Figure 4.

Cell necrosis induced by cationic carriers involves impairment of Na⁺/K⁺-ATPase activity. (A) Representative images of A549 cells after the addition of DOTAP liposomes (50 μg/ml). Cells were loaded with fluorescence sodium indicator CoroNa Green and stained with PI. Scale bar, 20 μm. (B) The fluorescence intensities of CoroNa Green in one field were recorded and analyzed using ImageJ software. (C) A549 cells were cultured in medium with or without sodium for 1 h and then treated with cationic carriers for 10 min. DOTAP liposome (50 μg/ml); PEI (10 μg/ml); Chitosan (10 μg/ml). (D) A549 cells were pretreated with inhibitors including ouabain (2 μM), eosin (5 μM), Gd³⁺(20 μM), NiCl₂ (1 mM), LaCl₃(0.1 mM) or 2-APB (50 μM) for 30 min and DOTAP liposomes (100 μg/ml) were added. Necrotic cells were detected by flow cytometry with PI staining in 10 min. (E, F) Na⁺/K⁺-ATPase activity in cultured cells and in tissues treated with cationic carriers. A549 cells were treated with DOTAP liposome (50 μg/ml), PEI (10 μg/ml), Chitosan (50 μg/ml), Neutral liposomes (NeutralL, 50 μg/ml) or Anionic liposomes (AnionicL, 50 μg/ml) for 5 min and heavy-membrane fraction of the cells were used for Na⁺/K⁺-ATPase activity assay (E). Mice were injected with DOTAP liposome (25 mg/kg), PEI (5 mg/kg), Chitosan (25 mg/kg), Neutral liposomes (25 mg/kg) or Anionic liposomes (25 mg/kg) and 20 min later homogenates of lungs were prepared for Na⁺/K⁺-ATPase activity assay (F). (G) Uptake of ⁸⁶Rb⁺ in A549 cells treated with cationic nanocarriers. A549 cells were incubated with DOTAP liposome (50 μg/ml), PEI (10 μg/ml), Chitosan (50 μg/ml), Neutral liposomes (50 μg/ml) or Anionic liposomes (50 μg/ml) for 2 min and treatment of 5 μM ouabain was used as positive control. n = 3. (H) Mice were pretreated with or without ouabain (5 μg/mice) for 10 min and subsequently injected with DOTAP liposomes (100 mg/kg) through tail veins every 24 h for two days and mouse survival were recorded every 24 h, n = 10. (I) Complex structures were calculated. (a) for Na⁺/K⁺-ATPase-DOTAP and (b) for Na⁺ /K⁺-ATPase-ouabain/DOTAP. (J) Control-shRNA, Na⁺/K⁺-ATPase-shRNA (ATP1A1-shRNA) and TRPM7-shRNA transfected A549 cells were treated with DOTAP liposomes (50 μg/ml) for 5 min before analysis by flow cytometry. Data are mean ± SEM; n = 3.^**P< 0.01,^***P< 0.001 compared with control group by Student's t-test.