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When optimizing nanocarriers, structural motifs that are beneficial for the respective type of cargo need to be identified. Here, succinoyl tetraethylene pentamine (Stp)-based lipo-oligoaminoamides (OAAs) were optimized for the delivery of plasmid DNA (pDNA). Structural variations comprised saturated fatty acids with chain lengths between C2 and C18 and terminal cysteines as units promoting nanoparticle stabilization, histidines for endosomal buffering, and disulfide building blocks for redox-sensitive release. Biophysical and tumor cell culture screening established clear-cut relationships between lipo-OAAs and characteristics of the formed pDNA complexes. Based on the optimized alternating Stp-histidine backbones, lipo-OAAs containing fatty acids with chain lengths around C6 to C10 displayed maximum gene transfer with around 500-fold higher gene expression than that of C18 lipo-OAA analogues. Promising lipo-OAAs, however, showed only moderate in vivo efficiency. In vitro testing in 90% full serum, revealing considerable inhibition of lytic and gene-transfer activity, was found as a new screening model predictive for intravenous applications in vivo.

Human transferrin protein (Tf ) modified polyplexes have already displayed encouraging potential for receptor-mediated nucleic acid delivery into tumors. The use of a blood-derived targeting protein and polydisperse macromolecular cationic subunits however presents a practical challenge for pharmaceutical grade production. Here, Tf receptor (TfR) targeted small interfering RNA (siRNA) polyplexes are designed that are completely composed of synthetic, monodisperse, and sequence-defined subunits generated by solid-phase supported synthesis. An optimized cationizable lipo-oligoaminoamide (lipo-OAA) is used for siRNA core polyplex formation, and a retro-enantio peptide (reTfR) attached via a monodisperse polyethylene glycol (PEG) spacer via click chemistry is applied for targeting. Improved gene silencing is demonstrated in TfR-expressing KB and DU145 cells. Analogous plasmid DNA (pDNA) polyplexes are successfully used for receptor-mediated gene delivery in TfR-rich K562 cells and Neuro2a cells. Six lipo-OAAs differing in their lipidic domain and redox-sensitive attachment of lipid residues are tested in order to evaluate the impact of core polyplex stability on receptor-dependent gene transfer.

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