Figure 4.

(Ai and ii) Measurement of apoptosis induced by DOX formulations. 10⁶ promastigotes were treated with 0.5 μg·mL⁻¹ of each of the free drug, NE-DOX and SA-NCs-DOX for 24 h, stained with PI and cell cycle analysis was done by flow cytometer. B, C, D and E gates represent cells in sub G1, G0/G, S and G2M phase respectively. Bar graph represents the percentage of cells in sub G1 region undergoing apoptosis after treatment with different formulations. (B) Decrease in Δψm in promastigotes following treatment with SA-NCs-DOX, NE-DOX and free DOX for the indicated times and staining with the potentiometric probe JC-1 (10 mM). Δψm values are expressed as the ratio of 590:530 nm fluorescence. Assessment of the generation of NO in infected macrophages (C) and ROS generation in promastigotes (Di) and infected macrophages (Dii) at different times after treatment. *P < 0.05; **P < 0.01; ***P < 0.001, significantly different from Free DOX and from NE–DOX-treated cells.