Article pubs.acs.org/Biomac
Temperature-Controlled Masking/Unmasking of Cell-Adhesive Cues with Poly(ethylene glycol) Methacrylate Based Brushes Solenne Desseaux and Harm-Anton Klok* Institut des Matériaux et Institut des Sciences et Ingénierie Chimiques, Laboratoire des Polymères, École Polytechnique Fédérale de Lausanne (EPFL), Bâtiment MXD, Station 12, CH-1015 Lausanne, Switzerland S Supporting Information *
ABSTRACT: Thin, thermoresponsive polymer coatings that allow to reversibly modulate cell adhesion and detachment are attractive substrates for cell sheet engineering. Usually, this is accomplished by applying thin poly(N-isopropylacrylamide) (PNIPAM) coatings, which allow cell adhesion via nonspecific interactions above the collapse temperature (TT) of the surface-attached polymer and cell detachment upon cooling below TT. This Article presents an alternative, thermoresponsive polymer platform that is based on 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA) containing copolymer brushes prepared via surface-initiated atom transfer radical polymerization (SI-ATRP). These brushes are interesting as they gradually collapse and dehydrate upon increasing the temperature from 10 to 40 °C, yet resist nonspecific adhesion of cells over this entire temperature window. The MEO2MA based brushes presented here were modified via a twostep postpolymerization modification protocol to introduce cell-adhesive RGD containing peptide ligands. The possibility to reversibly control the swelling and collapse of these brush films by varying temperature allows to modulate the effectively available surface concentration of these cell-adhesive cues and thus provides a way to mask/unmask their biological activity. As a first proof of concept, this Article demonstrates that these MEO2MA brush copolymer films enable integrin-mediated adhesion of 3T3 fibroblasts at 37 °C and allow release of these cells by cooling to 23 °C. The use of cell-adhesive ligands, which can be thermoreversibly masked/ unmasked, is attractive as it enables the use of serum-free cell culture conditions. This is advantageous since it avoids possible concerns regarding eventual toxicity and immunological side effects of serum proteins and also provides opportunities to select for particular cell types and for enhanced control over cell stimulation and differentiation.
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INTRODUCTION Thermoresponsive polymer coatings that can transition from a swollen, hydrophilic state at one temperature into a more collapsed and hydrophobic state at another temperature are very attractive substrates to produce cell sheets that can be used to repair or regenerate tissue. The basic principle behind this strategy, which is commonly referred to as cell sheet engineering, is that cells attach to and proliferate on these surfaces above the transition temperature (TT), whereas swelling of the coating upon decreasing the temperature below TT leads to detachment of the resulting cell sheet. One of the great advantages of the cell sheet detachment strategy is that it obviates the need for proteolytic enzymes such as trypsin and dispase, which are used to harvest cell sheets generated on conventional surfaces. This is attractive since the use of these enzymes can lead to cell damage.1 Most of the thermoresponsive polymer coatings that have been used for cell sheet engineering are based on poly(Nisopropylacrylamide) (PNIPAM). These coatings are most frequently produced using electron-beam irradiation, but have also been prepared via plasma polymerization2 and surfaceinitiated controlled radical polymerization (SI-CRP) techniques.3 The successful generation and harvesting of cell sheets using PNIPAM based substrates requires precise control of film thickness.4 PNIPAM films generated via e-beam irradiation © 2014 American Chemical Society
with thicknesses of 30 nm or more, for example, did not support cell attachment above the TT.5 For PNIPAM coatings prepared via SI-ATRP, the optimal film thickness was determined to be 20−45 nm. Thicker PNIPAM layers did not support cell attachment, whereas PNIPAM coatings of 20 nm or less did not allow effective detachment below the TT.6 The need for thin polymer coatings (98%) were obtained from GLBiochem (Shanghai, China). The ATRP initiator (6-(2bromo-2-methyl)propionyloxy) hexyldimethylchlorosilane was synthesized as previously reported.19 The inhibitor was removed from HEMA, MEO2MA, and PEGMA6 by passing the monomer through a column of basic alumina. Water was obtained from a Millipore Milli-Q gradient machine equipped with a 0.22 μm filter. Silicon (100) substrates (8 mm × 10 mm) covered with a native silicon oxide layer, SiO2 coated quartz crystals purchased from Q-Sense, and glass coverslips (Menzel-Gläzer, 18 mm × 18 mm) were used as substrates for surface-initiated polymerization. Methods. Atomic force microscopy was performed on a Multimode instrument (Bruker Nano Surface, Santa Barbara, CA) in tapping mode. To determine layer thicknesses, cross-sectional height profiles of micropatterned polymer brushes on silicon substrates were analyzed. X-ray photoelectron spectroscopy (XPS) was carried out using an Axis Ultra instrument from Kratos Analytical equipped with a conventional hemispheric analyzer. The X-ray source employed was a monochromatic Al Kα (1486.6 eV) source operated at 100 W and 10−9 mbar. Fluorescence images were taken with a Zeiss Axioplan microscope. Quartz crystal microbalance with dissipation monitoring (QCM-D) experiments were conducted on a Q-Sense E4 system. The quartz sensor was mounted in a flow module with one side exposed to the solvent. The cells were equilibrated at 10 °C before starting the measurement. The frequency and dissipation shifts were recorded between 10 and 40 °C after at least 20 min equilibration time between each temperature change. The temperature was increased gradually between each step (heating rate 1 °C/min). A clean quartz sensor was used as reference. The third harmonic of the resonance frequency was recorded and data were obtained after subtracting the background. Procedures. ATRP-Initiator Modified Substrates. First, the substrates were sonicated successively in water, acetone, and ethanol. After drying, the slides were exposed to an oxygen plasma (180 W) for 10 min. Subsequently, the slides were immersed in a 0.1% v/v solution of (6-(2-bromo-2-methyl)propionyloxy)hexyldimethylchlorosilane in dry toluene for 16 h at room temperature. After that, the slides were removed from the reaction mixture and rinsed extensively with toluene and dichloromethane. Finally, the initiator-functionalized substrates were dried under a flow of nitrogen and transferred to the appropriate reaction vessel for the ATRP experiment. Surface-Initiated ATRP of MEO2MA/HEMA/PEGMA6. Surfaceinitiated ATRP of MEO2MA/HEMA/PEGMA6 was performed using a reaction system composed of CuCl/CuCl2/bipy/MEO2MA/ HEMA/PEGMA6 at the following molar ratios adapted from a published protocol:20 2/0.2/5/50/30/20. First, the initiator-modified 3860
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Scheme 1. Schematic Outline of the Synthesis of RGD-Functionalized Poly(MEO2MA-co-HEMA-co-PEGMA) Copolymer Brushes
substrates were introduced in a reaction vessel. A separate vessel was charged with the monomers (total concentration 2.1 M), water, and methanol (water/methanol = 2/1 v/v) as well as bipy and CuCl2 and subsequently degassed by bubbling nitrogen gas for 30 min. After quick addition of CuCl under N2 flow, bubbling was continued for another 30 min and the resulting solution was transferred via a cannula to the nitrogen purged reaction vessel containing the initiator-modified slides. The polymerization was allowed to proceed for a defined period of time at room temperature. Afterward, the reaction mixture was removed, and the substrates rinsed with ethanol, water and finally dried in a stream of nitrogen. Peptide Functionalization. First, the hydroxyl side chain functional groups of the copolymer brushes were activated by reacting with 4nitrophenylchloroformate (NPC) following a previously published protocol.21 The NPC-activated PHEMA brushes were subsequently functionalized by treatment with a dry DMF solution containing the peptide as well as 2.5 mM 4-(dimethylamino)pyridine (DMAP) for 18 h at room temperature under gentle shaking in the dark. After that, a sufficient amount of ethanolamine to obtain a 0.5 M solution was injected in the reactor and allowed to react for 15 min in order to remove any residual unreacted NPC groups. Next, the substrates were removed from the reactor and extensively rinsed with DMF and water and finally dried with a stream of nitrogen. Cell Experiments. 3T3 fibroblasts were cultured in polystyrene flasks in Dulbecco’s modified Eagle’s medium (DMEM) (Lifetech) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (Lifetech). Cells were removed from the flasks by trypsinization with trypsin/EDTA (Lifetech) and centrifuged before resuspension in DMEM without serum. Cell Seeding. Polymer brush coated coverslips were placed in 6well-plates, incubated for 15 min in a solution with 70% ethanol and 30% water, and rinsed two times for 5 min with PBS and once for 20 min at 37 °C to equilibrate the brushes at this temperature. Then, 3T3 fibroblasts were seeded on the coverslips at a density of 20 000 cells/ cm2 in DMEM in the absence of serum for 30 min in an incubator at 37 °C in 5% CO2 atmosphere. Subsequently, the medium was replaced by supplemented serum DMEM. The slides were then placed back in the incubator for the time of the experiment. Quantification of Cell Adhesion. After 24 h, cells were rinsed three times with PBS, fixed for 15 min with 4% paraformaldehyde in PBS, rinsed two times with PBS, permealized with 0.1% Triton X-100 in PBS for 2 min, rinsed two times with PBS, incubated for 5 min in a solution of 4′,6′-diamidino-2-phenylindole (DAPI) (1 μg/mL PBS, 1
mL per well) to stain the nuclei, rinsed three times for 5 min in PBS containing 0.05% Tween 20, and mounted on a microscopy slide with an antifade mounting solution (ProLong Gold). From each slide, 10 fluorescence images were taken using a 10× objective and a blue filter. The images were then analyzed with ImageJ to determine the cell density. Each experiment was run in triplicate. Statistical analysis was done with the one-way analysis of variance (ANOVA) test followed by a Tukey’s posthoc test. Focal Adhesion Staining. At 24 h after seeding, cells were stained with Focal Adhesion Staining Kit (FAK100, Micropore), which allows one to visualize the actin cytoskeleton (red dye) and vinculin (green dye). Staining was performed as described in the manual of the kit. The slides were mounted on microscope slides with ProLong Gold. The slides were then analyzed with a fluorescence microscope equipped with blue, red, and green filters. Cell Detachment. After 48 h, the medium was replaced with serumfree DMEM equilibrated at room temperature. The cells were kept at room temperature (23 °C) in an incubator in a 5% CO2 atmosphere, and pictures were taken with an inverted microscope (Olympus CKX41 equipped with a digital camera) at different times.
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RESULTS AND DISCUSSION Polymer Brush Synthesis and Characterization. The synthesis of the thermoresponsive polymer brush substrates investigated in this study is outlined in Scheme 1. The brushes were prepared by surface-initiated atom transfer radical polymerization (SI-ATRP) of HEMA, PEGMA 6 , and MOE2MA from substrates modified with (6-(2-bromo-2methyl)propionyloxy)hexyldimethylchlorosilane. All polymerizations were performed using MOE2MA, HEMA, and PEGMA6 at a 50/30/20 molar ratio. This particular feed composition was selected as it has been shown to result in brush films that possess a collapse temperature of ∼35 °C (which is close to the 37 °C at which cells are usually grown) and which preserve their nonbiofouling properties up to 38 °C (i.e., above the collapse temperature).17 Furthermore, the HEMA and PEGMA6 comonomers contain hydroxyl side chain functional groups that can be used for further postpolymerization modification reactions, for example, to introduce celladhesive peptide ligands. The SI-ATRP protocol outlined in Scheme 1 allows the controlled synthesis of MEO2MA based 3861
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the peptide-modified brush, the intensity of which increases with increasing concentration of the peptide in the solution that was used for the postpolymerization modification reaction. The surface concentrations of the peptides were estimated by comparison of the N and O atomic percentages obtained from XPS and are expressed as percentage of modified side chain hydroxyl groups in Table 1. The thermoresponsive properties of the MEO2MA copolymer brush films, both before as well as after peptide modification, were investigated by QCM-D. For these experiments, copolymer brushes were grown via SI-ATRP from silicon oxide coated quartz crystals using the same process that was used to modify silicon wafer substrates. Figure 2 plots the temperature dependence of the third overtone of the frequency shift (Δf) and the dissipation shift (ΔD) recorded between 10 and 40 °C for MEO2MA copolymer brushes without and with the RGD peptide (samples A and F in Table 1) in 3 different media. The results on the unmodified brushes in Milli-Q water are qualitatively in agreement with earlier studies on MEO2MA brushes as well as PNIPAM brushes (Figure 2A).22,23 The increase in Δf and decrease in ΔD with increasing temperature reflect the temperature-induced dehydration and associated collapse of the polymer brush film with increasing temperature. Figure S3 in the Supporting Information presents the results of temperature-dependent cross-sectional height analysis experiments that were performed on a micropatterned nonmodified MEO2MA copolymer brush. Figure S3 shows a 30−35 nm decrease in film thickness, which illustrates the temperatureinduced collapse of the polymer film, upon increasing the temperature from 10−40 °C. In salt containing media, the unmodified MEO2MA containing copolymer brushes reveal similar temperature dependencies of Δf and ΔD, albeit with smaller absolute variations in Δf and ΔD. The latter is probably related to a small decrease in the collapse temperature of the polymer brushes in these salt containing media, as it has also been reported for solution studies on these copolymers.24 Due to the fact that the collapse of the surface-tethered polymer chains does not take at a sharp transition temperature as it is the case in homogeneous solution, the slight decrease in TT effectively results in a lower degree of hydration at any given temperature. This decreased hydration of the MEO2MA containing copolymer brush in PBS and DMEM is reflected in the smaller absolute changes in Δf and ΔD upon increasing the temperature from 10 to 40 °C. Figure 2B shows the temperature dependence of Δf and ΔD for the RGD peptide modified MEO2MA containing copolymer brush F in Milli-Q water as well as PBS and DMEM. In all three media, a gradual increase in Δf with increasing temperature is observed, which indicates that these peptide functionalized brushes also dehydrate and slowly transition into a more collapsed state with increasing temperature. In contrast, the peptide functionalized copolymer brush does not show any significant change in ΔD with increasing temperature, except in DMEM at temperature above 30 °C. The results on the peptide functionalized brush may be interpreted in terms of an increase in the collapse temperature upon introduction of the RGD peptide, which is negatively charged in the investigated media (pI ∼ 5.5). Similar observations have been made in solution studies on random copolymers of NIPAM and propyl acrylic acid, which also revealed an increase in LCST at pH values above the pKa of the propyl acrylic acid comonomer.25 Incorporation of, for example, only 3 mol % propyl acrylic acid resulted in an increase of ∼10 °C in the LCST as
copolymer brushes with thicknesses of up to 120−150 nm (Figure 1).
Figure 1. Evolution of film thickness as a function of reaction time for the SI-ATRP of a mixture of comonomers MEO2MA/HEMA/ PEGMA (50/30/20).
A series of MEO2MA based copolymer brushes that present the cell-adhesive RGD peptide at a range of surface concentrations was obtained in a two-step process that involves activation of the brush hydroxyl side chain functional groups with 4-nitrophenyl chloroformate (NPC) followed by reaction with a DMF solution containing the GGGRGDS or GGGRDGS peptide (the latter was used as a control).21 These experiments were performed starting from MEO2MA based copolymer brushes that were obtained after a polymerization time of 5 h (d = 105 nm). By variation of the peptide concentration in the DMF solution that was used for the postpolymerization modification reaction, a series of polymer brush coated substrates presenting a range of RGD peptide surface concentrations was prepared (Table 1). To validate the Table 1. XPS Surface Chemical Characterization of the Polymer Brush Samples Investigated in This Studya sample
peptide
peptide concentration in reaction solution (mM)
A B C D E F G
GGGRGDS GGGRGDS GGGRGDS GGGRGDS GGGRGDS GGGRDGS
0 0.01 0.02 0.05 0.1 1 1
N/O ratio (−)
hydroxyl group modification (%)
0 0 0.03 ND 0.10 0.26 ND
0 0 6 ND 21 54 ND
a
Samples B−G were obtained by postpolymerization modification of an unmodified brush (A) with a thickness of 105 nm (ND = not determined).
incorporation of the peptide and estimate the percentage of hydroxyl side chain functional groups that have been modified with these biochemical cues, the brushes were analyzed by XPS. XPS survey spectra as well as C1s and N1s high resolution scans of samples A−F are included in the Supporting Information (Figure S1). The successful incorporation of the peptide is evident from the presence of an N1s signal in the spectrum of 3862
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Figure 2. Temperature dependence of the third overtone of the frequency shift Δf (■) and the dissipation shift ΔD (□) for (A) the unmodified MEO2MA containing copolymer brush A and (B) RGD peptide modified MEO2MA containing copolymer brush F in three different media.
Figure 3. (A) Comparison of 3T3 fibroblast adhesion at 37 °C 24 h postseeding on different polymer brush substrates (initial cell seeding density 20 000 cells/cm2). Asterisks indicate statistically significant differences between the indicated samples (*p < 0.05; **p < 0.01). (B) Fluorescent micrograph of 3T3 fibroblasts on brush substrate F at 37 °C 24 h postseeding (scale bar = 20 μm) (red, actin; green, vinculin; blue, nucleus).
compared with the NIPAM homopolymer in pH 6.5 solutions of these polymers, which illustrates the dramatic effect of incorporating a small fraction of comonomer units. The collapse temperature of the unmodified MEO2MA copolymer brush has been estimated to ∼35 °C.17 An increase in TT due to incorporation of the RGD peptide would mean that for most of the investigated temperature range (10−40 °C) these brush films are significantly below their TT and thus in a relatively well hydrated state, which is reflected in the relatively small increase in Δf (and thus only minor dehydration) with increasing temperature. The relative insensitivity of ΔD of the peptide modified brush toward changes in temperature is attributed to the negatively charged nature of the peptide at the pH of the different media. The resulting repulsive intra- and interchain interactions lead to chain stretching and a decrease in the viscous character of the films (i.e., the brush becomes stiffer). Cell Adhesion and Detachment. Figure 3 compares the attachment of 3T3 fibroblasts on the different polymer brush substrates at 37 °C 24 h postseeding. The results in Figure 3 highlight the influence of peptide surface concentration. The highest cell densities were observed on sample F, which presents the highest peptide surface concentration (see Table
1). Decreasing the peptide surface concentration also results in a decrease in the number of attached cells; see, for example, sample D. The cell densities on substrates D and F were higher than those on the unmodified (sample A) and the RDG functionalized negative control substrate (sample G), which demonstrates that cell attachment is due to integrin binding of the RGD peptides and not mediated by nonspecific adsorption. Cell attachment on the lowest RGD surface concentration brushes B and C was very limited and not significantly different from the control surfaces (samples A and G). Cell detachment was investigated 48 h postseeding at 23 °C. Decreasing the temperature from 37 to 23 °C results in swelling of the polymer brush film, thereby masking the celladhesive RGD ligands and reducing their availability for cell surface integrin binding. This results in cell detachment, which is observed as a rounding up of the 3T3 fibroblasts that are well spread at 37 °C. Figure 4 plots for polymer brush substrates C, D, and F the change in the percentage of spread cells as a function of incubation time at 23 °C. Figures 5 and 6 show optical micrographs of 3T3 fibroblasts on these three substrates, both at 37 °C, as well as at different incubation times at 23 °C. Cell detachment from substrates C and D is 3863
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substrate proceeds at a much slower rate as compared to substrates C and D that were discussed above. The lower magnification images in Figure 6 show that at the edge of the sample cells detach in the form of continuous sheets, which is illustrated by the dark band that is visible, the width of which increases with increasing incubation time at 23 °C. In a final experiment, to verify the viability of the detached cells, a polymer brush substrate F that had been kept at 23 °C for 4 h to induce cell detachment was placed into serum containing media at a temperature of 37 °C. After 48 h, a confluent cell layer had formed on the substrate, indicating that the incubation at 23 °C does not affect cell viability (see Figure S4 in the Supporting Information).
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CONCLUSIONS
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ASSOCIATED CONTENT
Thermoresponsive, thin polymer films that allow reversible cell adhesion and detachment are attractive substrates for cell sheet engineering. This Article has demonstrated the feasibility of RGD-peptide functionalized 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA) based copolymer brushes prepared by surface-initiated atom transfer radical polymerization as an promising, alternative thermoresponsive substrate to modulate cell adhesion and detachment. These MEO2MA based copolymer brush films gradually dehydrate and adopt a more collapsed chain conformation upon increasing the temperature from 10 to 40 °C and swell and become hydrated again upon cooling. The ability of these polymer brush films to reversibly transition from a swollen, stretched state to a more collapsed state over a temperature range that is relevant to cell culture conditions allows to reversibly modulate (mask and unmask) the effective surface concentration of cell-adhesive peptide ligands that have been incorporated in these polymer films. Since the MEO2MA based polymer brushes used here resist nonspecific protein and cell adhesion over the investigated range of temperatures, these films enable specific (i.e., integrin mediated) temperature-modulated adhesion and release of cells. This is attractive as it allows the use of serum free media, which provides additional opportunities to select certain cell types and facilitate control over the stimulation and differentiation of cells.
Figure 4. Percentage of spread cells as a function of time on polymer brush substrates C (■), D (▲), and F (●) at 23 °C 48 h postseeding (the data points for substrates C and D are overlapping).
Figure 5. Optical micrographs of 3T3 fibroblasts on polymer brush substrates C and D at 37 °C and after 15 min at 23 °C (scale bar = 1 mm).
S Supporting Information *
XPS characterization of the peptide-functionalized polymer brush films; Temperature-dependent AFM height analysis as well as optical micrographs of 3T3 fibroblasts on polymer brush substrate F. This material is available free of charge via the Internet at http://pubs.acs.org.
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Figure 6. Optical micrographs of 3T3 fibroblasts on polymer brush substrate F at 37 °C (t = 0 min), 48 h postseeding, and at different times at 23 °C (scale bar = 1 mm, except for micrographs taken after 180 and 240 min where the scale bar = 2 mm).
AUTHOR INFORMATION
Corresponding Author
*E-mail: harm-anton.klok@epfl.ch. Fax: + 41 21 693 5650. Tel: + 41 21 693 4866.
relatively rapid with 99% of the cells being detached after 30 min. Due to the relatively low peptide ligand surface concentration on these substrates, cells do not grow to confluency, not even after 48 h. On the highest RGD surface concentration presenting brush substrate F, in contrast, 3T3 fibroblasts formed a confluent layer at 37 °C 48 h postseeding. Figure 6 presents optical micrographs of 3T3 fibroblasts on this substrate that were taken after different periods of time at 23 °C to monitor cell detachment. Cell detachment from this
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported within the European Union Seventh Framework Programme (FP7/2007-2013) under Grant Agreement No. NMP4-LA-2009-229289 NanoII. 3864
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