BPS2026 – Functional consequences of nanoscale heterogeneity in RNA-loaded lipid nanoparticles

Lipid nanoparticles (LNPs) are the leading delivery vehicle for RNA therapeutics, yet their performance remains variable and incompletely understood. A central challenge is to define how nanoscale structure governs the efficiency of RNA release into cells. Conventional methods report only ensemble averages, obscuring particle-to-particle variability. Even advanced single-particle approaches rarely provide both size and cargo content at the throughput needed to resolve functional subpopulations. As a result, it has remained unclear whether higher RNA loading per particle directly translates into improved biological activity. Here, we present a high-throughput single-particle fluorescence assay that quantifies diameter and RNA cargo across ∼1 million particles per condition. Combined with cryo-electron microscopy and live-cell imaging, this approach provides an integrated framework to connect structural heterogeneity with uptake and functional release. Our analysis revealed previously hidden correlations between RNA loading, lipid composition, and nanoparticle dimension that profoundly affect biological outcome. Importantly, we find that formulations with similar average encapsulation can display strikingly different efficiencies of functional release, demonstrating that bulk measurements are poor predictors of delivery. This toolbox establishes a scalable strategy for mapping heterogeneity across large particle populations, enabling mechanistic insights that are inaccessible with conventional ensemble approaches. More broadly, it highlights that nanoscale diversity within nominally identical formulations can dictate delivery performance, providing new levers for the rational design of next-generation nucleic acid medicines.