On the interactions between RNA and titrateable lipid layers: Implications for RNA delivery with lipid nanoparticles

Characterising the interaction between cationic ionisable lipids (CIL) and nucleic acids (NA) is key to understand the process of RNA lipid nanoparticle (LNP) formation and release of NAs from LNPs. Here, we have used different surface techniques to reveal the effect of pH and NA sequence on the interaction with a model system of DOPC and the CIL DLin-MC3-DMA (MC3). At only 5 % MC3, differences in the structure and dynamics of the lipid layer were observed. Looking at the structure in detail with molecular dynamics (MD) simulations, for protonated MC3, a bilayer-like structure was observed, whereas for neutral conditions, the MC3 tended to cluster closer to the layer centre. Increasing clustering of MC3 was observed for both protonation states with increasing %MC3. Both pH and %MC3 were shown to affect the absorption behaviour of erythropoietin mRNA, polyadenylic acid (polyA) and polyuridylic acid (polyU). The adsorbed amount of all studied NAs was found to increase with decreasing pH and increasing %MC3 but with different effects on the lipid layer, which could be linked to the NA secondary structure. For polyA at pH 6, adsorption to the surface of the layer was observed, whereas for other conditions and NAs, penetration of the NA into the layer resulted in the formation of a multilayer structure. By comparison to simulations excluding the secondary structure, difference in adsorption behaviours between polyA and polyU could be observed, indicating that there is a combined effect of primary and secondary structure on MC3-NA interactions.