Lipid monolayers adsorbing mRNA as models for mRNA enclosed in lipid nanoparticles for transfection

In lipid-based mRNA pharmaceuticals applicable for vaccination, cancer therapy, and other types of therapeutics, the negatively charged RNA is embedded in nanoparticles containing positively charged or ionizable lipids. The local cohesion at the lipid-RNA interface plays an important role for the stability and biological activity of the resulting nanoparticles. Ions and other buffer components in the RNAs immediate surroundings may affect RNA stability against hydrolysis and its binding strength to the oppositely charged lipid layers, which is relevant for release after cellular uptake and endosomal processing. Here, we use Langmuir monolayers at the air/water interface as well-defined experimental models to study the local molecular organization of mRNA adsorbed to lipid layers. Binding of mRNA in the presence of monovalent and divalent ions from the aqueous phase to monolayers consisting of cationic transfection lipids and zwitterionic phospholipids is investigated with synchrotron-based x-ray fluorescence and scattering techniques. The experiments provide detailed insights into the structure of the adsorbed layers as well as the fractions of all molecular moieties contributing to the interfacial electrostatic balance, specifically phosphates from RNA, phosphates from phospholipids in the monolayer, and elemental counterions. The mRNA forms discrete electron-dense layers tightly bound to the lipid membrane, where cationic ions accumulate at the interface together with the mRNA. This leads to an excess of anionic nucleotides relative to cationic lipids at the interface, which is more pronounced in the presence of divalent compared to monovalent cations. The quantitative information on local interfacial moieties obtained here may constitute a valuable basis for the evaluation of quality aspects of nanoparticles comprising mRNA in the bulk phase.