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 R ₂ 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 R ₂ value and the number of intracellular endosomes harboring intact IO@LNPs. In vivo MRI from mice demonstrated a gradual decrease in R ₂ 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.