Intracellular trafficking of lipid nanoparticles is hindered by cholesterol.

The intracellular trafficking of lipid nanoparticles (LNPs) leading to endosomal escape is critical for delivery efficiency. How components of LNP affect its intracellular trafficking and delivery efficiency remains unknown. Here, we developed a highly sensitive LNP/nucleic acid tracking platform based on streptavidin-biotin-DNA complex and high throughput imaging. Naked nucleic acids were found to be retained in the endocytotic vesicles proportional to endocytosis activity. With the help of LNP, nucleic acids were transported along the endolysosomal pathway with N/P ratio as low as 2 amongst very weak nucleic acid and LNP interaction. As the N/P ratio increases (concomitant concentration increase of all lipids), the monophasic endocytosis of LNP-DNA demonstrated biphasic characteristics, as shown by accumulation of LNP-DNA trapped in early endosomes in the peripheral of cells. Through a series of specifically designed LNPs, we found increase in N/P ratio alone, i.e. increase of ionizable lipid content, had no effect on the formation of peripheral LNP-endosomes. Importantly, increase in cholesterol content, via dose or concentration increase, positively correlated with formation and aggregation of peripheral LNP-endosomes. Meanwhile, helper lipid such as DSPC alleviated the detrimental effect of cholesterol on aggregation of peripheral LNP-endosomes. The trapping of LNP-nucleic acids in peripheral early endosomes hindered their intracellular trafficking along the endolysosomal pathway, thus reducing their reach to releasing compartments and diminishing cargo delivery efficiency. Our results demonstrate that high cholesterol content hinders LNP intracellular trafficking, which is detrimental for intracellular delivery of cargo.