Endocytic Transport of Polyplex and Lipoplex siRNA Vectors in HeLa Cells
| Autor: | Mihael Lazebnik, Rahul K. Keswani, Daniel W. Pack |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Cell Survival media_common.quotation_subject Endocytic cycle Cell Culture Techniques Pharmaceutical Science Endocytosis Fatty Acids Monounsaturated 03 medical and health sciences chemistry.chemical_compound Drug Delivery Systems Humans Polyethyleneimine Gene silencing Pharmacology (medical) Gene Silencing RNA Small Interfering Internalization media_common Pharmacology Polyethylenimine Gene knockdown Phosphatidylethanolamines Pinocytosis Organic Chemistry Molecular biology Cell biology Quaternary Ammonium Compounds Drug Liberation Cholesterol 030104 developmental biology Endocytic vesicle chemistry Liposomes Molecular Medicine lipids (amino acids peptides and proteins) HeLa Cells Biotechnology |
| Zdroj: | Pharmaceutical Research. 33:2999-3011 |
| ISSN: | 1573-904X 0724-8741 |
| DOI: | 10.1007/s11095-016-2022-1 |
| Popis: | siRNA may be delivered as electrostatic complexes with cationic lipids (lipoplexes) or polycations (polyplexes). The purpose of this project was to determine the effect of cellular internalization mechanism(s) on siRNA-mediated gene silencing efficiency. Lipoplexes were formed comprising siRNA and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP), cholesterol and dioleoyl phosphatidylethanolamine (DOPE), and polyplexes comprised siRNA with polyethylenimine (PEI). During transfections, specific uptake mechanisms were inhibited by pharmacological agents and RNAi-mediated knockdown of proteins involved in various endocytosis pathways. Confocal fluorescence microscopy further elucidated the predominant endocytic pathways of siRNA delivery via colocalization of vectors with endocytic vesicle markers. Inhibition of macropinocytosis (MP), caveolin-mediated endocytosis (CvME), flotillin-mediated endocytosis (FME) and knockdown of ARF6 significantly decreased PEI/siRNA-mediated gene silencing. Inhibition of endocytosis pathways, however, had negligible effect on lipoplex uptake and gene silencing mediated by lipoplexes. Rather, internalization of lipoplexes and subsequent siRNA-mediated gene silencing occurred via an energy-independent process. MP, CvME and FME, but not the acidified clathrin-mediated pathway, lead to effective gene silencing by PEI/siRNA polyplexes. Lipoplexes, in contrast, deliver siRNA primarily by direct fusion of the liposomal and cellular membranes. These results provide a new understanding of the mechanisms of siRNA delivery materials in HeLa cells and may aid in design of more effective RNAi strategies. |
| Databáze: | OpenAIRE |
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