Transmembrane routes of cationic liposome-mediated gene delivery using human throat epidermis cancer cells

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).

Fig. 1

Effect of concentrations of chlorpromazine (10–100 μM) and wortmannin (10–100 nM) on GFP expression mediated by Lipofecamine 2000/pGFP-N2 complexes against Hep-2 cells. a, c The expression of GFP was imaged by inverted fluorescence microscope (10 × 20); b, d contrast cells in bright field. Scale bar = 50 μm

Fig. 2

Luciferase reporter gene expression and cell survival rate. a, c The evaluation of transfection efficiency and cell survival rate with chlorpromazine (10–100 μM) and wortmannin (10–100 nM) treated Hep-2 cells. Lipofectamine 2000 was used as positive control in transfection. Cell survival rate was determined with the MTT method with non-treated cells as control. All results are expressed as mean ± SD (n = 4). b, d RLU/survival rate with chlorpromazine (10–100 μM) and wortmannin (10–100 nM) treated Hep-2 cells

Fig. 3

Effect of concentrations of genistein (10–200 μM) on GFP expression mediated by Lipofecamine 2000/pGFP-N2 complexes. a Inverted fluorescent microscope images (10 × 20); b contrast cells in bright field. Scale bar = 50 μm

Fig. 4

Luciferase reporter gene expression and cell survival rate. a The evaluation of transfection efficiency and cell survival with genistein (10–200 μM) treated Hep-2 cells. Lipofectamine 2000 was used as positive control. Cell survival rate was determined with the MTT method with non-treated cells as control. All results are expressed as mean ± SD (n = 4). b RLU/survival rate with genistein (10–200 μM) treated Hep-2 cells

Fig. 5

Effect of concentrations of vinblastine (10–200 nM) on GFP expression mediated by Lipofecamine 2000/pGFP-N2 complexes. a Inverted fluorescent microscope images (10 × 20); b contrast cells in bright field. Scale bar = 50 μm

Fig. 6

Luciferase reporter gene expression and cell survival rate. a The evaluation of transfection efficiency and cell viability with vinblastine (10–200 nM) treated Hep-2 cells. Lipofectamine 2000 was used as positive control. Cell viability was determined with the MTT method with non-treated cells as control. All results are expressed as mean ± SD (n = 4). b RLU/survival rate with vinblastine (10–200 nM) treated Hep-2 cells