Green synthesis of silver nanoparticles via Cynara scolymus leaf extracts: The characterization, anticancer potential with photodynamic therapy in MCF7 cells

Abstract

In this study, we report on the synthesis of silver nanoparticles (AgNPs) from the leaf extracts of Cynara scolymus (Artichoke) using microwave irradiation and the evaluation of its anti-cancer potential with photodynamic therapy (PDT). Silver nanoparticles formation was characterized by scanning electron microscopy with energy dispersive x-ray spectroscopy and Fourier transform infrared (FTIR) spectroscopy. Silver nanoparticles formation was also investigated the surface charge, particle size and distribution using zetasizer analysis. The cytotoxic effect of AgNPs and/or PDT was studied by MTT assay and migration by the scratch assay. The apoptotic inducing ability of the AgNPs and/or PDT was investigated by intracellular ROS analysis, antioxidant enzyme levels (SOD, CAT, GPx and GSH), Hoechst staining and Bax/Bcl-2 analysis using western blotting. The mean particle size of produced AgNPs was found 98.47±2.04 nm with low polydispersity (0.301±0.033). Zeta potential values of AgNPs show -32.3± 0.8 mV. These results clearly indicate the successful formation of AgNPs for cellular uptake. Mitochondrial damage and intracellular ROS production were observed upon treatment with AgNPs (10μg/mL) and PDT (0.5 mJ/cm2) showed significant reducing cell migration, expression of Bax and suppression of Bcl-2. Significantly, biosynthesized AgNPs showed a broad-spectrum anti-cancer activity with PDT therapy and therefore represent promoting ROS generation by modulating mitochondrial apoptosis induction in MCF7 breast cancer cells.

Fig 1. Schematic representation of nanoparticle synthesis with green chemistry.

Fig 2. Characterization of AgNPs.

a) UV spectrum, b)FT-IR spectrum, c)SEM images at different magnitudes and d) Energy-dispersive X-ray spectroscopy spectrum of AgNPs.

Fig 3. Effect of AgNPs and PDT therapy on the cell survival in MCF7 breast cancer cells.

a) Cell viability measurement with MTT assay after AgNPs and/or PDT treatment (0–500 10 μg/mL) of MCF7 cell lines for 24 h. b) Cell morphological changes after treatment AgNPs and/or PDT treatment MCF7 cell lines for 24 h. c) Cell growing stained with crystal violet d) Ratio of cell growing in MCF7 cells with treated of AgNPs (10 μg/mL) and/or PDT for 24h (*p<0.05 compare to control) for the three biological replicates within each group.

Fig 4. ROS generation following incubation of the MCF7 cells with AgNPs (10 μg/mL) and/or PDT for 24h.

a)SOD activity b)CAT activity c)GPx activity d)GSH levels e-f)Percentage of ROS generation plots on Muse Cell Analyzer (*p<0.05, **p<0.01, ***p<0.001 compare to control).

Fig 5. Effects of AgNPs and PDT therapy on cell migration in MCF7 breast cancer cells.

a) Cell migration measurement with scratch wound assay in AgNPs (10 μg/mL) and/or PDT for 24h treated MCF7 cells. b) The rate of wound closure was calculated differences of cells filling the scratched area (**p<0.01 compare to control in MCF7 cells) for the three biological replicates within each group.

Fig 6. Effects of AgNPs and PDT therapy on apoptosis in MCF7 breast cancer cells.

a) Hoechst 33342/PI Double Staining of AgNPs (10 μg/mL) and/or PDT for 24h treatment in MCF7 cells. b) Percentage of PI staining cells of AgNPs (10 μg/mL) and/or PDT for 24h treatment (*p<0.05, **p<0.01 compare to control). c) Caspase-3 activity of AgNPs and PDT therapy for 24h in MCF7 cells (*p<0.05, ***p<0.001 compare to control). d) Western blot bands of expression levels of Bax and Bcl-2 proteins in AgNPs (10 μg/mL) and/or PDT for 24h treatment in MCF7 cells. Each protein band was normalized to the intensity of β-actin used. Western blot densitometry analysis of ratio of Bax/Bcl-2 protein expression levels in MCF7 cells. (**p<0.01 compare to control cells) for the three biological replicates within each group.