Quantitative Control of Protein and Cell Interaction with Nanostructured Surfaces by Cluster Assembling

Abstract

The development of smart prosthetics, scaffolds, and biomaterials for tissue engineering and organ-on-a-chip devices heavily depends on the understanding and control of biotic/abiotic interfaces. In recent years, the nanometer scale emerged as the predominant dimension for processes impacting on protein adsorption and cellular responses on surfaces. In this context, the extracellular matrix (ECM) can be seen as the prototype for an intricate natural structure assembled by nanoscale building blocks forming highly variable nanoscale configurations, dictating cellular behavior and fate. How exactly the ECM nanotopography influences mechanotransduction, that is, the cellular capacity to convert information received from the ECM into appropriate responses, remains partially understood due to the complexity of the involved biological structures, limiting also the attempts to artificially reproduce the nanoscale complexity of the ECM. In this Account, we describe and discuss our strategies for the development of an efficient and large-scale bottom-up approach to fabricate surfaces with multiscale controlled disorder as substrates to study quantitatively the effect of nanoscale topography on biological entities. Our method is based on the use of supersonic cluster beam deposition (SCBD) to assemble, on a substrate, neutral clusters produced in the gas phase and accelerated by a supersonic expansion. The assembling of clusters in the ballistic deposition regime follows simple scaling laws, allowing the quantitative control of surface roughness and asperity layout over large areas. Due to their biocompatibility, we focused on transition metal oxide nanostructured surfaces assembled by titania and zirconia clusters. We demonstrated the engineering of structural and functional properties of the cluster-assembled surfaces with high relevance for interactions at the biotic/abiotic interface. We observed that isoelectric point and wettability, crucial parameters for the adhesion of biological entities on surfaces, are strongly influenced and controlled by the nanoscale roughness. By developing a high-throughput method (protein surface interaction microarray, PSIM), we characterized quantitatively the capacity of the nanostructured surfaces to adsorb proteins, showing that with increasing roughness the adsorption rises beyond what could be expected by the increase in specific area, paralleled by an almost linear decrease in protein binding affinity. We also determined that the spatial layout of the surface asperities effectively perceived by the cells mimics at the nanoscale the topographical ECM characteristics. The interaction with these features consequently regulates parameters significant for cell adhesion and mechanotransductive signaling, such as integrin clustering, focal adhesion maturation, and the correlated cellular mechanobiology, eventually impacting the cellular program and differentiation, as we specifically showed for neuronal cells.

Controlling Protein Adsorption through Nanostructured Polymeric Surfaces

“…According to Schulte et al this incoherent picture can be partially understood by large number of parameters affecting the adsorption process, for example, protein concentration, and by a lack of suitable tools for studying adsorption on rough surfaces. [60] To offset this problem and to correlate adsorption data with topographical surface parameters, Scopelliti et al proposed a quantitative, high-throughput method to conduct a parallel analysis of the protein-surface interaction, while systematically varying surface roughness as well as protein concentration and protein type. [61] The results indicate that the surface nanoscale topography drastically influences the amount of adsorbed proteins; in particular, the saturation uptake increases nonlinearly with increasing nanoroughness.…”

Adhesion force spectroscopy with nanostructured colloidal probes reveals nanotopography-dependent early mechanotransductive interactions at the cell membrane level

“…film rq = 15 nm. This particular value of rq was chosen because we recently demonstrated that it induces in PC12 cells mechanotransductive modulations at the level of integrin adhesion complexes and cytoskeleton (Figure 1b), as well as differentiative events, such as neuritogenesis, and a vast change in the cellular program 44,58,64 . Figure 4b; the cross section highlights distinct peaks and valleys at the nanoscale.…”

“…The obtained disordered nanostructured films possess nanotopographical features that resemble those that can be observed in in vivo ECM (e.g. in basement membranes or in the brain 5,6 ) and with nano-3D configurations (in terms of asperity dimensionality and distances) that have a potential to modulate integrin-dependent mechanotransductive processes and signalling 30,44,58,[63][64][65][66][67] ( Figure 1a). Indeed, also in those ECMs whose structure is dominated by fibrillar features, there are often irregularities at the nanoscale, e.g.…”

“…Using these nanostructured thin films as substrates ( Figure 1a), we have recently shown that the interaction of cells, in particular neuronal cells, with the nanotopographical features, impacts decisively on mechanotransductively relevant events, such as FA maturation, cytoskeletal organisation/mechanics and integrin signalling, as well as the cellular program and differentiative behaviour 44,58,63,64,68 . Instead of plating the cells on the nanostructured substrates, here we exploited the nanotopographical colloidal probes (nt-CPs) to stimulate the cells and to characterise the interfacial integrin dynamics by means of adhesion force spectroscopy ( Figure 1b).…”

“…CRL-1721.1TM). These cells interact with ns-ZrO2 films, reacting with mechanotransductive responses to the provided nanotopographical stimuli44,58,64 .For routine cell culture (subculturing every 2-3 days), the cells were kept in an incubator (Galaxy S, RS Biotech) at 37°C and 5% CO2, in RPMI-1640 medium supplemented with 10% horse serum, 5% fetal bovine serum, 2 mM L-Glutamine, 10 mM HEPES, 100 units/mL penicillin, 1 mM pyruvic acid, and 100 µg/mL streptomycin (all reagents from Sigma Aldrich, if not stated otherwise). plated in low concentration of 4.000 cells/cm 2 (to guarantee the presence of single separated cells) on Ø 40 mm glass-bottom dishes for cell culture (Willco Wells) the day before the experiment.…”