Early Endosomal Escape of a Cyclic Cell-Penetrating
Peptide Allows Effective Cytosolic Cargo Delivery
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Abstract
Cyclic
heptapeptide cyclo(FΦRRRRQ) (cFΦR<sub>4</sub>, where Φ
is l-2-naphthylalanine) was recently found
to be efficiently internalized by mammalian cells. In this study,
its mechanism of internalization was investigated by perturbing various
endocytic events through the introduction of pharmacologic agents
and genetic mutations. The results show that cFΦR<sub>4</sub> binds directly to membrane phospholipids, is internalized into human
cancer cells through endocytosis, and escapes from early endosomes
into the cytoplasm. Its cargo capacity was examined with a wide variety
of molecules, including small-molecule dyes, linear and cyclic peptides
of various charged states, and proteins. Depending on the nature of
the cargos, they may be delivered by endocyclic (insertion of cargo
into the cFΦR<sub>4</sub> ring), exocyclic (attachment of cargo
to the Gln side chain), or bicyclic approaches (fusion of cFΦR<sub>4</sub> and cyclic cargo rings). The overall delivery efficiency
(i.e., delivery of cargo into the cytoplasm and nucleus) of cFΦR<sub>4</sub> was 4–12-fold higher than those of nonaarginine, HIV
Tat-derived peptide, or penetratin. The higher delivery efficiency,
coupled with superior serum stability, minimal toxicity, and synthetic
accessibility, renders cFΦR<sub>4</sub> a useful transporter
for intracellular cargo delivery and a suitable system for investigating
the mechanism of endosomal escape