Uptake of cell-penetrating peptides is dependent on peptide-to-cell ratio rather than on peptide concentration

The influence of the peptide-to-cell ratio and energy depletion on uptake and degradation of the cell-penetrating peptides (CPPs) MAP (model amphipathic peptide) was investigated. The intracellular concentration of the CPPs, MAP and penetratin was monitored while varying the number of cells at fixed peptide concentration and incubation volume, or changing the concentration and incubation volume at fixed cell number. The uptake of CPPs was shown to be dependent on the peptide/cell ratio. At given peptide concentration and incubation volume, the intracellular concentration of peptide increased with lower cell number. At given cell number, doubling of the incubation volume increased intracellular peptide concentration to a similar extent as the doubling in incubation concentration. From a practical view, this means that the peptide/cell ratio has at least the same importance for the uptake of CPPs as the used peptide concentration. No influence of the peptide/cell ratio was found for the cellular uptake of peptide nucleic acid (PNA), or a non-amphipathic MAP analogue, investigated in parallel for comparison purposes. Energy depletion resulted in significantly reduced quantities of intracellular fluorescence label. Moreover, we show that this difference is mainly due to a membrane-impermeable fluorescent-labelled degradation product, which is lacking in energy-depleted cells. The mechanism of its generation is not likely to be endosomal degradation of endocytosed material, as it is not chloroquine- or brefeldin-sensitive. (C) 2004 Elsevier B.V. All rights reserved.