Magnetic Resonance Imaging-Based Quantification of Endosomal Escape Using Iron Oxide Nanoparticle-Loaded Lipid Nanoparticles

Lipid nanoparticles (LNPs) are considered promising and advanced nucleic acid-based therapeutic delivery platforms. The therapeutic efficacy of LNP-based drugs depends heavily on endosomal escape. However, few methods are available for quantifying the efficiency of endosomal escape in vivo. Herein, a novel method for quantifying endosomal escape efficiency using magnetic resonance imaging (MRI) is presented. In this method, ultrasmall iron oxide nanoparticles (IONPs) are synthesized and incorporated into LNPs, generating IONPs-loaded LNPs (IO@LNPs). After cellular internalization of IO@LNPs, the R₂ relaxation rate is reduced over time, suggesting the dispersal of free IONPs owing to endosomal escape. Data from electron microscopy further corroborated this finding, showing a strong correlation between the R₂ value and the number of intracellular endosomes harboring intact IO@LNPs. In vivo MRI from mice demonstrated a gradual decrease in R₂ signals at the tissue site where IO@LNPs are injected, indicating the potential application of the proposed method in vivo. These findings can advance LNP-based nucleic acid delivery research by enhancing the understanding of endosomal escape in vivo.