Guanidinium-Functionalized Flexible Azaproline Transporter for Efficient Intracellular Delivery of Proapoptotic Peptide and PDL1 Antisense Morpholino Oligo in Human Carcinoma Cells In Vitro

Cell-penetrating peptides (CPPs) are structurally diverse sophisticated tools endowed with high arginine content, amphipathicity, and well-adopted suitable secondary structures. Despite its capability of breaching the lipid barriers, CPP has major limitations such as in vivo metabolic instability, poor bioavailability, and reduced endosomal escape tendency, which are yet to be improved. In this context, we first have introduced a new class of cellular transporter having a guanidinium-functionalized δ-azaproline (δ-azp)-containing peptide where the δ-azp structurally resembles the “proline” amino acid having an additional “N” at the δ-position. This non-natural peptidic backbone was found to impart proteolytic stability, as reported earlier by our group. Herein, we report the synthesis of a flexible azaproline-tetraguanidinium transporter named FAT along with a revised scalable methodology for δ-azp compared to our previously reported procedure. FAT shows a random-coil-like structure as determined by CD spectroscopy, and is hence structurally different from the polyproline PPII helix. Direct translocation is predicted to be the possible mode of the cellular entrance of FAT into CHO cells when the “Bodipy” fluorophore is covalently attached as the cargo. Simultaneously, two other macromolecular therapeutics, e.g., proapoptotic domain peptide (PAD, a 14-mer peptide) and programmed death ligand 1 (PDL1) morpholino (a 25-mer antisense oligo), were successfully conjugated with FAT and delivered into human carcinoma cells, and their efficacy was analyzed by MTT assay and western blot technique, respectively. Having obtained promising results in internalizing different types of cargos, FAT could be envisaged as a potential drug delivery agent as an alternative to natural CPPs for future application.