title: Endocytic Transport of Polyplex and Lipoplex siRNA Vectors in HeLa Cells.
abstract: 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.
authors: Department of Chemical and Biomolecular Engineering, University of Illinois, Urbana-Champaign, Urbana, Illinois, USA.|Department of Early Stage Cell Culture, Genentech, Inc., South San Francisco, California, USA.
authors: Mihael
authors: M
authors: Lazebnik
authors: Department of Chemical and Biomolecular Engineering, University of Illinois, Urbana-Champaign, Urbana, Illinois, USA.|Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan, USA.
authors: Rahul K
authors: RK
authors: Keswani
authors: Department of Chemical & Materials Engineering, University of Kentucky, Lexington, Kentucky, USA. dan.pack@uky.edu.|Department of Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky, USA. dan.pack@uky.edu.|University of Kentucky, 789 South Limestone, Lexington, Kentucky, 40536-0596, USA. dan.pack@uky.edu.
authors: Daniel W
authors: DW
authors: Pack
mesh_terms: D018929:Cell Culture Techniques; D002470:Cell Survival; D002784:Cholesterol / Q000737:chemistry; D016503:Drug Delivery Systems; D065546:Drug Liberation; D004705:Endocytosis; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry* / Q000378:metabolism; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D008081:Liposomes; D010714:Phosphatidylethanolamines / Q000737:chemistry* / Q000378:metabolism; D011094:Polyethyleneimine / Q000737:chemistry*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*
chemical_list: D005229:Fatty Acids, Monounsaturated; D008081:Liposomes; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; D034741:RNA, Small Interfering; C094877:dioleoyl phosphatidylethanolamine; C020888:1,2-dielaidoylphosphatidylethanolamine; D011094:Polyethyleneimine; D002784:Cholesterol; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane
keywords: endocytosis; liposomes; polyethylenimine; siRNA delivery