Cellular uptake mechanisms of novel anionic siRNA lipoplexes.
Pharm Res
; 30(4): 1161-75, 2013 Apr.
Article
em En
| MEDLINE
| ID: mdl-23239010
ABSTRACT
PURPOSE:
To investigate cellular uptake pathways of novel anionic siRNA-lipoplexes as a function of formulation composition.METHODS:
Anionic formulations with anionic lipid/Ca(2+)/siRNA ratio of 1.3/2.5/1 (AF1) and 1.3/0.3/1 (AF2) were utilized. Uptake mechanisms were investigated using uptake inhibition and co-localization approaches in breast cancer cells. Actin-mediated uptake was investigated using actin polymerization and rearrangement assays. Silencing efficiency and endosomal escaping capability of lipoplexes were evaluated. The cationic formulation Lipofectamine-2000 was used as a control.RESULTS:
Anionic lipoplexes entered the breast cancer cells via endocytosis specifically via macropinocytosis or via both macropinocytosis and HSPG (heparin sulfate proteoglycans) pathways, depending on the Ca(2+)/siRNA ratio. Additionally, uptake of these lipoplexes was both microtubule and actin dependent. The control cationic lipid-siRNA complexes (Lipofectamine-2000) were internalized via both endocytic (phagocytosis, HSPG) and non-endocytic (membrane fusion) pathways. Their uptake was microtubule independent but actin dependent. Silencing efficiency of the AF2 formulation was negligible mainly due to poor endosomal release (rate-limiting step).CONCLUSIONS:
Formulation composition significantly influences the internalization mechanism of anionic lipoplexes. Uptake mechanism together with formulation bioactivity helped in identification of the rate-limiting steps to efficient siRNA delivery. Such studies are extremely useful for formulation optimization to achieve enhanced intracellular delivery of nucleic acids.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA Interferente Pequeno
/
Lipídeos
/
Ânions
Tipo de estudo:
Prognostic_studies
Limite:
Female
/
Humans
Idioma:
En
Revista:
Pharm Res
Ano de publicação:
2013
Tipo de documento:
Article
País de afiliação:
Estados Unidos