Ionizable Lipids Drive Subcellular Localization and Immune Cell Targeting of Barcoded Nanoparticles in Lung Cancer

To accurately predict the effect of a drug and enhance its potency, it is essential to examine not only the arrival of the carrier and its payload at the target cell but also the final destination of the subcellular organelle because a considerable number of diseases are associated with the malfunctioning of cellular organelles. Here, we present nanoparticle (NP) microscopy via signal amplification of DNA barcodes combined with the multiplexed cyclic immunofluorescence technique for quantifying multiple NP types simultaneously. This technique enhanced the fluorescence signal-to-noise by 15-fold compared to standard fluorescence <i>in situ</i> hybridization, thereby providing a more precise means of analyzing the intra- and interdistribution of three core-shell NPs (G0-P5, 7C1-F5, and C12-D) <i>in vitro</i> and in <i>vivo</i>. The <i>in vitro</i> results demonstrated that in macrophages, nucleic acids condensed with G0-C14 cationic lipids were often located in lysosomes, whereas in tumor cells, nucleic acids were mainly located in mitochondria, regardless of the type of cationic lipid. Together, the <i>in vivo</i> results reveal that nucleic acids condensed with G0-C14 cationic lipids demonstrated the greatest uptake by CD206+ immune cells, whereas nucleic acids condensed with 7C1 and C12-200 cationic lipids exhibited the highest level of uptake by CD206+CD11c+Arg1+ immune cells.