title: Mechanisms of cellular uptake and endosomal escape of calcium-siRNA nanocomplexes.
abstract: Ca2+-siRNA nanocomplexes represent a simple yet an effective platform for siRNA delivery into the cell cytoplasm, with subsequent successful siRNA-induced target gene silencing. Herein, we aimed to elucidate the roles played by calcium ions in siRNA nanocomplex formation, cell uptake, and endosomal escape. We investigated whether the replacement of Ca2+in the nanocomplex by other bivalent cations would affect their cell entry and subsequent gene silencing. Our results indicate that Mg2+and Ba2+lead to the formation of nanocomplexes of similar physical features (size=100nm, surface charge ζ=-8mV) as the Ca2+-siRNA nanocomplexes. Yet, these nanocomplexes were not uptaken by the cells to the same extent as those prepared with Ca2+, and siRNA-induced target gene silencing was not obtained. Cell internalization of Ca2+--siRNA nanocomplexes, examined by employing chemical inhibitors to clathrin-, caveolin- and dynamin-mediated endocytosis pathways, indicated the involvement of all mechanisms in the process. Inhibition of endosome acidification by bafilomycin completely abolished the siRNA-mediated silencing by Ca2+-siRNA nanocomplexes. Collectively, our results indicate that Ca2+promotes cell internalization and rapid endosomal escape, thus leading to the efficient siRNA-induced target gene silencing elicited by the Ca2+-siRNA nanocomplexes.
authors.affiliation: Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, Israel. Electronic address: goldshllager@gmail.com.
authors.forename: Matan
authors.initials: M
authors.lastname: Goldshtein
authors.affiliation: Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
authors.forename: Efrat
authors.initials: E
authors.lastname: Forti
authors.affiliation: Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
authors.forename: Emil
authors.initials: E
authors.lastname: Ruvinov
authors.affiliation: Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, Israel; Regenerative Medicine and Stem Cell (RMSC) Research Center, Ben-Gurion University of the Negev, Beer-Sheva, Israel; The Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva, Israel. Electronic address: scohen@bgu.ac.il.
authors.forename: Smadar
authors.initials: S
authors.lastname: Cohen
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002412:Cations / Q000378:metabolism; D022461:Caveolins / Q000378:metabolism; D045744:Cell Line, Tumor; D002966:Clathrin / Q000378:metabolism; D034281:Dynamins / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D011992:Endosomes / Q000378:metabolism*; D020868:Gene Silencing / Q000502:physiology; D051379:Mice; D053758:Nanoparticles / Q000378:metabolism*; D034622:RNA Interference / Q000502:physiology; D034741:RNA, Small Interfering / Q000378:metabolism*
chemical_list: D002412:Cations; D022461:Caveolins; D002966:Clathrin; D034741:RNA, Small Interfering; D034281:Dynamins; D002118:Calcium
keywords: Calcium; Cellular uptake; Nano particles; siRNA