Lipid nanoparticles using cationic ionisable lipids: Effect of cargo on structure.

Abstract

DLin-MC3-DMA (MC3), currently the most potent cationic ionisable lipid in research, is widely used in the most popular formulation for lipid nanoparticles (LNPs) for mRNA delivery, along with cholesterol, the neutral helper lipid DSPC and the PEG lipid DMPE-PEG2000. Although many reports have investigated the in vitro efficacy of these LNPs, less is known about the behaviour of the MC3 lipid and the LNP structure, especially with respect to its dependence upon the LNP cargo. In this work, we have investigated the structure and stability of LNPs formulated with three different cargos, double stranded DNA (dsDNA) and the model mRNAs polyadenylic acid (polyA) and polyuridylic acid (polyU), at two different concentrations. Small angle x-ray and neutron scattering (SAXS and SANS) measurements were performed to investigate the LNP structure, highlighting their core-shell structure. In the SAXS patterns, the peak corresponding to the internal structure is in a similar position for all the cargos, however different in shape for the polyU loaded LNPs. Through in situ SAXS measurements of the microfluidic mixing process, we were able to follow the self-assembly of the LNP, especially the internal structure and observe the appearance of the corresponding peak. These structural differences can be linked to the colloidal stability of the LNPs, measured by dynamic light scattering (DLS). For all samples, the size increased with cargo concentration and initial PDI was reasonable. In the case of the polyU LNPs, aggregation was observed within 24 hrs after preparation, whereas the other LNPs remained stable over the 41 day measurement period. In conclusion, we have characterised the effect of different cargos on the particle structure in order to contribute to the understanding of the cargo's role, aiming to link this to LNP efficacy.