title: siRNA delivery by a transferrin-associated lipid-based vector: a non-viral strategy to mediate gene silencing.
abstract: BACKGROUND
  RNA interference provides a powerful technology for specific gene silencing. Therapeutic applications of small interfering RNA (siRNA) however require efficient vehicles for stable complexation, protection, and extra- and intracellular delivery of these nucleic acids. Here, we evaluated the potential of transferrin (Tf)-associated liposomes for siRNA complexation and gene silencing.
  
  
  METHODS
  Cationic liposomes composed of DOTAP : Cholesterol associated with or without transferrin (Tf) were complexed with siRNA at different lipid/siRNA charge ratios. Complexation and protection of siRNA from enzymatic degradation was assessed with the PicoGreen intercalation assay and gel electrophoresis. Cellular internalization of these siRNA Tf-lipoplexes was detected by confocal microscopy. Luciferase assay, immunoblot and fluorescence-activated cell sorting (FACS) analysis were used to evaluate reporter gene silencing in Huh-7 hepatocarcinoma and U-373 glioma cells. c-Jun knockdown in HT-22 cells was evaluated by quantitative real-time polymerase chain reaction (RT-PCR). Cytotoxicity of the siRNA complexes was assessed by Alamar blue, lactate dehydrogenase and MTT assays.
  
  
  RESULTS
  Complexation of siRNA with the cationic liposomes in the presence of Tf results in the formation of stable particles and prevents serum-mediated degradation. Confocal microscopy showed fast cellular internalization of the Tf-lipoplexes via endocytosis. In the GFP glioma cells Tf-lipoplexes showed enhanced gene silencing at minimum toxicity in comparison to Tf-free lipoplexes. Targeting luciferase in the hepatocarcinoma cell line resulted in more than 70% reduction of luciferase activity, while in HT-22 cells 50% knockdown of endogenous c-Jun resulted in a significant protection from glutamate-mediated toxicity.
  
  
  CONCLUSIONS
  Cationic liposomes associated with Tf form stable siRNA lipoplexes with reduced toxicity and enhanced specific gene knockdown activity compared to conventional lipoplexes. Thus, such formulations may constitute efficient delivery systems for therapeutic siRNA applications.
authors.affiliation: Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal.
authors.forename: A L C
authors.initials: AL
authors.lastname: Cardoso
authors.forename: S
authors.initials: S
authors.lastname: Simões
authors.forename: L P
authors.initials: LP
authors.lastname: de Almeida
authors.forename: J
authors.initials: J
authors.lastname: Pelisek
authors.forename: C
authors.initials: C
authors.lastname: Culmsee
authors.forename: E
authors.initials: E
authors.lastname: Wagner
authors.forename: M C
authors.initials: MC
authors.lastname: Pedroso de Lima
mesh_terms: D002412:Cations; D045744:Cell Line, Tumor; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry; D005453:Fluorescence; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism; D009369:Neoplasms / Q000628:therapy*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000737:chemistry / Q000627:therapeutic use; D014168:Transferrin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002412:Cations; D005229:Fatty Acids, Monounsaturated; D008055:Lipids; D008081:Liposomes; D000644:Quaternary Ammonium Compounds; D034741:RNA, Small Interfering; D014168:Transferrin; D049452:Green Fluorescent Proteins; D048031:JNK Mitogen-Activated Protein Kinases; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane