Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling

The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Nature Biotechnology (Impact Factor: 41.51). 06/2013; 31(7). DOI: 10.1038/nbt.2614
Source: PubMed


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.

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    • "Thus, mannosylation of PBAEs is clearly triggering uptake and processing mechanisms beneficial to eventual gene delivery. At the same time, studies have indicated that gene delivery can be significantly improved by designing synthetic vectors that are capable of avoiding degradative compartments of the cell [45e48] or by escaping endocytic recycling mechanisms [49]. For example, lipid nanoparticles have been associated with a " gentler " endosomal escape mechanism [50]; whereas, cationic polyplexes' pore formation and subsequent burst provide a " rougher " means of compartment escape and cargo release. "
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    • "It is also useful to note that lipid/siRNA complexes appear to use mainly the clathrin-mediated endocytosis and macropinocytosis pathways for internalization (Gilleron et al., 2013; Sahay et al., 2013). Interestingly, high-resolution images reveal that the majority of the endocytosed lipid nanoparticles typically fail to escape from the endosome before it fuses with an exosome and eventually undergoes exocytosis (Sahay et al., 2013) (or before it merges with a lysosome for the degradation of the enclosed material (Gilleron et al., 2013)). To our knowledge, the exact intracellular fate of micelleplextype siRNA delivery systems has not been determined. "
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    • "within cells [37] or may perturb mixing of πPEI polyplexes with the membrane phospholipid bilayer [38], both effects leading eventually to poor cytosolic delivery. Nonetheless, this experiment confirms that entry of the πPEI polyplexes within the vacuolar pathway plays a significant role in the efficiency of the πPEI-mediated cytosolic protein delivery. "
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