Tuning the Thickness of Polymer Brushes Grafted from Nonlinearly

Apr 9, 2009 - Tuning the Thickness of Polymer Brushes Grafted from Nonlinearly Growing Multilayer Assemblies. Erik Wischerhoff†, Stefan Glatzel†, ...
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Tuning the Thickness of Polymer Brushes Grafted from Nonlinearly Growing Multilayer Assemblies Erik Wischerhoff,† Stefan Glatzel,† Katja Uhlig,‡ Andreas Lankenau,‡ Jean-Franc-ois Lutz,† and Andre Laschewsky*,†,§ †

Fraunhofer Institute for Applied Polymer Research, Geiselbergstrasse 69, ‡Fraunhofer Institute for Biomedical :: Engineering, Am Muhlenberg 13, and §University of Potsdam, Institute of Chemistry, Karl-Liebknecht-Str. 24-25, D-14476 Potsdam-Golm, Germany Received December 19, 2008. Revised Manuscript Received March 6, 2009

A new versatile method for tuning the thickness of surface-tethered polymer brushes is introduced. It is based on the combination of polyelectrolyte multilayer deposition and surface-initiated atom transfer radical polymerization. To control the thickness of the brushes, the nonlinear growth of certain polyelectrolyte multilayer systems is exploited. The method is demonstrated to work with different polyelectrolytes and different monomers. The relevance for applications is demonstrated by cell adhesion experiments on grafted thermoresponsive polymer layers with varying thickness.

Introduction Surfaces coated with so-called “polymer brushes” offer unique properties because of the high number of polymer chains per unit of area.1,2 To reach the desired high chain density, they are most often prepared by the “grafting from” method. This requires the immobilization of initiators for polymerization, most often free radical polymerization, at the surface to be coated. Frequently, silane or thiol chemistries are used for this initial functionalization. However, the choice of surfaces amenable to these chemistries is limited. Methods like polymerization by electron beam or γ-irradiation are more generally applicable but require expensive and potentially hazardous equipment and give rather undefined polymerization conditions. By virtue of their versatility and ease of application, polyelectrolyte macroinitiators provide an attractive alternative,3 as many materials bear surface charges. Moreover, a wide variety of materials can be furnished with a charged surface via alternating deposition of oppositely charged polyelectrolytes.4 On all of these surfaces, polyelectrolyte macroinitiators can be conveniently immobilized by electrostatic adsorption. Consequently, polyelectrolyte macroinitiators are;directly or via simple intermediate steps;capable of modifying virtually any surface of interest. While early examples of polymer brushes grafted from polyelectrolyte multilayers were used for modification of planar surfaces,5 in the following, polyelectrolyte macroinitiators exhibiting groups suitable for ATRP (atom transfer radical polymerization) were employed for the modification of colloidal particles with polymer brushes.3,6 Recently, macroinitiator deposition and *To whom correspondence should be addressed. Tel: +493315681327. Fax: +493315683000. E-mail: [email protected]. (1) Edmondson, S.; Osborne, V. L.; Huck, W. T. S. Chem. Soc. Rev. 2004, 33, 14–22. :: (2) Advincula, R. C., Brittain, W. J., Caster, K. C., Ruhe, J., Eds. Polymer Brushes; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, 2004. (3) Chen, X. Y.; Armes, S. P.; Greaves, S. J.; Watts, J. F. Langmuir 2004, 20, 587–595. (4) Bertrand, P.; Jonas, A.; Laschewsky, A.; Legras, R. Macromol. Rapid Commun. 2000, 21, 319–348. (5) See ref 4 and references therein; cf. page 340. (6) Chen, X.; Armes, S. P. Adv. Mater. 2003, 15, 1558–1562.

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“grafting from” polymerization procedures via surface-initiated ATRP were also applied to planar substrates.7-9 Until now, control of the brush thickness is either achieved by varying the polymerization time,7,8 which, however, is a parameter that one may not want to vary in an industrial application, or by the assembly of macroinitiator multilayers.9 The latter is a rather lengthy process when aiming at thicker brushes. Also, brush thickness can be tuned by the choice of the polymerization solvent or by variation of the monomer concentration. Still, this may be undesirable, because components required for the reaction may lose solubility, for instance, when changing the solvent or the concentration. By the combination of appropriate polyelectrolyte multilayer systems with the “grafting from” technique, it is possible to leave all of the aforementioned experimental parameters constant and yet adjust a grafted polymer layer to a desired thickness. Control of the brush layer thickness is a key factor to obtain defined systems and is particularly important for applications such as the control of cell adhesion, as will be shown in the following. Specifically, the lower critical solution temperature (LCST) exhibited by certain copolymers in aqueous media10,11 can be exploited to modulate the adhesion of cells in cell culture by simply changing the temperature. Advantageously, the LCST is designed to be near physiological temperature, so that the cells adhere to the collapsed polymer on the surface at temperatures near human body temperature, as typically used during cell cultivation. When the temperature is lowered below the LCST, the previously collapsed, tethered polymer chains expand. As the cells tend to minimize the contact with the now more hydrated surface, the cells can be harvested after culturing without the need to apply digesting enzymes, which is an advantage for the integrity (7) Edmondson, S.; Vo, C. D.; Armes, S. P.; Unali, G. F. Macromolecules 2007, 40, 5271–5278. (8) Jain, P.; Dai, J.; Grajales, S.; Saha, S.; Baker, G. L.; Bruening, M. L. Langmuir 2007, 23, 11360–11365. (9) Edmondson, S.; Vo, C.-D.; Armes, S. P.; Unali, G.-F.; Weir, M. P. Langmuir 2008, 24, 7208–7215. (10) Hudson, S. M.; Gil, E. S. Prog. Polym. Sci. 2004,:: 29, 1173–1222. (11) Laschewsky, A.; Lutz, J.-F.; Hoth, A.; Akdemir, O.; Kristen, J.; Skrabania, K. Langmuir 2007, 23, 84–93.

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of the important cell surface proteins. The mechanism of cell adhesion to artificial polymer surfaces is still not completely understood, but most likely, it is mediated by surface-adsorbed proteins12 like serum components or the extracellular matrix. However, control of cell adhesion on such thermoresponsive surfaces is only achieved if the polymer brushes have an optimized thickness,13 which is explained by reduced mobility of the polymer chains under steric restrictions.14 Here, we introduce a new convenient method to easily tune the thickness of a polymer brush, while leaving crucial parameters like solvent, monomer concentration, reaction time, as well as the number of initiator layers constant. For this, we have assembled multilayer structures on Si/SiO2 and on glass, which consist of an alternating polyelectrolyte multilayer system with a nonlinear growth regime, only a single macroinitiator layer, and the polymer brush grown on the macroinitiator layer using the “grafting from” method. Different from previous reports, which focus on macroinitiator adsorption on a silane-modified surface,7 multiple layers of macroinitiator,8,9 or a single macroinitiator layer on a linearly growing polyelectrolyte multilayer assembly,15 the nonlinear dependence of the thickness of the polyelectrolyte multilayer assembly on the number of layer pairs is exploited to control the thickness of the grafted polymer layer. In this specific configuration, we found that the thickness of the macroinitiator layer as well as the thickness of the polymer brush grow with the number of polycation/polyanion layer pairs in the interlayer system. This dependence can be exploited to tune the thickness of the polymer brush layer conveniently. When conditions are adjusted appropriately, polymer brushes with thicknesses of more than 100 nm are accessible with reaction times of less than 1 h. To explore the scope of the new strategy, we built multilayer systems incorporating different polyelectrolytes and used them to grow polymer brushes of different monomers. To underline its relevance for practical purposes, we used the ability to tune the brush layer thickness to produce coatings, which are optimized for the control of cell adhesion.15,16 For this purpose, surfaceinitiated ATRP was performed with an appropriate mixture of the monomers 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA) and oligo(ethylene glycol) methyl ether methacrylate (OEGMA 475). We grew polymer brushes with a suitable thickness and exhibiting a LCST in the desired temperature region from a surface having an appropriate number of alternating polyelectrolyte interlayers, that is, on top of a hydrogel film, different from a previous study, where these monomers were used for surfaceinitiated ATRP from a monolayer on silicon.17

Experimental Section Chemicals. MEO2MA (Aldrich, 95%), OEGMA 475 (Aldrich, Mn = 475 g mol-1), 2-hydroxyethyl methacrylate (Aldrich, 98%), copper(II) bromide (Aldrich, 99%), sodium Lascorbate (Aldrich, 99%), 2,20 -bipyridyl (Fluka, 98%), poly (styrene sulfonate) (PSS; Aldrich, average Mw = 70000), poly (ethyleneimine) (Aldrich, high molecular weight, water-free), poly(allylamine hydrochloride) (Aldrich, average Mw = 70000), poly(glutamic acid) (PGA; Sigma, average Mw = (12) Allen, L. T.; Tosetto, M.; Miller, I. S.; O’Connor, D. P.; Penney, S. C.; Lynch, I.; Keenana, A. K.; Pennington, S. R.; Dawson, K. A.; Gallagher, W. M. Biomaterials 2006, 27, 3096–3108. (13) Matsuda, N.; Shimizu, T.; Yamato, M.; Okano, T. Adv. Mater. 2007, 19, 3089–3099. (14) Kikuchi, A.; Okano, T. J. Controlled Release 2005, 101, 69–84. :: (15) Wischerhoff, E.; Uhlig, K.; Lankenau, A.; Borner, H. G.; Laschewsky, A.; Duschl, C.; Lutz, J.-F. Angew. Chem., Int. Ed. 2008, 47, 5666–5668. (16) Lutz, J.-F. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 3459–3470. (17) Jonas, A. M.; Glinel, K.; Oren, R.; Nysten, B.; Huck, W. T. S. Macromolecules 2007, 40, 4403–4405.

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Figure 1. Simplified schematic representation of the multilayer assemblies prepared in this study. 17000), 2-acrylamido-2-methyl-propylsulfonic acid (AMPS) (Lubrizol), 4,4’-azobis(4-cyanovalerianic acid) (V-501, Wako), diallyldimethylammonium chloride solution (65% in H2O, Aldrich), diallylamine (Aldrich, 99%), and 2,20 -azobis (2-methylpropionamidine) dihydrochloride (V-50, Wako) were used as received. The macroinitiator MA01 (DP = 65, Mn ≈ 20000 g mol-1) was synthesized as described in a previous publication.15 The synthesis of polycation PCM10:1 bearing hydrophobic groups is described elsewhere.18 Prior to use, all surfaces were cleaned by immersion in a solution of 1.0 g of potassium permanganate in 100 mL of concentrated H2SO4 for at least 1 h. (Caution: The mixture of KMnO4 and concentrated H2SO4 can be explosive.)

Synthesis of Poly(2-acrylamido-2-methyl-propylsulfonic acid) (PAMPS). AMPS (7.00 g, 33.8 mmol) was neutralized

with aqueous NaOH solution (1.0 mol L-1), and water was added until the final volume of the solution was 50 mL. After 4,4’-azobis(4-cyanovaleric acid) (335 mg, 1.2 mmol) was added, the solution was degassed by bubbling nitrogen for 30 min. The degassed solution was heated in a sealed flask to 60 °C for 15 h. After this time, a highly viscous solution had formed, which was diluted with 500 mL of water. The product was purified by dialysis (cut off 3500 g mol-1) against water and then freezedried. Yield, 1.44 g (20.6%). Synthesis of F-PDADMAC. Diallyldimethylammonium chloride solution (25.0 g, 0.157 mol, 38.5 g of a 65% aqueous solution), diallylamine (1.52 g, 0.016 mol), 2,2’-azobis(2-methylpropionamidine) dihydrochloride (V-50, Wako) (6.0 g, 0.022 mol), and 1.55 g of HCl (37%) were dissolved in 225 mL of H2O. The mixture was degassed by bubbling nitrogen through it for 30 min. Then, it was heated to 85 °C in a sealed tube for 3.5 h. The product was purified by ultrafiltration (cut off 5000 g mol-1) and then freeze-dried. Yield, 23.9 g (95.6%). After this purification step, 12.0 g of the formed copolymer was dissolved in 30 mL of H2O and heated to 50 °C. After 20 min, 44 mg of rhodamine isothiocyanate was added under vigorous stirring. The mixture was kept at 50 °C for 3 h. Purification was accomplished by dialysis against H2O. Yield, quantitative. Polyelectrolyte Multilayer Deposition. All layer structures were built on two initial layers of poly(ethylene imine) (PEI, deposited by immersion of the Si/SiO2 substrate in an aqueous solution with a polymer concentration of 2.0 mg mL-1, pH 1, for :: (18) Koberle, P.; Laschewsky, A.; van den Boogaard, D. Polymer 1992, 33, 4029–4039.

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Figure 2. Polyelectrolytes used in this study. 20 min) and PSS (deposited by immersion of the Si/SiO2 substrate in an aqueous solution with a polymer concentration of 1.0 mg mL-1, pH 1, for 20 min). The polyions forming the nonlinearly growing multilayer structure were deposited from acidic aqueous solutions (pH 1 or 3) containing 1.0 mg mL-1 of polymer and 0.5 mol mL-1 of sodium chloride. In the case of the PAH/PGA multilayer system, the conditions described in the literature were applied.19 The final multilayers were dried with a blower at 50 °C and then handled at ambient conditions.

Surface-Initiated Polymerization. Grafting of Thermoresponsive P(MEO2MA-co-OEGMA) Brushes9,16. A solution of CuBr2 (375 mg, 1.65 mmol), oligo(ethylene glycol) methacrylate (OEGMA 475) (1845 mg, 3.88 mmol), 2-(20 -methoxyethoxy)ethyl methacrylate (MEO2MA) (9705 mg, 51.6 mmol), 2,20 -bipyridyl (435 mg, 2.85 mmol), and sodium Lascorbate (195 mg, 0.98 mmol) in 15 mL of H2O and 15 mL of ethanol was prepared in a flask sealed with a septum and degassed by bubbling argon through it for 15 min. Then, the reagent solution and the substrates coated with the interlayer system and the macroinitiator MA01 were transferred into a glovebox with a nitrogen atmosphere. The reagent solution was transferred into a beaker, and subsequently, the substrates were immersed in the reagent solution for 1 h at ambient temperature. The reaction was stopped by removing the substrates from the

(19) Boulmedais, F.; Ball, V.; Schwinte, P.; Frisch, B.; Schaaf, P.; Voegel, J.-C. Langmuir 2003, 19, 440–445.

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reagent solution and rinsing them first with ethanol and then with H2O under ambient atmospheric conditions. Grafting of PHEMA Brushes. The substrates coated with the interlayer system and the macroinitiator MA01 were immersed in a degassed solution of CuBr2 (500 mg, 2.24 mmol), HEMA (13.4 g, 103 mmol), bipyridyl (751 mg, 4,81 mmol), and sodium L-ascorbate (341 mg, 1.72 mmol) in 6.25 mL of H2O and 6.25 mL of methanol in an inert atmosphere at ambient temperature. After the substrates were kept in the reaction mixture for 1 h, they were first immersed in ethanol and then three times in H2O under ambient atmospheric conditions for at least 1 min. The final polymer films were macroscopically homogeneous; they were optically clear and showed homogeneous interference colors. Characterization Methods. Layer thicknesses were determined on dry samples by ellipsometry on a Multiscope from Optrel GbR (Kleinmachnow, Germany). The instrument was used in a null ellipsometer configuration, with an angle of incidence of 70°. Film thicknesses were calculated by the software “Elli”, version 5.2 (Optrel GbR), using a four-layer model with the following parameters: layer 1, air (n = 1.0000, k = 0); layer 2, organic layer (n = 1.5000, k = 0); layer 3, SiO2 (d = 1.0 nm, n = 1.4580, k = 0); and layer 4, silicon (n = 3.8858, k = -0.0200). On each sample, measurements were performed on three randomly chosen spots; the reported thickness values represent averages derived from these measurements. Typically, the single values were in a range of (2 nm from the average. Atomic force microscopy (AFM) measurements were performed DOI: 10.1021/la804197j

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Figure 3. Characterization of the layer system {PEI/PSS/(F-PDADMAC/PSS)n/F-PDADMAC/MA01/graft-MEO2MA-co-OEGMA475}: increasing thickness of interlayer assembly, macroinitiator layer, and grafted polymer layer with an increasing number of interlayer pairs n. (a) Dependence of the thickness of the interlayer assembly on n (b). (b) Dependence of the thickness of the macroinitiator (MA01) layer on n (4). (c) Dependence of the graft polymer layer thickness on n (+). (d) Correlation of the graft polymer layer thickness with the thickness of the macroinitiator (MA01) layer (). with an AFM from Veeco Instruments and a Nanoscope III controller in tapping mode with a resonance frequency of ca. 300 kHz. To determine the thickness of the multilayer assembly, the sample on a silicon wafer was first scratched with a pair of tweezers. Then, the height difference between the bottom of the trench and the surface of the multilayer assembly was determined via a cross-section analysis. This height difference corresponds to the film thickness. Cell adhesion experiments were performed as previously reported.15,20

Results and Discussion Figure 1 depicts the fundamental features of the multilayer assemblies with polymer brush described in this work. They consist of the following essential components: (i) a solid substrate with surface charges; (ii) an alternating polyelectrolyte multilayer assembly exhibiting an increase of the thickness of layer pairs with an increasing distance from the surface (i.e., nonlinear growth), with the topmost layer being oppositely charged to the macroinitiator; (iii) a macroinitiator layer, composed of a macroinitiator exhibiting initiator groups for a polymerization reaction and charged groups; and (iv) a polymer brush layer. Whereas linear multilayer growth is typically observed for strongly interacting polyelectrolytes, nonlinear growth is found when the interactions between the oppositely charged polyelectrolytes are weakened. The detailed mechanism(s) are still under debate. Thermodynamic reasons have been proposed,21 but mostly, nonlinear growth is attributed to an increased mobility (20) Ernst, O.; Lieske, A.; Hollaender, A.; Lankenau, A.; Duschl, C. Langmuir 2008, 24, 10259–10264. (21) Biesheuvel, P. M.; Cohen Stuart, M. A. Langmuir 2004, 20, 4764–4770.

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of polyelectrolyte chains in the assemblies. The diffusion of at least one of the polyelectrolytes out of and back into the layer system seems to be involved.22,23 Recent results suggest that diffusion is limited to a zone close to the film-solution interface. The thickness of this zone approaches a limiting value after a certain number of deposition steps, so that the growth regime eventually becomes linear.24 More recently, nonlinear growth was explained by the interaction of one constituent with nonstoichiometric polyelectrolyte complexes in the zone close to the filmsolution interface.25 Independent of the exact mechanism, relatively weak interactions between the oppositely charged polyelectrolytes seem to be a prerequisite for nonlinear growth. Interactions can be weakened in several ways: “dilution” of charged groups within one of the polyelectrolytes,26 incorporation of hydrophobic groups27 in one or more of the polymers forming the interlayer system, or use of certain combinations of weak polyelectrolytes.19,28 Furthermore, when appropriate materials and conditions are chosen, such as high ionic strength, nonlinear growth is even encountered in multilayer systems (22) Picart, C.; Mutterer, J.; Richert, L.; Luo, Y.; Prestwich, G. D..; Schaaf, P.; Voegel, J.-C.; Lavalle, P. Proc. Nat. Acad. Sci. U.S.A. 2002, 99, 12531–12535. :: (23) Salomaki, M.; Vinokurov, I. A.; Kankare, J. Langmuir 2005, 21, 11232– 11240. :: (24) Hubsch, E.; Ball, V.; Senger, B.; Decher, G.; Schaaf, P.; Voegel, J.-C.; Lavalle, P. Langmuir 2004, 20, 1980–1985. (25) Porcel, C.; Lavalle, P.; Senger, B.; Decher, G.; Schaaf, P.; Voegel, J.-C. Langmuir 2007, 23, 1898–1904. (26) Zhang, F.; Wu, Q.; Chen, Z.-C.; Li, X.; Jiang, X.-M.; Lin, X.-F. Langmuir 2006, 22, 8458–8464. (27) Laschewsky, A.; Wischerhoff, E.; Bertrand, P.; Delcorte, A. Macromol. Chem. Phys. 1997, 198, 3239–3253. (28) Jourdainne, L.; Arntz, Y.; Senger, B.; Debry, C.; Voegel, J.-C.; Schaaf, P.; Lavalle, P. Macromolecules 2007, 40, 316–321.

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Figure 4. Characterization of the layer system {PEI/PSS/(PCM10:1/PAMPS)n/PCM10:1/MA01/graft-MEO2MA-co-OEGMA475}: increasing thickness of interlayer assembly, macroinitiator layer, and grafted polymer layer with an increasing number of interlayer pairs n. (a) Dependence of the thickness of the interlayer assembly on n (b). (b) Dependence of the thickness of the macroinitiator layer on n (4). (c) Dependence of the graft polymer layer thickness on n (+). (d) Correlation of the graft polymer layer thickness with the thickness of the macroinitiator (MA01) layer ().

assembled from strong polyelectrolytes, which are devoid of hydrophobic groups and exhibit a high charge density.29 As an example for the incorporation of aromatic hydrophobic groups, the interlayer system was built with a fluorescently labeled copolymer incorporating diallyl-dimethylammonium chloride units (F-PDADMAC, Figure 2) and PSS (Figure 2). Incorporation of aliphatic hydrophobic groups was tested with a poly (choline methacrylate) copolymer exhibiting counits with long alkyl chains (PCM 10:1) and PAMPS (Figure 2). As a representative for assemblies built from weak polyelectrolyte pairs, poly (allylamine) (PAH)/PGA was used. Poly(diallyl-dimethylammonium chloride)/PSS (PDADMAC/PSS) was chosen as a representative nonlinearly growing layer system made from strong polyelectrolytes exhibiting high charge density and no hydrophobic groups. Finally, for a control experiment with a linearly growing interlayer system, the combination PAH/PSS was used. The employed macroinitiator (MA01, Figure 2) is a polyanion made by sequential esterification of poly(hydroxyethyl methacrylate) with 2-bromo isobutyryl bromide and 2-sulfobenzoic acidcyclo-anhydride (SBA). 2,15 Silicon wafers were used as solid substrates to enable the monitoring of the layer assembly and brush growth by ellipsometry. Sequential electrostatic adsorption of poly(ethyleneimine) and PSS from salt-free acidic aqueous solutions at pH 1 provided a primer coating with a negative surface charge. On the resulting charged substrate, the nonlinear interlayer structure was constructed by alternating electrostatic adsorption of the appropriate polycations and polyanions. As an example for an interlayer (29) Liu, G.; Sou, S.; Fu, L.; Zhang, G. J. Phys. Chem. B 2008, 112, 4167–4171.

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system incorporating aromatic hydrophobic groups, F-PDADMAC and PSS were successively deposited. Then, the anionic macroinitiator MA01 was adsorbed on the top polycation layer. Figure 3a shows the increase in thickness vs the number of deposition cycles n for an assembled coating with the sequence {PEI/PSS/(F-PDADMAC/PSS)n}, visualizing the nonlinear growth. In Figure 3b, the thickness of the anionic macroinitiator layer deposited on top of this multilayer system is plotted vs the number of interlayer pairs n. With increasing n, that is, with increasing thickness of the interlayer system, the macroinitiator layer becomes thicker. Following the general explanation for nonlinear multilayer growth, this behavior can be attributed to weakened interactions of the macroinitiator with the underlying multilayer, as the macroinitiator is a polyelectrolyte incorporating hydrophobic repeat units and is deposited from a saline solution. Thus, the macroinitiator molecules are prone to nonlinear growth, and consequently, with increasing n, that is, with increasing thickness of the interlayer system, the macroinitiator layer becomes thicker. When, however, the given polyelectrolyte interlayer system shows linear growth, the interaction with the top film zone is always the same, independent of the number of deposition cycles, and accordingly, the amount of adsorbed macroinitiator will be constant (see control experiments below and Figure 7). From these assemblies with varying n, thermoresponsive polymers P(MEO2MA-co-OEGMA 475) were grown using ATRP in a subsequent step.15 The thickness of the grafted thermoresponsive polymer layer increases with the thickness of the macroinitiator layer (Figure 3d). This means that the thickness of the DOI: 10.1021/la804197j

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Figure 5. Characterization of the layer system {PEI/PSS/(PDADMAC/PSS)n/PDADMAC/MA01/graft-MEO2MA-co-OEGMA475}: increasing thickness of interlayer assembly, macroinitiator layer, and grafted polymer layer with an increasing number of interlayer pairs n. (a) Dependence of the thickness of the interlayer assembly on n (b). (b) Dependence of the macroinitiator layer thickness on n (4). (c) Dependence of the graft polymer layer thickness on n (+). (d) Correlation of the graft polymer layer thickness with the thickness of the macroinitiator (MA01) layer ().

Figure 6. Layer system {PEI/PSS/(PAH/PGA)n/PAH/MA01/graft-MEO2MA-co-OEGMA475}: increasing thickness of the macroinitiator layer with increasing number of interlayer pairs n: After surface-initiated ATRP, a further increase can only be observed for n = 2. (a) Dependence of the macroinitiator layer thickness on n (4). (b) Difference in total layer thickness after surface-initiated ATRP in dependence of n (+).

grafted polymer layer can be controlled by the number n of layer pairs in the interlayer (Figure 3c). Consequently, with appropriately thick macroinitiator layers, polymer layers exceeding a thickness of 100 nm can be grafted within 1 h under mild reaction conditions. Likely, the lengths of the polymer chains grown from the differently thick macroinitiator layers will not vary much. Therefore, we hypothesize that the increase in thickness of the polymer brushes as measured by ellipsometry is mainly caused by a higher density of grafted chains per unit area, due to an increased number of initiating sites per unit area in the thicker 5954

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macroinitiator layer. This explanation implies that initiating sites remain active even if they are covered with tens of nanometers of additional soft matter. This observation is consistent with results of grafting experiments employing initiator multilayers.9 Thickness determination by ellipsometry is a convenient and relatively rapid method, which also enables the characterization of macroscopic areas. Still, the determination of absolute thickness values is not trivial. Nevertheless, the fundamental conclusions remain valid in any case, even if a systematical error in the thickness determination by ellipsometry occurred, as the relative Langmuir 2009, 25(10), 5949–5956

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Figure 7. Layer thicknesses of the layer system {PEI/PSS/(PAH/ PSS)n/PAH/MA01/graft-MEO2MA-co-OEGMA475} (4, macroinitiator layers; +, graft polymer layers). There is an increase of neither the macroinitiator layer thickness nor the graft polymer thickness with increasing n within the limits of experimental accuracy ((2 nm).

values would be comparable to each other. Still, thickness determination by ellipsometry was verified by AFM exemplarily on a sample with the sequence {PEI/PSS/(F-PDADMAC/PSS)4/ F-PDADMAC/MA01/graft-MEO2MA-co-OEGMA475}. The measured total thickness of 97 nm with a rms roughness of ca. 20 nm was in good agreement with the thickness determined by ellipsometry of 92 nm, simultaneously corroborating the homogeneous appearance of the film at the microscopic level. In an analogous experiment, the interlayer system was built from PCM 10:1 and PAMPS, thus from a system incorporating aliphatic hydrophobic groups, while keeping all other experimental parameters constant. Again, a nonlinear increase of the interlayer’s thickness (Figure 4a) was observed with the number of deposition cycles n. Also, the thicknesses of the macroinitiator layer and of the grafted polymer layer increased with n (Figure 4b,c). Next, an interlayer system composed of PDADMAC and PSS was built. Although both polymers have a high charge density and no hydrophobic units, nonlinearly growing layers are formed, when they are deposited from solutions containing high amounts of inorganic salts. When the macroinitiator MA01 was deposited on top of this multilayer system, the thickness of the MA01 layer increased again with the number of layer pairs in the interlayer (Figure 5a). Subsequent surface-initiated ATRP yielded polymer brushes with a thickness, which increases with n, as well. Hence, the thickness of the polymer brush films is correlated to the thickness of the macroinitiator layer (Figure 5b,c). To verify whether the new strategy could also be transferred to polyelectrolyte multilayers, which are exclusively built from weak polyelectrolytes, we chose the well-characterized example PAH/ PGA. For this system, a nonlinear increase of the assembly thickness with the number of deposition cycles was found. However, macroinitiator deposition on the assembly {PEI/PSS/ (PAH/PGA)n/PAH} revealed a clear difference to the previously discussed examples. While there was a noticeable increase in the macroinitiator layer thickness when going from n = 2 to n = 4, very little growth occurred when increasing n any further (Figure 6a). The differences to the previously discussed examples became even more striking, when these assemblies were used for surface-initiated ATRP. Except for n = 2, surface initiated ATRP resulted in a loss of material, which became more important with increasing n (Figure 6b). This finding suggests that the structure of the interlayer, which is exclusively made of weak polyelectrolytes, Langmuir 2009, 25(10), 5949–5956

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is weaker than in the previously studied cases. Hence, the strain imposed on the system by the grafting process probably leads to partial disintegration of the assemblies. As a control, the interlayer system was built from PAH and PSS, while keeping all other conditions constant. The system (PAH/PSS)n is well-characterized and known to form layer systems with a linear dependence of the thickness on the number of deposition cycles.30 Thus, layer systems with the sequence {PEI/PSS/(PAH/PSS)n/PAH/MA01/graft polymer} were built. When the number of PAH/PSS interlayers was varied, the thickness of both the macroinitiator layer and the grafted polymer layer (Figure 7) remained constant within the limits of experimental error. This indicates that a nonlinearly growing interlayer system is most probably required to allow the tuning of the thickness of the grafted polymer layer. To demonstrate that the strategy is not limited to one particular case and other monomers can be used, the layer sequence {PEI/ PSS/(F-PDADMAC/PSS)n/F-PDADMAC/MA01} with n = 4 and n = 10 was prepared, using the same compounds and conditions as described above. Then, a graft layer consisting of the hydroxy-functionalized poly(2-hydroxyethyl methacrylate) (PHEMA) was polymerized on top, and after drying, the layer thickness was determined by ellipsometry. Again, the thickness of the macroinititor layer and the thickness of the grafted brush layer increased with the number of layer pairs in the interlayer system. Here, the system with n = 4 produced a grafted polymer brush of 84 nm thickness, while a polymer brush with 170 nm thickness was grafted from the system with n = 10. As an example for a potential application, cell adhesion experiments were performed on glass slides, using the sequence {PEI/PSS/(F-PDADMAC/PSS)n/F-PDADMAC/MA01} with n = 2, 4, and 10. Grafted polymer brushes of a 93:7 (mol:mol) mixture of the monomers MEO2MA and OEGMA 475, which are grown from this layer system, exhibit a LCST of ca. 34 °C in PBS, so that the cell adhesion can be switched by changing the temperature in physiologically interesting range.16,31,32 Figure 8 shows images of L 929 mouse fibroblasts on these surfaces after 2 days of incubation at 37 °C and then after removing them from the incubator and leaving them for 30 min at 22 °C. The influence of the polymer brush thickness and ultimately the number of interlayer pairs n on the control of cell adhesion is obvious. For n = 2, the fibroblasts adhere well to the surface at elevated temperature, but their rounding at 22 °C is incomplete. For n = 10, the adhesion at elevated temperature is hampered, and consequently, there is no significant difference to the situation at 22 °C. However, for n = 4, the cells adhere well at 37 °C to the substrate, and the cells are well-rounded at 22 °C. This represents the desired scenario. Neither detachment nor rounding of spread L929 fibroblasts was observed upon a temperature decrease from 37 to 22 °C, when the cells were cultivated on a plain, purified glass surface or on a cell culture polystyrene dish (data not shown). When the effect of repeated temperature cycles on cell adhesion and vitality was studied for PNIPAM-grafted gold surfaces,20 cell adhesion and release were found to be reversible. Because oligo(ethylene glycol)-based polymers as used in this study are well-known for their biocompatibility,16 these thermoresponsive coatings are expected not to affect cell viability either.

(30) Ramsden, J. J.; Lvov, Y. M.; Decher, G. Thin Solid Films 1995, 254, 246– 251. :: (31) Lutz, J.-F.; Akdemir, O.; Hoth, A. J. Am. Chem. Soc. 2006, 128, 13046– 13047. (32) Lutz, J.-F.; Hoth, A. Macromolecules 2006, 39, 893–896.

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Figure 8. Control of L 929 mouse fibroblast adhesion on a MEO2MA-co-OEGMA 475 polymer brush surface grown on top of the layer sequence {PEI/PSS/(F-PDADMAC/PSS)n/F-PDADMAC/MA01}. For n = 2 (left), cell adhesion at elevated temperature (top) is effective, but the rounding at 22 °C is incomplete (bottom); for n = 4 (middle), there is good cell adhesion at 37 °C (top) and good rounding at 22 °C (bottom); and for n = 10 (right), cell adhesion at 37 °C (top) is obstructed, and there is little difference to the situation at 22 °C (bottom).

Conclusions An anionic macroinitiator was deposited on top of alternating polyelectrolyte interlayer structures; these multilayer assemblies were successfully used for surface-initiated ATRP. Exclusively, if the polyelectrolyte interlayer structures had nonlinear growth characteristics, the thickness of the macroinitiator layer and of the grafted polymer layer depended on the number of deposition cycles. While assemblies incorporating the weak polyelectrolytes PAH and PGA disintegrated when exposed to ATRP conditions, it was possible to implement the concept with polyelectrolyte multilayer assemblies incorporating aromatic or aliphatic groups. Furthermore, interlayer systems comprising only strong polyelectrolytes devoid of hydrophobic groups are suited if the deposition is tuned to nonlinear growth by a high salt concentration. The method was shown to be applicable for different types of monomers. Thus, the combination of nonlinearly growing multilayer assemblies and macroinitiator-based surface-initiated ATRP is a convenient and versatile way to control the thickness of graft polymer brushes. As an example for an application, the assembly

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method was used to graft thermoresponsive polymer brushes with appropriate transition temperature and thickness for cell culturing. At 37 °C, as typically used for this technique, L 929 mouse fibroblasts spread and grow on the modified surface, whereas when reducing the temperature below the LCST, the cells minimize contact with the surface and round up. :: Acknowledgment. We thank Dr. I. Donch (Max-Planck Institute of Colloids & Interfaces, Potsdam) for AFM experiments. This research was supported by the interdisciplinary network of excellence “Synthetic bioactive surfaces” of the Fraunhofer Society and the Max-Planck Society. Supporting Information Available: 1H NMR spectrum of the macroinitiator MA 01. AFM image and height profile of a multilayer assembly {PEI/PSS/(F-PDADMAC/PSS)4/FPDADMAC/MA01/graft-MEO2MA-co-OEGMA475}. This material is available free of charge via the Internet at http:// pubs.acs.org.

Langmuir 2009, 25(10), 5949–5956