Polymeric Substrates with Tunable Elasticity and Nanoscopically

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Polymeric Substrates with Tunable Elasticity and Nanoscopically Controlled Biomolecule Presentation )

Daniel Aydin,†,^ Ilia Louban,†,^ Nadine Perschmann,† Jacques Bl€ummel,† Theobald Lohm€uller,† Elisabetta Ada Cavalcanti-Adam,† Tobias L. Haas,‡ Henning Walczak,§ Horst Kessler, Roberto Fiammengo,*,† and Joachim P. Spatz*,†

)

† Department of New Materials and Biosystems, Max Planck Institute for Metals Research, Stuttgart, Germany, and Department of Biophysical Chemistry, University of Heidelberg, Germany, Postal Address: Heisenbergstrasse 3, 70569 Stuttgart, Germany, ‡Fondazione Istituto Oncologico del Mediterraneo, Via Penninazzo 795029, Viagrande (CT), Italy, §Tumour Immunology Unit, Division of Medicine, Imperial College London, Hammersmith Hospital Campus, 10N6-Commonwealth Building, Du Cane Road, London W12 ONN, United Kingdom, and Institute for Advanced Study and Center of Integrated Protein Science, Technische Universit€ at M€ unchen Lichtenbergstr. 4, 85747 Garching, Germany. ^Both authors contributed equally to this work.

Received August 2, 2010. Revised Manuscript Received August 27, 2010 Despite tremendous progress in recent years, nanopatterning of hydrated polymeric systems such as hydrogels still represents a major challenge. Here, we employ block copolymer nanolithography to arrange gold nanoparticles on a solid template, followed by the transfer of the pattern to a polymeric hydrogel. In the next step, these nanoparticles serve as specific anchor points for active biomolecules. We demonstrate the engineering of poly(ethylene glycol) hydrogel surfaces with respect to elasticity, nanopatterning, and functionalization with biomolecules. For the first time, biomolecule arrangement on the nanometer scale and substrate stiffness can be varied independently from each other. Young’s moduli, a measure of the compliance of the substrates, can be tuned over 4 orders of magnitude, including the values for all of the different tissues found in the human body. Structured hydrogels can be used to pattern any histidinetagged protein as exemplified for his-protein A as an acceptor for immunoglobulin. When cell-adhesion-promoting peptide cRGDfK is selectively coupled to gold nanoparticles, the surfaces provide cues for cell-surface interaction and allow for the study of the modulation of cellular adhesion by the mechanical properties of the environment. Therefore, these substrates represent a unique multipurpose platform for studying receptor/ligand interactions with adhering cells, mechanotransduction, and cell-adhesion-dependent signaling.

Introduction In the last few years, numerous methods have been developed to arrange active biomolecules on surfaces.1-5 However, because of fabrication requirements, many of these techniques are limited to solid inorganic materials such as silicon and glass. In contrast, polymeric substrates, especially those consisting of a highly hydrated material such as poly(ethylene glycol) (PEG), exhibit a number of advantageous properties such as being transparent, deformable, biocompatible, and permeable to nutrients and gases.6 At present, PEG hydrogels are widely used in biomedical *Corresponding authors. E-mail: [email protected], spatz@ mf.mpg.de. (1) Agheli, H.; Malmstrom, J.; Larsson, E. M.; Textor, M.; Sutherland, D. S. Large area protein nanopatterning for biological applications. Nano Lett. 2006, 6, 1165–1171. (2) Christman, K. L.; Enriquez-Rios, V. D.; Maynard, H. D. Nanopatterning proteins and peptides. Soft Matter 2006, 2, 928–939. (3) Salaita, K.; Wang, Y.; Mirkin, C. A. Applications of dip-pen nanolithography. Nat. Nanotechnol. 2007, 2, 145–155. (4) Staii, C.; Wood, D. W.; Scoles, G. Verification of biochemical activity for proteins nanografted on gold surfaces. J. Am. Chem. Soc. 2008, 130, 640–646. (5) Tinazli, A.; Piehler, J.; Beuttler, M.; Guckenberger, R.; Tampe, R. Native protein nanolithography that can write, read and erase. Nat. Nanotechnol. 2007, 2, 220–225. (6) Cruise, G. M.; Scharp, D. S.; Hubbell, J. A. Characterization of permeability and network structure of interfacially photopolymerized poly(ethylene glycol) diacrylate hydrogels. Biomaterials 1998, 19, 1287–1294. (7) Krsko, P.; Libera, M. Biointeractive hydrogels. Mater. Today 2005, 8, 36–44. (8) Fedorovich, N. E.; Alblas, J.; de Wijn, J. R.; Hennink, W. E.; Verbout, A. J.; Dhert, W. J. Hydrogels as extracellular matrices for skeletal tissue engineering: state-of-the-art and novel application in organ printing. Tissue Eng. 2007, 13, 1905–1925.

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applications including implants, wound healing, drug delivery, and contact lenses.7 Structured polymer hydrogels have potential relevance in tissue engineering and high-throughput sensor devices.8-10 Such highly hydrated polymer systems bearing specific biofunctionality resemble the natural environment of cells in tissue, the so-called extracellular matrix (ECM), from both a chemical and a physical perspective.11,12 In vivo, the elasticity and presentation of cellular epitopes vary significantly from one ECM site to another. For example, very soft hyaluronic acid (HA) gels consisting of 98% water offer binding sites for aggrecanes that modulate the degree of cross linking and thereby the gels’ mechanical properties. In contrast, collagen fibers are solid and form a rather stiff and elastic scaffold to which cells adhere via specialized transmembrane proteins. It is well known that besides the biochemical, the mechanical properties of the ECM also dramatically influence cellular morphology and behavior such as motility, differentiation, and proliferation.11,13,14 Numerous (9) Hultschig, C.; Kreutzberger, J.; Seitz, H.; Konthur, Z.; Bussow, K.; Lehrach, H. Recent advances of protein microarrays. Curr. Opin. Chem. Biol. 2006, 10, 4–10. (10) Khetani, S. R.; Bhatia, S. N. Engineering tissues for in vitro applications. Curr. Opin. Biotechnol. 2006, 17, 524–531. (11) Engler, A. J.; Sen, S.; Sweeney, H. L.; Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 2006, 126, 677–689. (12) Geiger, B.; Bershadsky, A.; Pankov, R.; Yamada, K. M. Transmembrane crosstalk between the extracellular matrix--cytoskeleton crosstalk. Nat. Rev. Mol. Cell Biol. 2001, 2, 793–805. (13) Geiger, B.; Spatz, J. P.; Bershadsky, A. D. Environmental sensing through focal adhesions. Nat. Rev. Mol. Cell Biol. 2009, 10, 21–33. (14) Vogel, V.; Sheetz, M. Local force and geometry sensing regulate cell functions. Nat. Rev. Mol. Cell Biol. 2004, 2006, 265–275.

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biophysical models have been developed to reconstruct and understand the signaling pathways involved in the conversion of mechanical into biochemical information.15-17 Thus, an effective model for the ECM should comprise not only proper compliance but also surface patterning and functionalization on the micrometer and nanometer scales. In this context, artificial ECM systems are powerful tools for influencing,18 visualizing,19 and quantifying20 cellular activity in a controlled manner. In this way, it is possible to specifically address relevant questions such as the required density of ECM ligands necessary for the interaction and clustering of cell-surface receptors. However, patterning of hydrogels on the nanometer and micrometer scales remains challenging for now. Most approaches used to structure polymeric material over an area of >1 cm2 entail relatively strong topographic alterations of the surface, which potentially influence the cell response to the material.21-24 In previous work, we demonstrated the feasibility of the transfer of extended gold nanoparticle arrays from a glass template to polymer surfaces25 such as polystyrene, poly(dimethylsiloxane), and poly(ethylene glycol)-diacrylate-based hydrogels (PEG-DA). These PEGDA hydrogels are very promising materials for a number of biological applications such as cell experiments where unspecific protein adsorption needs to be prevented.26,27 In fact, PEG-DA macromolecules retain the protein-repellent properties of PEG, and the acryl groups allow the polymerization of the macromers to a hydrogel.25,28,29 Nevertheless, it would be a great advantage to have hydrogel substrates with a broad range of elasticity and to introduce further flexibility into the fabrication method, resulting in the possibility of additional patterning of the substrates on the micrometer length scale. Here we present our results for the (15) Bershadsky, A.; Kozlov, M.; Geiger, B. Adhesion-mediated mechanosensitivity: a time to experiment, and a time to theorize. Curr. Opin. Cell Biol. 2006, 18, 472–481. (16) Nicolas, A.; Safran, S. A. Limitation of cell adhesion by the elasticity of the extracellular matrix. Biophys. J. 2006, 91, 61–73. (17) Schwarz, U. S.; Erdmann, T.; Bischofs, I. B. Focal adhesions as mechanosensors: the two-spring model. BioSystems 2006, 83, 225–232. (18) Engler, A.; Bacakova, L.; Newman, C.; Hategan, A.; Griffin, M.; Discher, D. Substrate compliance versus ligand density in cell on gel responses. Biophys. J. 2004, 86, 617–628. (19) Balaban, N. Q.; Schwarz, U. S.; Riveline, D.; Goichberg, P.; Tzur, G.; Sabanay, I.; Mahalu, D.; Safran, S.; Bershadsky, A.; Addadi, L.; Geiger, B. Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates. Nat. Cell Biol. 2001, 3, 466–472. (20) Schwarz, U. S.; Balaban, N. Q.; Riveline, D.; Bershadsky, A.; Geiger, B.; Safran, S. A. Calculation of forces at focal adhesions from elastic substrate data: the effect of localized force and the need for regularization. Biophys. J. 2002, 83, 1380–1394. (21) Hong, Y.; Krsko, P.; Libera, M. Protein surface patterning using nanoscale PEG hydrogels. Langmuir 2004, 20, 11123–11126. (22) Lee, B. K.; Lee, H. Y.; Kim, P.; Suh, K. Y.; Seo, J. H.; Cha, H. J.; Kawai, T. Stepwise self-assembly of a protein nanoarray from a nanoimprinted poly(ethylene glycol) hydrogel. Small 2008, 4, 342–348. (23) Suh, K. Y.; Seong, J.; Khademhosseini, A.; Laibinis, P. E.; Langer, R. A simple soft lithographic route to fabrication of poly(ethylene glycol) microstructures for protein and cell patterning. Biomaterials 2004, 25, 557–563. (24) Yu, T.; Wang, Q.; Johnson, D. S.; Wang, M. D.; Ober, C. K. Functional hydrogel surfaces: binding kinesin-based molecular motor proteins to selected patterned sites. Adv. Funct. Mater. 2005, 15, 1303–1309. (25) Graeter, S. V.; Huang, J.; Perschmann, N.; Lopez-Garcia, M.; Kessler, H.; Ding, J.; Spatz, J. P. Mimicking cellular environments by nanostructured soft interfaces. Nano Lett. 2007, 7, 1413–1418. (26) Bl€ummel, J.; Perschmann, N.; Aydin, D.; Drinjakovic, J.; Surrey, T.; Lopez-Garcia, M.; Kessler, H.; Spatz, J. P. Protein repellent properties of covalently attached PEG coatings on nanostructured SiO2-based interfaces. Biomaterials 2007, 28, 4739–4747. (27) Lussi, J. W.; Falconnet, D.; Hubbell, J. A.; Textor, M.; Csucs, G. Pattern stability under cell culture conditions--a comparative study of patterning methods based on PLL-g-PEG background passivation. Biomaterials 2006, 27, 2534–2541. (28) Lin, H.; Kai, T.; Freeman, B. D.; Kalakkunnath, S.; Kalika, D. S. The effect of cross-linking on gas permeability in cross-linked poly(ethylene glycol diacrylate). Macromolecules 2005, 38, 8381–8393. (29) Moon, J. J.; Lee, S. H.; West, J. L. Synthetic biomimetic hydrogels incorporated with ephrin-A1 for therapeutic angiogenesis. Biomacromolecules 2007, 8, 42–49.

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Article Scheme 1. Schematic of the Fabrication Process of Micronanostructured PEG-DA Hydrogelsa

a

(a) Extended gold nanoparticle arrays are fabricated by block copolymer micellar nanolithography. (b) Microstructuring of the nanostructure with a polymer resist followed by the removal of noncovered nanoparticles creates a microstructure of hexagonally ordered gold nanoparticles. (c) N,N0 -Bis(acryloyl)cysteamine-functionalized gold nanoparticles are covered with PEG-DA initiator solution. During UV irradiation, polymerization takes place and the transfer linker is copolymerized within the PEG-DA matrix. During the swelling process, PEG hydrogel and gold nanoparticles detach from the glass support.

production and characterization of such micro-nanostructured hydrogel substrates. Our preliminary studies were carried out using only PEG-700- and PEG-20000-DA macromers (molecular weights of 700 g/mol and 20000 g/mol, respectively) and no attempt was made to fine-tune the elasticity of the hydrogels.25 We have now used PEG-10000- and PEG-35000-DA macromers as well, and we have varied the concentration of macromer during the polymerization reaction. We show that the elasticity of the substrate is now finely tunable over 4 orders of magnitude from 0.6 kPa up to 6 MPa, covering the elasticity of the extracellular space in all tissues.30 Because of the variability of polymerization conditions (the selected macromer and its concentration in the prepolymer solution) and the additional microstructuring of the molds, we reinvestigated the transfer protocol to ensure highfidelity transfer in each case. In particular, a single-step functionalization of the gold nanoparticles with the transfer linker, N,N0 -bis(acryloyl)cysteamine, affords excellent results for the fabrication of all investigated micro-nanostructered hydrogels. Histidine-tagged, Fc-tagged, and thiol-terminated proteins and peptides can be attached to the surface via site-directed immobilization, and the spacing of the biomolecules on the nanometer scale as well as their geometric arrangement on the micrometer scale can be precisely adjusted. Different substrate systems and biofunctionalization techniques used within the life science community feature good elasticity control but in general poor variability in terms of ligand density and no control in terms of ligand-to-ligand spacing on the nanometer length scale.18 Moreover, the elasticity and ligand density usually depend directly or indirectly on each other. To mimic the huge variety of different tissues and to disentangle the relationship between substrate elasticity and biomolecule presentation, more sophisticated materials are needed. In this study, we present the first cell-biology-aimed approach to a substrate system where crucial parameters such as substrate compliance (30) Discher, D. E.; Janmey, P.; Wang, Y. L. Tissue cells feel and respond to the stiffness of their substrate. Science 2005, 310, 1139–1143.

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Figure 1. SU8-based photolithography on nanostructured surfaces. (a, b) Scanning electron micrographs of SU8 resist structures. (c) A higher-magnification image shows the underlying gold nanoparticle structure. (d-f) After a 2 h treatment with reactive hydrogen plasma, the 1-μm-thick epoxy structures are removed, uncovering the protected nanostructure, where gold nanoparticles have a diameter of approximately 6 ( 1 nm. Scheme 2. Polymerization Process of PEG-DAs (in Aqueous Solution) Initiated by 4-(2-Hydroxyethoxy)phenyl-(2-propyl)ketone upon UV Irradiation and Copolymerization of N,N0 -Bis(acryloyl)cysteamine-Functionalized Gold Nanoparticles into the Resulting PEG-DA Hydrogel

and the spacing and density of numerous biomolecules can be varied fully independently from each other.

Results and Discussion Fabrication of Micro-nanostructured PEG Hydrogels. Block copolymer micellar nanolithography is capable of producing extended quasi-hexagonally ordered arrays of gold nano15474 DOI: 10.1021/la103065x

particles on solid inorganic supports such as silicon and glass with adjustable interparticle spacing (Scheme 1a).31,32 This method (31) Glass, R.; M€oller, M.; Spatz, J. P. Block copolymer micelle nanolithography. Nanotechnology 2003, 14, 1153–1160. (32) Spatz, J. P.; Lohm€uller, T.; Arnold, M. Method for the creation of planar variations in size or distance in nanostructure patterns on surfaces. International Patent Application PCT/EP2008/001981, October 23, 2008.

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Figure 2. Cryo-scanning electron micrographs of a micro-nanostructured PEG-700-DA hydrogel surface. (a) Overview and (b) close-up micrographs demonstrate the accuracy and efficiency of the transfer process. The inset in image b is 1 μm  1 μm.

takes advantage of the hexagonal assembly of spherical PS-blockPVP micelles on solid substrates. A metal precursor salt such as HAuCl4 can be loaded into the micellar core forming nanoparticles on the surface upon treatment with a reactive gas plasma. To structure these particle arrays on the micrometer scale, we applied standard photolithography to gold nanopatterns (Figure 1a-c). The removal of nonprotected particles followed by uncovering the protected particles by the elimination of the photoresist yielded micropatches of gold nanoparticles on glass (Scheme 1b, Figure 1d-f). Both positive and negative photoresists were used to achieve more flexibility in choosing the chromium photomasks. As a positive resist, we used a novolak-based photoresist as described previously.33 With the latter, we were able to produce structure sizes down to 3 μm. Smaller feature sizes, in the submicrometer regime, could be obtained using well-established negative resist SU8. However, a major drawback of the SU8 system is represented by its poor removability after photo-crosslinking.34 By employing microwave-induced hydrogen plasma, we could completely remove SU8, as evidenced by XPS measurements (Supporting Information Figure 1) and SEM imaging (Figure 1d-f). Transfer of the micro-nanopatterns from the glass support to the hydrogel was achieved via a one-step functionalization of the gold nanoparticles with transfer linker N,N0 -bis(acryloyl)cysteamine (1 mM in ethanol for 1 h). This is a significant advance compared to the procedure reported in our previous work, where high-fidelity transfer of the nanoparticle arrays on “soft” PEG20000-DA hydrogels could be obtained only with a longer, twostep procedure.25 Additionally, the procedure works well for each macromer employed in hydrogel production including PEG-700-DA, thereby avoiding the use of the very volatile, smelly propene-thiol. After functionalization, an aqueous prepolymer solution con(33) Aydin, D.; Schwieder, M.; Louban, I.; Knoppe, S.; Ulmer, J.; Haas, T. L.; Walczak, H.; Spatz, J. P. Micro-nanostructured protein arrays - a tool for geometrically controlled ligand presentation. Small 2009, 5, 1014–1018. (34) Dentinger, P. M.; Clift, W. M.; Goods, S. H. Removal of SU-8 photoresist for thick film applications. Microelectron. Eng. 2002, 61, 993–1000.

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Figure 3. Compliance and nanoparticle decoration properties of PEG-DA hydrogels. (a) The Young’s modulus (EY) of PEG-10000DA, PEG-20000-DA, PEG-35000-DA, and PEG-700-DA hydrogels polymerized with different initial water content is compared to the Young’s modulus of different tissues. The complete range of biologically relevant material compliance can be covered. (b) Overview of the adjustable material properties of nanopatterned PEGDA hydrogels. The variations of bioactive ligand spacing on the hydrogel and the compliance of the material are fully independent of each other. The number associated to the various symbols denotes the polymer used for preparation of the micellar solutions.

taining the desired PEG-DA macromer and photoinitiator (4-(2-hydroxyethoxy)phenyl-(2-propyl)ketone) was cast on the mold substrates (Scheme 1c).35 Polymerization was induced by standard UV irradiation, resulting in covalent attachment of the gold nanoparticles to the surface of the hydrogels via copolymerization of the transfer linker immobilized on their surface (Scheme 2). Subsequently, the samples were placed in water until they spontaneously detached from the glass support and were further swollen for a total of at least 48 h. Lateral forces induced by the swelling process are responsible for the detachment of gold nanoparticles from the glass surface with a transfer efficiency of almost 100% as shown in Figure 2. Four different PEG-DA macromers with molecular weights (Mw) of 700 g/mol (PEG-700-DA), 10 000 g/mol (PEG-10000-DA), 20 000 g/mol (PEG-20000-DA), and 35 000 g/mol (PEG-35000DA) were used to finely tune the elasticity of the resulting hydrogels. Furthermore, the elasticity could also be varied by adjusting the concentration of the chosen macromer in the aqueous prepolymer solution (Figure 3a). As expected, macroscopic swelling of the micro-nanostructured hydrogels after fabrication leads to an increase in interparticle spacing. However, the macroscopic and nanoscopic changes in the hydrogel during swelling corresponded very well. In fact, the ratio of the edge lengths of a swollen and an unswollen gel was the same as the ratio of the interparticle distance between the template and the swollen gel (data not shown). Swelling ratios were found to be between (35) Bryant, S. J.; Nuttelman, C. R.; Anseth, K. S. Cytocompatibility of UV and visible light photoinitiating systems on cultured NIH/3T3 fibroblasts in vitro. J. Biomater. Sci., Polym. Ed. 2000, 11, 439–457.

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almost 1 for stiff PEG-DA-700 and 1.8 for very soft PEGDA-35000. The broad distribution of interparticle spacing on PEG-DA hydrogels shown in Figure 3b demonstrates the flexibility in choosing spacing and substrate compliance independently. Because the swelling ratios were found to be very reproducible as well as consistent with the nanoscopic changes on the gel surface, it is possible to know a priori the interparticle distance needed on the glass template to obtain the desired spacing on the swollen hydrogel. Viscoelastic Characterization of PEG Hydrogels. To quantify the mechanical properties of PEG-DA hydrogels (i.e., the Young’s modulus EY), we performed AFM indentation measurements based on the Hertz model36 and adjusted for conically shaped tips with a semivertical opening angle R. In this case, the total force F is a function of the indentation Δz and is described by 

Fcone ðΔzÞ ¼ 2

EY Esample Δz2 , where E ¼ π tanðRÞ 1 - μsample 2

ð1Þ

if the EY of the indenter (e.g., the cantilever tip) is much higher than the EY of the measured sample.37 The Poisson ratio μ was assumed to be 0.5 because of the high water content of PEG-DA hydrogels. The EY values of PEG-700-DA, PEG-10000-DA, PEG-20000-DA, and PEG-35000-DA hydrogels are presented in Figure 3a. The AFM indentation measurements confirm that the mechanical properties (EY) of the hydrogels can be effectively controlled by choosing a certain PEG-DA macromer of appropriate molecular weight (Mw) and by varying the water content (cH2O) of the macromer/initiator solution before the polymerization process. As a result, the elasticity of PEG-DA hydrogels can be gradually adjusted within 4 orders of magnitude (0.6 kPa e EY (Mw, cH2O) e 6 MPa), including all known compliance of tissues in vivo.38 Tuning of the Particle Size. Gold particles allow the optical observation of cell-surface interactions by utilizing their intriguing plasmonic properties.39 These properties are advantageous for biosensing and spectroscopy applications because the excitation of surface plasmons by light, referred to as localized surface plasmon resonance (LSPR), is not subject to bleaching and is very sensitive. Furthermore, numerous biofunctionalization methods are available.40,41 Because of their intense light scattering, single gold nanoparticles can even be resolved by using darkfield optical microscopy, as reported previously.42 In this context, recent studies showed that single plasmonic nanoparticles are useful tools for the analysis of protein interaction with biological :: (36) Hertz, H. Uber die ber€uhrung fester elastischer k€orper. J. Reine Angew. Math. 1881, 92, 156–171. (37) Weisenhorn, A. L.; Khorsandi, M.; Kasas, S.; Gotzos, V.; Butt, H. J. Deformation and height anomaly of soft surfaces studied with an AFM. Nanotechnology 1993, 4, 106–113. (38) Zajac, A. L.; Discher, D. E. Cell differentiation through tissue elasticitycoupled, myosin-driven remodeling. Curr. Opin. Cell Biol. 2008, 20, 609–615. (39) Giebel, K.; Bechinger, C.; Herminghaus, S.; Riedel, M.; Leiderer, P.; Weiland, U.; Bastmeyer, M. Imaging of cell/substrate contacts of living cells with surface plasmon resonance microscopy. Biophys. J. 1999, 76, 509–516. (40) Shipway, A.; Katz, E.; Willner, I. Nanoparticle arrays on surfaces for electronic, optical, and sensor applications. ChemPhysChem 2000, 1, 18–52. (41) S€onnichsen, C.; Geier, S.; Hecker, N.; Von Plessen, G.; Feldmann, J.; Ditlbacher, H.; Lamprecht, B.; Krenn, J.; Aussenegg, F.; Chan, V. Spectroscopy of single metallic nanoparticles using total internal reflection microscopy. Appl. Phys. Lett. 2000, 77, 2949–2951. (42) S€onnichsen, C.; Reinhard, B. M.; Liphardt, J.; Alivisatos, A. P. A molecular ruler based on plasmon coupling of single gold and silver nanoparticles. Nat. Biotechnol. 2005, 23, 741–745. (43) Baciu, C. L.; Becker, J.; Janshoff, A.; Sonnichsen, C. Protein-membrane interaction probed by single plasmonic nanoparticles. Nano Lett. 2008, 8, 1724– 1728.

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Figure 4. Cryoscanning electron micrographs of the micro-nanostructured PEG-10000-DA hydrogel (a) before and (b) after the electroless deposition of additional gold from solution. (c) Overview and (d) close-up of a circle-shaped patch demonstrating the accuracy and efficiency of the particle growth.

membranes.43 An important issue, however, is that these optical properties are mainly related to the particle size.44 The presence or prevention of nanoclustered molecular multimers at the surface of cellular membranes or in intracellular complexes is controversially discussed and is known to play a crucial role in various inside-out or outside-in signaling events.45,46 The immobilization of ligands on gold nanoparticles with tunable particle size allows for the indirect generation of molecular multimers and their controlled presentation to cellular systems. Methods to control the particle diameter and shape precisely would thus greatly improve the general applicability of the aforementioned approach. Gold nanoparticles covalently tethered to the hydrogel matrix were enlarged by using a seeded growth procedure.47,48 The gold nanopatterned hydrogels were therefore immersed in an aqueous solution of HAuCl4 in the presence of NH2OH 3 HCl as a reducing agent.49 Cryo-SEM measurements were performed to investigate the particle growth in detail as shown in Figure 4. After 3 min of reaction time, the particles had grown to a size of around 20 nm. As shown in Figure 4b,d, the quasi-hexagonal order of the nanopattern was not affected by the process. This indicates that the covalent attachment of the nanoparticles to the hydrogel via (44) Link, S.; El-Sayed, M. Size and temperature dependence of the plasmon absorption of colloidal gold nanoparticles. J. Phys. Chem. B 1999, 103, 4212–4217. (45) Kiessling, L. L.; Gestwicki, J. E.; Strong, L. E. Synthetic multivalent ligands as probes of signal transduction. Angew. Chem., Int. Ed. 2006, 45, 2348–2368. (46) Ma, Z.; Sharp, K. A.; Janmey, P. A.; Finkel, T. H. Surface-anchored monomeric agonist pMHCs alone trigger TCR with high sensitivity. PLoS Biol. 2008, 6, e43. (47) Brown, K. R.; Natan, M. J. Hydroxylamine seeding of colloidal Au nanoparticles in solution and on surfaces. Langmuir 1998, 14, 726–728. (48) Turkevich, J.; Stevenson, P. C.; Hillier, J. The nucleation and growth processes in the synthesis of colloidal gold. Discuss. Faraday Soc. 1951, 55-75. (49) Lohm€uller, T.; Bock, E.; Spatz, J. P. Synthesis of quasi-hexagonal ordered arrays of metallic nanoparticles with tuneable particle size. Adv. Mater. 2008, 20, 2297–2302.

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Figure 6. Alexa Fluor 555 conjugated mouse IgG coupled to a gold nanoparticle array through the site-directed immobilization of histidine-tagged protein A on NTA. The spacing of gold nanoparticles within the 5 μm circles is 70 nm, and the hydrogel consists of PEG-700-DA.

Figure 5. Electroless deposition of gold on micro-nanostructured PEG hydrogels ((a) PEG-700-DA and (b-d) PEG-20000-DA). The localized plasmon resonance of grown particle patches is visible (a) with the naked eye and (b) with phase-contrast microscopy. (c, d) Nanoparticles within the patches grow together into a cohesive gold plaque.

the copolymerized N,N0 -bis(acryloyl)cysteamine remains stable during the growth process. Particle enlargement proceeds as long as Au3þ ions are present in solution. Immersing the substrate in water can stop the reaction. Thus, the particle size can be precisely adjusted depending on the reaction time. With ongoing reaction time, the particle size increases until a cohesive metal plaque is formed as shown in Figure 5. In this case, single particles could no longer be detected. However, it is evident from Figure 5b,c that the deposition of Au from solution occurred only on the prestructured areas of the hydrogel surface, namely, those containing Au nanoparticles that served as seeds. The strong red color due to plasmon absorbance of the enlarged micro-nanopattern is visible with the naked eye (Figure 5a). Protein Immobilization. Because poly(ethylene glycol) is very protein-repellent, we aimed at the selective immobilization of proteins onto surface areas decorated with gold nanoparticles, which can be used as anchor points. To control protein orientation at the interface and to preserve protein function and activity, we used the well-established NTA/his-tag system. In this very versatile and broadly used approach, a certain number of histidine residues (usually six) are fused to the N- or C-terminal end of the protein of interest. Nitrilotriacetic acid (NTA), which provides a strong coordination environment for histidine in a hexagonal complex with transition-metal ions such as Ni2þ and Cu2þ, serves as the surface-bound capture molecule.50 (50) Hochuli, E.; D€obeli, H.; Schacher, A. New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J. Chromatogr. 1987, 411, 177–184. (51) Groll, J.; Albrecht, K.; Gasteier, P.; Riethmueller, S.; Ziener, U.; Moeller, M. Nanostructured ordering of fluorescent markers and single proteins on substrates. ChemBioChem 2005, 6, 1782–1787. (52) Wolfram, T.; Belz, F.; Sch€on, T.; Spatz, J. P. Site-specific presentation of single recombinant proteins in defined nanoarrays. Biointerphases 2007, 2, 44–48.

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We coupled a thiol-terminated NTA derivative (Experimental Section) to the gold nanoparticles33,51,52 on the hydrogel surface. After the NTA was charged with Ni2þ ions, histidine-tagged protein A, which is known to bind the Fc portion of immunoglobulins, was attached to the hydrogel in a site-directed manner. The success of this selective immobilization of protein A onto the nanostructured area could be confirmed by developing the surface with Alexa Fluor 555 conjugated mouse IgG. The confocal fluorescence microscopy images clearly show that IgG binding took place as expected only onto areas covered by gold nanoparticles and therefore functionalized with protein A (Figure 6). Very interestingly, this result paves the way for the development of biosensors based on selectively immobilized antibodies. Cell Adhesion to Micro-nanopatterned Soft Substrates. Having demonstrated the selective immobilization of proteins onto our micro-nanopatterned soft substrates, we decided to investigate their function as substrates for studying cell adhesion. In fact, our fabrication strategy allows for the precise and independent tuning of materials compliance, ligand functionalization, and interligand spacing affording materials that are ideally suited for studying cell adhesion mediated by integrin receptors. Cell adhesion to extracellular matrix proteins, such as fibronectin and collagen, is a key event in regulating many cellular functions including mechanosensing and cell growth.53 These responses are often mediated by integrin transmembrane receptors, which cluster54 and recruit several intracellular proteins into micrometer-sized structures, termed focal adhesions.55 Fibroblasts that were stably transfected with YFP-paxillin (REF52-YFP-paxillin), a relevant protein of focal adhesion plaques,12 were seeded onto soft micro-nanostructured substrates where the gold nanoparticles were functionalized with cyclic RGDfK pentapeptide (cRGDfK). This peptide was shown to be a selective ligand for integrin type Rvβ3,56 which is highly expressed in fibroblasts. The formation of focal adhesions could be visualized by observing the formation of paxillin clusters in fluorescence microscopy. The effect of material compliance was investigated (53) Schwartz, M. A. Integrin signaling revisited. Trends Cell Biol. 2001, 11, 466–470. (54) Arnold, M.; Cavalcanti-Adam, E. A.; Glass, R.; Bl€ummel, J.; Eck, W.; Kantlehner, M.; Kessler, H.; Spatz, J. P. Activation of integrin function by nanopatterned adhesive interfaces. ChemPhysChem 2004, 5, 383–388. (55) Petit, V.; Thiery, J. P. Focal adhesions: structure and dynamics. Biol. Cell. 2000, 92, 477–494. (56) Dechantsreiter, M. A.; Planker, E.; Matha, B.; Lohof, E.; Holzemann, G.; Jonczyk, A.; Goodman, S. L.; Kessler, H. N-Methylated cyclic RGD peptides as highly active and selective RVβ3 integrin antagonists. J. Med. Chem. 1999, 42, 3033–3040.

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Figure 7. Cell adhesion of REF52-YFP-paxillin fibroblasts to cRGDfK-functionalized micro-nanostructured PEG-DA hydrogels. (a) Localization of YFP-paxillin over 2 μm square patches of nanostructures on a PEG-700-DA hydrogel. The patches were separated by 4 μm prior to swelling. (b) The same as in image a but on a PEG-10000-DA hydrogel, where the separation and size of the patches are increased because of a higher swelling ratio (1.03 ( 0.06 for PEG-700-DA and 1.25 ( 0.07 for PEG-10000-DA). (c) The same as in images a and b but on a soft PEG-35000-DA hydrogel. The formation of focal adhesion clusters is impaired. (d) Immunofluorescence micrograph of a REF52-YFP-paxillin fibroblast on a PEG-10000-DA hydrogel. The microstructure consists of 5 μm circles separated by 5 μm. Cells are stained for the nucleus (blue) and the actin cytoskeleton (green), and stably expressed YFP-paxillin localized at focal adhesions is shown in red. (e) Atomic force micrograph (vertical deflection signal, with corresponding height and phase signals shown in Supporting Information Figure 2) on the PEG-700-DA hydrogel substrate. The micro-nanostructure consisting of 5 μm circles separated by 5 μm is clearly visible (white arrowheads).

using micro-nanostructured PEG-700-DA-, PEG-10000-DA-, and PEG-35000-DA-derived substrates with EY 700 = 6.0 ( 0.3 MPa, EY 10000 = 60 ( 5 kPa, and EY 35000 = 3.9 ( 0.4 kPa, respectively. The interparticle spacing on all substrates was kept below the critical limit of 73 nm in order to avoid effects originating from insufficient integrin clustering.54 Figure 7 shows micrographs of (a-c, e) living and (d) fixed cells 24 h after seeding on micro-nanostructured hydrogels. The protein-repellent characteristics of polymerized PEG-DA result 15478 DOI: 10.1021/la103065x

in selective cell-substrate interactions established via the cyclic RGDfK peptide immobilized on the gold nanoparticles in the micro-nanostructured area. As demonstrated in Figure 7a,b, local maxima of the YFPpaxillin concentration corresponded well to the surface-pattern geometry indicating the colocalization of focal adhesions with micro-nanopatterns. During the fabrication process, identical 2 μm squares separated by 4 μm of micro-nanopatterned particle arrays on glass supports were used to fabricate structured hydrogel samples. Langmuir 2010, 26(19), 15472–15480

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Because of the swelling of the hydrogels, the separation of the patches and their size in the final material are larger than for those on the glass substrate used during fabrication (Scheme 1). The interparticle spacing on the templates was chosen such that 50 nm was not exceeded after swelling to stay well below the critical distance of 73 nm.54 Larger differences in spacing between the swollen hydrogel and the glass template are observed for softer substrates compared to less-compliant ones (e.g., PEG-10000-DA vs PEG-700-DA, Figure 7a,b). As demonstrated in Figure 7c, the compliance of the substrate has a major impact on cell morphology and no clear focal adhesion clusters could be observed on soft substrates (EY 35000 = 3.9 ( 0.4 kPa). To demonstrate that PEG-DA substrates do not interfere with standard staining protocols, we performed immunofluorescence imaging of the cytoskeletal orientation and focal contact formation on micro-nanopatterned hydrogels (Figure 7d). A clear microstructure is observable by the localization of YFP-fluorescence (Figure 7d, white arrowheads), and the actin cytoskeleton is clearly oriented toward the micropatches. To visualize the actin stress fibers of the cytoskeleton and the underlying micro-nanostructure at the same time, live-cell atomic force microscopy was performed. Figure 7e displays the vertical deflection image of a 70  70 μm2 scan of an REF52-YFP-paxillin cell on a PEG-700-DA hydrogel substrate. Bundles and thinner filaments are visible and appear as a fine weblike structure beneath the cell surface. The cytoskeleton is clearly oriented toward the micro-nanostructure, which features 5 μm circles separated by 5 μm (white arrowheads). We could demonstrate that structured PEG-DA hydrogels are very suitable substrates for cell adhesion studies by live-cell fluorescence and atomic-force microscopy as well as for immunofluorescence staining. The compliance of the substrate had a major impact on cell adhesion, and the threshold value for the breakdown of focal adhesion formation was in good accordance with that reported by others.57 Micro-nanostructured PEG-DA hydrogels represent the first tool that allows for the study of cellular adhesion depending on the spatial distribution of anchor points and the mechanical properties of the material and where these parameters are fully independent of each other.

Conclusions We report on an efficient and reliable strategy for the preparation of soft micro-nanostructured PEG-hydrogels. The fabrication process, based on transfer nanolithography, is very versatile and allows for the preparation of materials where the compliance can be independently varied from the distance between the micrometer-sized patches of gold nanoparticles. The shape and distance between the patches is also independent of the interparticle spacing within the ordered nanoparticle array constituting a single patch. In addition, we show that the size of the nanoparticles can be adjusted as desired. We also show that the prepared substrates can be selectively biofunctionalized with proteins and peptides by the most commonly used immobilization systems using the gold nanoparticles as anchor points. As a proof of principle, we demonstrate that these substrates can be used to investigate the adhesion of fibroblasts as a function of material compliance. In conclusion, these artificial matrices possess very well defined mechanical and biochemical properties and will certainly find application in cell biology and tissue engineering. (57) Yeung, T.; Georges, P. C.; Flanagan, L. A.; Marg, B.; Ortiz, M.; Funaki, M.; Zahir, N.; Ming, W.; Weaver, V.; Janmey, P. A. Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell Motil. Cytoskeleton 2005, 60, 24–34.

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Further studies currently underway in our laboratories are aimed at using PEG-DA hydrogels to understand how different cell types such as osteoblasts, fibroblasts, myocytes, and neurons are generating, sensing, and modifying the extracellular matrix.

Experimental Section Synthesis of Poly(ethylene glycol)-diacrylates. Poly(ethylene glycol)-diacrylates (PEG-DAs) were synthesized according to a slightly modified literature procedure.58 In a typical synthesis, PEG-10000 (50 g, 5 mmol) was dried by codistillation with toluene (3  250 mL). The residue was redissolved in dichloromethane (125 mL) and toluene (75 mL) under a nitrogen atmosphere. From this point, all the following operations were performed with protection from light. Triethylamine (2.1 mL, 15 mmol) and acryloyl chloride (1.25 mL, 15 mmol) were sequentially added, and stirring was continued overnight at room temperature. The reaction mixture was then filtered over a plug of alumina (neutral). K2CO3 (12.5 g) was added to the filtrate, and the mixture was stirred for 1.5 h. The solid was removed by filtration, and the filtrate was concentrated under vacuum to remove as much dichloromethane as possible from the mixture. The product was then precipitated by the addition of diethylether (350-400 mL) under vigorous stirring. After 2 h, the solid was recovered by filtration, washed with diethylether (2  100 mL), and dried under vacuum. Pure product (40-45 g) was obtained (80-90% yield). PEG-20000-DA and PEG-35000-DA were prepared accordingly. PEG-10000-DA: 1H NMR (300 MHz, CDCl3, δ): 6.39 (dm, J = 17.3 Hz, 2H), 6.12 (ddd, J = 1.1, 10.3, 17.3 Hz, 2H), 5.80 (dm, J = 10.3 Hz, 2H), 4.28 (m, 4H), 3.88-3.34 (m, 988H). PEG-20000-DA: 1H NMR (300 MHz, CDCl3, δ): 6.39 (dm, J = 17.3 Hz, 2H), 6.12 (ddd, J = 1.1, 10.3, 17.3 Hz, 2H), 5.80 (dm, J = 10.3 Hz, 2H), 4.28 (m, 4H), 3.87-3.33 (m, 1980H). PEG-35000-DA: 1H NMR (300 MHz, CDCl3, δ): 6.38 (dd, J = 1.4, 17.3 Hz, 2H), 6.10 (dd, J = 10.3, 17.3 Hz, 2H), 5.78 (dd, J = 1.4, 10.3 Hz, 2H), 4.26 (m, 4H), 3.86-3.31 (m, 4780H). Photolithography. SU8-2 (MicroChem, Newton, MA) was spin-coated onto nanopatterned 20 mm  20 mm glass coverslips (Roth GmbH, Germany) to yield a 1-μm-thick resist layer. (For nanopatterning, see the Supporting Information.) Contact illumination with a 50 mJ/cm2 dose from an HBO 350 mercury lamp was carried out under a mask aligner (S€ uss Microtec GmbH, Garching, Germany), and the structure was developed according to the manufacturer’s instructions. Subsequently, noncovered parts of the nanostructure were removed by sonication of the substrate in an aqueous solution of cysteamine (100 mM), followed by removal of the SU8 structure by applying a reactive hydrogen plasma (TePla 100-E, 0.4 mbar H2, 150 W, 120 min). Photolithography using a Novolak-based positive resist (ARP 5350, Allresist GmbH, Strausberg, Germany) was performed as described previously.33

PEG-DA Polymerization and Transfer Lithography. PEG-700-DA was mixed with water to a final concentration of 1100 mg/mLwater. PEG-10000-DA, PEG-20000-DA, or PEG35000-DA was dissolved in water at a chosen concentration between 200 and 1400 mg/mLwater. An aqueous, cold saturated solution of 4-(2-hydroxyethoxy)phenyl-(2-propyl)ketone acted as the initiator and was added to the polymer solution before the polymerization process. The concentration of added initiator solution was 0.22 μL/mgPEG-DA for PEG-700-DA and 0.15 μL/mgPEG-DA for PEG-10000-DA, PEG-20000-DA, and PEG-35000-DA, respectively. Prior to the transfer and polymerization process, gold micro-nanostructures were incubated in an ethanolic solution of N,N0 -bis(acryloyl)cysteamine (1 mM) for 1 h. To remove excess (58) Elbert, D. L.; Pratt, A. B.; Lutolf, M. P.; Halstenberg, S.; Hubbell, J. A. Protein delivery from materials formed by self-selective conjugate addition reactions. J. Controlled Release 2001, 76, 11–25.

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N,N0 -bis(acryloyl)cysteamine, the glass substrates were rinsed with ethanol three times for 10 min. The functionalized glass substrates were assembled in a sandwich with thin (∼0.2 mm) spacers, and the empty space was filled with the prepolymer initiator solution. Polymerization was performed by irradiation with UV light (365 nm, 15 W) for 60 min. In the last step, samples were detached from the support by immersing them in deionized water and swelling them for at least 48 h. Cryo-scanning Electron Microscopy. To verify the success of transfer lithography to PEG-DA hydrogels, we performed scanning electron microscopy under low-temperature conditions (cryo SEM; toperation=-120 to -140 C). A Zeiss Ultra 55 fieldemission electron microscope (FE-SEM) equipped with in-lens, secondary electron (SE), and energy- and angle-selective backscattered electron (ESB) detectors was used for image acquisition. Low acceleration voltages of 1-2.5 kV were applied because of the low conductivity of the investigated samples. A BAL-TECH VLC 100 shuttle and loading system and a BAL-TECH MED 020 preparation device were used to cool down and transfer the PEGDA hydrogels into the SEM chamber. AFM Indentation Measurements. Atomic force micrographs and indentation measurements were performed with a Nano Wizard I AFM that was commercially provided by JPK Instruments AG. The instrument is mounted on an optical microscope (Zeiss Axiovert 200) and is suitable for simultaneous phase contrast or fluorescence measurements. Additionally, an incubator box driven by an EMBL GPI68 IV controller is installed to provide physiological conditions during AFM measurements. Silicon nitride cantilevers with a cone-shaped tip (μmasch NSC 35 ALBS) were used for indentation measurements. Spring constants were determined by the thermal noise calibration method and ranged between 2.3 and 8.2 N/m.59 Because the opening angle of the tip is a crucial parameter for measuring the Young’s modulus by the Hertz method, it was determined for each cantilever used by scanning electron microscopy.60 Atomic force micrographs of living cells were obtained under physiological conditions (37 C, 5% CO2) in cell culture medium (DMEM supplemented with 10% FBS v/v) using intermittent contact mode. Soft, backside-coated, tilt-stabilized silicone nitride cantilevers (Veeco Instruments Inc., NP-S, k ≈ 0.05) were used. Height, phase, and vertical deflection information were recorded.

Electroless Deposition. Electroless deposition was performed by immersing the samples into an aqueous mixture of hydroxylamine hydrochloride (0.2 mM) and HAuCl4 (0.1% w/w). After 3 min of reaction time, the samples were extensively rinsed with ultrapure water to remove the excess seeding solution and prevent further particle enlargement. Protein Immobilization. The hydrogel sample was incubated with a solution of HS-(CH2)11-EG3-NTA (500 μM in ethanol/water 1:1, Prochimia, Sopot, Poland) at 4 C for 12 h. After repeated rinsing with water, the NTA was loaded with NiCl2 (100 mM), followed by repeated rinsing with water and PBS. A histidine-tagged variant of protein A was incubated at 4 C for 1 h (50 μg/mL in PBS), samples were rinsed three times with PBS, and Alexa Fluor 555 conjugated mouse IgG (50 μg/mL in PBS) was applied for 45 min. Subsequently, samples were rinsed three times with PBS before mounting them upside down on a glass coverslip using Elvanol. Functionalization with Cyclic RGDfK. To provide cell adhesion on PEG-DA hydrogel substrates, cRGDfK-thiol was immobilized on gold micro-nanopatterns.54 Therefore, substrates were immersed in an aqueous solution of cRGDfK-thiol (25 μM) for 45 min. To remove the noncovalently bound cRGDfK-thiol, the substrates were rinsed with Milli-Q water every 10 min for a period of 40 min. Before cells were seeded onto the substrates, PEG-DA hydrogels were transferred into sterile PBS solution and from there into warm (37 C) cell culture medium (DMEM supplemented with 10% FBS v/v). Immunofluorescence Imaging. Samples were gently rinsed with prewarmed PBS and slightly permeabilized with 0.01% Triton-X100 (Sigma-Aldrich) in PBS containing 3.8% paraformaldehyde (PFA). After a short rinse with PBS, cells were treated with PFA (3.8% w/w in PBS) for another 30 min, rinsed again with PBS, and then stained for actin and the nucleus using phalloidin-TRITC (Sigma) and DAPI (Roche), respectively. Immunofluorescence was visualized by a DeltaVision microscope (Applied Precision, Issaqua, WA) equipped with an oil immersion objective (60/1.3 NA plan-Neofluar, Olympus, Tokyo, Japan).

(59) Cleveland, J. P.; Manne, S.; Bocek, D.; Hansma, P. K. A nondestructive method for determining the spring constant of cantilevers for scanning force microscopy. Rev. Sci. Instrum. 1993, 64, 403–405. (60) Vinckier, A.; Semenza, G. Measuring elasticity of biological materials by atomic force microscopy. FEBS Lett. 1998, 430, 12–16.

Supporting Information Available: Material used for micellar nanolithography and XP spectra of the SU8 removal process. This material is available free of charge via the Internet at http:// pubs.acs.org.

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Acknowledgment. We thank Nicole Plath for help with XPS measurements and Julia Svozil and Michael Dieckmann for technical assistance. This work was funded by grants from the Landesstiftung Baden-W€ urttemberg. The Max Planck Society is acknowledged for financial support.

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