Liposomes and lipid nanoparticles: a tutorial for advanced chemical and structural characterisation

Liposomes and lipid nanoparticles (LNPs) are central to modern drug‑delivery strategies, yet their reliable characterisation remains challenging due to their structural complexity and the limited availability of harmonised analytical standards. In this study, we assess an extensive set of established and novel dimensional, structural, and chemical characterisation methods across a panel of well‑defined LNP formulations and two liposomal systems with distinct compositions and stabilisation mechanisms. Cryogenic transmission electron microscopy provided direct visualisation of particle morphology and lamellarity, revealing clear structural differences between homogeneous, predominantly unilamellar liposomes and heterogeneous, multilamellar liposomes, and confirming the structural consistency of LNP samples. Dimensional methods for particles in fluids generated complementary size metrics and highlighted differences in resolution as well as method‑dependent artefacts, particularly for polydisperse samples. Among these, small angle X-ray scattering unlocked structural information including bilayer thickness and multilamellar spacing under native liquid conditions, while ribonucleic acid (RNA)-specific assays provided robust quantification of total and encapsulated RNA. Advanced electron- and mass‑spectrometric techniques, delivered additional insight into surface chemistry and, critically, enabled molecular‑level analysis at the single‑particle scale. Together, these results show that no single technique can fully capture the complexity of lipid‑based delivery systems. Instead, a metrologically informed, multimodal approach is essential for generating reliable, reproducible datasets and for supporting the development of future standards for the characterisation and quality control of nanomedicine formulations.