Structural Modifications to the Internal Oligoguanidinium Transporter Uncover Two Potent Analogues that Effectively Deliver the Proapoptotic Peptide in Multiple Cancer Cell Lines.

Efficient cytosolic delivery with serum-independent kinetics and low toxicity are the ultimate challenges towards the transformation of an antisense oligonucleotide or a therapeutic peptide to a suitable drug candidate for clinical trials. Most delivery vehicles falter on at least one of the above requirements, which hinders their potential in in vivo models as well. Our previous reports on internal guanidinium transporters (IGTs) have established the diversity of this particular class of molecule with the efficient delivery of antisense phosphorodiamidate morpholino oligonucleotides. In this paper, we report twenty IGTs with different types of evidence-backed structural modifications with different types of head-group linkage R, which significantly change the transfection, toxicity, and endosomal escape. Based on these three criteria, the analogues were sorted systematically to find the more promising IGTs, which were then further examined by LysoTracker studies. Finally, two analogues, with cholesteryl linkage (R = Chol) and pentafluorobenzyl linkage (R = PF Cbz), were selected for a proapoptotic peptide delivery as the final validation using a long-chain di-acid linker conjugation. Detailed mechanistic studies also revealed that the primary pathway of endocytosis is macropinocytosis, and that other pathways play different roles depending on the head group of the IGT. Since endocytosis pathways for entry depend on the nature of the cell line, we have shown the mechanistic variations in two cell lines for validation.