Reconstitution of lipid nanoparticle-mediated siRNA transfer across endosomal membrane mimics

Endosomal escape limits RNA delivery by lipid nanoparticles (LNPs). If escape requires membrane rupture, productive delivery would be intrinsically coupled to membrane damage and its potential toxicity. Here, we establish a cell-free system based on giant unilamellar vesicles (GUVs) with an early endosome-like lipid composition to dissect the mechanism of siRNA transfer and determine whether it can occur while membrane barrier function is preserved. Acidification promoted LNP-membrane association, lipid redistribution and siRNA translocation into the receiving lumen without detectable dissipation of transmembrane pH gradient. Complementary cryo-electron tomography revealed local interfacial lipid reorganisation without detectable membrane rupture. Fluorescence correlation spectroscopy revealed heterogeneous diffusion of translocated siRNA, consistent with free and lipid-associated species. Pre-formed RNA-lipid complexes exhibited pH-dependent dissociation and heterogeneous diffusion, supporting lipid association as a possible origin of slower luminal diffusion. Serum or apolipoprotein-E preincubation reduced siRNA translocation into the GUV lumen. Together, these findings support a model where siRNA-lipid association facilitates membrane crossing without sustained loss of barrier function. This platform provides a means to dissect LNP-mediated RNA delivery and develop formulations with improved efficacy while limiting membrane damage.