Journal
PHARMACEUTICAL RESEARCH
Volume 30, Issue 4, Pages 1161-1175Publisher
SPRINGER/PLENUM PUBLISHERS
DOI: 10.1007/s11095-012-0952-9
Keywords
anionic lipoplexes; endocytosis; formulation composition; siRNA; uptake mechanism/pathway
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To investigate cellular uptake pathways of novel anionic siRNA-lipoplexes as a function of formulation composition. Anionic formulations with anionic lipid/Ca2+/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. 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 Ca2+/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). 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.
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