title: Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape.
abstract: Delivery of short interfering RNAs (siRNAs) remains a key challenge in the development of RNA interference (RNAi) therapeutics. A better understanding of the mechanisms of siRNA cellular uptake, intracellular transport and endosomal release could critically contribute to the improvement of delivery methods. Here we monitored the uptake of lipid nanoparticles (LNPs) loaded with traceable siRNAs in different cell types in vitro and in mouse liver by quantitative fluorescence imaging and electron microscopy. We found that LNPs enter cells by both constitutive and inducible pathways in a cell type-specific manner using clathrin-mediated endocytosis as well as macropinocytosis. By directly detecting colloidal-gold particles conjugated to siRNAs, we estimated that escape of siRNAs from endosomes into the cytosol occurs at low efficiency (1-2%) and only during a limited window of time when the LNPs reside in a specific compartment sharing early and late endosomal characteristics. Our results provide insights into LNP-mediated siRNA delivery that can guide development of the next generation of delivery systems for RNAi therapeutics.
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
authors.forename: Jerome
authors.initials: J
authors.lastname: Gilleron
authors.forename: William
authors.initials: W
authors.lastname: Querbes
authors.forename: Anja
authors.initials: A
authors.lastname: Zeigerer
authors.forename: Anna
authors.initials: A
authors.lastname: Borodovsky
authors.forename: Giovanni
authors.initials: G
authors.lastname: Marsico
authors.forename: Undine
authors.initials: U
authors.lastname: Schubert
authors.forename: Kevin
authors.initials: K
authors.lastname: Manygoats
authors.forename: Sarah
authors.initials: S
authors.lastname: Seifert
authors.forename: Cordula
authors.initials: C
authors.lastname: Andree
authors.forename: Martin
authors.initials: M
authors.lastname: Stöter
authors.forename: Hila
authors.initials: H
authors.lastname: Epstein-Barash
authors.forename: Ligang
authors.initials: L
authors.lastname: Zhang
authors.forename: Victor
authors.initials: V
authors.lastname: Koteliansky
authors.forename: Kevin
authors.initials: K
authors.lastname: Fitzgerald
authors.forename: Eugenio
authors.initials: E
authors.lastname: Fava
authors.forename: Marc
authors.initials: M
authors.lastname: Bickle
authors.forename: Yannis
authors.initials: Y
authors.lastname: Kalaidzidis
authors.forename: Akin
authors.initials: A
authors.lastname: Akinc
authors.forename: Martin
authors.initials: M
authors.lastname: Maier
authors.forename: Marino
authors.initials: M
authors.lastname: Zerial
mesh_terms: D000818:Animals; D004705:Endocytosis / Q000235:genetics*; D018014:Gene Transfer Techniques; D006046:Gold / Q000008:administration & dosage / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008055:Lipids / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics*; D053768:Metal Nanoparticles / Q000008:administration & dosage / Q000737:chemistry; D051379:Mice; D008854:Microscopy, Electron; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics*
chemical_list: D008055:Lipids; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D006046:Gold