Abstract
Three cell-penetrating peptides (CPPs), Tat, Pep-3 and penetratin, were split into two parts and each fragment was terminated with a cysteine residue, to allow disulfide bridge formation, as well as a fluorescent label, for visualization and quantitative analysis. After disulfide formation between two complementary CPP fragments, cellular uptake of the resulting conjugates was observed. As confirmed by in vitro experiments, the conjugated peptides showed uptake activity comparable to the native CPP sequences, while the truncated peptides were hardly active. Until now, this split CPP strategy has only been demonstrated for oligo-arginine CPPs, but here we demonstrate that it is also applicable to other cell-penetrating peptides. This wider applicability may help in the design of new activatable cell-penetrating peptides for, e.g., targeted drug delivery.
Highlights
In the search for new, efficient delivery methods of therapeutic molecules and particles into cells, cell-penetrating peptides (CPPs) emerged 30 years ago as extraordinary vector molecules (Copolovici et al 2014)
We labeled all three N-terminally with 5(6)-carboxyfluorescein (Cbfl) in combination with an ε-aminohexanoic acid (6Ahx) spacer to yield CbFl-Tat, CbFl-Pep-3 and CbFl-Pen, respectively, that serve as reference CPPs
The C-terminal parts, c-TatB, c-Pep-3B and c-PenB, had a cysteine residue added to their N-terminus with a 3-nitro-2-pyridinesulfenyl (Npys) group on the thiol group that allowed for a facile preparation of the corresponding reconstituted peptides, CbFl-TatA+B, CbFl-Pep-3A+B and CbFl-PenA+B
Summary
In the search for new, efficient delivery methods of therapeutic molecules and particles into cells, cell-penetrating peptides (CPPs) emerged 30 years ago as extraordinary vector molecules (Copolovici et al 2014).
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