Over the past decades, many new therapeutic approaches have been developed for several conditions, including neurodegenerative diseases. However, efficient biodistribution and delivery at biological target sites are hampered by the presence of cell and tissue barriers, and a clinical therapy is prevented by the requirement of invasive administration routes. Candidate drug conjugation to cell-penetrating
peptides, which are able to cross cellular membranes and reach biological targets even when administered systemically, represents a promising tool to overcome this issue. Here, we review the biology, classification and mechanisms of internalization of cell-penetrating peptides. We focus our attention on the cell-penetrating peptide: HIV-derived Tat peptide, and discuss its efficient but controversial use in basic, preclinical and clinical research from its discovery to the present day.