Tuning of Thermo-responsive Self-Assembly Monolayers on Gold for

Reaction with cysteamine: polymer (1.0 g) synthesized as described above was dissolved ... performed with a molar mixture ratio of 1:6 (0.1 mM/0.56 mM...
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Langmuir 2008, 24, 10259-10264

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Tuning of Thermo-responsive Self-Assembly Monolayers on Gold for Cell-Type-Specific Control of Adhesion Oliver Ernst,† Antje Lieske,‡ Andreas Holla¨nder,‡ Andreas Lankenau,*,† and Claus Duschl† Fraunhofer Institute for Biomedical Engineering, Am Mu¨hlenberg 13, 14476 Potsdam, and Fraunhofer Institute for Applied Polymer Research, Geiselbergstrasse 69, 14476 Potsdam, Germany ReceiVed April 2, 2008. ReVised Manuscript ReceiVed May 23, 2008 Self-assembled monolayers (SAMs) on gold containing a thermo-responsive poly(N-isopropylacrylamide)poly(ethylene glycol)-thiol copolymer were formed. These layers show considerable potential for inducing enzymefree and gentle detachment of cultivated cells. In an effort to optimize detachment of cells, including strongly adhering ones, two approaches are presented. First, two thermo-responsive copolymers with different poly(ethylene glycol) (PEG) contents of 15 wt % (“P15”) and 19 wt % (“P19”) were grafted to Au surfaces. Second, mixed monolayers were formed containing P19 and various concentrations of thiol bearing PEG. X-ray photoelectron spectroscopy (XPS) on pure and mixed P19 containing layers confirmed the expected layer compositions. Contact angle measurements showed good functionality of all surfaces prepared. Upon a temperature decrease below the lower critical solution temperature (LCST), the duration until cultivated fibroblasts detached from pure P19 surfaces was half of the one determined on P15. Strongly adherent human osteosarcoma cells could not be detached from pure P19 layers. Through co-adsorption of P19 and thiol-bearing PEG of a molar composition of 1:6, layers were formed that allowed good spreading of osteosarcoma cells above LCST and their efficient detachment below LCST.

Introduction A key procedure in cell biology and cellular biotechnology is the detachment of cells from their culture substrate for further processing. For example, the harvesting of single cells, separation of cells, or the detachment of cell sheets for tissue engineering are crucial steps for making cells available for biomedical application. Most currently available protocols still depend upon enzymatic treatment using trypsin, which affects the viability of the cells because it damages cell surface proteins and has adverse effects on the functioning of many signal transduction pathways.1 Recently, thermo-responsive polymers, e.g., poly(N-isopropylacrylamide) (PNIPAAm), have been introduced as an alternative for controlling the adhesion of cells on solid substrates.2-6 These polymers exhibit a phase transition from a dehydrated coil state to a highly hydrated state upon passing the lower critical solution temperature (LCST).7-9 When grafted onto solid substrates, the polymers facilitate cell adhesion in their coil-like * To whom correspondence should be addressed. Fax: +49-331-58187399. E-mail: [email protected]. † Fraunhofer Institute for Biomedical Engineering. ‡ Fraunhofer Institute for Applied Polymer Research. (1) Canavan, H. E.; Cheng, X.; Graham, D. J.; Ratner, B. D.; Castner, D. G. J. Biomed. Mater. Res., Part A 2005, 75(1), 1–13. (2) Bae, Y.; Okano, T.; Kim, S. J. Polym. Sci., Part B: Polym. Phys. 1990, 28(6), 923–936. (3) von Recum, H. A.; Kim, S. W.; Kikuchi, A.; Okuhara, M.; Sakurai, Y.; Okano, T. J. Biomed. Mater. Res. 1998, 40(4), 631–639. (4) Kwon, O. H.; Kikuchi, A.; Yamato, M.; Sakurai, Y.; Okano, T. J. Biomed. Mater. Res. 2000, 50(1), 82–89. (5) Yamato, M.; Konno, C.; Utsumi, M.; Kikuchi, A.; Okano, T. Biomaterials 2002, 23(2), 561–567. (6) Yang, J.; Yamato, M.; Shimizu, T.; Sekine, H.; Ohashi, K.; Kanzaki, M.; Ohki, T.; Nishida, K.; Okano, T. Biomaterials 2007, 28(34), 5033–5043. (7) Takezawa, T.; Mori, Y.; Yoshizato, K. Biotechnology 1990, 8(9), 854– 856. (8) Yamada, N.; Okano, T.; Sakai, H.; Karikusa, F.; Sawasaki, Y.; Sakurai, Y. Makromol. Chem., Rapid Commun. 1990, 11(11), 571–576. (9) Hirose, M.; Kwon, O. H.; Yamato, M.; Kikuchi, A.; Okano, T. Biomacromolecules 2000, 1(3), 377–381. (10) Schmaljohann, D. e-Polymers 2005, 21, 1–17. (11) Harris, J. M. Introduction to biotechnical and biomedical application of poly(ethylene glycol). In Poly(ethylene glycol) Chemistry; Harris, J. M., Ed.; Plenum Press: New York, 1992.

state at high temperatures and induce cell detachment below their LCST. The LCST of the polymer can be adjusted to be close to the optimal culture temperature of the cells by the introduction of PEG chains to the polymer.10,11 Thus far, most work has been dedicated to coatings produced by surface-initiated atom transfer radical polymerization (ATRP) or by electron beam irradiation.12,13 Although this method produces highly functional layers, the fine-tuning of their properties toward the adhesion behavior of specific cell lines is rather difficult. In particular, the variation of the molar composition and the precise positioning of molecular entities within the layer require demanding preparation protocols. To this end, selfassembled monolayers (SAMs) of sulfur bearing compounds on gold are robust systems that can be fabricated with an enormous degree of complexity and functionality.14,15 Recently, we have demonstrated that this approach is also well-suited for the formation of functional thermo-responsive layers that allow for the control of the cell adhesion in microfluidic channels.16 We employed a copolymer PNIPPAAm-PEG-SH (P15) that contains 15 wt % PEGMA of a molecular weight of 475 Da as side groups. Although the switching of the layer upon reducing the temperature from 37 to 25 °C induced cell rounding and detachment, this process required approximately 30 min, which is rather long and inconvenient for numerous cell protocols. In an effort to improve time and efficiency of cell detachment, here, we present two approaches for the tuning of layer properties and compare the performance of the films formed. First, we synthesized another thermo-responsive copolymer PNIPPAAmPEG-SH (P19) that contains 19 wt % PEGMA and formed SAM on gold. Second, we produced mixed films containing P19 and (12) Kim, D.; Kong, B.; Jung, Y.; Kim, K.; Kim, W.; Lee, K.; Kang, S.; Jeon, S.; Choi, I. Bull. Korean Soc. 2004, 25(11), 1629–1630. (13) Ebara, M.; Yamato, M.; Aoyagi, T.; Kikuchi, A.; Sakai, K.; Okano, T. Biomacromolecules 2004, 5(2), 505–510. (14) Cornell, B. A.; Braach-Maksvytis, V. L. B.; King, L. G.; Osman, P. D. J.; Raguse, B.; Wieczorek, L.; Pace, R. J. Nature 1997, 387(6633), 580. (15) Bieri, C.; Ernst, O. P.; Heyse, S.; Hofmann, K. P.; Vogel, H. Nat. Biotechnol. 1999, 17(11), 1105–1108. (16) Ernst, O.; Lieske, A.; Jager, M.; Lankenau, A.; Duschl, C. Lab Chip 2007, 7(10), 1322–1329.

10.1021/la801026y CCC: $40.75  2008 American Chemical Society Published on Web 08/21/2008

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various concentrations of a thiol bearing PEG. The introduction of PEG chains leads to stronger hydration of the film and thus is supposed to accelerate cell detachment upon decreasing the temperature below LCST. We tested this assumption using two cell types with different capacities to adhere to solid substrates. In this context, we performed a detailed functional characterization also including contact angle measurement. To correlate these results to the molar composition of the films, X-ray photoelectron spectroscopy (XPS) was employed.

Experimental Section Starting Materials. Double deionized water (0.054 µS) was obtained from a Millipore-Q-Synthesis system. DMEM/HEPES cell culture medium, fetal calve serum (FCS), penicillin/streptomycin, trypsin/EDTA, stabilized L-glutamine, and Ca2+- and Mg2+-free Hanks buffer were purchased from Biochrom, Germany. Ethanol (EtOH) for liquid chromatography, H2SO4 (95-98%), H2O2 (30%), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride were obtained from Merck, Germany. N-Hydroxysuccinimide, N-isopropylacrylamide, poly(ethylene-glycol)-monomethylethermonomethacrylate, thioglycolic acid, and cysteamine were from Sigma-Aldrich, Germany. 2,2′-Azo-bis(isobutyronitrile) was obtained from WAKO Chemicals, Germany. CH3O-PEG(750)-SH with a molecular mass of 750 Da was purchased from Rapp Polymers, Germany. Synthesis of PNIPAAm-PEG(19%)-SH (P19). The synthesis of PNIPAAm-PEG(15%)-SH (P15) was described previously.16 Synthesis of the PNIPAAm-PEG(19%)-COOH: N-isopropylacrylamide (NIPAAm) (15.1 g), poly-(ethylene glycol)-monomethylethermonomethacrylate (4.55 g) with a molecular weight of 475 g/mol (PEGMA 475), and thioglycolic acid (0.45 g) were dissolved in dioxane (165 g) and purged with nitrogen for 30 min. The mixture was then heated to 60 °C, and 2,2′-azo-bis(isobutyronitrile) (AIBN) (0.45 g) dissolved in dioxane (10 mL) was added rapidly. The polymerization proceeded overnight at 60 °C under nitrogen. Afterward, about 70% of the dioxane was evaporated on a rotary evaporator, and the residual solution was precipitated in diethyl ether. The precipitated polymer was collected, dried under vacuum, and purified by dialysis against deionized water (cutoff of dialysis membrane 1000D). The polymer solid was obtained by freeze-drying. The molecular weight was determined by titration of the COOH end groups and found to be about 11.000 g/mol. Reaction with cysteamine: polymer (1.0 g) synthesized as described above was dissolved in dioxane (10 g) and transferred to a roundbottom-flask equipped with a stirrer, reflux condenser, and nitrogen inlet. The mixture is purged with nitrogen for 15 min. Dicyclohexylcarbodiimide (DCC) (0.18 g) in dioxane (15 g) was added. After 5 min, cysteamine hydrochloride (0.4 g) in 5 mL of dimethylsulfoxide (DMSO) and 200 µL of pyridine were added, and the mixture was stirred overnight at room temperature under nitrogen. The reaction results in the precipitation of a solid (pyridine hydrochloride). Deionized water (40 g) was added, and the turbid solution was dialysed at 8 °C for 1 week against a diluted aqueous HCl solution (pH 3). The precipitated solid was filtered off, and the remaining solution was lyophilized to collect the polymer. The amount of terminal thiol groups was determined colorimetrically using Ellman’s reagent, 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), which reacts specifically with thiol groups and found to be 22.3 µM/g of polymer. The molar composition of the copolymer as determined by 13C nuclear magnetic resonance (NMR) was NIPAAm/PEGMA ) 93:7, which corresponds to a weight fraction of 19% PEG in the polymer (Figure 1). The LCST of the NIPAM-PEG copolymer was measured for the COOH-terminated polymer. The determination of the LCST was performed by measuring the particle sizes in an aqueous solution of the polymer by means of dynamic light scattering (Malvern HPPSET). Above the LCST, the polymer becomes insoluble in water and forms aggregates. Preparation of Copolymeric-Grafted Gold Surfaces. The 22 × 22 mm gold-coated slides were purchased from GeSiM, Germany.

Figure 1. Determination of the LCST of the PNIPAAm-PEG copolymer with different PEG content before the reaction with cysteamine. PNIPAAm-PEG(15%)-COOH (2) shows a LCST between 35 and 36 °C, and PNIPAAm-PEG(19%)-COOH (9) exhibits a LCST in the range of 36-37 °C. Error bars are not shown because they are smaller than the size of the symbols.

The cleaning of the gold surface included washing in a piranha solution (3:1 H2SO4/H2O2) for 30 min and a subsequent rinsing with double deionized water 10 times. The cleaned gold surface was grafted in a solution of 0.014 mM P15 or P19 and EtOH for 3 h at room temperature without shaking. Unbound polymer molecules were removed by repeated washing of the grafted gold surface in EtOH for 1 h. The co-adsorption of PNIPAAm-PEG(19%)-SH and CH3O-PEG(750)-SH on gold was performed with a molar mixture ratio of 1:6 (0.1 mM/0.56 mM) and 1:9 (0.066 mM/0.56 mM) in EtOH for 3 h at room temperature without shaking. Unbound molecules were removed by repeated washing of the grafted gold surface in EtOH for 1 h. The grafted surface was dried with nitrogen. Measurements of Dynamic Water Contact Angles. The dynamic contact angles of uncoated, cleaned gold and copolymer-coated gold were measured at different temperatures ranging from 20 to 45 °C with a contact angle measuring system G10 (Kru¨ss Surface Science, Germany) including a microscopy heating plate Linkam MS100 (Linkam Scientific Instruments, U.K.). Each sample was measured 5 times, and the results were averaged. The standard error of mean was calculated for each sample. XPS Analysis of the Copolymer Surface. For the XPS measurements, we used an Axis 165 instrument (Kratos Analytical, U.K.) with monochromatic Al KR radiation (anode: 15 kV, 20 mA) in hybrid mode, i.e., with electrostatic and magnetic lenses. Thermal electrons from a filament were used to compensate the charging of the sample. The survey spectra for the determination of the elemental composition were recorded with a pass energy of 80 eV, while for the high-resolution spectra, we used 20 eV. The data were processed using the Kratos Vision2 software. For the quantification of the elemental concentrations and the binding states, a linear background was subtracted. The high-resolution spectra were fitted with Gaussian functions to determine the concentrations of the atoms in the various binding states. The ratio between PNIPPAm and PEGMA was determined from the fits of the C1s and the O1s data. These two fitted spectra contain information on the theoretical molecular structure of PNIPPAm and PEGMA and allow for the determination of the ratio of these two species. Cell Culture and Microscopy. L929 mouse fibroblasts (ACC 2, DSMZ, Germany) and MG63 human osteosarcomas (CRL-1427, ATCC, kindly provided by Mark Rosowski, DRFZ, Germany) were cultivated in DMEM medium containing 25 mM HEPES, 10% FCS, 2 mM L-glutamine, and 1% penicillin/ streptomycin at 37 °C and 5% CO2. The 90% confluent cell layer was passaged every 2 days using trypsin/EDTA (0.05%/0.02%, w/v). The dissociated cells were resuspended in cell medium and seeded at 2 × 104 cells cm-2. The cell detachment was triggered through the exposure of the substrate with the cultured cells to a temperature of 25 °C for 30 min and mounted on a Leica DMIL microscope, including a high plan 10×/ 0.25 objective. The cell behavior was monitored using the microscope in phase contrast mode. For the documentation of the optical information, a Nikon Digital Sight DS-L1 (Nikon, Germany) was

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attached to the microscope setup. The number of adherent, rounded, and detached cells was counted manually.

Results The content of PEG moieties in PNIPPAm-based polymer coatings has a strong influence on its ability to promote cell adhesion or cell detachment. We pursued two approaches for the variation of the PEG content in SAMs to tailor their properties with respect to efficient detachment behavior of cells. We chose two cell types with contrasting adhesion behavior, fibroblasts showing average adherance to most technical substrates, and osteosarcoma cells that represent cell lines with strong adhesion to culture substrates. In the first approach, we synthesized two polymers with different PEG content of 15 or 19% of the total molecular weight: PNIPAAm-PEG(15%)-SH (“P15”) and PNIPAAm-PEG(19%)-SH (“P19”). It was expected that adhesion of a given cell type would be tighter to P15 than to P19. The second approach to tune the cell adhesion capacity of our surfaces was based on the formation of mixed layers of PNIPAAm-PEGSH (here, we used P19) and the antiadhesive polymer CH3OPEG-SH (“PG”). The resulting surfaces were found to be better suited for those cell types that would adhere to pure P19 so strongly that temperature switching would not easily release them from the surface. XPS Analysis of P19 and P19/PG. X-ray photoelectron spectroscopy is an extremely versatile method for the analysis of the chemical composition of organic coatings. From our instrument setting, we would assume a penetration depth of 7 nm from where information on our layers can be obtained. The elemental analysis yielded a value of approximately 30 atomic % of gold from the substrate. This percentage allows for an estimation of the thickness of the coating if we ignore the influence of its molecular density. On the basis of the assumption that the monolayers show a homogeneous coverage and neglecting surface roughness, we estimate the layer thickness to be 3-4 nm. The value corresponds well to thickness values derived from surface plasmon resonance measurements, which were taken in aqueous medium: Taking 1.4 and 1.45 as minimal and maximal limits of the range of probable refractive indices of the film, thicknesses of 4.7 and 2.1 nm are obtained, respectively. The carbon C1s spectrum comprises features that prominently represent four different types of carbon signals. Three of those can be attributed to the PNIPPAm moiety: carbon bonded to carbon and hydrogen only (binding energy BE ) 285.0 eV), carbon bonded to one nitrogen atom (binding energy BE ) 285.7 eV), and the carbon of the amide group (binding energy BE ) 287.8 eV). The signal at a BE ) 286.4 eV is assigned to the carbon with one single bond to oxygen because it is present in the PEG moiety. On the basis of these prominent signals, we were able to perform a quantitative analysis to derive relative concentrations of the two moieties of the copolymer. A total of 98% of the entire spectrum can be assigned to the PNIPPAm and PEGMA moieties and used for the determination of the composition of the layer. As a result, we obtain 88 mol% PNIPPAm and 12 mol% PEGMA (moles related to monomeric units). The remaining 2% of the spectrum result from the slightly broadened C-C peak at 285.0 eV and a rather unstructured tail on the high-energy side of this peak at approximately 289.5 eV (not visible in Figure 2). Both features are typical for nonspecific organic adsorbates. They do not affect the determination of the composition of the layer. The O1s spectrum was fitted in the same manner, and we obtained the best results when assuming relative concentration values for PNIPPAm and PEGMA that were identical to those

Figure 2. Carbon C1s spectrum of a P19 sample and a calculated spectrum for a molar PEGMA fraction of 12 mol %.

Figure 3. Advancing contact angles of water on Au surfaces modified with P15 (O), P19 (0), a mixture of P19/PG in a molar ratio of 1:6 (×), and plain, purified Au substrate (4) as a function of the temperature. The data are expressed as the mean of five measurements on one substrate. Error bars are not shown because the standard error of the mean never exceeded the symbol heights.

found from the C1s spectrum (88 mol% PNIPPAm and 12 mol% PEGMA, respectively). This composition deviates slightly form the one chosen for the synthesis (93 mol% PNIPPAm and 8 mol% PEGMA, see the Experimental Section). However, such a small difference may be due to an uneven distribution of PEG in the layer. For example, PEG could be more exposed in the film under the conditions present in the vacuum chamber. From the mixed layer sample containing P19 and PG, we obtained a spectrum that gave relative molar concentrations values of 38 and 63 mol %, respectively. This mass ratio of the two components of 6.8 is fully compatible with the molar composition of the self-assembly solution of 6.0. Contact Angle Measurements. Because the variation of the PEG content in the layers was expected to show up in the wettability of the polymer coating, contact angle measurements were performed. Such measurements also allow for the monitoring of changes of the architecture of the thermo-responsive films that are induced through temperature variations. The results of these measurements are summarized in Figure 3. Deposition of P19 versus P15 resulted in significantly lower contact angles, which is consistent with the higher content of hydrophilic PEG within the P19 molecule. The temperature dependence course is parallel for the two surfaces, indicating a similar switching behavior. Figure 3 shows the contact angles for grafted and plain Au surfaces as a function of the temperature. These contact angles increase for the P15-grafted Au surface (O) from 50° at 10 °C to 65° at 45 °C. For the P19-grafted Au surface (0), the contact angles rise from 45° at 10 °C to 61° at 45 °C. Expectedly, uncoated

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Figure 4. (A) Rounding of adherent cells upon temperature shift from 37 to 25 °C after 3 days of culture. In comparison are the two polymers P15 and P19 with different ratios of PEG. Phase contrast pictures were taken at 0, 14, and 28 min after temperature drop (only representative sections are shown). Scale bar: 100 µM. (B) Rounded cells on P15 (white bar) and P19 (gray bar) were counted per recorded field of vision and plotted as a percentage of total cell number at the time points indicated. Data are corrected for background (numbers of round cells at 37 °C) and expressed as the mean of independent measurements on three different samples with a standard error of the mean.

Au surfaces (4) show no significant change of wettablilty for any temperature tested. Also in Figure 3, the effect of co-adsorption of PG with P19 on hydrophilicity becomes evident. In comparison to adsorption of pure P19, the 1:6 P19/PG molar mix creates a more hydrophilic surface, with contact angles of 37° at 10 °C and 46° at 45 °C (×). The contact angle changes upon temperature, switching for all grafted surfaces between 8° and 15°. The polymer molecules in the layer show a transition behavior that is different with respect to what has been determined in solution. The transition is considerably broadened toward lower temperatures. Cooperative effects and the presence of the gold surface may play a role for such an alteration of the switching behavior. In the mixed P19/PG film, the LCST of P19 can be estimated from the contact angle data to be in the range of 30-36 °C. The response of all three layer systems seems well compatible with their use in cell culture. Accelerated Detachment of L929 Fibroblasts from a PNIPAAm-PEG Surface with Increased Molecular PEG Content. The intramolecular PEG contents of P15 and P19 were found to have a clear impact on the cell release time upon temperature switching. In Figure 4A, L929 cells (3 days after seeding) are adherent on surfaces treated with P15 or P19 at the initial culture temperature of 37 °C. Morphologically, cells look marginally less spread on P19. However, the difference is negligible, and the fraction of rounded cells is low in both cases. After 14 min at a temperature of 25 °C, the difference between the two surfaces becomes evident, because on P19, the switching process is already near completion (74% rounded), whereas on P15, the majority of cells still look almost unchanged (23% rounded). Later on, at 28 min after the temperature decrease, this difference disappears. Cells on P15 are now also rounded (73%) and comparable with those on P19 after 14 min. Figure 4B quantifies these results as a percentage of rounded cells within the whole population (after background correction). It illustrates that the switching efficiencies of P15 and P19 are about

comparable if cells are given a sufficiently long time to react. However, there is a major difference in the switching kinetics between the two polymer surfaces. Controllable Release of MG-63 Human Osteosarcoma Cells by Co-adsorption of Cell-Repellent and Switchable Polymers. Cell adhesion greatly varies between different cell types. In an effort to explore the potential of our approach to tune cell-typespecific adhesion, we employed MG-63 osteosarcomas, which are a good example for cell lines displaying a strong adhesion to culture substrates.17 Upon reducing the temperature, they were not released from a switchable PNIPAAm-PEG surface formed by P15 (data not shown) or P19 (Figure 5). Instead, they remained adherent and could not be removed by physical treatment, such as gentle rinsing. To address this problem, we co-adsorbed the commonly known anti-cell-adhesive polymer CH3O-PEG-SH (“PG”) together with P19 in different molar ratios to find a surface composition where MG-63s would both adhere to the surface and release upon switching. In Figure 5, the results of this optimization are summerized. On a pure P19-coated surface, MG-63 cells adhered well but can not be removed from the surface by temperature switching (Figure 5A). Pure PG surfaces (data not shown) and surfaces comprising a high PG content from a co-adsorption mixing ratio of 1:9 P19/PG (Figure 5C) prevented initial cell adhesion. Only the surface derived from a P19/PG deposition ratio of 1:6 turned out to mediate both a satisfying initial cell adhesion and sufficient cell rounding (74%) upon switching (Figure 5B).

Discussion The gentle detachment of cells from their culture substrate is a important requirement in cellular biotechnology. The great variety of adherence properties of different cell types requires strategies for the straightforward optimization and modification (17) Kieswetter, K.; Schwartz, Z.; Hummert, T. W.; Cochran, D. L.; Simpson, J.; Dean, D. D.; Boyan, B. D. J. Biomed. Mater. Res. 1996, 32(1), 55–63.

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Figure 5. Phase contrast images of MG-63 on surfaces produced by co-adsorption of P19 and PG in different mixing ratios. Cells were allowed to settle on the polymer coatings for 36 h before the temperature was decreased for 30 min. (A) On pure P19, cells adhere well and no visible switching effect can be induced. (B) On a surface produced from a P19/PG mixture with a molar ratio of 1:6, cells show satisfactory initial adhesion and spreading. Upon switching of P19, a majority of the population becomes spherical and/or detaches. (C) The surface produced using a P19/PG mixture with a 1:9 molar ratio is already, to such a degree, cell-repellent that it does not allow MG-63 cells to adhere initially. Scale bar: 100 µM.

of layer architectures to tailor their functionality toward an efficient, gentle, and specific cell detachment. Recently, we demonstrated that with a newly synthesized thermo-responsive polymer (P15) containing 15 wt % PEG side chains and a thiol anchor group, layers can be produced that allow for the control of the adhesion of cells in the relevant temperature range.16 In this work, we extended this approach by presenting two ways of introducing additional PEG chains to address various limitations of the previous work, namely, the long response times after decreasing the temperature below LCST and the suitability of these coatings only for moderately adhering cells. In this respect, we take advantage of the ease and the flexibility of gold sulfur chemistry with which layers can be produced with different molar compositions and structural details. The introduction of additional PEG side chains into a thermoresponsive layer based on PNIPAAm is supposed to have two effects. First, it is known from numerous studies that PEG groups suppress the adsorption of proteins and partly related to it generally weaken the adhesion of cells.11 This relation is believed to result from the fact that cells contacting the surface produce a number of proteins that promote cell adhesion. Second, PEG affects the intramolecular interaction between the NIPAAm groups, and hence, its relative amount can be used to control the transition temperature of the polymer. However, increasing the content of PEG introduced to the layer as side chains of the thermoresponsive polymer is limited because it shifts its LCST to higher temperatures that may not be acceptable for cell culture. As a result of these considerations, we have chosen to synthesize a polymer with 19 wt % PEG (P19). The measurement of the LCST of P19 using dynamic light scattering gives a value of 36 °C, just below the optimal cell culture temperature. This is also in accordance with work of others who have obtained comparable results.10 The contact angle measurements of the P19 copolymer layer show an increased hydrophilicity over the entire temperature range measured, compared to the one produced from P15. The contact angle difference between the layers formed from P15

and P19 is approximately constant over the investigated temperature range, indicating a similar structure and switching behavior of the two layers. In particular, there are no indications that the relative exposure of PEG chains below and above LCST is in any way different in the two layers. Interestingly, the contact angle measurements on the mixed P19/PG layer (mixing ratio in solution of 1:6) show an approximately parallel temperature course with respect to the pure layers, albeit at angles that are 10° lower. This suggests that the behavior of the thermoresponsive copolymer is very similar in the pure and mixed coatings upon the reduction of the temperature from 45 to 20 °C. This supports our argument, that the production of layers through co-adsorption of different molecules in a wide range of mixing ratios is a powerful tool for obtaining systems with complex functionalities in a predictive manner. Approaches that use a “grafting from” strategy are certainly more difficult to adapt to the requirements of a process as complex as cell adhesion control. Finally, it is worth noting that the contact angle of the hydrated layer at 20 °C shows a contact angle even below the one measured on a pure CH3O-PEG(750)-S-Au surface. This indicates that the PEG chains in the mixed films are similarly organized as in the pure PEG film and a kind of saturation of hydrophilicity through the presence of PEG chains has been achieved. The functionality of the coatings was tested with two different cell types. The introduction of additional PEG chains onto to the PNIPAAm backbone leads to a considerable reduction of the response time by a factor of 2 on P19 over P15 upon a decrease of the culture temperature from 37 to 25 °C using the moderately adhering L929 fibroblasts. The cause of this reduction remains unclear to us, because we believe that the response time is determined by cellular processes and not by the kinetics of the polymer layer. Because we do find only a marginal difference of the time required until the cells spread on the P19 and P15 coating after cell seeding at 37 °C, we are tempted to speculate that the increased content of PEG in P19 does not substantially contribute to the interaction between cells and the layer at 37 °C when the copolymer is in the collapsed state.

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To test whether layers of the copolymer P19 are also suited to induce detachment of cells with strong adhesion properties, we employed MG-63 human osteosarcoma cells as a wellcharacterized representative of this class of cells.17 These cells adhere, spread, and proliferate well on the P19 copolymer coating at 37 °C. However, upon a temperature decrease below LCST, the cells do not detach from this surface. We did not consider the option of synthesizing another copolymer with an even higher content of PEG chains compared to P19 for two reasons. First, the synthesis and the subsequent analysis of a new copolymer is tedious and labor-intensive. Second, we were not sure whether the extra content of PEG would shift the LCST to unacceptable high temperatures. Rather, we tested the possibility to introduce additional PEG chains into the layer through the co-adsorption of PEG with P19. A molar composition of P19 and PG of 1:6 in solution turned out to produce optimal results in the sense that cell spreading was efficient at 37 °C and cell rounding was induced in the biggest part of the adherent cells. The XPS measurements indicate that this ratio is also present in the coating. It is surprising that such a high dilution of the thermo-responsive polymer in the layer does still produce films that undergo a transition from a cell-adherent to a cell-repellent state. When the cell adhesion experiments and the contact angle measurements on the mixed layers are taken together, then it seems that the addition of PG just shifts the overall wettability of the layer toward greater hydrophilicty and leaves the switching mechanism unaffected.

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Conclusion Here, we present two approaches for the fine-tuning of layer properties using sulfur chemistry with thermo-responsive copolymers grafted onto gold surfaces. The two synthesized copolymers consist of a PNIPAAm backbone and a terminal thiol group and vary with respect to the content of PEG side chain. As one approach, the significant acceleration of the cell detachment is enabled by increasing the intramolecular PEG content up to 19 wt % (P19). The polymer exhibits an excellent switching behavior between a cell-adhesive (at 37 °C) and a cell-repulsive state (at 25 °C). Second, the fine-tuning of the layer properties by the co-adsorption of P19 and thiol-terminated PEG enables the controlled adhesion and detachment of cells with strong adhesion, e.g., human osteosarcoma cells. These two approaches exhibit the potential of fine-tuning switchable surface properties for the control of different cell adhesion types. This technology can be used for gentle handling of different cell types in critical applications without harmful enzymatic treatment of cellular material. Acknowledgment. The authors thank Beate Morgenstern for assistance with cell culture work and Mark Rosowski, DRFZ, Germany, for providing MG63 cells. This work is financially supported through the MAVO program of the Fraunhofer Society and the network of the excellence initiative of the Fraunhofer Society and the Max-Planck Society. LA801026Y