankenau, A.; Lutz, J.-F.; Laschewsky, A. Langmuir 2009, 25(10), 5949–5956. (19) Lutz, J.-F.; Akdemir, O.; Hoth, A. J. Am. Chem. Soc. 2006, 128(40), 13046– 13047. (20) Lutz, J.-F.; Hoth, A. Macromolecules 2006, 39(2), 893–896. (21) Lutz, J.-F.; Andrieu, J.; Uzgun, S.; Rudolph, C.; Agarwal, S. Macromolecules 2007, 40(24), 8540–8543. (22) Lutz, J.-F. J. Polym. Sci., Part A: Polym. Chem. 2008, 46(11), 3459–3470. (23) Pyun, J.; Kowalewski, T.; Matyjaszewski, K. Macromol. Rapid Commun. 2003, 24(18), 1043–1059. (24) Edmondson, S.; Osborne, V. L.; Huck, W. T. S. Chem. Soc. Rev. 2004, 33 (1), 14–22. (25) Cappella, B.; Dietler, G. Surf. Sci. Rep. 1999, 34(1-3), 1–2. (26) Ducker, W. A.; Senden, T. J.; Pashley, R. M. Nature 1991, 353(6341), 239– 241. (27) Butt, H. J. Biophys. J. 1991, 60(6), 1438–1444. (28) Butt, H. J.; Cappella, B.; Kappl, M. Surf. Sci. Rep. 2005, 59(1-6), 1–152. (29) Cho, E. C.; Kim, Y. D.; Cho, K. Polymer 2004, 45(10), 3195–3204. (30) Ishida, N.; Kobayashi, M. J. Colloid Interface Sci. 2006, 297(2), 513–519.
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In the present study, the colloidal probe AFM technique was utilized to characterize thermoresponsive and biocompatible P(MEO2MA-co-OEGMA) surfaces. To understand the cell adhesion process, we perform a temperature-dependent investigation of the surface forces. The AFM force-distancemeasurements are performed on P(MEO2MA-co-OEGMA) brushes prepared via a grafting-onto approach.16 The different surface properties (hydration state) of the polymer chains above and below the LCST result in different interactions between the surface and probe. Below the LCST, at 25 C, repulsive forces dominate the interaction. Above the LCST, at 37 C, attractive forces appear and stronger adhesion forces between surface and probe are detected. These results correlate with the adherent and repellent states of fibroblasts on the polymermodified surface. In addition, we show the influence of the medium in the interaction force and also investigate the kinetics of the switching process.
2. Materials and Methods Preparation of Polymer Layer on Gold Substrate. The synthesis of the thermoresponsive copolymer and its attachment onto the gold surface have been described in detail in previous publications.16,20 Briefly, the copolymer P(MEO2MAco-OEGMA) was synthesized by atom transfer radical polymerization in ethanol solution and in the presence of a disulfide initiator. The copolymer formed contained about 10 mol % OEGMA and 90 mol % MEO2MA. Thus, this copolymer exhibits a cloud point at approximately 35 C (i.e., in between room and body temperature) in physiological buffer. The disulfide-functionalized P(MEO2MA-co-OEGMA) was dissolved in ethanol and adsorbed on freshly prepared gold surfaces for 3 h at room temperature. Unbound macromolecules were carefully removed by washing the surfaces with ethanol for 1 h. AFM Force-Distance Measurements. The force spectroscopy measurements were performed with a Nanowizard I setup (JPK instruments, Germany) using the colloidal probe technique. Prior to the measurement, colloidal particles (glass beads, diameter ∼ 30-50 μm, Polyscience Inc.) were attached to tipless silicon cantilevers (CSC12, MikroMasch, Estonia) utilizing a two component epoxy glue (UHU GmbH & Co.KG, Germany) and a micromanipulator (Suttner Instrument Co.). The spring constants of the cantilevers were derived from both the thermal noise method31 and the Sader method32 before attaching the colloidal probes. Both methods agreed within 10%, and values of the spring constants were in the range reported by the manufacturer. All measurements were performed in aqueous media (distilled water, standard PBS buffer, and cell culture medium). The composition of the cell culture medium was 25 mM N-(2hydroxyethyl)piperazine-N0 -ethanesulfonic acid (HEPES), 2 mM L-glutamine, and 1% penicillin/streptomycin (all from Biochrom, Germany). To avoid nonspecific interactions with the colloidal probe, no serum proteins were added to the media. To control the solution temperature and simultaneously the surface temperature during the measurement, an AFM environmental cell (BioCell, JPK instruments, Germany) was used. The solution temperature in the fluid cell was controlled and held at constant values with variations not exceeding 0.1 C. The force measurements were carried out in a temperature range from 20 to 40 C. The heating rate of the environmental cell was set to 5 C/min. For each distinct temperature in a temperature cycle, the measurement (approach and retract) was done on two independent spots, which were randomly chosen on the substrate. (31) Hutter, J. L.; Bechhoefer, J. Rev. Sci. Instrum. 1993, 64(7), 1868–1873. (32) Sader, J. E.; Chon, J. W. M.; Mulvaney, P. Rev. Sci. Instrum. 1999, 70(10), 3967–3969.
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Coating of Glass Beads. The coating of the glass beads with fibronectin (fibronectin from human plasma, cell culture tested, Sigma-Aldrich) was performed after attaching the glass beads by dipping the cantilever into an aqueous fibronectin solution (10 μg/mL) for 30 min. Unbound fibronectin was then carefully washed away with an excess of water. Cell Assay. L929 mouse fibroblasts (ACC 2, DSMZ, Germany) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM), 25 mM HEPES, 10% fetal calf serum, 2 mM L-glutamine, and 1% penicillin/streptomycin (all from Biochrom, Germany) at 37 C and 5% CO2. After incubation for three days at 37 C the cells were imaged and temperature was decreased to 25 C. The cell behavior was monitored in phase contrast mode using a 20 0.75 NA objective and a Nikon Digital Sight DS-L1 camera (Nikon, Germany).
3. Results and Discussion 3.1. Characterization of the P(MEO2MA-co-OEGMA) Layer on Gold. Biocompatible polymer brush coatings of oligo(ethylene glycol) methacrylates can be readily prepared by, for example, atom transfer radical polymerization (ATRP).20 Yet, brushes with side chains longer than five ethylene oxide units are rather passive; that is, they offer no response upon any biocompatible stimuli. To obtain switchable surfaces, we followed the method of Wischerhoff et al.16 and prepared random copolymers of short (only two) and longer ethylene oxide side chains. Here, an atom transfer radical copolymerization of MEO2MA and OEGMA in solution starting from a disulfide initiator (Figure 1a, 1) results in a defined P(MEO2MA-co-OEGMA) coating (Figure 1a, 2). With the usage of 10% OEGMA monomer, the LCST of the resulting copolymer could be adjusted to 35 C in a phosphate buffered saline (PBS) solution.16,20 The coated surface was prepared by adsorbing the disulfide-functionalized P(MEO2MA-co-OEGMA) onto freshly prepared gold substrates and characterized by surface plasmon resonance (SPR) and contact angle measurements as recently published.16 The estimated calculations of the grafting density of these surfaces seem to indicate that the polymer grafts are probably not in a dense brush regime. For the cell experiments, L929 mouse fibroblasts were employed. These cells were transferred to the surface at 37 C, where strong adhesion and spreading of the cells could be observed (Figure 1b). Shortly after cooling down to 25 C, the cells rounded and could be removed by gentle rinsing (Figure 1b). These results confirmed that the polymer-coated surfaces can be efficiently switched from a cell-adherent to a cell-repellent state. 3.2. Temperature-Dependent Interaction Forces between Polymer Layer and Colloidal Probe. First, we present results in cell culture media which are more relevant as a comparison for the cell assay shown in Figure 1. After inserting the switchable polymer-coated surfaces into the fluid cell, it was filled with the medium. Then, after adjusting the temperature and equilibration, five colloidal probe approaching/retracting cycles were performed on each spot. The colloidal probe was approached onto the surface with a maximum load of 20 nN and then immediately retracted. While the approach curves showed pure repulsive behavior and no jump-to-contact in the whole range of temperatures, the characteristic adhesion forces could be detected in the retraction curves (Figure 2). As a measure of the adhesion, we used the pull-off force (also called adhesion force) which can be read from the AFM retraction curves: Upon retraction, the colloidal probe keeps in contact with the surface until the cantilever force overcomes the adhesion force. The adhesion force can be directly read from the minimum force in a retraction cycle (Figure 2). Figure 2 shows a typical force-distance curve upon retracting the cantilever at 25 and 37 C. We detected only small adhesion 3464 DOI: 10.1021/la903007v
Figure 1. (a) Chemical structure of the disulfide initiator (1) used for the atom transfer radical copolymerization of MEO2MA/OEGMA (90/10) and the resulting statistical copolymer P(MEO2MA-co-OEGMA) (2). (b) Phase contrast microscopy images of L929 mouse fibroblast cells 44 h after incubation at 37 C and 30 min after cooling the surface to 25 C.
forces of 0.28 ( 0.15 nN at 25 C. Heating up the polymer surface above the LCST (i.e., at 37 C) induced a different behavior as shown in Figure 2b. The retraction of the probe from the surface was accompanied by stronger attractive forces that result in an average adhesion force of 1.17 ( 0.40 nN. Above the LCST, the polymer chains exist in a collapsed, hydrophobic state; hence, surface interaction forces switched to attractive hydrophobic forces. In the retracting curves, the pull-off is not a single event (vertical jump off contact), but rather a continuous detachment is observed, as frequently found for polymer surfaces.33,34 Next, we present the adhesion forces for a systematic increase of the temperature from 25 to 40 C (Figure 3). Between each temperature step, the system was allowed to equilibrate for 15 min. At temperatures below the LCST, the adhesion forces were very weak and just slightly increasing with increasing temperature. We observed a drastic increase in adhesion around 37 C, close to the LCST. Here, the measured adhesion force reached values of about 1.4 nN. When heating the surface further to 40 C, the adhesion force values kept increasing to 2.9 nN. Interestingly, the phase transition (collapse) of the polymer in bulk solution is much sharper and also finishes at a temperature of 35 C.16 Here, the phase transition as measured via adhesion forces has broadened and the LCST seems to have slightly increased. Steric constrains that the polymer is subjected to when immobilized to the surface may explain this behavior. As a control for the measurements on the P(MEO2MA-coOEGMA)-based brushes, surfaces with covalently bound linear PEG-chains were investigated. It is well-known that PEG-coated surfaces have protein- and cell-repellent behavior.2,3 The AFM measurements confirm these findings. Linear PEG molecules with a thiol end group were assembled onto the gold substrates and measured under the same conditions as the P(MEO2MA-coOEGMA) surfaces. In cell culture medium, very weak adhesion forces below 100 pN were measured. In addition, no temperaturedependency of the adhesion forces was observed. (33) Senden, T. J.; di Meglio, J. M.; Auroy, P. Eur. Phys. J. B 1998, 3(2), 211– 216. (34) Papastavrou, G.; Kirwan, L. J.; Borkovec, M. Langmuir 2006, 22(26), 10880–10884.
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Figure 2. Force versus distance curves from the retraction of the cantilever. Characteristic curve measured in cell culture medium at 25 C (a) and 37 C (b).
Figure 3. Average adhesion force values determined from measurements with systematic increased temperatures in the range 25-40 C indicating the surface interaction transition point at ∼35 C. Average adhesion force values were calculated from five data points at two positions at each temperature.
Furthermore, in some cell-biology applications, it is necessary to seed and to remove the cells several times from the surface of the cell culture dish. Therefore, it is interesting to investigate the reproducibility of the surface adhesion, when exposing the surface to several heating/cooling cycles. For this purpose, the temperature of the surrounding medium was switched several times from 25 to 37 C and a measurement (approaching/ retracting for five times at two independent spots) was done at each temperature after allowing the system to equilibrate for at least 30 min. The whole procedure was iterated four times. The evaluated average adhesion force values are plotted in Figure 4. Repeatedly, in every cycle at 25 C, very small adhesion forces of 300 ( 30 pN were measured. Again, at 37 C, the adhesion forces were much larger. However, slightly different values were determined in every cycle. On average, the adhesion forces were 1.37 ( 0.2 nN in the cell culture medium. The slight variations in the calculated adhesion force values can be explained by small variation of the surface roughness. The surface roughness has a pronounced influence on adhesion force and is probably not homogeneous on the microscopic scale. Therefore, the microscopic-scale contact geometry varied for each cycle. These repeated measurements indicate that the switching of the surface properties by adjusting the temperature above/below the LCST can be done several times without a loss of performance in terms of switching the adhesion on and off. By contrast, PNIPAMcoated surfaces show changes in the shape of the force curves after compression via colloidal probe, as shown by Ishida and Kobayashi.30 In the present, case no systematic change of the force curve shape was detected. Using the outlined conditions, the adhesion force values are stable at each individual temperature Langmuir 2010, 26(5), 3462–3467
Figure 4. Graph showing the results of the force measurements that where obtained during four temperature cycles at 25/37 C. They confirm the reproducibility of the changes of the surface properties. The average adhesion force values were calculated from five data points at two positions at each temperature ((9) 37 C; (O) 25 C; the dotted lines indicate the heating and cooling steps).
and within several temperature cycles. Furthermore, Ishida and Kobayashi observed completely repulsive PNIPAM brushes above the LCST for hydrophilic colloidal probes. Here, we used similar hydrophilic colloidal probes, but in contrast to the PNIPAM brushes the PEG-based coating always showed strong adhesive interactions above the LCST. 3.3. Influence of the Medium. In the following, we examined whether the surrounding medium influences the interaction forces between probe and polymer layer. Figure 5 shows the adhesion forces for several temperature cycles performed in distilled water, PBS, and cell culture medium. In order to avoid uncontrolled adsorption of proteins to the probe, the cell medium did not contain serum proteins. In pure water and PBS, the adhesion forces at 25 C were ∼150 pN, whereas in the cell culture stronger forces of ∼300 pN were observed. In all three media, the adhesion force values at 37 C are roughly 5 times larger (1.0-1.4 nN). In PBS, the adhesion force was strongest in the first cycle (1.15 nN) and then stabilized at a constant value (0.95 nN) in the next cycles. In water and cell culture medium, no systematic change can be observed in the maximum adhesion values from cycle to cycle. Slight differences were detected in the adhesion forces, probably due to the roughness of the surface and the condition of the glass probe. In all three media used, the switching of the surface properties was possible and reversibility of switching was observed over several temperature cycles. As expected, since a noncharged polymer coating was investigated, the variation of ionic strength of the medium had only a small influence on the maximum values of the adhesion. Therefore, electrostatic DOI: 10.1021/la903007v
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Figure 5. Investigation of the influence of the medium in which the measurements were performed. Four temperature cycles with repeated heating to 37 C and cooling to 25 C were carried out in distilled water (9), cell culture medium (O), and PBS (g).
Figure 6. Time-dependent measurements to analyze the kinetics of the switching process. After heating to 37 C (9), the temperature was held for 60 min, and then the surface was cooled down to 25 C (O). Switching between both temperatures took 2 min. The data points denote average values of 10 retraction cycles; the error was in the range of 10-25%.
interactions are not relevant for the adhesion properties. This shows that P(MEO2MA-co-OEGMA)-coated surfaces are relatively insensitive to variations of the ionic milieu facilitating their application in various environments. 3.4. Kinetic of Temperature Induced Switching. So far, studies concerning the analysis of thermoresponsive PNIPAMcoated surfaces by colloidal probe AFM did not report on the kinetics of the phase transition. An insight into the kinetics would be helpful in understanding the cell adhesion process upon switching the surfaces. Here, we analyzed the time-dependence of the adhesion force after temperature induced switching. First, measurements were done on the sample surface at 25 C. Afterward, the surface was heated up and the temperature was held constant at 37 C. The probe was brought into contact with the surface after 5, 10, 20, 30, and 60 min. The adhesion force values extracted from the force-distance curves are depicted in Figure 6. In the first minutes after reaching the desired temperature of 37 C, the values for the adhesion forces increased significantly, and after 20 min the maximum was reached. Then, while holding the temperature constant at 37 C, the force values decreased slowly. After 60 min, the adhesion forces equilibrated and did not change further. These results indicate that the initial rearrangement of the polymer chains tethered to the surface takes 15-20 min. After 20 min, the polymer was situated in a state where it generates maximum adhesion. Interestingly, the adhesion forces decrease thereafter, which could be triggered by additional reorganization processes. When cooling down to 25 C, the phase transition process of the polymer chains was completed faster. A 3466 DOI: 10.1021/la903007v
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Figure 7. Graph illustrating the average adhesion force values at 25 and 37 C comparing fibronectin-coated beads (9) and pure glass beads (O). The two temperature cycles were measured in cell culture medium.
drastic decrease of the adhesion forces was observed within 10 min, and a constant minimal value was reached around 20 min. These observations were confirmed by consecutive heating/cooling cycles. In contrast to the cell adhesion process that lasted approximately 2 days until the cells were fully spread, the rounding of the cells after cooling to 25 C was usually finished within 30 min. The results obtained by time-resolved colloidal probe AFM measurements are in qualitative agreement with rounding of the cells after cooling. The different time scales of surface interaction changes and cell response clearly demonstrate that the cell response was coupled to the polymer kinetics, but probably limited by the cellular rearrangements. 3.5. Protein Coated Colloidal Probe. As mentioned before, the cell adhesion process on synthetic surfaces is strongly related to the adhesion properties of extracellular matrix proteins. Hence, it is interesting not only to analyze the surface forces between entirely synthetic interfaces but also to investigate the influence of extracellular matrix proteins. To get a first idea of how proteins alter the interaction forces, the colloidal probe surface was modified with fibronectin by dip-coating. We chose fibronectin as a model protein because it plays a very important role in the cell adhesion process. Afterward, the force measurements were done in a temperature cycle as described for the noncoated beads. In Figure 7, the temperature cycles of coated and uncoated probes performed in cell culture medium are illustrated. Whereas at 37 C the adhesion forces measured were weaker, they were stronger at 25 C compared to the forces obtained for the untreated glass beads. The differences in the adhesion forces when switching the surface from 25 C (220 pN) to 37 C (380 pN) are much smaller for the fibronectin covered beads. This clearly shows that the involved proteins change the surface interaction between polymer coatings and the analyzing probe. As can be seen in Figure 7, the adhesion forces show a slight increase for repeated measurements above the LCST with the fibronectin-coated colloidal probe. This indicates that part of the physisorbed fibronectin was transferred to the PEG coating upon contact with the substrate. Here, just a first example on the effect of an extracellular matrix protein on the adhesion force is reported. More research has to be done to get a broader understanding. 3.6. Responsiveness of the Polymer Brushes and Surface Interactions. In the present case, the P(MEO2MA-co-OEGMA) brushes are hydrated and extended below the LCST and collapsed on the surface above the LCST. Being controlled by entropic elasticity and osmotic pressure, the degree of swelling Langmuir 2010, 26(5), 3462–3467
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of in fact any polymer brush-solvent system is temperaturedependent; see Toomey and Tirrell for a recent review.35 This is well-known for so-called classic polymer brush systems, for example, polystyrene brushes in toluene, where only van der Waals interactions are present. However, due to tethering constrains, classic brush systems never show sharp deswelling as free polymers do at the upper critical solution temperature. In contrast, nonclassic, water swollen brush systems such as P(MEO2MA-co-OEGMA) can show sharp changes in the degree of swelling. For such systems, it is argued that the dissociation of hydrogen bonds with increasing temperature leads to a decrease of the second viral coefficient and thus to an increase of the effective interaction parameter (stronger segment interactions). This may result in perturbation of the solvation structure and brush collapse. Modeling approaches often use a two-state description where the segments exist in a hydrophobic or hydrophilic state,36 which can also be used to describe the interaction forces measured here. Above the LCST, the relatively large adhesion force can be attributed to van der Waals interactions between the colloidal probe and polymer layer. Attractive hydrophobic forces should not be present, as the hydrophilic silica particles were used as colloidal probes. Below LCST, the presence of water molecules stretches the PEG side chains toward the bulk, due to osmotic forces within the brush layer. This results in a repulsive force when the brush layer is compressed by the colloidal probe or cellular adhesion. Further, hydration of the PEG chains disables attractive van der Waals forces of the polymer with the colloidal probe, which adds to the overall repulsive interaction. The fibronectinmodified colloidal probes show reduced adhesion over the complete temperature range. This shows the effect of unspecific repulsive hydration forces of the protein layer against the bioinert polymer surface. The adhesion forces observed here significantly decrease but do not completely vanish in the swollen brush state. Thus, attractive van der Waals forces are not completely overcome by the repulsive steric interaction of these rather short polymer brushes. (35) Toomey, R.; Tirrell, M. Annu. Rev. Phys. Chem. 2008, 59, 493–517. (36) Dormidontova, E. E. Macromolecules 2002, 35(3), 987–1001.
Langmuir 2010, 26(5), 3462–3467
Article
4. Conclusions Here, we used AFM force-distance measurements to characterize novel thermoresponsive PEG-based polymer layers that allow control over cell adhesion. The colloidal probe technique was used to determine the interaction forces between the probe and the polymer-coated surfaces. Measurements were carried out at temperatures below and above the LCST of the copolymer P(MEO2MA-co-OEGMA). We observed temperature-dependent adhesion forces, showing a sharp transition around the LCST. Within four heating and cooling cycles, the properties of the polymer-coated surfaces can be switched without a loss of adhesive performance. High values for the adhesion force of about 1 nN were reproducibly obtained at 37 C, and lower values around 100 pN at 25 C. Generally, the results were independent of the ionic strength of the medium. Time-dependent measurements provided an insight in the kinetics of the switching process, showing that the maximum adhesion force was reached after 20 min upon heating, and minimum adhesion approximately 20 min after cooling. Besides, it has been shown that proteins adsorbed to the colloidal probe alter the surface-bead interaction. The nonspecific interactions analyzed between the thermoresponsive polymer layer and the colloidal probe can be related to the forces acting between the fibroblast cells and the surface. At 37 C where strong adhesion forces were detected, the cells also formed large contact areas. At 25 C where almost no adhesion forces were detected, the cells minimized their surface contact. The fact that adhesion forces were diminished, but not completely vanishing in the brush state, indicates that polymer coverage of the surface was relatively low. To achieve denser polymer coverage, we recently investigated alternative surface chemistries for preparing dense P(MEO2MA-co-OEGMA) surfaces.1 In connection with the results obtained, AFM force-distancemeasurements represent a powerful tool to characterize synthetic surfaces for biomedical applications and offer the possibility to optimize new systems without the need for cell experiments. Acknowledgment. We gratefully acknowledge financial support by the Fraunhofer Society and the Max-Planck-Society in the network of excellence “Synthetic Bioactive Surfaces”.
DOI: 10.1021/la903007v
3467
