Micellization of\nLipopeptides Containing Toll-like\nReceptor Agonist and Integrin Binding Sequences

Short bioactive peptide sequences are of great interest\nin biomaterials\ndevelopment. We investigate the self-assembly of a lipopeptide containing\nboth the highly cationic CSK<sub>4</sub> toll-like receptor agonist\nhexapeptide sequence and RGDS integrin-binding motif, i.e., C<sub>16</sub>-CSK<sub>4</sub>RGDS, as well as the control containing a\nscrambled terminal sequence C<sub>16</sub>-CSK<sub>4</sub>GRDS. Both\nlipopeptides are found to form micelles, as revealed by small-angle\nX-ray scattering and cryogenic transmission electron microscopy, and\nmodeled using atomistic molecular dynamics simulations. We carefully\nexamined methods to probe the aggregation of the molecules, i.e. to\nobtain the critical micelle concentration (CMC). Fluorescent probe\nassays using 1-anilino-8-naphthalenesulfonate (ANS) reveal low CMC\nvalues, 1–2 μM, which contrast with consistent values\nmore than 2 orders of magnitude larger obtained from surface tension\nand electrical conductivity as well as unexpected UV/vis absorption\nspectra discontinuities and fluoresccence probe assays using Nile\nred. The anomalous results obtained from an ANS fluorescence probe\nare ascribed to the effect of ANS binding to the cationic (lysine\nand arginine) residues in the lipopeptide, which leads to a conformational\nchange, as shown by circular dichroism, even at low concentrations\nbelow the actual CMC. Despite the small change in the peptide sequence\n(swapping of G and R residues), there is surprisingly a significant\ndifference in the aggregation propensity and association number, both\nof which are greater for C<sub>16</sub>-CSK<sub>4</sub>GRDS. Both\nlipopeptides are cytocompatible (with fibroblasts and myoblasts) at\nlow concentration, although cytotoxicity is noted at higher concentration.