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siRNA delivery by a transferrin-associated lipid-based vector: a non-viral strategy to mediate gene silencing
- Source :
- Repositório Científico de Acesso Aberto de Portugal, Repositório Científico de Acesso Aberto de Portugal (RCAAP), instacron:RCAAP
- Publication Year :
- 2007
-
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. Copyright © 2007 John Wiley & Sons, Ltd.
- Subjects :
- Small interfering RNA
Genetic Vectors
Green Fluorescent Proteins
Biology
Fluorescence
Fatty Acids, Monounsaturated
RNA interference
Genes, Reporter
Cations
Cell Line, Tumor
Neoplasms
Drug Discovery
Genetics
Gene silencing
Humans
Cationic liposome
RNA, Small Interfering
Cytotoxicity
Molecular Biology
Genetics (clinical)
chemistry.chemical_classification
Reporter gene
Gene knockdown
Gene Transfer Techniques
JNK Mitogen-Activated Protein Kinases
Transferrin
Genetic Therapy
Molecular biology
Lipids
Quaternary Ammonium Compounds
chemistry
Liposomes
Molecular Medicine
RNA Interference
Subjects
Details
- ISSN :
- 1099498X
- Volume :
- 9
- Issue :
- 3
- Database :
- OpenAIRE
- Journal :
- The journal of gene medicine
- Accession number :
- edsair.doi.dedup.....0851f2b37b84231ee5f41e5d937ea14c