Control of Cell Attachment and Spreading on Poly(acrylamide

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Control of Cell Attachment and Spreading on Poly(acrylamide) Brushes with Varied Grafting Density Inga Lilge and Holger Schönherr* Physical Chemistry I, Department of Chemistry and Biology & Research Center of Micro and Nanochemistry and Engineering (Cμ), University of Siegen, Adolf-Reichwein-Strasse 2, 57076 Siegen, Germany S Supporting Information *

ABSTRACT: To achieve spatial control of fibroblast cell attachment and spreading on a biocompatible polymer coating, the effect of poly(acrylamide) (PAAm) brushes with varied grafting density was investigated. The synthesis of the brushes was performed by surface-initiated atom transfer radical polymerization (SI-ATRP). Gold substrates were modified with binary self-assembled monolayers (SAMs) of an initiator and 16-mercaptohexadecanoic acid (MHDA) as an “inert” thiol to initiate the ATRP of AAm. By using different mixtures for the binary SAMs, a series of polymer brushes with varied grafting densities were prepared. The fractional coverage of surface bound initiator was determined by grazing incidence Fourier transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and contact angle measurements. A linear relationship between the Br/S ratio determined by XPS and ToF-SIMS versus the fraction of initiator on the surface determined by water contact angle measurements was observed. The varied initiation concentration on the gold substrates yielded PAAm brushes with different thicknesses, indicating a transition from mushroom to brush regimes with increasing grafting density. Thereby we achieved exquisite control of the degree of cell adhesion. Cell attachment experiments with NIH 3T3 fibroblast cells revealed cell spreading on PAAm brushes with low grafting densities (initiator fractional coverage 0.02) have a significance of ***p < 0.0001, except when it is especially indicated: *p < 0.05, **p < 0.01, and (ns) not significant. 844

DOI: 10.1021/acs.langmuir.5b04168 Langmuir 2016, 32, 838−847

Langmuir

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the transition regime and cell attachment and spreading is prevented. This is in line with the results of Liu et al., who observed hindered cell adhesion of thick PAAm brushes on gold as well as resistance to single protein solutions and inhibition of bacterial attachment.4 The results above suggest that cell attachment is controllable by the PAAm brush grafting density and indicate that the PAAm brushes were stable in the cell culture medium. A reduced cell density was also observed for an increasing grafting density of PHEMA brushes as reported by the group of Washburn.46 Cell attachment on surfaces is mediated by the interaction between cell membrane integrins and adhesive proteins on surfaces, whereas surface hydration is generally considered to be a key factor in achieving resistance to nonspecific protein adsorption. In this way, the nonfouling properties of PAAm brushes can be attributed to the surface hydration in aqueous media. Considering the high reproducibility of the PAAm brush synthesis, the ability to cross-link PAAm brushes to vary the mechanical properties48,53 and the low cost of the monomer, PAAm brushes are a promising alternative to hydrogels and PEG-based antifouling systems.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b04168. XPS survey spectra of binary SAM monolayers and polymer brushes as well as detailed FTIR spectra of block copolymer brush films and light microscopy images of long-term cell attachment studies on polymer brush films (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel +49 271 740 2806; Fax +49 271 740 2805; e-mail [email protected] (H.S.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Yvonne Voß, Dr. Mareike Müller, andDipl.-Ing. Gregor Schulte for their constant support, stimulating discussions, and technical advice as well as Dr. Jürgen Schnekenburger (Biomedical Technology Center of the Medical Faculty Münster, Germany), who kindly provided the cell lines. We also gratefully thank the group of Prof. Dr. Carsten Engelhardt for providing access to the ToF SIMS instrument and Dr. Lars Birlenbach for the performance of the analysis. The authors additionally acknowledge financial support from the European Research Council (ERC project ASMIDIAS, Grant No. 279202) and the University of Siegen.

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CONCLUSIONS We used a simple and reproducible method for the fabrication of surface tethered PAAm brush films that vary in grafting density, which were further utilized for cell adhesion studies. In the first part of this paper, it was shown that binary SAMs can be used to vary the amount of initiating groups on the surface. The solution mixture of two thiols used during monolayer formation has an influence on the fractional coverage of the initiator on the substrate. Despite the similarity in structure, CA, XPS, and TOF-SIMS measurements revealed that the surface ratio of the initiator was always lower than expected based on its fraction in the reaction mixture that was used to modify the substrate. It was determined that the amount of active chain ends could be varied by modifying a gold substrate with mixtures containing two different relative amounts of an ATRP initiating thiol and an inactive thiol. In the second part of the paper, it was shown that the polymer brush thickness is influenced by the initiator coverage on the substrate. Changes in the composition of the SAM yield polymer films with a transition from mushroom to brush regimes with increasing grafting densities. The film thickness dependency could be directly related to the underlying initiator concentration and initiating moieties (Br−), as determined by TOF-SIMS measurements. A kinetic study additionally revealed that the trend of the layer thickness from substrates presenting different initiator molecule densities is independent from the molar mass. Thus, the grafting density of polymer brushes, grown by SI-ATRP in aqueous media, can be adjusted by varying the surface density of the initiating groups on the surface. Finally the feasibility of the PAAm brushes with varied grafting densities to influence the cell adhesion of NIH 3T3 fibroblasts was investigated. It was found that PAAm brushes with various grafting densities affected the subsequent cell attachment. Interestingly, the adhesion of cells was only observed on low density PAAm brushes, while polymer brushes with higher grafting density remained cell resistant, which was demonstrated by the complete elimination of cellular adhesion. This methodology provides opportunities for the formation of welldefined, surface grafted polymer layers with independently varied grafting densities and thus tailored interactions with biological systems.

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ABBREVIATIONS PAAm, poly(acrylamide); SI-ATRP, surface-initiated atom transfer radical polymerization; SAM, self-assembled monolayers; OEG, oligo(ethylene glycol); PEG, poly(ethylene glycol); OEGMA, oligo(ethylene glycol methyl ether methacrylate); PAAm, poly(acrylamide); FTIR, Fourier transform infrared; XPS, X-ray photoelectron spectroscopy; ToF-SIMS, time-of-flight secondary ion mass spectrometry; PDEGMA, poly(diethylene glycol methyl ether methacrylate); PMPC, poly(2-methacryloxyethyl phosphorylcholine); PHEMA, poly(hydroxyethyl methacrylate); PMDETA, 1,1,4,7,7-pentamethyldiethylene triamine; MHDA, 16-mercaptohexadecanoic acid; MUBiB, ω-mercaptoundecyl bromoisobutyrate; DEMEM, Dulbecco’s modified Eagle’s media.

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REFERENCES

(1) Choi, C. K.; Breckenridge, M. T.; Chen, C. S. Engineered materials and the cellular microenvironment: a strengthening interface between cell biology and bioengineering. Trends Cell Biol. 2010, 20, 705−714. (2) Krishnamoorthy, M.; Hakobyan, S.; Ramstedt, M.; Gautrot, J. E. Surface-initiated polymer brushes in the biomedical field: applications in membrane science, biosensing, cell culture, regenerative medicine and antibacterial coatings. Chem. Rev. 2014, 114, 10976−11026. (3) Ayres, N. Polymer brushes: Applications in biomaterials and nanotechnology. Polym. Chem. 2010, 1, 769−777. (4) Liu, Q.; Singh, A.; Lalani, R.; Liu, L. Ultralow Fouling Polyacrylamide on Gold Surfaces via Surface-Initiated Atom Transfer Radical Polymerization. Biomacromolecules 2012, 13, 1086−1092. (5) Rodriguez-Emmenegger, C.; Brynda, E.; Riedel, T.; Houska, M.; Šubr, V.; Alles, A. B.; Hasan, E.; Gautrot, J. E.; Huck, W. T. S. Polymer brushes showing non-fouling in blood plasma challenge the currently

845

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Article

Langmuir accepted design of protein resistant surfaces. Macromol. Rapid Commun. 2011, 32, 952−957. (6) Fan, X.; Lin, L.; Messersmith, P. B. Cell Fouling Resistance of Polymer Brushes Grafted from Ti Substrates by Surface-Initiated Polymerization: Effect of Ethylene Glycol Side Chain Length. Biomacromolecules 2006, 7, 2443−2448. (7) Delaittre, G.; Greiner, A. M.; Pauloehrl, T.; Bastmeyer, M.; Barner-Kowollik, C. Chemical approaches to synthetic polymer surface biofunctionalization for targeted cell adhesion using small binding motifs. Soft Matter 2012, 8, 7323−7347. (8) Brandley, B. K.; Schnaar, R. L. Covalent attachment of an ArgGly-Asp sequece peptide to derivatizable polyacrylamide surfaces: Support of fibroblast adhesion and long-term growth. Anal. Biochem. 1988, 172, 270−278. (9) Arnold, M.; Cavalcanti-Adam, E. A.; Glass, R.; Blümmel, J.; Eck, W.; Kantlehner, M.; Kessler, H.; Spatz, J. P. Activation of Integrin Function by Nanopatterned Adhesive Interfaces. ChemPhysChem 2004, 5, 383−388. (10) Kankate, L.; Werner, U.; Turchanin, A.; Gölzhäuser, A.; Großmann, H.; Tampé, R. Protein resistant oligo(ethylene glycol) terminated self-assembled monolayers of thiols on gold by vapor deposition in vacuum. Biointerphases 2010, 5, 30−36. (11) Branch, D. W.; Wheeler, B. C.; Brewer, G. J.; Leckband, D. E. Long-term stability of grafted polyethylene glycol surfaces for use with microstamped substrates in neuronal cell culture. Biomaterials 2001, 22, 1035−1047. (12) Prime, K. L.; Whitesides, G. M. Adsorption of proteins onto surfaces containing end-attached oligo(ethylene oxide): a model system using self-assembled monolayers. J. Am. Chem. Soc. 1993, 115, 10714−10721. (13) Pale-Grosdemange, C.; Simon, E. S.; Prime, K. L.; Whitesides, G. M. Formation of self-assembled monolayers by chemisorption of derivatives of oligo(ethylene glycol) of structure HS(CH2)11(OCH2CH2)mOH on gold. J. Am. Chem. Soc. 1991, 113, 12−20. (14) Luk, Y.-Y.; Kato, M.; Mrksich, M. Self-Assembled Monolayers of Alkanethiolates Presenting Mannitol Groups Are Inert to Protein Adsorption and Cell Attachment. Langmuir 2000, 16, 9604−9608. (15) Capadona, J. R.; Collard, D. M.; García, A. J. Fibronectin Adsorption and Cell Adhesion to Mixed Monolayers of Tri(ethylene glycol)- and Methyl-Terminated Alkanethiols †. Langmuir 2003, 19, 1847−1852. (16) Mrksich, M.; Dike, L. E.; Tien, J.; Ingber, D. E.; Whitesides, G. M. Using microcontact printing to pattern the attachment of mammalian cells to self-assembled monolayers of alkanethiolates on transparent films of gold and silver. Exp. Cell Res. 1997, 235, 305−313. (17) Ma, H.; Wells, M. C.; Beebe; Thomas, P.; Chilkoti, A. SurfaceInitiated Atom Transfer Radical Polymerization of Oligo(ethylene glycol) Methyl Methacrylate from a Mixed Self-Assembled Monolayer on Gold. Adv. Funct. Mater. 2006, 16, 640−648. (18) Fan, X.; Lin, L.; Dalsin, J. L.; Messersmith, P. B. Biomimetic anchor for surface-initiated polymerization from metal substrates. J. Am. Chem. Soc. 2005, 127, 15843−15847. (19) Edmondson, S.; Osborne, V. L.; Huck, W. T. S. Polymer brushes via surface-initiated polymerizations. Chem. Soc. Rev. 2004, 33, 14−22. (20) Barbey, R.; Lavanant, L.; Paripovic, D.; Schüwer, N.; Sugnaux, C.; Tugulu, S.; Klok, H.-A. Polymer Brushes via Surface-Initiated Controlled Radical Polymerization: Synthesis, Characterization, Properties, and Applications. Chem. Rev. 2009, 109, 5437−5527. (21) Advincula, R. C.; Brittain, W. J.; Caster, K. C.; Rühe, J. Polymer Brushes: Synthesis, Characterization, Applications; Wiley-VCH: Weinheim, 2004. (22) Mittal, V. Polymer Brushes: Substrates, Technologies, and Properties; Taylor & Francis: Boca Raton, FL, 2012. (23) Bourgeat-Lami, E.; Vana, P. Controlled Radical Polymerization at and from Solid Surfaces; Springer: Cham, 2016.

(24) Tugulu, S.; Klok, H.-A. Stability and Nonfouling Properties of Poly(poly(ethylene glycol) methacrylate) Brushes under Cell Culture Conditions. Biomacromolecules 2008, 9, 906−912. (25) Zhou, X.; Liu, X.; Xie, Z.; Zheng, Z. 3D-patterned polymer brush surfaces. Nanoscale 2011, 3, 4929−4939. (26) Jennings, G. K.; Brantley, E. L. Physicochemical Properties of Surface-Initiated Polymer Films in the Modification and Processing of Materials. Adv. Mater. 2004, 16, 1983−1994. (27) Pyun, J.; Kowalewski, T.; Matyjaszewski, K. Synthesis of Polymer Brushes Using Atom Transfer Radical Polymerization. Macromol. Rapid Commun. 2003, 24, 1043−1059. (28) Golas, P. L.; Tsarevsky, N. V.; Sumerlin, B. S.; Matyjaszewski, K. Catalyst Performance in “Click” Coupling Reactions of Polymers Prepared by ATRP: Ligand and Metal Effects. Macromolecules 2006, 39, 6451−6457. (29) Barbey, R.; Lavanant, L.; Paripovic, D.; Schüwer, N.; Sugnaux, C.; Tugulu, S.; Klok, H.-A. Chem. Rev. 2009, 109 (11), 5437−5527. (30) Wu, T.; Efimenko, K.; Genzer, J. Combinatorial Study of the Mushroom-to-Brush Crossover in Surface Anchored Polyacrylamide. J. Am. Chem. Soc. 2002, 124, 9394−9395. (31) Ahmad, S. A.; Leggett, G. J.; Hucknall, A.; Chilkoti, A. Microand nanostructured poly[oligo(ethylene glycol)methacrylate] brushes grown from photopatterned halogen initiators by atom transfer radical polymerization. Biointerphases 2011, 6, 8−15. (32) Ma, H.; Hyun, J.; Stiller, P.; Chilkoti, A. ″Non-Foulung” Oligo(ethylene glycol)-functionalized Polymer Brushes Synthesized by Surface-Initiated Atom Transfer Radical Polymerization. Adv. Mater. 2004, 16, 338−341. (33) Ma, H.; Li, D.; Sheng, X.; Zhao, B.; Chilkoti, A. ProteinResistant Polymer Coatings on Silicon Oxide by Surface-Initiated Atom Transfer Radical Polymerization. Langmuir 2006, 22, 3751− 3756. (34) Zhang, Z.; Vaisocherová, H.; Cheng, G.; Yang, W.; Xue, H.; Jiang, S. Nonfouling behavior of polycarboxybetaine-grafted surfaces: structural and environmental effects. Biomacromolecules 2008, 9, 2686−2692. (35) Fang, Y.; Xu, W.; Meng, X.-L.; Ye, X.-Y.; Wu, J.; Xu, Z.-K. Poly(2-hydroxyethyl methacrylate) Brush Surface for Specific and Oriented Adsorption of Glycosidases. Langmuir 2012, 28, 13318− 13324. (36) Zhang, N.; Pompe, T.; Amin, I.; Luxenhofer, R.; Werner, C.; Jordan, R. Tailored Poly(2-oxazoline) Polymer Brushes to Control Protein Adsorption and Cell Adhesion. Macromol. Biosci. 2012, 12, 926−936. (37) Al-Haque, S.; Miklas, J. W.; Feric, N.; Chiu, W. L. Y.; Chen, W. L. K.; Simmons, C. A.; Radisic, M. Macromol. Biosci. 2012, 12 (10), 1342−1353. (38) Hale, N. A.; Yang, Y.; Rajagopalan, P. Cell Migration at the Interface of a Dual Chemical-Mechanical Gradient. ACS Appl. Mater. Interfaces 2010, 2, 2317−2324. (39) Huang, X.; Doneski, L. J.; Wirth, M. J. Surface-Confindes Living Radical Polymerization for Coatings in Capillary Electrophoresis. Anal. Chem. 1998, 70, 4023−4029. (40) Fundeanu, I.; van der Mei, H. C.; Schouten, A. J.; Busscher, H. J. Polyacrylamide brush coatings preventing microbial adhesion to silicone rubber. Colloids Surf., B 2008, 64, 297−301. (41) Fundeanu, I.; van der Mei, H. C.; Schouten, A. J.; Busscher, H. J. Microbial adhesion to surface-grafted polyacrylamide brushes after long-term exposure to PBS and reconstituted freeze-dried saliva. J. Biomed. Mater. Res., Part A 2010, 94, 997−1000. (42) Singh, N.; Cui, X.; Boland, T.; Husson, S. M. The role of independently variable grafting density and layer thickness of polymer nanolayers on peptide adsorption and cell adhesion. Biomaterials 2007, 28, 763−771. (43) Wu, T.; Gong, P.; Szleifer, I.; Vlček, P.; Šubr, V.; Genzer, J. Behavior of Surface-Anchored Poly(acrylic acid) Brushes with Grafting Density Gradients on Solid Substrates: 1. Experiment. Macromolecules 2007, 40, 8756−8764. 846

DOI: 10.1021/acs.langmuir.5b04168 Langmuir 2016, 32, 838−847

Article

Langmuir (44) Lilge, I.; Steuber, M.; Tranchida, D.; Sperotto, E.; Schönherr, H. Tailored (Bio)Interfaces via Surface Initiated Polymerization: Control of Grafting Density and New Responsive Diblock Copolymer Brushes. Macromol. Symp. 2013, 328, 64−72. (45) Feng, W.; Brash, J. L.; Zhu, S. Non-biofouling materials prepared by atom transfer radical polymerization grafting of 2methacryloloxyethyl phosphorylcholine: Separate effects of graft density and chain length on protein repulsion. Biomaterials 2006, 27, 847−855. (46) Mei, Y.; Wu, T.; Xu, C.; Langenbach, K. J.; Elliott, J. T.; Vogt, B. D.; Beers, K. L.; Amis, E. J.; Washburn, N. R. Tuning Cell Adhesion on Gradient Poly(2-hydroxyethyl methacrylate)-Grafted Surfaces. Langmuir 2005, 21, 12309−12314. (47) Missirlis, D.; Spatz, J. P. Combined effects of PEG hydrogel elasticity and cell-adhesive coating on fibroblast adhesion and persistent migration. Biomacromolecules 2014, 15, 195−205. (48) Lilge, I.; Schö nherr, H. Covalently cross-linked poly(acrylamide) brushes on gold with tunable mechanical properties via surface-initiated atom transfer radical polymerization. Eur. Polym. J. 2013, 49, 1943−1951. (49) Fan, X.; Lin, L.; Messersmith, P. B. Cell Fouling Resistance of Polymer Brushes Grafted from Ti Substrates by Surface-Initiated Polymerization: Effect of Ethylene Glycol Side Chain Length. Biomacromolecules 2006, 7, 2443−2448. (50) Jones, D. M.; Brown, A. A.; Huck, W. T. S. Surface-Initiated Polymerizations in Aqueous Media: Effect of Initiator Density. Langmuir 2002, 18, 1265−1269. (51) Mrksich, M.; Whitesides, G. M. Patterning self-assembled monolayers using microcontact printing: a new technology for biosensors? Trends Biotechnol. 1995, 13, 228−235. (52) Israelachvili, J. N.; Gee, M. L. Contact angles on chemically heterogeneous surfaces: A New Look at Cassie’s and Wenzel’s Laws. Langmuir 1989, 5, 288−289. (53) Li, A.; Benetti, E. M.; Tranchida, D.; Clasohm, J. N.; Schoenherr, H.; Spencer, N. D. Surface-Grafted, Covalently CrossLinked Hydrogel Brushes with Tunable Interfacial and Bulk Properties. Macromolecules 2011, 44, 5344−5351. (54) Bain, C. D.; Whitesides, G. M. Formation of Monolayers by the Coadsorption of Thiols on Gold: Variation in the Length of the Alkyl Chain; American Chemical Society: 1989; Vol. 111, pp 7164−7175. (55) Israelachvili, J. N.; Gee, M. L. Contact angles on chemically heterogeneous surfaces: A New Look at Cassie’s and Wenzel’s Laws. Langmuir 1989, 5, 288−289. (56) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. Spontaneously organized molecular assemblies. 4. Structural characterization of n-alkyl thiol monolayers on gold by optical ellipsometry, infrared spectroscopy, and electrochemistry. J. Am. Chem. Soc. 1987, 109, 3559−3568. (57) Bhat, R. R.; Genzer, J.; Chaney, B. N. Controlling the assembly of nanoparticles using surface grafted molecular and macromolecular gradients. Nanotechnology 2003, 14, 1145−1152. (58) Akkahat, P.; Mekboonsonglarp, W.; Kiatkamjornwong, S.; Hoven, V. P. Surface-Grafted Poly(acrylic acid) Brushes as a Precursor Layer for Biosensing Applications: Effect of Graft Density and Swellability on the Detection Efficiency. Langmuir 2012, 28, 5302− 5311. (59) Cringus-Fundeanu, I.; Luijten, J.; van der Mei, H. C.; Busscher, H. J.; Schouten, A. J. Synthesis and Characterization of Surface-Grafted Polyacrylamide Brushes and Their Inhibition of Microbial Adhesion. Langmuir 2007, 23, 5120−5126. (60) Tsao, M.-W.; Rabolt, J. F.; Schönherr, H.; Castner, D. G. Semifluorinated/Hydrogenated Alkylthiol Thin Films: A Comparison between Disulfides and Thiol Binary Mixtures. Langmuir 2000, 16, 1734−1743. (61) Myshakina, N. S.; Ahmed, Z.; Asher, S. A. Dependence of amide vibrations on hydrogen bonding. J. Phys. Chem. B 2008, 112, 11873− 11877.

(62) Wang, S.; Zhu, Y. Facile Method to Prepare Smooth and Homogeneous Polymer Brush Surfaces of Varied Brush Thickness and Grafting Density. Langmuir 2009, 25, 13448−13455. (63) Xue, C.; Choi, B.-C.; Choi, S.; Braun, P. V.; Leckband, D. E. Protein Adsorption Modes Determine Reversible Cell Attachment on Poly(N-isopropyl acrylamide) Brushes. Adv. Funct. Mater. 2012, 22, 2394−2401. (64) Zhou, D.; Gao, X.; Wang, W.-j.; Zhu, S. Termination of Surface Radicals and Kinetic Modeling of ATRP Grafting from Flat Surfaces by Addition of Deactivator. Macromolecules 2012, 45, 1198−1207. (65) Bao, Z.; Bruening, M. L.; Baker, G. L. Control of the Density of Polymer Brushes Prepared by Surface-Initiated Atom Transfer Radical Polymerization. Macromolecules 2006, 39, 5251−5258.

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DOI: 10.1021/acs.langmuir.5b04168 Langmuir 2016, 32, 838−847