Tuning siRNA packing order in lipid nanoparticles modulates oligonucleotide functional delivery

Abstract Efficient siRNA delivery by lipid nanoparticles (LNPs) is widely attributed to carrier composition, yet how intraparticle packing governs function remains unclear. Here, we developed a single-particle fluorescence microscopy assay that simultaneously quantifies size and siRNA loading of individual, chromophore-labeled LNPs. Imaging ~0.5M particles per condition per hour uncovered two major packing modes: a high and a low order corroborated by cryo-EM. Quantitative live cell imaging combined with systematic variation of LNPs lipid composition and N/P ratio allowed deconvolution of siRNA packing, internalization, and silencing and its dependance on lipid composition and electrostatics. Surprisingly, low-order particles while encapsulating modest RNA mediate more efficient knockdown of a fluorescent reporter than their high-order counterparts. Guided by these findings we predicted and experimentally validated that tuning composition and N/P ratio to favor less compact siRNA packing enhances silencing potency. This framework offers actionable guidance for the rational optimization of LNP formulations for RNA therapeutics.