Cellular and biophysical barriers to lipid nanoparticle mediated delivery of RNA to the cytosol.

Lipid nanoparticles (LNPs) are clinically approved for mRNA-based vaccines and liver-targeted siRNA delivery. However, poor nucleic acid delivery efficiency limits their application in extrahepatic tissues and tumors. Here, using live-cell and super-resolution microscopy, we identify multiple distinct steps of inefficiencies in the cytosolic delivery of both siRNA and mRNA cargoes. Membrane damages marked by galectin recruitment are conducive to cytosolic RNA release, whereas membrane perturbations recruiting the ESCRT machinery do not permit endosomal escape. Notably, only a small fraction of RNA is released from galectin-marked endosomes and, unexpectedly, many damaged endosomes contain no detectable RNA cargo. Using LNPs with both fluorescently labeled ionizable lipid and RNA, we show that these components segregate during endosomal sorting - both within single endosomes and across endosomal compartments. Finally, we visualize localized ionizable lipid enrichment in endosomal membranes and membrane damage in direct proximity to siRNA-LNPs tethered to luminal vesicle membranes. Taken together, our findings reveal multiple mechanistic barriers limiting intracellular RNA delivery by LNPs.