Lipid nanoparticle properties explored using online asymmetric flow field-flow fractionation coupled with small angle X-ray scattering: Beyond average characterisation

[Display omitted] •Asymmetric flow field-flow fraction with small angle x-ray scattering is useful for studying lipid-based drug delivery systems.•Encapsulation of polyA caused a significant change in particle size and size distribution.•LNP shell thickness increases as particle size increases.•Lipid composition closely matches the initial, with minor size-based variations; lipid 5 and cholesterol varying the most.•Over the size distribution, polyA loading shows a positive correlation with the abundance of lipid 5. This study employs asymmetric flow field-flow fractionation online coupled with small angle X-ray scattering at a synchrotron beamline, along with multiple downstream detectors, including multi-angle light scattering, dual wavelength UV and dRI. This setup enables size-resolved characterization of lipid nanoparticles, allowing for a detailed comparison between empty and cargo-loaded lipid nanoparticles intended for nucleic acid delivery. Batch-mode characterization techniques, including cryogenic transmission electron microscopy and dynamic light scattering, alongside collection of fractions for offline characterization with liquid chromatography-charged aerosol detection, allowed for determination of the particle morphology, hydrodynamic radius, and the lipid composition over the size distribution. Cargo-containing and empty lipid nanoparticles show differences in density, and loaded particles exhibit a broader size distribution and a higher frequency of blebs at the surface. Both samples consist of spherical core–shell structured particles, with no distinguishable internal structure. A pivotal finding, often assumed until now, is that the mole fraction of each individual lipid component closely matches the original formulation. This work contributes to a more detailed understanding of lipid nanoparticles, supporting their continued development and rational design in medical applications.