Tuning siRNA packing order in lipid nanoparticles modulates oligonucleotide functional delivery

2026 · doi:10.64898/2026.02.06.704289
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This study used single-particle microscopy to reveal that less compact siRNA packing within lipid nanoparticles enhances gene silencing potency, providing guidance for LNP formulation optimization.

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⚙ AI-generated deep summary by claude@2026-07, 2026-07-17 · read from full text ⓘ

The paper investigates how intraparticle siRNA packing order within lipid nanoparticles (LNPs) affects functional delivery, using a single-particle fluorescence microscopy assay to quantify individual LNP size and siRNA loading and relate these to packing modes inferred by cryo-EM. By performing quantitative live-cell imaging in an eGFP reporter cell line with destabilization, and systematically varying lipid composition and N/P ratio, the authors deconvolve how siRNA packing, internalization, and silencing interact. They find two major packing modes (high and low order) and report that low-order particles, despite modest RNA encapsulation, drive more efficient knockdown than high-order particles, with silencing potency improved by tuning composition and N/P ratio to favor less compact packing, which they predict and experimentally validate. The paper does not explicitly discuss endometriosis or adenomyosis; it was included in the corpus via a keyword match in the upstream search index.

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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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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 hour uncovered two major packing modes: a high and a low order corroborated by cryo-EM. Quantitative live cell imaging on destabilized eGFP reporter cell line combined with systematic variation of LNPs lipid composition and N/P ratio allowed deconvolution of the interplay between siRNA packing, cell internalization and silencing and its dependance on lipid composition and electrostatics. Our findings surprisingly revealed that low-order particles while encapsulating modest RNA, they mediate more efficient knockdown of the destabilized eGFP 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 guiding for the rational optimization of LNP formulations for RNA therapeutics. Competing Interest Statement N.S.H. is the CSO and co-founder of EDGE Biotechnologies. A.B. is part time employee of EDGE Biotechnologies. Footnotes minor modification in one figure and text editing/sharpening

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last seen: 2026-05-20T01:45:00.602351+00:00