Abstract
For studying the mechanism of cationic liposome-mediated transmembrane routes for gene delivery, various inhibitors of endocytosis were used to treat human throat epidermis cancer cells, Hep-2, before transfection with Lipofectamine 2000/pGFP-N2 or Lipofectamine 2000/pGL3. To eliminate the effect of inhibitor toxicity on transfection, the RLU/survival rate was used to represent the transfection efficiency. Chlorpromazine and wortmannin, clathrin inhibitors, decreased transfection efficiency by 44 % (100 μM) and 31 % (100 nM), respectively. At the same time, genistein, a caveolin inhibitor, decreased it by 30 % (200 μM). Thus combined transmembrane routes through the clathrin and caveolae-mediated pathways were major mechanisms of cell uptake for the cationic liposome-mediated gene delivery. After entering the cells, microtubules played an important role on gene delivery as vinblastine, a microtubulin inhibitor, could reduce transfection efficiency by 41 % (200 nM).
Highlights
Non-viral vectors are inferior compared to viral vectors in terms of transfection efficiency, they have the advantages over viral ones as they are nonimmunogenic, easy to produce and are not oncogenic
Because the cell membrane is the first barrier for liposome/ DNA complexes to enter into cells, the mechanism of cationic liposome-mediated transmembrane gene delivery is important as a study of it could contribute to improving transfection efficiency
This study was aimed to elucidate the transmembrane pathways of gene delivery mediated by a cationic liposome, Lipofectamine 2000, while the inhibitors such as chlorpromazine, genistein and vinblastine for inhibiting clathrin, caveolin and microtubulin pathways, respectively, were used to treat Hep-2 cells before transfection
Summary
Non-viral vectors are inferior compared to viral vectors in terms of transfection efficiency, they have the advantages over viral ones as they are nonimmunogenic, easy to produce and are not oncogenic. Because the cell membrane is the first barrier for liposome/ DNA complexes (lipoplexes) to enter into cells, the mechanism of cationic liposome-mediated transmembrane gene delivery is important as a study of it could contribute to improving transfection efficiency. The relative contribution of each pathway in lipoplexes internalization has been poorly defined to date (Mayor and Pagano 2007; Howes et al 2010) Endocytotic inhibitors, such as chloroquine, chlorpromazine, NaN3 or filipin, are helpful for studying drug intracellular release or elucidating a specific endocytosis route (Blanchard et al 2006). This study was aimed to elucidate the transmembrane pathways of gene delivery mediated by a cationic liposome, Lipofectamine 2000, while the inhibitors such as chlorpromazine (wortmannin), genistein and vinblastine for inhibiting clathrin, caveolin and microtubulin pathways, respectively, were used to treat Hep-2 cells before transfection
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