Weaker Hydrophobic Collapse in Peptide Amphiphile Supramolecular Assemblies Enhances Permeation Across a Blood-Brain...

The blood-brain barrier (BBB) prevents diffusion of most molecules from the vasculature into the brain tissue, and therefore is of great interest for therapeutic delivery into the central nervous system. We investigated the potential of supramolecular assemblies to cross the BBB using peptide amphiphiles (PAs) in which the lipid tail length was varied to modify supramolecular cohesion but maintaining the same tetrapeptide sequence VVEE. Alkyl tails containing 16, 14, or 12 carbon atoms in the PAs studied were found to contain decreasing amounts of β-sheet secondary structure by wide-angle X-ray scattering and Fourier transform infrared spectroscopy. We also found diminishing pKa values as the lipid tails were shortened, and therefore concluded that longer tails resulted in the most cohesive assemblies. Using confocal microscopy and flow cytometry, we demonstrated that PAs with longer tails had significantly higher accumulation in brain endothelial cells, whereas an in vitro BBB transwell assay showed that C12 assemblies were endocytosed but were also able to exocytose most efficiently from confluent layers of the cells. Live cell imaging demonstrated that all assemblies entered lysosomes, but transcytosis occurred to the greatest extent in C12 assemblies and to a lesser degree in the C14 PA. We conclude that strong hydrophobic collapse, combined with high pKa values, creates stable nanostructures that remain sequestered within brain endothelial cells without the possibility of permeation. Our work also indicates that PA supramolecular nanostructures retain the capacity to reassemble after transcytosis, thus offering potential for their development into therapeutic delivery platforms to the brain.