Early Endosomal Escape of a Cyclic Cell-Penetrating Peptide Allows Effective Cytosolic Cargo Delivery

Abstract

Cyclic heptapeptide cyclo(FΦRRRRQ) (cFΦR4, 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 cFΦR4 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 cFΦR4 ring), exocyclic (attachment of cargo to the Gln side chain), or bicyclic approaches (fusion of cFΦR4 and cyclic cargo rings). The overall delivery efficiency (i.e., delivery of cargo into the cytoplasm and nucleus) of cFΦR4 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 cFΦR4 a useful transporter for intracellular cargo delivery and a suitable system for investigating the mechanism of endosomal escape.

Not Just for Tumor Targeting: Unmet Medical Needs and Opportunities for Nanomedicine

“…Unfortunately, the endosomal membrane has proven to be a significant barrier toward cytoplasmic delivery by these CPPs; often a negligible fraction of the peptides escapes into the cell interior. However, in a recent study [89], the cyclic heptapeptide cyclo(FΦRRRRQ) (cFΦR4, where Φ is L-2-naphthylalanine) showed an efficient escape from early endosomes into the cytoplasm. It appears that compared with other cationic CPPs, cFΦR4 is less dependent on endosomal acidification for release and thus able to exit from the less acidic early endosomes.…”

Neuroprotective peptides fused to arginine-rich cell penetrating peptides: Neuroprotective mechanism likely mediated by peptide endocytic properties

“…It is important to note that any neuroprotective peptide fused to a CPP and internalised by endocytosis must escape the endosome to interact with its intended cytoplasmic target. However, endosomal escape appears to be a highly inefficient process (Appelbaum et al, 2012;Qian et al, 2014) (and rarely confirmed) and as a result, due to the cargo's inability to engage with its intracellular target it is unlikely to have a significant impact within the cytoplasm. In light of our recent findings, the aim of this review is to critically re-examine studies in the literature that have used neuroprotective peptides fused to cationic CPPs (i.e.…”

Inhibition of Ras Signaling by Blocking Ras–Effector Interactions with Cyclic Peptides

“…Gln-8 was therefore replaced with L-or D-arginine, to potentially improve the membrane permeability of the peptide. [17,18] To our delight, the resulting peptides (9A5 and 9A6) showed both improved cell permeability and a ~4-fold higher affinity for K-Ras (IC 50 = 0.12 and 0.17 μM, respectively) ( Figure 2a). Further modifications of 9A5 and 9A6 produced mixed results, with a few resulting in significant improvements (Table S2).…”