A Novel Mechanism Is Involved in Cationic Lipid-Mediated Functional siRNA Delivery

A key challenge for therapeutic application of RNA interference is to efficiently deliver synthetic small interfering RNAs (siRNAs) into target cells that will lead to the knockdown of the target transcript (functional siRNA delivery). To facilitate rational development of nonviral carriers, we have...

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Bibliographic Details
Published in:Molecular pharmaceutics 2009-06, Vol.6 (3), p.763-771
Main Authors: Lu, James J, Langer, Robert, Chen, Jianzhu
Format: Article
Language:English
Subjects:
Animals
Caveolins - metabolism
Cell Line
Cercopithecus aethiops
Chlorpromazine - pharmacology
Cholesterol - metabolism
Clathrin - metabolism
Cytochalasin D - pharmacology
Dopamine Antagonists - pharmacology
Endocytosis - drug effects
Endocytosis - physiology
HeLa Cells
Humans
Lipids - chemistry
Nucleic Acid Synthesis Inhibitors - pharmacology
RNA, Small Interfering - administration & dosage
RNA, Small Interfering - chemistry
RNA, Small Interfering - pharmacokinetics
Temperature
Transfection - methods
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Summary:A key challenge for therapeutic application of RNA interference is to efficiently deliver synthetic small interfering RNAs (siRNAs) into target cells that will lead to the knockdown of the target transcript (functional siRNA delivery). To facilitate rational development of nonviral carriers, we have investigated by imaging, pharmacological and genetic approaches the mechanisms by which a cationic lipid carrier mediates siRNA delivery into mammalian cells. We show that ∼95% of siRNA lipoplexes enter the cells through endocytosis and persist in endolysosomes for a prolonged period of time. However, inhibition of clathrin-, caveolin-, or lipid-raft-mediated endocytosis or macropinocytosis fails to inhibit the knockdown of the target transcript. In contrast, depletion of cholesterol from the plasma membrane has little effect on the cellular uptake of siRNA lipoplexes, but it abolishes the target transcript knockdown. Furthermore, functional siRNA delivery occurs within a few hours and is gradually inhibited by lowering temperatures. These results demonstrate that although endocytosis is responsible for the majority of cellular uptake of siRNA lipoplexes, a minor pathway, probably mediated by fusion between siRNA lipoplexes and the plasma membrane, is responsible for the functional siRNA delivery. Our findings suggest possible directions for improving functional siRNA delivery by cationic lipids.
ISSN:1543-8384
1543-8392
DOI:10.1021/mp900023v