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Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape

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... Full description

Journal Title: Nature biotechnology 2013-07, Vol.31 (7), p.638-646
Main Author: Gilleron, Jerome
Other Authors: Querbes, William , Zeigerer, Anja , Borodovsky, Anna , Marsico, Giovanni , Schubert, Undine , Manygoats, Kevin , Seifert, Sarah , Andree, Cordula , Stöter, Martin , Epstein-Barash, Hila , Zhang, Ligang , Koteliansky, Victor , Fitzgerald, Kevin , Fava, Eugenio , Bickle, Marc , Kalaidzidis, Yannis , Akinc, Akin , Maier, Martin , Zerial, Marino
Format: Electronic Article Electronic Article
Language: English
Subjects:
RNA
Publisher: United States: Nature Publishing Group
ID: ISSN: 1087-0156
Link: https://www.ncbi.nlm.nih.gov/pubmed/23792630
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title: Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape
format: Article
creator:
  • Gilleron, Jerome
  • Querbes, William
  • Zeigerer, Anja
  • Borodovsky, Anna
  • Marsico, Giovanni
  • Schubert, Undine
  • Manygoats, Kevin
  • Seifert, Sarah
  • Andree, Cordula
  • Stöter, Martin
  • Epstein-Barash, Hila
  • Zhang, Ligang
  • Koteliansky, Victor
  • Fitzgerald, Kevin
  • Fava, Eugenio
  • Bickle, Marc
  • Kalaidzidis, Yannis
  • Akinc, Akin
  • Maier, Martin
  • Zerial, Marino
subjects:
  • Animals
  • Biological transport
  • Biotechnology
  • Cellular biology
  • Endocytosis - genetics
  • Gene Transfer Techniques
  • Gold - administration & dosage
  • Gold - chemistry
  • Green Fluorescent Proteins - antagonists & inhibitors
  • Green Fluorescent Proteins - genetics
  • HeLa Cells
  • Humans
  • Lipids
  • Lipids - administration & dosage
  • Lipids - chemistry
  • Lipids - genetics
  • Metal Nanoparticles - administration & dosage
  • Metal Nanoparticles - chemistry
  • Mice
  • Microscopy, Electron
  • Nanoparticles
  • Physiological aspects
  • Research
  • Ribonucleic acid
  • RNA
  • RNA, Small Interfering - administration & dosage
  • RNA, Small Interfering - chemistry
  • RNA, Small Interfering - genetics
ispartof: Nature biotechnology, 2013-07, Vol.31 (7), p.638-646
description: 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.
language: eng
source:
identifier: ISSN: 1087-0156
fulltext: no_fulltext
issn:
  • 1087-0156
  • 1546-1696
url: Link


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titleImage-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape
creatorGilleron, Jerome ; Querbes, William ; Zeigerer, Anja ; Borodovsky, Anna ; Marsico, Giovanni ; Schubert, Undine ; Manygoats, Kevin ; Seifert, Sarah ; Andree, Cordula ; Stöter, Martin ; Epstein-Barash, Hila ; Zhang, Ligang ; Koteliansky, Victor ; Fitzgerald, Kevin ; Fava, Eugenio ; Bickle, Marc ; Kalaidzidis, Yannis ; Akinc, Akin ; Maier, Martin ; Zerial, Marino
creatorcontribGilleron, Jerome ; Querbes, William ; Zeigerer, Anja ; Borodovsky, Anna ; Marsico, Giovanni ; Schubert, Undine ; Manygoats, Kevin ; Seifert, Sarah ; Andree, Cordula ; Stöter, Martin ; Epstein-Barash, Hila ; Zhang, Ligang ; Koteliansky, Victor ; Fitzgerald, Kevin ; Fava, Eugenio ; Bickle, Marc ; Kalaidzidis, Yannis ; Akinc, Akin ; Maier, Martin ; Zerial, Marino
descriptionDelivery 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.
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subjectAnimals ; Biological transport ; Biotechnology ; Cellular biology ; Endocytosis - genetics ; Gene Transfer Techniques ; Gold - administration & dosage ; Gold - chemistry ; Green Fluorescent Proteins - antagonists & inhibitors ; Green Fluorescent Proteins - genetics ; HeLa Cells ; Humans ; Lipids ; Lipids - administration & dosage ; Lipids - chemistry ; Lipids - genetics ; Metal Nanoparticles - administration & dosage ; Metal Nanoparticles - chemistry ; Mice ; Microscopy, Electron ; Nanoparticles ; Physiological aspects ; Research ; Ribonucleic acid ; RNA ; RNA, Small Interfering - administration & dosage ; RNA, Small Interfering - chemistry ; RNA, Small Interfering - genetics
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descriptionDelivery 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.
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abstractDelivery 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.
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