Modulating mRNA carrier efficacy through the molecular catwalk of ionizable nitrogens in lipidic domains

Double pH-responsive lipo-xenopeptides (lipo-XPs) with varying lipo amino fatty acid (LAF) to succinoyl tetraethylene pentamine (Stp) ratios and two distinct bundle and U-shape topologies were identified as highly effective carriers for mRNA delivery. Physicochemical properties and transfection efficacies of the carriers are strongly influenced by the lipid tail length and position of the ionizable nitrogen within the LAF domain, defined as the "molecular catwalk". Bundles containing short LAFs (8Oc, 12Bu) and U-shapes with medium-length LAFs (12Oc) and centrally placed tertiary amines exhibited strongest activity, while extreme catwalk variants (2Hd, 16Et) proved ineffective. These trends establish clear structure-activity relationships which were subjected to further mechanistic investigation. Mechanistic studies revealed topology- and LAF-specific differences in requirement for endosomal acidification and corresponding escape, indicating that different molecular arrangements trigger distinct biological mechanisms. All-atom molecular dynamics simulations at the water-octanol interface further contextualized these behaviors by resolving how protonation reshapes carrier polarity, hydration and interfacial localization. Protonation-induced relocation was more pronounced in bundles, consistent with their higher LAF content, whereas 12Oc-based U-shapes displayed more moderate shifts and retained greater octanol affinity at neutral pH. Variations in nitrogen placement modulated interfacial enrichment and hydrogen bonding, providing molecular comprehension for the experimentally observed activity profiles across the LAF-Stp carrier library.