Nano Research

, Volume 7, Issue 3, pp 399–409 | Cite as

Large-scale dendrimer-based uneven nanopatterns for the study of local arginine-glycine-aspartic acid (RGD) density effects on cell adhesion

  • Anna LagunasEmail author
  • Albert G. Castaño
  • Juan M. Artés
  • Yolanda Vida
  • Daniel Collado
  • Ezequiel Pérez-Inestrosa
  • Pau Gorostiza
  • Silvia Claros
  • José A. Andrades
  • Josep Samitier
Research Article


Cell adhesion processes are governed by the nanoscale arrangement of the extracellular matrix (ECM), being more affected by local rather than global concentrations of cell adhesive ligands. In many cell-based studies, grafting of dendrimers on surfaces has shown the benefits of the local increase in concentration provided by the dendritic configuration, although the lack of any reported surface characterization has limited any direct correlation between dendrimer disposition and cell response. In order to establish a proper correlation, some control over dendrimer surface deposition is desirable. Here, dendrimer nanopatterning has been employed to address arginine-glycine-aspartic acid (RGD) density effects on cell adhesion. Nanopatterned surfaces were fully characterized by atomic force microscopy (AFM), scanning tunneling microscopy (STM) and X-ray photoelectron spectroscopy (XPS), showing that tunable distributions of cell adhesive ligands on the surface are obtained as a function of the initial dendrimer bulk concentration. Cell experiments showed a clear correlation with dendrimer surface layout: Substrates presenting regions of high local ligand density resulted in a higher percentage of adhered cells and a higher degree of maturation of focal adhesions (FAs). Therefore, dendrimer nanopatterning is presented as a suitable and controlled approach to address the effect of local ligand density on cell response. Moreover, due to the easy modification of dendrimer peripheral groups, dendrimer nanopatterning can be further extended to other ECM ligands having density effects on cells.


dendrimer arginine-glycine-aspartic acid atomic force microscopy scanning tunneling microscopy cell adhesion focal adhesions 


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Supplementary material

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  1. [1]
    Mager, D. M.; LaPointe, V.; Stevens, M. M. Exploring and exploiting chemistry at the cell surface. Nat. Chem. 2011, 3, 582–589.CrossRefGoogle Scholar
  2. [2]
    Geiger, B.; Spatz, J. P.; Bershadsky, A. D. Environmental sensing through focal adhesions. Nat. Rev. Mol. Cell Biol. 2009, 10, 21–33.CrossRefGoogle Scholar
  3. [3]
    Geiger, B.; Bershadsky, A.; Pankov, R.; Yamada, K. M. Transmembrane extracellular matrix-cytoskeleton crosstalk. Nat. Rev. Mol. Cell Biol. 2001, 2, 793–804.CrossRefGoogle Scholar
  4. [4]
    Vogel, V.; Sheetz, M. Local force and geometry sensing regulate cell functions. Nat. Rev. Mol. Cell Biol. 2006, 7, 265–275.CrossRefGoogle Scholar
  5. [5]
    Smith, M. L.; Gourdon, D.; Little, W. C.; Kubow, K. E.; Eguiluz, R. A.; Luna-Morris, S.; Vogel, V. Force-induced unfolding of fibronectin in the extracellular matrix of living cells. PLoS Biol. 2007, 5, 2243–2254.Google Scholar
  6. [6]
    Jiang, F.; Hörber, H.; Howard, J.; Müller, D. J. Assembly of collagen into microribbons: Effects of pH and electrolytes. J. Struct. Biol. 2004, 148, 268–278.CrossRefGoogle Scholar
  7. [7]
    Abrams, G. A.; Goodman, S. L.; Nealey, P. F.; Franco, M.; Murphy, C. J. Nanoscale topography of the basement membrane underlying the corneal epithelium of the rhesus macaque. Cell Tissue Res. 2000, 299, 39–46.CrossRefGoogle Scholar
  8. [8]
    Christman, K. L.; Enriquez-Rios, V. D.; Maynard, H. D. Nanopatterning proteins and peptides. Soft Matter 2006, 2, 928–939.CrossRefGoogle Scholar
  9. [9]
    Falconnet, D.; Csucs, G.; Grandin, H. M.; Textor, M. Surface engineering approaches to micropattern surfaces for cell-based assays. Biomaterials 2006, 27, 3044–3063.CrossRefGoogle Scholar
  10. [10]
    Arnold, M.; Schwieder, M.; Blümmel, J.; Cavalcanti-Adam, E. A.; López-Garcia, M.; Kessler, H.; Geiger, B.; Spatz, J. P. Cell interactions with hierarchically structured nano-patterned adhesive surface. Soft Matter 2009, 5, 72–77.CrossRefGoogle Scholar
  11. [11]
    Malmström, J.; Christensen, B.; Jakobsen, H. P.; Lovmand, J.; Foldbjerg, R.; Sørensen, E. S.; Sutherland, D. S. Large area protein patterning reveals nanoscale control of focal adhesion development. Nano Lett. 2010, 10, 686–694.CrossRefGoogle Scholar
  12. [12]
    Deeg, J. A.; Louban, I.; Aydin, D.; Selhuber-Unkel, C.; Kessler, H.; Spatz, J. P. Impact of local versus global ligand density on cellular adhesion. Nano Lett. 2011, 11, 1469–1476.CrossRefGoogle Scholar
  13. [13]
    Rolland, O.; Turrin, C. O.; Caminade, A. M.; Majoral, J. P. Dendrimers and nanomedicine: Multivalency in action. New J. Chem. 2009, 33, 1809–1824.CrossRefGoogle Scholar
  14. [14]
    Saovapakhiran, A.; D’Emanuele, A.; Attwood, D.; Penny, J. Surface modification of PAMAM dendrimers modulates the mechanism of cellular internalization. Bioconjug. Chem. 2009, 20, 693–701.CrossRefGoogle Scholar
  15. [15]
    Albertazzi, L.; Fernandez-Villamarin, M.; Riguera, R.; Fernandez-Megia, E. Peripheral functionalization of dendrimers regulates internalization and intracelular trafficking in living cells. Bioconjug. Chem. 2012, 23, 1059–1068.CrossRefGoogle Scholar
  16. [16]
    Mikhail, A. S.; Jones, K. S.; Sheardown, H. Dendrimer-grafted cell adhesion peptide-modified PDMS. Biotechnol. Prog. 2008, 24, 938–944.CrossRefGoogle Scholar
  17. [17]
    Kino-oka, M.; Kim, J.; Kurisaka, K.; Kim, M. H. Preferential growth of skeletal myoblasts and fibroblasts in co-culture on a dendrimer-immobilized surface. J. Biosci. Bioeng. 2013, 115, 96–99.CrossRefGoogle Scholar
  18. [18]
    Lomba, M.; Oriol, L.; Sánchez-Somolinos, C.; Grazú, V.; Moros, M.; Serrano, J. L.; Martínez De la Fuente, J. Cell adhesion on surface patterns generated by the photocrosslinking of hyperbranched polyesters with a trisdiazonium salt. React. Funct. Polym. 2013, 73, 499–507.CrossRefGoogle Scholar
  19. [19]
    Kim, M. H.; Kino-oka, M.; Morinaga, Y.; Sawada, Y.; Kawase, M.; Yagi, K.; Taya, M. Morphological regulation and aggregate formation of rabbit chondrocytes on dendrimer-immobilized surfaces with D-glucose display. J. Biosci. Bioeng. 2009, 107, 196–205.CrossRefGoogle Scholar
  20. [20]
    Kim, M. H.; Kino-oka, M.; Kawase, M.; Yagi, K.; Taya, M. Synergistic effect of D-glucose and epidermal growth factor display on dynamic behaviors of human epithelial cells. J. Biosci. Bioeng. 2007, 104, 428–431.CrossRefGoogle Scholar
  21. [21]
    Maheshwari, G.; Brown, G.; Lauffenburger, D. A.; Wells, A.; Griffith, L. G. Cell adhesion and motility depend on nanoscale RGD clutering. J. Cell Sci. 2000, 113, 1677–1686.Google Scholar
  22. [22]
    Pericet-Camara, R.; Cahill, B. P.; Papastavrou, G.; Borkovec, M. Nano-patterning of solid substrates by adsorbed dendrimers. Chem. Commun. 2007, 3, 266–268.CrossRefGoogle Scholar
  23. [23]
    Tokuhisa, H.; Zhao, M. Q.; Baker, L. A.; Phan, V. T.; Dermody, D. L.; Garcia, M. E.; Peez, R. F.; Crooks, R. M.; Mayer, T. M. Preparation and characterization of dendrimer monolayers and dendrimer-alkanethiol mixed monolayers adsorbed to gold. J. Am. Chem. Soc. 1998, 120, 4492–4501.CrossRefGoogle Scholar
  24. [24]
    Pericet-Camara, R.; Papastavrou, G.; Borkovec, M. Atomic force microscopy study of the adsorption and electrostatic self-organization of poly(amidoamine) dendrimers on mica. Langmuir 2004, 20, 3264–3270.CrossRefGoogle Scholar
  25. [25]
    Li, J.; Piehler, L. T.; Qin, D.; Baker, J. R.; Tomalia, D. A. Visualization and characterization of poly(amidoamine) dendrimers by atomic force microscopy. Langmuir 2000, 16, 5613–5616.CrossRefGoogle Scholar
  26. [26]
    Mertz, L.; Hitz, J.; Hubler, U.; Weyermann, P.; Diederich, F.; Murer, P.; Seebach, D.; Widmer, I.; Stöhr, M.; Güntherodt, H. J., et al. STM investigation on single, physisorbed dendrimers. Single Mol. 2002, 5, 295–299.CrossRefGoogle Scholar
  27. [27]
    Horcas, I.; Fernández, R.; Gómez-Rodríguez, J. M.; Colchero, J.; Gómez-Herrero, J.; Baro, A. M. WSXM: A software for scanning probe microscopy and a tool for nanotechnology. Rev. Sci. Instrum. 2007, 78, 13705–13713.CrossRefGoogle Scholar
  28. [28]
    Prats-Alfonso, E.; García-Martín, F.; Bayo, N.; Cruz, L. J.; Pla-Roca, M.; Samitier, J.; Errachid, A.; Albericio, F. Facile solid-phase synthesis of biotinylated alkyl thiols. Tetrahedron 2006, 62, 6876–6881.CrossRefGoogle Scholar
  29. [29]
    Boas, U.; Heegaard, P. M. Dendrimers in drug research. Chem. Soc. Rev. 2004, 33, 43–63.CrossRefGoogle Scholar
  30. [30]
    Zhou, M.; Bentley, D.; Ghosh, I. Helical supramolecules and fibers utilizing leucine zipper-displaying dendrimers. J. Am. Chem. Soc. 2004, 126, 734–735.CrossRefGoogle Scholar
  31. [31]
    Zhou, M.; Ghosh, I. Noncovalent multivalent assembly of Jun peptides on a leucine zipper dendrimer displaying Fos peptides. Org. Lett. 2004, 20, 3561–3564.CrossRefGoogle Scholar
  32. [32]
    Huang, J. H.; Gräter, S. V.; Corbellini, F.; Rinck, S.; Bock, E.; Kemkemer, R.; Kessler, H.; Ding, J. D.; Spatz, J. P. Impact of order and disorder in RGD nanopatterns on cell adhesion. Nano Lett. 2009, 9, 1111–1116.CrossRefGoogle Scholar
  33. [33]
    Xiong, J. P.; Stehle, T.; Zhang, R. G.; Joachimiak, A.; Frech, M.; Goodman, S. L.; Arnaout, M. A. Crystal structure of the extracellular segment of integrin αVβ3 in complex with an Arg-Gly-Asp ligand. Science 2002, 296, 151–155.CrossRefGoogle Scholar
  34. [34]
    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.CrossRefGoogle Scholar
  35. [35]
    Liu, L.; Chen, S.; Giachelli, C. M.; Ratner, B. D.; Jiang, S. Controlling osteopontin orientation on surfaces to modulate endothelial cell adhesion. J. Biomed. Mater. Res. A 2005, 74A, 23–31.CrossRefGoogle Scholar
  36. [36]
    Tatkiewicz, W. I.; Seras-Franzoso, J.; García-Fruitós, E.; Vazquez, E.; Ventosa, N.; Peebo, K.; Ratera, I.; Villaverde, A.; Veciana, J. Two-dimensional microscale engineering of protein-based nanoparticles for cell guidance. ACS Nano 2013, 7, 4774–4784.CrossRefGoogle Scholar
  37. [37]
    Lehnert, D.; Wehrle-Haller, B.; David, C.; Welland, U.; Ballestrem, C.; Imhol, B. A.; Bastmeyer, M. Cell behavior on micropatterned substrata: Limits of extracellular matrix geometry for spreading and adhesion. J. Cell Sci. 2004, 117, 41–52.CrossRefGoogle Scholar
  38. [38]
    Schaller, M. D. Paxillin: A focal adhesion-associated adaptor protein. Oncogene 2001, 20, 6459–6472.CrossRefGoogle Scholar
  39. [39]
    Cavalcanti-Adam, E. A.; Volberg, T.; Micoulet, A.; Kessler, H.; Geiger, B.; Spatz, J. P. Cell spreading and focal adhesion dynamics are regulated by spacing of integrin ligands. Biophys. J. 2007, 92, 2964–2974.CrossRefGoogle Scholar
  40. [40]
    Irvine, D. J.; Hue, K. A.; Mayes, A. M.; Griffith, L. G. Simulations of cell-surface integrin binding to nanoscale- clustered adhesion ligands. Biophys. J. 2002, 82, 120–132.CrossRefGoogle Scholar
  41. [41]
    Comisar, W. A.; Mooney, D. J.; Linderman, J. J. Integrin organization: Linking adhesion ligand nanopatterns with altered cell responses. J. Theor. Biol. 2011, 274, 120–130.CrossRefGoogle Scholar

Copyright information

© Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Anna Lagunas
    • 1
    • 2
    Email author
  • Albert G. Castaño
    • 1
    • 2
  • Juan M. Artés
    • 2
    • 3
  • Yolanda Vida
    • 4
    • 5
  • Daniel Collado
    • 4
    • 5
  • Ezequiel Pérez-Inestrosa
    • 4
    • 5
  • Pau Gorostiza
    • 1
    • 2
    • 6
  • Silvia Claros
    • 1
    • 7
  • José A. Andrades
    • 1
    • 7
  • Josep Samitier
    • 1
    • 2
    • 8
  1. 1.Networking Biomedical Research Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN)BadajozSpain
  2. 2.Institute for Bioengineering of Catalonia (IBEC)BarcelonaSpain
  3. 3.Physical Chemistry DepartmentUniversity of Barcelona (UB)BarcelonaSpain
  4. 4.Andalusian Centre for Nanomedicine and Biotechnology (BIONAND)MálagaSpain
  5. 5.Organic Chemistry DepartmentUniversity of Málaga (UMA)MálagaSpain
  6. 6.Institució Catalana de Recerca i Estudis Avançats (ICREA)BarcelonaSpain
  7. 7.Cell Biology, Genetics and Physiology DepartmentUniversity of Málaga (UMA)MálagaSpain
  8. 8.Electronics DepartmentUniversity of Barcelona (UB)BarcelonaSpain

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