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. Owing 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) analog ((D)-PEN). These effects were investigated when exerted on hepatocellular (HepG2) or intestinal (Caco-2 and 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, a 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 pronounced 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), although (D)-PEN exhibited a longer half-life when compared with 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.