Counterion-mediated membrane penetration:: Cationic cell-penetrating peptides overcome Born energy barrier by ion-pairing with phospholipids

Arginine-rich cell-penetrating peptides (CPPs) can enter cells non-endocytotically, despite that transport of charge across a membrane should be formally associated with an extremely high Born energy barrier. We studied partitioning of several derivatives of the CPP penetratin in a water-octanol two-phase system in presence of natural phospholipids to explore if solvation by ion-pairing to hydrophobic counter-ions may serve as a mechanism for cell internalisation. We demonstrate that anionic lipids can aid peptide partitioning into octanol. Particularly efficient partitioning into octanol is observed with an arginine-rich penetratin compared to a lysine-rich derivative. Substituting tryptophans for phenylalanines results in poor partitioning into octanol, due to decreased overall peptide hydrophobicity. Partitioning into octanol is dependent of phospholipid type and the peptides induced structural changes in the lipid assemblies found in octanol. Attachment of carboxyfluorescein as a model cargo was found to enhance peptide partitioning into octanol. We discuss our results with respect to theoretical electrostatic energies, empirical hydrophobicity scales and in terms of implications for CPP uptake mechanisms. An important improvement of the theoretical transfer energies is obtained when, instead of singular ions, the insertion of ion-paired dipolar species is considered. (c) 2007 Elsevier B.V. All rights reserved.