Stereochemistry as a determining factor for the effect of a cell-penetrating peptide on cellular viability and epithelial integrity

Cell-penetrating peptides (CPPs) comprise efficient peptide-based delivery vectors. Due to the inherent poor enzymatic stability of peptides, CPPs displaying partial or full replacement of L-amino acids with the corresponding D-amino acids might possess advantages as delivery vectors. Thus, the present study aims to elucidate the membrane- and metabolism-associated effects of L-Penetratin (L-PEN) and its corresponding all-D analogue (D-PEN). These effects were investigated when exerted on hepatocellular (HepG2) or intestinal (Caco-2, IEC-6) cell culture models. The head-to-head comparison of these enantiomeric CPPs included evaluation of their effects on cell viability and morphology, epithelial membrane integrity, and cellular ultrastructure. In all investigated cell models, rapid decrease in cell viability, pronounced membrane perturbation and an altered ultrastructure were detected upon exposure to D-PEN. At equimolar concentrations, these observations were less prononced or even absent for cells exposed to L-PEN. Both CPPs remained stable for at least 2 h during exposure to proliferating cells (cultured for 24 h), albeit D-PEN exhibited a longer half-life as compared to that of L-PEN when exposed to well-differentiated cell monolayers (cultured for 18-20 days). Thus, the stereochemistry of the CPP penetratin significantly influences its effects on cell viability and epithelial integrity when profiled against a panel of mammalian cells.