A peptide for transcellular cargo delivery: Structure-function relationship and mechanism of action

The rate of transport of small molecule drugs across biological barriers, such as the blood-brain barrier, is often a limiting factor in achieving a therapeutic dose. One proposed strategy to enhance delivery across endothelial or epithelial monolayers is conjugation to cell-penetrating peptides (CPPs); however, very little is known about the design of CPPs for efficient transcellular transport. Here, we report on transcellular transport of a CPP, designated the CL peptide, that increases the delivery of small-molecule cargoes across model epithelium approximately 10-fold. The CL peptide contains a helix-like motif and a polyarginine tail. We investigated the effect of cargo, helix-like motif sequence, polyarginine tail length, and peptide stereochemistry on cargo delivery. We showed that there is an optimal helix-like motif sequence (RLLRLLR) and polyarginine tail length (R₇) for cargo delivery. Furthermore, we demonstrated that the peptide-cargo conjugate is cleaved by cells in the epithelium at the site of a two-amino acid linker. The cleavage releases the cargo with the N-terminal linker amino acid from the peptide prior to transport out of the epithelium. These studies provide new insight into the sequence requirements for developing novel CPPs for transcellular delivery of cargo.