Probing the structure and pH responsiveness of next-generation mRNA vaccines based on lipid-polymer hybrid nanoparticles

RNA therapeutics are expected to revolutionize medicine. However, one of the biggest barriers to RNA therapy is to safely deliver the drugs into the correct cells. Lipid-polymer hybrid nanoparticles (LPNs), composed of ionizable cationic lipid and a polymer, have an ability to deliver RNA to target tissues, but little is known about the structure-activity relationship of these LPNs and how they interact with the biological environment after administration. We have designed monodisperse mRNA-loaded LPNs using the microfluidics manufacturing method that display very high in vitro and in vivo transfection efficiency and immunogenicity in initial vaccination studies in mice. CryoTEM imaging suggests that these LPNs display structural characteristics that depend on: (i) the lipid:polymer ratio, (ii) the lipid:mRNA ratio, and (iii) the solvents used for preparation. For an optimized mRNA-loaded LPN formulation, we aim to reveal the structure and component distribution of LPNs for a range of formulations (lipid weight % and type of RNA cargo) in conditions relevant to the extracellular and endosomal compartments (physiological and acidic pH).