Cargo delivery kinetics of cell-penetrating peptides

Abstract

A diversity of cell-penetrating peptides (CPPs), is known, but so far the only 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 well as to quantitatively compare the uptake efficiency of different CPPs, we have studied the cellular uptake and cargo delivery kinetics of penetratin, transportan, Tat (48-60) and MAP (KLAL). The respective CPPs (labelled with the fluorescence quencher, 3-nitrotyrosine) are coupled to small a pentapeptide cargo (labelled with the 2-amino benzoic acid fluorophore) via a disulfide bond. The cellular uptake of the cargo is registered as an increase in fluorescence intensity when the disulfide bond of the CPP-S-S-cargo construct is reduced in the intracellular milieu. Our data show that MAP has the fastest uptake, followed by transportan, Tat(48-60) and, last, penetratin. Similarly, MAP has the highest cargo delivery efficiency, followed by transportan, Tat (48-60) and, last, penetratin. Since some CPPs have been found to be toxic at high concentration, we characterized the influence of CPPs on cellular 2-[(3)H]deoxyglucose-6-phosphate leakage. Measurements on this system show that the membrane-disturbing potential appears to be correlated with the hydrophobic moment of the peptides. In summary, the yield and kinetics of cellular cargo delivery for four different CPPs has been quantitatively characterized.

Cell-penetrating and cell-targeting peptides in drug delivery

“…Surprisingly, the disulfide conjugate also showed a higher nuclear accumulation compared to the thioether conjugate [72]. One would have expected the opposite result since the intracellular reducing environment should be strong enough to cause the disulfide bridge reduction as shown in a previous study with a CPP-peptide conjugate [73]. Therefore, as a nuclear localization sequence (NLS) is present within the Tat peptide sequence [74], it is difficult to understand why the Fab fragment enters more efficiently the nucleus with a reducible linker than with a stable one.…”

In vivo identification of ribonucleoprotein-RNA interactions

“…An efficient strategy for the delivery of bioactive compounds into living cells is the use of CPPs. The CPP transportan can cross the extracellular lipid bilayer (21) and facilitate the translocation of proteins, small molecules (22), and PNA antisense oligomers as cargoes (23). Transportan 10 (TP10), used in this study, is a truncated analogue of the full-length transportan protein lacking the GTPase activity found in the native sequence (24).…”

“…1). After transport into the cell, the disulfide bond between the CPP and PNA is reduced (22), and the PNA will hybridize to its complementary ank mRNA target. Subsequently administered UV irradiation will release the benzoyl moiety of the Bpa, creating a free phenylalanine radical able to crosslink the nearest molecules (i.e., the RBPs).…”

Calpastatin Exon 1B-Derived Peptide, a Selective Inhibitor of Calpain: Enhancing Cell Permeability by Conjugation with Penetratin

“…Here, as in most previously reported applications of penetratin, we disulfide-linked full-length Cys-penetratin to Cys-CP1B. The disulfide-linked cargo is supposed to be rapidly released in the reductive intracellular milieu (Hällbrink et al, 2001).…”