Chemical topology of lipo-amino fatty acid mRNA carriers triggers distinct lipid bulk phases and endosomal escape mechanisms

LAF-Stp xenopeptides (XPs) comprising lipoamino fatty acid (LAF) units connected with the tetraethylene pentamine derived polyamino acid Stp were identified as a novel class of amphiphilic mRNA carriers. A ‘chameleon-like’ polarity switch is observed upon transition from neutral physiological to endosomal acidic pH. Protonation of the lipidic tertiary amine of LAF results in an ∼2 log unit change in the octanol/water partition coefficient (logD), consistent with all-atom molecular dynamics simulations. mRNA delivery efficacy is tunable by the chemical topology (either a flexible ‘U-shape’ or a constrained ‘Bundle’ structure) and specific modifications in the LAF-Stp linker domains. Specifically, replacing lysine with shorter ornithine linkers further increases the structural rigidity of Bundles. For all LAF-XPs, protonation-induced solubilization of carriers triggers endosomal destabilization (evidenced by galectin-8 recruitment) and successful cytosolic mRNA transfection. Carriers with U-shape topology strongly depend on endosomal acidification, as demonstrated by an up to ∼20-fold drop in efficacy upon treatment with v-ATPase inhibitor bafilomycin, similar as found for the ionizable polymer polyethylenimine. In contrast, Bundles are far less affected (<4-fold reduction) by bafilomycin treatment. Small-angle X-ray scattering (SAXS) reveals that lipid bulk phase structures are largely pH-independent for both topologies. Interestingly, U-shapes form lamellar phases, whereas Bundle topology induces non-lamellar bicontinuous phases with negative membrane curvature. These findings suggest distinct endosomal escape mechanisms for Bundle and U-shapes. Both carriers benefit from endosomal protonation and solubilization, but only Bundles can adopt pH-independent membrane-destabilizing fusogenic phases, reflected by faster kinetics and characteristic cellular morphology in a galectin-8 assay.