Twenty years ago, the discovery of peptides able to cross cellular membranes launched a novel field in molecular delivery based on these non-invasive vectors, most commonly called cell-penetrating peptides (CPPs) or protein transduction domains (PTDs). These peptides were shown to efficiently transport various biologically active molecules inside living cells, and thus are considered promising devices for medical and biotechnological developments. Moreover, CPPs emerged as potential tools to study the prime mechanisms of cellular entry across the plasma membrane. This review is dedicated to CPP fundamentals, with an emphasis on the molecular requirements and mechanism of their entry into eukaryotic cells.

Cell-penetrating peptides (CPPs) share the property of cellular internalization. The question of how these peptides reach the cytoplasm of cells is still widely debated. Herein, we have used a mass spectrometry-based method that enables quantification of internalized and membrane-bound peptides. Internalization of the most used CPP was studied at 37°C (endocytosis and translocation) and 4°C (translocation) in wild type and proteoglycandeficient Chinese hamster ovary cells. Both translocation and endocytosis are internalization pathways used by CPP. The choice of one pathway versus the other depends on the peptide sequence (not the number of positive changes), the extracellular peptide concentration, and the membrane components. There is no relationship between the high affinity of these peptides for the cell membrane and their internalization efficacy. Translocation occurs at low extracellular peptide concentration, whereas endocytosis, a saturable and cooperative phenomenon, is activated at higher concentrations. Translocation operates in a narrow time window, which implies a specific lipid/peptide co-import in cells.Cell-penetrating peptides (CPPs) 3 that share the activity of cellular entry are usually short peptides of less than 20 amino acids highly enriched in basic residues. Among them, Antp, Tat-(48 -60), and oligoarginine peptides are the most intensively studied. Despite the wide use of these CPPs as macromolecular delivery devices, the internalization mechanism of these peptides in cells still remains largely controversial. The energy dependence of the internalization mechanism is unique because all endocytotic pathways are inhibited at low temperature. Consequently, at low temperature, internalization likely reflects a direct translocation mechanism. Early studies proposed that Antp enters cells by an energy-independent membrane translocation mechanism (1). This first analysis was then contradicted by other studies that suggested, with the use of inhibitors, the involvement of endocytosis in the cellular internalization of cell-penetrating peptides (2-4).The hypothesis that endocytosis was the only internalization mechanism of CPP resulted from studies examining whether the temperature or the binding to cell-surface glycosaminoglycans (GAGs) were critical for peptide internalization. Most of these interpretations resulted from fluorescence microscopy data. For instance, it was reported that Antp, Tat, and oligoarginine peptides were not efficiently internalized in the Chinese hamster ovary (CHO) mutant pgsA-745 cell line, which does not produce cell-surface heparan sulfate or chondroitin sulfate (5, 6). However, recent data indicates that Tat-mediated transduction occurs in the absence of heparan sulfate and chondroitin sulfate (7). The discrepancies observed between studies may be explained in part by different incubation conditions (peptide/cells ratio and peptide concentration) (8), limits in fluorescence imaging, such as quenching (9), or fluorophore-dependent intracellular trafficking (10), as...