Ion Mobility-Mass Spectrometry\nCaptures the Structural\nConsequences of Lipid Nanoparticle Encapsulation on Ribonucleic Acid\nCargo

Ribonucleic acids (RNAs) are becoming increasingly significant\nin our search for improved biotherapeutics. RNA-based treatments offer\nhigh specificity, targeted delivery, and potentially lower-cost options\nfor various debilitating human diseases. Despite these benefits, there\nare still relatively few FDA-approved RNA-based therapies, with the\nnotable exceptions being the mRNA (mRNA) COVID-19 vaccines, which\nare delivered using lipid nanoparticle (LNP) systems. LNPs are distinctive\ndrug delivery systems (DDSs) because of their ability to target specific\ncells, their biocompatibility, and their efficiency in merging with\ncellular membranes to enhance treatment effectiveness. While the biophysical\nlandscapes of RNA structures in solution are relatively well understood,\nthe impact of the LNP environment on RNA remains less clear. This\nstudy uses native ion mobility-mass spectrometry (IM-MS) and collision-induced\nunfolding (CIU) techniques to investigate how LNP encapsulation affects\nRNA structure and stability. We examine how various factors, such\nas ionization polarity, cofactor binding, lipid types, and lipid ratios,\ninfluence LNP-released RNA cargo. Our findings reveal that LNP DDSs\ninduce significant changes in the structures and stabilities of their\nRNA cargo. However, the extent of these changes strongly depends on\nthe type and composition of the lipids used. We conclude by discussing\nhow IM-MS and CIU can aid in the continued development of more efficient\nLNP DDSs and improve DDS selection methodologies overall.