Molecular orientation and stratification revealed in RNA-lipid nanoparticles using Cryogenic Orbitrap Secondary Ion Mass Spectrometry (Cryo-OrbiSIMS)

Lipid nanoparticle-RNA formulations are used for the delivery of vaccines and other therapies. RNA molecules are encapsulated within their interior through electrostatic interactions with positively charged lipids. The identity of the lipids that present at their surface play a role in how they interact with and are perceived by the body and their resultant potency. To investigate the surface chemistry of two formulations we develop cryogenic sample preparation for transmission electron microscopy (TEM) prior to depth profiling Orbitrap secondary ion mass spectrometry (Cryo-OrbiSIMS). It is found that the depth distribution of individual lipid components is revealed relative to the surface and the RNA cargo defining the core. A preferential lipid orientation can be determined for the 1,2-Dimyristoyl-glycero-3-methox-polyethylene glycol 2000 (DMG-PEG2k) molecule, by comparing the profiles of different fragments from the same molecule, from PEG or DMG. PEG fragments are found at the LNP surface, while the DMG fragments are deeper, coincident with the RNA fragment from the core, in agreement with established models of LNPs. This laboratory-based de novo analysis technique requires no labelling, providing advantages over large facility neutron scattering characterisation.