Figure 1

Acute cell necrosis induced by cationic carriers in vivo and in vitro. (A) The detection of propidium iodide (PI)-positive necrotic cells in mouse lungs. Various particles were injected through tail vein of mouse, 2 h later, PI and 4% formaldehyde were perfused through tail vein for the detection of the necrotic cells. (B) A representative experiment of the detection of the necrotic cells induced by the injection of cationic liposomes in vivo by flow cytometry with Annexin-V and PI staining. C57BL/6 mice were injected with DOTAP liposomes (25 mg/kg). Necrotic cells in BAL fluid were detected 4 h after injection by flow cytometry with Annexin-V and PI staining. n = 3/group. (C) The morphological change of the cells treated with various nanocarriers in vitro. Cells were treated in vitro with DOTAP liposome (50 μg/ml), PEI (10 μg/ml), chitosan (50 μg/ml), anionic or neutral liposomes (abbreviated as AnionicL and NeutralL, 50 μg/ml) for 30 min. Cells were subjected to inverted microscope observation. (D) The detection of the necrotic cells induced in vitro by flow cytometry with Annexin-V and PI staining. Primary lung cells of C57BL/6 mice (left) and A549 cells (right) were treated with cationic carriers for 10 min. Percentages of necrotic cells in PI-positive region are shown. (E) A representative experiment of immunofluorescense of Cathepsin-B (green) and Caspase-3. A549 cells were treated with DOTAP liposomes (20 μg/ml) for 30 min. Diffused cytoplasmic cathepsin-B immunoreactivity was evident after the treatment of DOTAP liposome. In contrast, the activation of Caspase-3 was observed after 24 h of treatment. (F) A549 cells were treated with DOTAP liposomes, and intracellular Ca²⁺ concentration and ROS levels were detected with Fluo-3/AM and H₂DCF-DA by flow cytometry, respectively. Data are mean ± SEM; n = 3.^**P< 0.01,^***P< 0.001 compared with control group by Student's t-test.