Cargo delivery kinetics of cell-penetrating peptides

A diversity of cell-penetrating peptides (CPPs), is known, but so far the o nly common denominator for these peptides is the ability to gain cell entry in an energy-independent manner. The mechanism used by CPPs for cell entry is largely unknown, and data comparing the different peptides are lacking. In order to gain more information about the cell-penetrating process, as w ell as to quantitatively compare the uptake efficiency of different CPPs, w e have studied the cellular uptake and cargo delivery kinetics of penetrati n, transportan, Tat (48-60) and MAP (KLAL). The respective CPPs (labelled w ith the fluorescence quencher, 3-nitrotyrosine) are coupled to small a pent apeptide cargo (labelled with the 2-amino benzoic acid fluorophore) via a d isulfide bond. The cellular uptake of the cargo is registered as an increas e in fluorescence intensity when the disulfide bond of the CPP-S-S-cargo co nstruct is reduced in the intracellular milieu. Our data show that MAP has the fastest uptake, followed by transportan, Tat(48-60) and, last, penetrat in. Similarly, MAP has the highest cargo delivery efficiency, followed by t ransportan, Tat (48-60) and, last, penetratin. Since some CPPs have been fo und to be toxic at high concentration, we characterized the influence of CP Ps on cellular 2-[H-3]deoxyglucose-6-phosphate leakage. Measurements on thi s system show that the membrane-disturbing potential appears to be correlat ed with the hydrophobic moment of the peptides. In summary, the yield and k inetics of cellular cargo delivery for four different CPPs has been quantit atively characterized. (C) 2001 Elsevier Science B.V. All rights reserved.