Time Resolved Inspection of Ionizable-Lipid Facilitated Lipid Nanoparticle Disintegration and Cargo Release at an Endosomal Membrane Mimic

ABSTRACT This study investigates pH-triggered fusion dynamics of lipid nanoparticles (LNPs) with an endosomal membrane mimic, addressing mechanistic aspects of a crucial yet elusive process for effective mRNA delivery. Utilizing time-resolved total internal reflection fluorescence (TIRF) imaging, we observed a delayed onset of LNP fusion upon pH drop, lasting seconds to minutes depending on pH and LNP size. Once initiated, LNP fusion and cargo escape occurred rapidly within tens to hundreds of milliseconds. While LNP disintegration is observed to lead to release of a significant portion of mRNA into the acidic environment, some mRNA molecules remained mobile on the endosomal membrane mimic due to deprotonation-resistant complex salt formation. Comparison of the fusion efficiency of two LNP formulations correlated with protein translation in human primary cell transfection data, emphasizing the importance of biophysical investigations in understanding ionizable-lipid-containing LNP-assisted mRNA delivery mechanisms and providing insights for optimizing mRNA-LNP design for enhanced endosomal escape.