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bioRxiv · 10.64898/2026.02.06.704289

Tuning siRNA packing order in lipid nanoparticles modulates oligonucleotide functional delivery

Breuer, A.·Kyriakakis, G.·Dreisler, M. W.·Schulz, F. H.·Bolis, G.·Margaritaki, S.·Papageorgiou, V.·Spacho, N.·Hatzakis, N. S.
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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.

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Breuer, A., Kyriakakis, G., Dreisler, M. W., Schulz, F. H., Bolis, G., Margaritaki, S., Papageorgiou, V., Spacho, N., Hatzakis, N. S.. 2026-02-06. Tuning siRNA packing order in lipid nanoparticles modulates oligonucleotide functional delivery. https://doi.org/10.64898/2026.02.06.704289

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