title: Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling.
abstract: 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.
authors.affiliation: The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Gaurav
authors.initials: G
authors.lastname: Sahay
authors.forename: William
authors.initials: W
authors.lastname: Querbes
authors.forename: Christopher
authors.initials: C
authors.lastname: Alabi
authors.forename: Ahmed
authors.initials: A
authors.lastname: Eltoukhy
authors.forename: Sovan
authors.initials: S
authors.lastname: Sarkar
authors.forename: Christopher
authors.initials: C
authors.lastname: Zurenko
authors.forename: Emmanouil
authors.initials: E
authors.lastname: Karagiannis
authors.forename: Kevin
authors.initials: K
authors.lastname: Love
authors.forename: Delai
authors.initials: D
authors.lastname: Chen
authors.forename: Roberto
authors.initials: R
authors.lastname: Zoncu
authors.forename: Yosef
authors.initials: Y
authors.lastname: Buganim
authors.forename: Avi
authors.initials: A
authors.lastname: Schroeder
authors.forename: Robert
authors.initials: R
authors.lastname: Langer
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Anderson
mesh_terms: D002352:Carrier Proteins; D004705:Endocytosis / Q000235:genetics*; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008055:Lipids / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics; D008562:Membrane Glycoproteins; D053768:Metal Nanoparticles / Q000008:administration & dosage* / Q000737:chemistry; D018613:Microscopy, Confocal; D000077296:Niemann-Pick C1 Protein; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008055:Lipids; D008562:Membrane Glycoproteins; C106881:NPC1 protein, human; D000077296:Niemann-Pick C1 Protein; D034741:RNA, Small Interfering; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases