Despite efforts to understand the interactions between nanoparticles and cells, the cellular processes that determine the efficiency of intracellular drug delivery remain unclear. Here we examine cellular uptake of short interfering RNA (siRNA) delivered in lipid nanoparticles (LNPs) using cellular trafficking probes in combination with automated high-throughput confocal microscopy. We also employed defined perturbations of cellular pathways paired with systems biology approaches to uncover protein-protein and protein-small molecule interactions. We show that multiple cell signaling effectors are required for initial cellular entry of LNPs through macropinocytosis, including proton pumps, mTOR and cathepsins. siRNA delivery is substantially reduced as ≅70% of the internalized siRNA undergoes exocytosis through egress of LNPs from late endosomes/lysosomes. Niemann-Pick type C1 (NPC1) is shown to be an important regulator of the major recycling pathways of LNP-delivered siRNAs. NPC1-deficient cells show enhanced cellular retention of LNPs inside late endosomes and lysosomes, and increased gene silencing of the target gene. Our data suggest that siRNA delivery efficiency might be improved by designing delivery vehicles that can escape the recycling pathways.
Bibliographical noteFunding Information:
We dedicate this work to the memory of MIT police officer Sean Collier who valiantly gave his life for the protection of the MIT community. We would like to thank P. Lobel and L. Huang from Rutgers University for NPC1 primary and immortalized cell lines. We wish to thank W. Salmon and N. Watson of the Whitehead Institute Core facility at MIT for help with confocal imaging. We would also like to thank D. Brown (Harvard MGH), K. Whitehead, R. Bogorad (MIT) and H. Yin (MIT) for healthy discussion. We would also like to thank J. Maraganore (Alnylam) and M. Invernale (MIT) for critical reading of the manuscript. Special thanks to D. Alakhova at University of Nebraska Medical Center for her help with graphic design. We would also like to thank Alnylam Pharmaceuticals, Control release grant EB000244 for funding. This work was supported by the National Heart, Lung, and Blood Institute, US National Institutes of Health, as a Program of Excellence in Nanotechnology (PEN) Award, Contract #HHSN268201000045C.