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

Autores
Perez, Ana Paula; Cosaka, María Luz; Romero, Eder Lilia; Morilla, María José
Año de publicación
2011
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Gene silencing using small interfering RNA (siRNA) is a promising new 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-beta-cyclodextrin, 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-beta-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.
Fil: Perez, Ana Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina
Fil: Cosaka, María Luz. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina
Fil: Romero, Eder Lilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Morilla, María José. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Materia
SILENCING
DENDRIMERS
CLATHRIN
CAVEOLIN
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/193973
Acceder
id CONICETDig_8596bee676f24bbc25d9b8edede7d153
oai_identifier_str oai:ri.conicet.gov.ar:11336/193973
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophagesPerez, Ana PaulaCosaka, María LuzRomero, Eder LiliaMorilla, María JoséSILENCINGDENDRIMERSCLATHRINCAVEOLINhttps://purl.org/becyt/ford/2.10https://purl.org/becyt/ford/2Gene silencing using small interfering RNA (siRNA) is a promising new 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-beta-cyclodextrin, 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-beta-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.Fil: Perez, Ana Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Cosaka, María Luz. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Romero, Eder Lilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Morilla, María José. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaDove Press2011-11info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/193973Perez, Ana Paula; Cosaka, María Luz; Romero, Eder Lilia; Morilla, María José; Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages; Dove Press; International Journal of Nanomedicine; 6; 11-2011; 2715-27281176-91141178-2013CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.2147/IJN.S25235info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T12:16:56Zoai:ri.conicet.gov.ar:11336/193973instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-10-22 12:16:56.577CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages
title Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages
spellingShingle Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages
Perez, Ana Paula
SILENCING
DENDRIMERS
CLATHRIN
CAVEOLIN
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
dc.creator.none.fl_str_mv Perez, Ana Paula
Cosaka, María Luz
Romero, Eder Lilia
Morilla, María José
author Perez, Ana Paula
author_facet Perez, Ana Paula
Cosaka, María Luz
Romero, Eder Lilia
Morilla, María José
author_role author
author2 Cosaka, María Luz
Romero, Eder Lilia
Morilla, María José
author2_role author
author
author
dc.subject.none.fl_str_mv SILENCING
DENDRIMERS
CLATHRIN
CAVEOLIN
topic SILENCING
DENDRIMERS
CLATHRIN
CAVEOLIN
purl_subject.fl_str_mv https://purl.org/becyt/ford/2.10
https://purl.org/becyt/ford/2
dc.description.none.fl_txt_mv Gene silencing using small interfering RNA (siRNA) is a promising new 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-beta-cyclodextrin, 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-beta-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.
Fil: Perez, Ana Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina
Fil: Cosaka, María Luz. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina
Fil: Romero, Eder Lilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Morilla, María José. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
description Gene silencing using small interfering RNA (siRNA) is a promising new 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-beta-cyclodextrin, 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-beta-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.
publishDate 2011
dc.date.none.fl_str_mv 2011-11
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/193973
Perez, Ana Paula; Cosaka, María Luz; Romero, Eder Lilia; Morilla, María José; Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages; Dove Press; International Journal of Nanomedicine; 6; 11-2011; 2715-2728
1176-9114
1178-2013
CONICET Digital
CONICET
url http://hdl.handle.net/11336/193973
identifier_str_mv Perez, Ana Paula; Cosaka, María Luz; Romero, Eder Lilia; Morilla, María José; Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages; Dove Press; International Journal of Nanomedicine; 6; 11-2011; 2715-2728
1176-9114
1178-2013
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.2147/IJN.S25235
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Dove Press
publisher.none.fl_str_mv Dove Press
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 12.982451

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