Sequence-defined four-arm oligo(ethanamino)amides for pDNA and siRNA delivery: Impact of building blocks on efficacy

Abstract

Cationic oligomers were assembled by solid-phase supported synthesis in few coupling steps based on C-terminal alanine and two lysine branchings, followed by elongation of the four arms with two to five repeats of artificial oligoamino acids containing the 1,2-diaminoethane motif, and ended by N-terminal cysteines or alanines. These sequence-defined oligomers, containing between 28 and 68 protonatable nitrogens, were evaluated for complex formation with plasmid DNA (pDNA) and short interfering RNA (siRNA), followed by reporter gene transfer and gene silencing experiments in Neuro2A cells. By two simple variations, the pDNA gene transfer activity could be thousand-fold improved, exceeding the gold standard linear PEI up to >50-fold. Firstly, the N-terminal cysteines introduced for bioreversible stabilization of polyplexes by internal disulfide links after complex formation greatly enhanced gene transfer. Secondly, variation of the artificial oligoamino acid building blocks containing either triethylene tetramine (Gtt), tetraethylene pentamine (Stp), or pentaethylene hexamine (Sph) disclosed a clear ranking in the order of Sph>Stp>>Gtt for both pDNA compaction and transfection activity. Extending the chain lengths of the arms beyond three building blocks had marginal impact on the performance. For the much smaller siRNA cargo, polyplex stabilization by cysteine disulfides presents a strict requirement. Sph and Stp based cysteine-ended four-arms displayed similar binding activity, with Stp providing best gene silencing efficiency.