Laminin-1 is a 900kD glycoprotein that is a major component of the extracellular matrix (ECM). Laminin is known to be involved in many cellular processes including cell adhesion, migration and differentiation in vivo and is often used in in vitro experiments to encourage neuronal attachment. While laminin contains some integrin-receptor binding sites, multiple domains on the protein including the peptide sequences IKVAV, YIGSR and RGD have also been shown to bind to non-integrin neuronal receptors, encourage cell adhesion, and encourage neurite outgrowth in the absence of the full protein structure. However, previous experiments performed using these peptides as substrates for neuronal culture fail to consider the effects of excretion of the cells own ECM proteins onto the surface, which we have shown can occur on surfaces with high peptide concentrations. By utilizing self-assembled monolayers (SAMs), we have created a substrate that prevents the non-specific adsorption of proteins excreted by neurons, while introducing a low-concentration of the peptide sequences to encourage neuronal attachment.

 

A pair of alkane-thiol monomers for assembly on a gold substrate have been designed which contain a tetra-ethylene glycol moiety at the tail to prevent the non-specific adsorption of proteins. One of the monomers is terminated with an azide moiety that can be used to specifically attach molecules with an alkyne group by a copper-mediated azide-alkyne cycloaddition (CuAAC) reaction, also known as the “click” reaction. By introducing 5-pentynoic acid at the N-terminus of each of the peptides during synthesis, we have introduced a bioorthogonal attachment point for reaction with the azide-terminated monomers on the surface. Once prepared, these surfaces have been confirmed to be resistant to adsorption of proteins by quartz-crystal microbalance (QCM) experiments. Primary culture neurons dissected from embryonic mice have been cultured on the surface and the effects of the peptides on neurite outgrowth have been quantified. To better understand the role of these peptides in cell adhesion, neurons transfected with a plasmid encoding EGFP-vinculin or EGFP-paxillin have been cultured on the surface and the focal adhesion morphologies have been observed in live-cell imaging experiments. These results are compared to those observed for a surface where the full laminin protein has been adsorbed. Together, these experiments help to design a surface resistant to non-specific protein adsorption that allows for neuronal adhesion, sheds light on laminin-mediated attachment, and aids in developing better small-molecule mimics of receptor-mediated cellular adhesion.