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Visualization of self-delivering hydrophobically modified siRNA cellular internalization

siRNAs are a new class of therapeutic modalities with promising clinical efficacy that requires modification or formulation for delivery to the tissue and cell of interest. Conjugation of siRNAs to lipophilic groups supports efficient cellular uptake by a mechanism that is not well characterized. He...

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Autores principales: Ly, Socheata, Navaroli, Deanna M., Didiot, Marie-Cécile, Cardia, James, Pandarinathan, Lakshmipathi, Alterman, Julia F., Fogarty, Kevin, Standley, Clive, Lifshitz, Lawrence M., Bellve, Karl D., Prot, Matthieu, Echeverria, Dimas, Corvera, Silvia, Khvorova, Anastasia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5224471/
https://www.ncbi.nlm.nih.gov/pubmed/27899655
http://dx.doi.org/10.1093/nar/gkw1005
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author Ly, Socheata
Navaroli, Deanna M.
Didiot, Marie-Cécile
Cardia, James
Pandarinathan, Lakshmipathi
Alterman, Julia F.
Fogarty, Kevin
Standley, Clive
Lifshitz, Lawrence M.
Bellve, Karl D.
Prot, Matthieu
Echeverria, Dimas
Corvera, Silvia
Khvorova, Anastasia
author_facet Ly, Socheata
Navaroli, Deanna M.
Didiot, Marie-Cécile
Cardia, James
Pandarinathan, Lakshmipathi
Alterman, Julia F.
Fogarty, Kevin
Standley, Clive
Lifshitz, Lawrence M.
Bellve, Karl D.
Prot, Matthieu
Echeverria, Dimas
Corvera, Silvia
Khvorova, Anastasia
author_sort Ly, Socheata
collection PubMed
description siRNAs are a new class of therapeutic modalities with promising clinical efficacy that requires modification or formulation for delivery to the tissue and cell of interest. Conjugation of siRNAs to lipophilic groups supports efficient cellular uptake by a mechanism that is not well characterized. Here we study the mechanism of internalization of asymmetric, chemically stabilized, cholesterol-modified siRNAs (sd-rxRNAs(®)) that efficiently enter cells and tissues without the need for formulation. We demonstrate that uptake is rapid with significant membrane association within minutes of exposure followed by the formation of vesicular structures and internalization. Furthermore, sd-rxRNAs are internalized by a specific class of early endosomes and show preferential association with epidermal growth factor (EGF) but not transferrin (Tf) trafficking pathways as shown by live cell TIRF and structured illumination microscopy (SIM). In fixed cells, we observe ∼25% of sd-rxRNA co-localizing with EGF and <5% with Tf, which is indicative of selective endosomal sorting. Likewise, preferential sd-rxRNA co-localization was demonstrated with EEA1 but not RBSN-containing endosomes, consistent with preferential EGF-like trafficking through EEA1-containing endosomes. sd-rxRNA cellular uptake is a two-step process, with rapid membrane association followed by internalization through a selective, saturable subset of the endocytic process. However, the mechanistic role of EEA1 is not yet known. This method of visualization can be used to better understand the kinetics and mechanisms of hydrophobic siRNA cellular uptake and will assist in further optimization of these types of compounds for therapeutic intervention.
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spelling pubmed-52244712017-01-17 Visualization of self-delivering hydrophobically modified siRNA cellular internalization Ly, Socheata Navaroli, Deanna M. Didiot, Marie-Cécile Cardia, James Pandarinathan, Lakshmipathi Alterman, Julia F. Fogarty, Kevin Standley, Clive Lifshitz, Lawrence M. Bellve, Karl D. Prot, Matthieu Echeverria, Dimas Corvera, Silvia Khvorova, Anastasia Nucleic Acids Res Chemical Biology and Nucleic Acid Chemistry siRNAs are a new class of therapeutic modalities with promising clinical efficacy that requires modification or formulation for delivery to the tissue and cell of interest. Conjugation of siRNAs to lipophilic groups supports efficient cellular uptake by a mechanism that is not well characterized. Here we study the mechanism of internalization of asymmetric, chemically stabilized, cholesterol-modified siRNAs (sd-rxRNAs(®)) that efficiently enter cells and tissues without the need for formulation. We demonstrate that uptake is rapid with significant membrane association within minutes of exposure followed by the formation of vesicular structures and internalization. Furthermore, sd-rxRNAs are internalized by a specific class of early endosomes and show preferential association with epidermal growth factor (EGF) but not transferrin (Tf) trafficking pathways as shown by live cell TIRF and structured illumination microscopy (SIM). In fixed cells, we observe ∼25% of sd-rxRNA co-localizing with EGF and <5% with Tf, which is indicative of selective endosomal sorting. Likewise, preferential sd-rxRNA co-localization was demonstrated with EEA1 but not RBSN-containing endosomes, consistent with preferential EGF-like trafficking through EEA1-containing endosomes. sd-rxRNA cellular uptake is a two-step process, with rapid membrane association followed by internalization through a selective, saturable subset of the endocytic process. However, the mechanistic role of EEA1 is not yet known. This method of visualization can be used to better understand the kinetics and mechanisms of hydrophobic siRNA cellular uptake and will assist in further optimization of these types of compounds for therapeutic intervention. Oxford University Press 2017-01-09 2016-11-29 /pmc/articles/PMC5224471/ /pubmed/27899655 http://dx.doi.org/10.1093/nar/gkw1005 Text en © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Chemical Biology and Nucleic Acid Chemistry
Ly, Socheata
Navaroli, Deanna M.
Didiot, Marie-Cécile
Cardia, James
Pandarinathan, Lakshmipathi
Alterman, Julia F.
Fogarty, Kevin
Standley, Clive
Lifshitz, Lawrence M.
Bellve, Karl D.
Prot, Matthieu
Echeverria, Dimas
Corvera, Silvia
Khvorova, Anastasia
Visualization of self-delivering hydrophobically modified siRNA cellular internalization
title Visualization of self-delivering hydrophobically modified siRNA cellular internalization
title_full Visualization of self-delivering hydrophobically modified siRNA cellular internalization
title_fullStr Visualization of self-delivering hydrophobically modified siRNA cellular internalization
title_full_unstemmed Visualization of self-delivering hydrophobically modified siRNA cellular internalization
title_short Visualization of self-delivering hydrophobically modified siRNA cellular internalization
title_sort visualization of self-delivering hydrophobically modified sirna cellular internalization
topic Chemical Biology and Nucleic Acid Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5224471/
https://www.ncbi.nlm.nih.gov/pubmed/27899655
http://dx.doi.org/10.1093/nar/gkw1005
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