Entrapment of lipid nanoparticles in peripheral endosomes but not lysosomes impairs intracellular trafficking and endosomal escape

The uptake and intracellular trafficking of lipid nanoparticles (LNPs) along the endolysosomal pathway leading to releasing compartments is critical for delivery efficiency. How the players of the processes interact with each other to affect LNP delivery remains unclear. Here, we employed a recently developed, highly sensitive LNP labeling platform in combination with defined-state of endolysosomal activity of cells to address this outstanding question with spatiotemporal analysis. We found the endolysosomal activity (endolyosomal pH, endolysosomal protease activation), which was regulated by nutrients, determines the active endocytosis activity of cells. Elevated internalization of DNA and LNP alike by cells correlated with increased endolysosomal activity. Similar to naked DNA, elevated internalization of LNP resulted in entrapment of LNPs in peripheral endosomes, which significantly impaired the intracellular trafficking of LNP to the perinuclear lysosome region and cytosolic release of LNP cargo. On the other hand, we found the extent of perinuclear lysosomal LNP accumulation positively correlated with the level of transgene expression. Moreover, we found continuous internalization of LNP was necessary not only to saturate the degradation compartments to overcome rapid degradation of LNP but also to maintain a necessary pool of releasing compartments, shuttling between peripheral endosomes and lysosomes via anterograde transport and retrograde transport along microtubules respectively, for meaningful endosomal release. Our results suggest the balance between endocytosis and intracellular trafficking needs to be fine-tuned according to endolysosomal activity of target cell to achieve optimal cytosol release.