The application of cell-penetrating peptides (CPPs) for delivering various cargo molecules with biologicalfunctions into cells has gained much attention in recent years. However, the internalizationmechanisms and delivery properties of CPP–cargo remains controversial. In this study, low- and highmolecular-weight cargoes attached to arginine-rich CPPs were employed: the former was the fluoresceinisothiocyanate-labeled nona-arginine (CPP–FITC), and the latter was the fluorescently labeled nonaarginine–avidin complex (CPP–avidin). We measured the intracellular trafficking of CPP–FITC andCPP–avidin in four cancer cell lines in a series of microenvironments altered by the presence or absenceof serum, different temperatures and different incubation times. The results revealed that CPP–cargodelivery exhibited no specificity toward any cell line, but the levels were found to be related to cell
type and cargo. Furthermore, their endocytic mechanisms were investigated via incubation with relatedendocytic inhibitors. Two different types of CPP–cargo were required to cross the plasma membraneto bind to cell surface-associated heparan sulfate proteoglycans in a time-dependent manner. CPPs andsmall cargoes attached to CPP may enter cells rapidly via direct translocation in addition to the endocyticroute. Translocation of large components linked to CPP tended to be mediated by macropinocytosis in anenergy-dependent manner with slower rates for larger compounds. In contrast, the clathrin-dependentpathway is not essential to the translocation of either type of CPP–cargo.