ABSTRACTThe aim of the present study was to synthesize silver (Ag) and titanium dioxide (TiO2) nanoparticles (NPs) using green synthesis from aqueous leaf extract ofEuphorbia prostrataas antileishmanial agents and to explore the underlying molecular mechanism of induced cell death.In vitroantileishmanial activity of synthesized NPs was tested against promastigotes ofLeishmania donovaniby alamarBlue and propidium iodide uptake assays. Antileishmanial activity of synthesized NPs on intracellular amastigotes was assessed by Giemsa staining. The leishmanicidal effect of synthesized Ag NPs was further confirmed by DNA fragmentation assay and by cell cycle progression and transmission electron microscopy (TEM) of the treated parasites. TEM analysis of the synthesized Ag NPs showed a spherical shape with an average size of 12.82 ± 2.50 nm, and in comparison to synthesized TiO2NPs, synthesized Ag NPs were found to be most active againstLeishmaniaparasites after 24 h exposure, with 50% inhibitory concentrations (IC50) of 14.94 μg/ml and 3.89 μg/ml in promastigotes and intracellular amastigotes, respectively. A significant increase in G0/G1phase of the cell cycle with a subsequent decrease in S (synthesis) and G2/M phases compared to controls was observed. The growth-inhibitory effect of synthesized Ag NPs was attributed to increased length of S phase. A decreased reactive oxygen species level was also observed, which could be responsible for the caspase-independent shift from apoptosis (G0/G1arrest) to massive necrosis. High-molecular-weight DNA fragmentation as a positive consequence of necrotic cell death was also visualized. We also report that the unique trypanothione/trypanothione reductase (TR) system ofLeishmaniacells was significantly inhibited by synthesized Ag NPs. The green-synthesized Ag NPs may provide promising leads for the development of cost-effective and safer alternative treatment against visceral leishmaniasis.