Spectroelectrochemical analysis of membrane permeation mechanisms of cell-penetrating peptide-modified fluorescent proteins

Abstract

Cell penetrating peptides (CPPs) can translocate substances into cells and have potential as molecular carriers in drug delivery system (DDS). In order to elucidate the cellular internalization mechanism of cargoes utilizing CPPs, the interfacial behavior of fluorescent protein, monomeric Azami-Green (mAG) modified with two types of CPPs, ε-poly-l-lysine (εPL) and octa-arginine (R8), was studied at the liquid|liquid interface as a model of biomembrane surfaces. CPP-unmodified mAG and R8-mAG showed the aqueous adsorption process at the water|1,2-dichloroethane interface. On the other hand, εPL-mAG was transferred across the interface accompanied by the adsorption steps at both sides of the interface. Spectroelectrochemical analysis indicated that the adsorption of mAG was facilitated at the phospholipid-modified biomimetic interface. In the presence of CPPs, the adsorption of mAG on the membrane surface was inhibited by competitive adsorption with εPL, whereas the phase transfer of mAG readily occurred due to the disturbance of the membrane structure by R8. Although the R8-mAG behaves in a similar membrane reaction mechanism to the unmodified mAG, the phase transfer efficiency of εPL-mAG was improved by suppressing the interaction with the phospholipid membrane. Therefore, the membrane permeation of mAG was successfully achieved by the modification with εPL. Present findings demonstrated that εPL is a promising CPP for the membrane permeation of proteins with a high hydrophilicity and molecular weight.