Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages

Ana Paula Perez, Maria Luz Cosaka, Eder Lilia Romero, Maria Jose Morilla Programa de Nanomedicinas, Departamento de Ciencia y Tecnolog&iacute;a, Universidad Nacional de Quilmes, Bernal, Buenos Aires, Argentina Background: Gene silencing using small interfering RNA (siRNA) is a promising new...

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Autores principales:	Perez AP, Cosaka ML, Romero EL, Morilla MJ
Formato:	article
Lenguaje:	EN
Publicado:	Dove Medical Press 2011
Materias:	Medicine (General) R5-920
Acceso en línea:	https://doaj.org/article/eaf168bb37e84aeca79309e82876736d
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spelling	oai:doaj.org-article:eaf168bb37e84aeca79309e82876736d2021-12-02T05:14:23ZUptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages1176-91141178-2013https://doaj.org/article/eaf168bb37e84aeca79309e82876736d2011-11-01T00:00:00Zhttp://www.dovepress.com/uptake-and-intracellular-traffic-of-sirna-dendriplexes-in-glioblastoma-a8600https://doaj.org/toc/1176-9114https://doaj.org/toc/1178-2013Ana Paula Perez, Maria Luz Cosaka, Eder Lilia Romero, Maria Jose Morilla Programa de Nanomedicinas, Departamento de Ciencia y Tecnolog&iacute;a, Universidad Nacional de Quilmes, Bernal, Buenos Aires, Argentina Background: Gene silencing using small interfering RNA (siRNA) is a promising new therapeutic approach for glioblastoma. The endocytic uptake and delivery of siRNA to intracellular compartments could be enhanced by complexation with polyamidoamine dendrimers. In the present work, the uptake mechanisms and intracellular traffic of siRNA/generation 7 dendrimer complexes (siRNA dendriplexes) were screened in T98G glioblastoma and J774 macrophages. Methods: The effect of a set of chemical inhibitors of endocytosis on the uptake and silencing capacity of dendriplexes was determined by flow cytometry. Colocalization of fluorescent dendriplexes with endocytic markers and occurrence of intracellular dissociation were assessed by confocal laser scanning microscopy. Results: Uptake of siRNA dendriplexes by T98G cells was reduced by methyl-&szlig;-cyclodextrin, and genistein, and cytochalasine D, silencing activity was reduced by genistein; dendriplexes colocalized with cholera toxin subunit B. Therefore, caveolin-dependent endocytosis was involved both in the uptake and silencing activity of siRNA dendriplexes. On the other hand, uptake of siRNA dendriplexes by J774 cells was reduced by methyl-&szlig;-cyclodextrin, genistein, chlorpromazine, chloroquine, cytochalasine D, and nocodazole, the silencing activity was not affected by chlorpromazine, genistein or chloroquine, and dendriplexes colocalized with transferrin and cholera toxin subunit B. Thus, both clathrin-dependent and caveolin-dependent endocytosis mediated the uptake and silencing activity of the siRNA dendriplexes. SiRNA dendriplexes were internalized at higher rates by T98G but induced lower silencing than in J774 cells. SiRNA dendriplexes showed relatively slow dissociation kinetics, and their escape towards the cytosol was not mediated by acidification independently of the uptake pathway. Conclusion: The extent of cellular uptake of siRNA dendriplexes was inversely related to their silencing activity. The higher silencing activity of siRNA dendriplexes in J774 cells could be ascribed to the contribution of clathrin-dependent and caveolin-dependent endocytosis vs only caveolin-dependent endocytosis in T98G cells. Keywords: silencing, dendrimers, clathrin, caveolinPerez APCosaka MLRomero ELMorilla MJDove Medical PressarticleMedicine (General)R5-920ENInternational Journal of Nanomedicine, Vol 2011, Iss default, Pp 2715-2728 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine (General) R5-920
spellingShingle	Medicine (General) R5-920 Perez AP Cosaka ML Romero EL Morilla MJ Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages
description	Ana Paula Perez, Maria Luz Cosaka, Eder Lilia Romero, Maria Jose Morilla Programa de Nanomedicinas, Departamento de Ciencia y Tecnolog&iacute;a, Universidad Nacional de Quilmes, Bernal, Buenos Aires, Argentina Background: Gene silencing using small interfering RNA (siRNA) is a promising new therapeutic approach for glioblastoma. The endocytic uptake and delivery of siRNA to intracellular compartments could be enhanced by complexation with polyamidoamine dendrimers. In the present work, the uptake mechanisms and intracellular traffic of siRNA/generation 7 dendrimer complexes (siRNA dendriplexes) were screened in T98G glioblastoma and J774 macrophages. Methods: The effect of a set of chemical inhibitors of endocytosis on the uptake and silencing capacity of dendriplexes was determined by flow cytometry. Colocalization of fluorescent dendriplexes with endocytic markers and occurrence of intracellular dissociation were assessed by confocal laser scanning microscopy. Results: Uptake of siRNA dendriplexes by T98G cells was reduced by methyl-&szlig;-cyclodextrin, and genistein, and cytochalasine D, silencing activity was reduced by genistein; dendriplexes colocalized with cholera toxin subunit B. Therefore, caveolin-dependent endocytosis was involved both in the uptake and silencing activity of siRNA dendriplexes. On the other hand, uptake of siRNA dendriplexes by J774 cells was reduced by methyl-&szlig;-cyclodextrin, genistein, chlorpromazine, chloroquine, cytochalasine D, and nocodazole, the silencing activity was not affected by chlorpromazine, genistein or chloroquine, and dendriplexes colocalized with transferrin and cholera toxin subunit B. Thus, both clathrin-dependent and caveolin-dependent endocytosis mediated the uptake and silencing activity of the siRNA dendriplexes. SiRNA dendriplexes were internalized at higher rates by T98G but induced lower silencing than in J774 cells. SiRNA dendriplexes showed relatively slow dissociation kinetics, and their escape towards the cytosol was not mediated by acidification independently of the uptake pathway. Conclusion: The extent of cellular uptake of siRNA dendriplexes was inversely related to their silencing activity. The higher silencing activity of siRNA dendriplexes in J774 cells could be ascribed to the contribution of clathrin-dependent and caveolin-dependent endocytosis vs only caveolin-dependent endocytosis in T98G cells. Keywords: silencing, dendrimers, clathrin, caveolin
format	article
author	Perez AP Cosaka ML Romero EL Morilla MJ
author_facet	Perez AP Cosaka ML Romero EL Morilla MJ
author_sort	Perez AP
title	Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages
title_short	Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages
title_full	Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages
title_fullStr	Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages
title_full_unstemmed	Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages
title_sort	uptake and intracellular traffic of sirna dendriplexes in glioblastoma cells and macrophages
publisher	Dove Medical Press
publishDate	2011
url	https://doaj.org/article/eaf168bb37e84aeca79309e82876736d
work_keys_str_mv	AT perezap uptakeandintracellulartrafficofsirnadendriplexesinglioblastomacellsandmacrophages AT cosakaml uptakeandintracellulartrafficofsirnadendriplexesinglioblastomacellsandmacrophages AT romeroel uptakeandintracellulartrafficofsirnadendriplexesinglioblastomacellsandmacrophages AT morillamj uptakeandintracellulartrafficofsirnadendriplexesinglioblastomacellsandmacrophages
_version_	1718400505237995520

Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages

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