The microenvironment of immobilized Arg-Gly-Asp peptides is an important determinant of cell adhesion.

This paper uses self-assembled monolayers on gold as a model system to demonstrate that the attachment and spreading of Swiss 3T3 fibroblasts depends strongly on the microenvironment of immobilized RGD peptides. This work utilized monolayers that present mixtures of Arg-Gly-Asp peptides, which are ligands for cellular integrin receptors, and oligo(ethylene glycol) groups, which resist the nonspecific adsorption of protein. The microenvironment of the peptide ligands was controlled by altering the length of the surrounding oligo(ethylene glycol) groups on the monolayer. By using thiols that present either tri-, tetra-, penta-, or hexa(ethylene glycol) units, the average distance separating the glycol groups and the peptide ligand is altered while the structure and properties of the background remain unchanged. Cell attachment to monolayers presenting a fixed density of peptide decreased as the length of the oligo(ethylene glycol) group increased. The average projected area of attached cells showed a similar trend. At lower densities of immobilized peptide, decreases in both cell attachment and projected cell area were more pronounced. Attachment and spreading did not depend on density of peptide on monolayers presenting tri(ethylene glycol) groups, but showed a high sensitivity to density of ligand on monolayers presenting longer glycol oligomers. Experiments that used a soluble peptide to inhibit the attachment of cells to monolayers demonstrated that the strength of the cell-substrate interaction decreased on monolayers presenting longer glycol groups. Together, these results suggest that the microenvironment of the peptide ligand influences the affinity of the integrin-peptide interaction and that weaker interactions display a density-dependent enhancement of binding during cell attachment and spreading. This finding is an important consideration in studies that correlate biological function with the composition of ligands on a substrate. This finding also represents an important principle for the design of biologically active materials because it illustrates the degree to which the presentation of adhesion motifs can modify the response of mammalian cells.