Abstract
In this paper, the relatived mechanism between lipofectamine 2000 mediated transmembrane gene delivery and endocytic pathway were investigated. Clathrin and caveolae-mediated endocytic pathway contributions to transfection efficiency were studied. The inhibitors of endocytosis were used to treat HEp-2 cells before lipofectamine 2000/pGFP-N2 transfection. Transfection efficiency was evaluated with green fluorescence protein (GFP) expression assays. Cell viability and cytotoxicity were evaluated with MTT method. The results indicated that inhibitors of clathrin (chlorpromazine or wortmannin) and caveolin (genistein) could reduce the cell transfection efficiency observably. Both clathrin and caveolae-mediated endocytic pathways play important roles in transmembrane gene delivery.
Highlights
Non-viral DNA delivery systems have been developed to facilitate gene entry into mammalian cells[1]
Chlorpromazine (10-100μM) could observably lower transfection efficiency 1-12-fold (Figure 1 a, b) and wortmannin (10-100nM) had 0.8-15-fold (Fig. 1 c, d) compared with control. These results suggested that both chlorpromazine and wortmannin could lower transfection efficiency through inhibiting clathrin pathway
Lipoplexes via which endocytic pathways entry into cell is of great importance for the design of further improved non-viral gene delivery systems
Summary
Non-viral DNA delivery systems have been developed to facilitate gene entry into mammalian cells[1]. Because cell membrane was the first barrier for lipoplexes entry into the cell, the mechanism of cationic liposomes mediated transmembrane gene delivery was important as the study could contribute to improving transfection efficiency. The relative contribution of each pathway in lipoplexes internalization has been poorly defined to date, involvement of the clathrin-mediated pathway has been firmly established, while evidence was emerging that entry may occur via macropinocytosis[4,5,6]. Endocytotic inhibitors such as chloroquine, chlorpromazine are very helpful for studying drug intracellular release or elucidating a specific endocytosis route[7]. Via evaluated inhibitory effects of lipoplexes transfection to explore the underlying transmembrane mechanism in HEp-2 cells
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