Cell interactions with hierarchically structured nano-patterned adhesive surfaces.

The activation of well-defined numbers of integrin molecules in predefined areas by adhesion of tissue cells to biofunctionalized micro-nanopatterned surfaces was used to determine the minimum number of activated integrins necessary to stimulate focal adhesion formation. This was realized by combining micellar and conventional e-beam lithography, which enabled deposition of 6 nm large gold nanoparticles on predefined geometries. Patterns with a lateral spacing of 58 nm and a number of gold nanoparticles, ranging from 6 to 3000 per adhesive patch, were used. For α(v) ß(3)-integrin activation, gold nanoparticles were coated with c(-RGDfK-)-thiol peptides, and the remaining glass surface was passivated to prevent non-specific protein adsorption and cell adhesion. Results show that focal adhesion formation is dictated by the underlying hierarchical nanopattern. Adhesive patches with side lengths of 3000 nm and separated by 3000 nm, or with side lengths of 1000 nm and separated by 1000 nm, containing approximately 3007 ± 193 or 335 ± 65 adhesive gold nanoparticles, respectively, induced the formation of actin-associated, paxillin-rich focal adhesions, comparable in size and shape to classical focal adhesions. In contrast, adhesive patches with side lengths of 500, 250 or 100 nm, and separated from adjacent adhesive patches by their respective side lengths, containing 83 ± 11, 30 ± 4, or 6 ± 1 adhesive gold nanoparticles, respectively, showed a significant increase in paxillin domain length, caused by bridging the pattern gap through an actin bundle in order to mechanically, synergistically strengthen each single adhesion site. Neither paxillin accumulation nor adhesion formation was induced if less than 6 c(-RGDfK-)-thiol functionalised gold nanoparticles per adhesion site were presented to cells.

Induction of cell polarization and migration by a gradient of nanoscale variations in adhesive ligand spacing.

Cell interactions with adhesive surfaces play a vital role in the regulation of cell proliferation, viability, and differentiation, and affect multiple biological processes. Since cell adhesion depends mainly on the nature and density of the adhesive ligand molecules, spatial molecular patterning, which enables the modulation of adhesion receptor clustering, might affect both the structural and the signaling activities of the adhesive interaction. We herein show that cells plated on surfaces that present a molecularly defined spacing gradient of an integrin RGD ligand can sense small but consistent differences in adhesive ligand spacing of about 1 nm across the cell diameter, which is approximately 61 mum when the spacing includes 70 nm. Consequently, these positional cues induce cell polarization and initiate cell migration and signaling. We propose that differential positional clustering of the integrin transmembrane receptors is used by cells for exploring and interpreting their environment, at high spatial sensitivity.

Cell adhesion and polarisation on molecularly defined spacing gradient surfaces of cyclic RGDfK peptide patches.

In vivo cell migration and location are orchestrally guided by soluble and bound chemical gradients. Here, gradients of extracellular matrix molecules are formed synthetically by the combination of a surface nanopatterning technique called block copolymer nanolithography (BCN) and a biofunctionalisation technique. A modified substrate dip-coating process of BCN allows for the formation of precise molecular gradients of cyclic RGDfK peptide patches at interfaces, which are presented to cells for testing cell adhesion and polarisation. Surfaces formed by BCN consist of hexagonally ordered gold dot patterns with a gradient in particle spacing. Each dot serves as a chemical anchor for the binding of cyclic RGDfK peptides, which are specifically recognised by alpha(v)beta(3) integrins. Due to steric hindrance only up to one integrin binds to one functionalised gold dot which forms a peptide patch spacing. We demonstrate how cell morphology, adhesion area, actin and vinculin distribution as well as cell body polarisation are influenced by the peptide patch spacing gradient. As a consequence, these gradients of adhesive ligands induce cell orientation towards smaller particle spacing when the gradient strength is 15nm/mm at least. This implicates that an adherent cell's sensitivity to differentiate between ligand patch spacing is approximately 1nm across the cell body.

Activation of integrin function by nanopatterned adhesive interfaces.

To study the function behind the molecular arrangement of single integrins in cell adhesion, we designed a hexagonally close-packed rigid template of cell-adhesive gold nanodots coated with cyclic RGDfK peptide by using block-copolymer micelle nanolithography. The diameter of the adhesive dots is < 8 nm, which allows the binding of one integrin per dot. These dots are positioned with high precision at 28, 58, 73, and 85 nm spacing at interfaces. A separation of > or = 73 nm between the adhesive dots results in limited cell attachment and spreading, and dramatically reduces the formation of focal adhesion and actin stress fibers. We attribute these cellular responses to restricted integrin clustering rather than insufficient number of ligand molecules in the cell-matrix interface since "micro-nanopatterned" substrates consisting of alternating fields with dense and no nanodots do support cell adhesion. We propose that the range between 58-73 nm is a universal length scale for integrin clustering and activation, since these properties are shared by a variety of cultured cells.