Probing the influence of RNA cargos and polymer ratios on the structure of mRNA vaccines based on lipid-polymer hybrid nanoparticles

RNA therapeutics are expected to revolutionize medicine. However, how to safely deliver RNA into the correct cells still a hindrance. Lipid-polymer hybrid nanoparticles (LPNs), composed of ionizable cationic lipid and a polymer, can deliver RNA to target tissues, but little is known about the structure-activity relationship of these LPNs. We have designed monodisperse mRNA-loaded LPNs using the microfluidics manufacturing method that displays very high in vitro and in vivo transfection efficiency and immunogenicity in initial vaccination studies in mice. CryoTEM imaging suggests that these LPNs show structural characteristics that depend on: (i) the lipid:polymer ratio, (ii) the lipid:RNA ratio. At the same time, based on the SAXS and SANS data we acquired, we found differences in the high-q regions, which may indicate a difference in probed structure due to the different contrasts that lipid and RNA have with x-rays and neutrons. For an optimised RNA-loaded LPN formulation, we aim to reveal the structure and component distribution of LPNs for a range of formulations (lipid:polymer ratio, lipid:RNA ratio and type of RNA cargo) in conditions relevant to the extracellular and endosomal compartment (physiological and acidic pH).