Structure-activity relationships of siRNA carriers based on sequence-defined oligo (ethane amino) amides

Sequence defined oligo (ethane amino) amides produced by solid-phase supported synthesis using different building blocks and molecular shapes were tested for structure-activity relationships in siRNA delivery. Efficient reporter gene knockdown was obtained in a variety of cell lines using either branched three-armed structures, or lipid-modified structures with i-shape, T-shape, U-shape configuration. For the majority of structures (apart from U-shapes), the presence of 2 or 3 cysteines was strictly required for polyplex stabilization and silencing activity. Although all four building blocks contain the ethylenediamine proton sponge motif, only oligomers assembled with the tetraethylenepentamine based amino acids (Stp, Gtp, Ptp) but not with the triethylenetetramine based amino acid (Gtt) were able to mediate efficient gene silencing. For the lipopolymeric structures, out of the tested saturated (from C4 to C18) and unsaturated (C18) fatty acid moieties, two proximate oleic acids or linolic acids provided the oligomers with the best gene silencing activity and also pH specific lytic activity at pH 5.5, presumably facilitating endosomal escape of the polyplexes. Evaluation of oligomer chain length revealed a minimal number of at least two oligo (ethane amino) building blocks per oligomer arm as necessary for the vast majority of structures, but only marginal changes were found with higher numbers (structures with up to 60 ethane amino nitrogens were evaluated). Two promising carriers (T-shape 49, i-shape 229) were also evaluated for EG5 siRNA delivery. This resulted in tumor cell cycle arrest, and appearance of mitotic monoastral spindles both in vitro and in vivo upon systemic delivery. Repeated intratumoral treatment with EG5 siRNA polyplexes significantly reduced Neuro2A-eGFPLuc tumor growth in a siRNA-specific manner.