Review Cell entry of cell penetrating peptides: tales of tails wagging dogs Arwyn T. Jones ⁎, Edward J. Sayers Cardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, Wales, CF10 3NB, United Kingdom abstract article info Article history: Received 13 March 2012 Accepted 2 April 2012 Available online 10 April 2012 Keywords: Cell penetrating peptides (CPPs) Fluorophores Photostimulation Plasma membrane Peptide therapeutics Cell penetrating peptides hold considerable potential for academic and pharmaceutical remits with an inter- est in delivering macromolecules to the insides of cells. Hundreds of sequences now fall within the cell pen- etrating peptide classification and HIV-Tat, penetratin, transportan, and octaarginine represent extensively studied variants. The process by which membrane translocation is achieved has received significant interest in an aim to exploit new mechanistic knowledge to gain higher efficiency of penetration. There is evidence that many of the most well studied peptides are able to deliver themselves, relatively small cargo and possi- bly large macromolecular structures directly across the plasma membrane but there is also support for the involvement of an endocytic pathway or pathways. This review focuses on recent findings relating to exper- imental protocols and cell penetrating peptide modifications or extensions that yield significant effects on penetration capability. Relatively small changes in extracellular peptide concentrations, the inclusion or ab- sence of serum from the incubation medium and the in vitro model exemplify variables that significantly in- fluence the capacity of CPPs to penetrate membranes. Attachment of any type of cargo to these entities has the potential to affect their interaction with cells. There is increasing evidence to suggest that this is true for relatively small molecules such as fluorescent probes and hydrophobic adducts such as lipids and short peptide sequences designed as peptide therapeutics. Information gained from these findings will improve our knowledge of, and capacity to study the interactions of CPPs with cells, and this will accelerate their translation as efficient vectors from the in vitro setting into the clinical arena. © 2012 Elsevier B.V. All rights reserved. Contents 1. Introduction and CPP classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583 2. Well studied CPPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583 3. Methodological parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584 3.1. Artefacts of fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584 3.2. Effective peptide concentration for cell interaction and entry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584 4. CPP membrane effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585 4.1. Peptide concentration and membrane disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585 4.2. Membrane repair responses and other downstream effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585 4.3. Cell surface sugars and CPP interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 586 5. Influence of fluorophores and other detection systems on CPP uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 586 5.1. Fluorophores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 586 5.2. Photostimulation in CPP mediated delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587 5.3. Alternative labelling of CPPs for cell uptake studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587 6. CPPs as vectors for peptide therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587 6.1. Peptide cargo effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587 7. Hydrophobicity and CPP uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588 7.1. Hydrophobic terminals enhancing cell entry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588 7.2. Influence of tryptophan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588 7.3. Lipidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588 Journal of Controlled Release 161 (2012) 582–591 ⁎ Corresponding author. Tel.: + 44 2920876431; fax: + 44 2920874536. E-mail address: jonesat@cardiff.ac.uk (A.T. Jones). 0168-3659/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2012.04.003 Contents lists available at SciVerse ScienceDirect Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel