Elucidating the underlying mechanism of mechanical stress-induced impact on mRNA-LNP structure

The COVID-19 pandemic has demonstrated the suitability of mRNA-lipid nanoparticle (LNP) drug product as an appropriate vaccine for emergency response during a global health crisis. Understanding of mRNA-LNPs stability and mechanisms of degradation is important; however a deeper mechanistic understanding of the impact of liquid-air interfaces on mRNA-LNP is still absent. This study used a combination of nanoparticle tracking analysis (NTA), nuclear magnetic resonance (NMR) spectroscopy and cryogenic electron microscopy (cryo-EM) to elucidate the dynamics occurring during shaking induced stress on mRNA-LNPs. Minimal impact is observed for mRNA-LNPs upon 30 min of shaking. However, a significant increase of particle sizes and heterogeneity, accompanied by a decrease of particle concentrations were observed by NTA upon 240 min of shaking. Cryo-EM imaging showed the formation of larger mRNA-LNP structures, which was consistent with the NTA results. Additionally, unencapsulated RNA was observed with RNA staining after prolonged shaking of mRNA-LNPs. NMR suggests that the mRNA-LNP surface structure changes significantly which was marked by changes in the lipid mobility of the PEGylated and ionizable lipids. NMR also detected distinct sucrose signals owing to the movement from the bulk solution into the larger mRNA-LNPs. Collectively, the suite of these techniques provides a deeper understanding of the dynamics leading to morphological changes of mRNA-LNPs under mechanical stress conditions by shaking.