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

Lipid nanoparticle RNA (LNP-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. Here, we use a model formulation to develop cryogenic sample preparation for molecular depth profiling Orbitrap secondary ion mass spectrometry (Cryo-OrbiSIMS) preceded by morphological characterisation using cryogenic transmission electron microscopy (Cryo-TEM). 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 PEG to DMG fragments. PEG fragments are found immediately during analysis of the LNP surface, while the DMG fragments are deeper, coincident with RNA ions located in 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.