Unveiling the cytotoxicity of phytosynthesised silver nanoparticles using Tinospora cordifolia leaves against human lung adenocarcinoma A549 cell line

Abstract

Biosynthesis of silver nanoparticles (AgNPs) using plant extract is a cheap, easily accessible and natural process in which the phyto-constituents of the plants act as capping, stabilising and reducing agent. The present study explored the biosynthesis of AgNPs using aqueous leaf extract of Tinospora cordifolia and characterised via various techniques such as Fourier transform infrared, scanning electron microscopy, transmission electron microscopy (TEM), energy dispersive X-ray analysis and X-ray diffraction. Here, TEM confirmed the spherical morphology with 25-50 nm size of synthesised AgNPs. Further, anticancer efficiency of AgNPs synthesised using T. cordifolia leaves were evaluated against human lung adenocarcinoma cell line A549 by MTT, trypan blue assay, apoptotic morphological changes using Annexin V-FITC and Propidium iodide (PI), nuclear morphological changes by DAPI (4, 6-diamidino-2-phenylindole dihydrochloride) staining, reactive oxygen species generation and mitochondrial membrane potential determination. Results confirmed the AgNPs synthesised using T. cordifolia leaves are found to be highly toxic against human lung adenocarcinoma cell line A549.

Fig. 1

Characterisation of AgNPs (a) UV–Vis spectrophotometer graph of biosynthesised AgNPs, (b) XRD pattern of AgNPs indicating FCC structure, (c) SEM micrographs of biosynthesised AgNPs showing surface morphology and spherical shape, (d) TEM micrographs of phytofabricated AgNPs describing their shape and size between 25–50 nm

Fig. 2

FTIR analysis of biosynthesised AgNPs

Fig. 3

Dose‐dependent cytotoxicity analysis by MTT assay (a) Cytotoxicity of different concentrations (25, 50, 75, 100, 150 ppm) of AgNPs was measured by MTT assay on A549 cells for 24 h, (b) IC₅₀ value was detected as 100 ppm by MTT assay in treated A549 cells for 24 h. Data is representative of three experiments. *p value<0.05

Fig. 4

Dose‐ and time‐dependent analysis of relative cell viability percentage by trypan blue dye exclusion method. Decrease in relative cell viability percentage in A549 cells up to 48 h after using AgNPs in different concentrations (25–150 ppm) was determined by microscopic observation of the cells after staining with trypan blue dye. Data is representative of three experiments. *p value<0.05

Fig. 5

Cytomorphological change analysis (a) 5×10⁵ cells/well (6‐well plate) A549 cells were treated with 100 ppm AgNPs for 24 h, shrinked and poorly adhered cells were observed under inverted microscope (40X magnification) in comparison with the untreated control cells, (b) Decrease in number of A549 cells after the same treatment (24 and 48 h) was observed under (20X magnification)

Fig. 6

Observation of apoptotic changes in treated cells after double staining with Annexin FITC/PI (a) Untreated control A549 cells showed no sign of apoptosis after staining with Annexin V‐FITC and PI, (b) Cells treated with AgNPs showed detection of apoptosis after staining with Annexin V‐FITC and PI, (1) AnnexinV‐FITC positive cells indicate early apoptosis and (2) PI positive cells indicate late apoptosis

Fig. 7

Nuclear damage detection assay (a) Untreated A549 cells showed undamaged nucleus after staining with nuclear stain DAPI, (b) (i) Cells treated with AgNPs 100 ppm for 24 h showed nuclear membrane rupture under fluorescence microscopy after staining with DAPI. (ii) After 48 h of treatment cells showed fragmented nuclei when stained with DAPI

Fig. 8

Detection of ROS in treated A549 cells (a) Time‐dependent (24 and 48 h) increase of green fluorescence intensity in AgNPs treated A549 cells when stained with DCF‐DA stain, clearly indicating ROS generation under fluorescent microscopy, (b) Fluorescence intensity (FI) was plotted against time. The graph indicates time‐dependent increment of F1 in treated cells in comparison with untreated cells. Data is representative of three experiments. *p ‐value <0.05

Fig. 9

Mitochondrial membrane potential (ψ_m) decreases in AgNPs treated A549 cells (a) (i) Untreated control A549 cells with no changes in ψ_m showing red colour under fluorescence microscopy (590 nm) after staining with JC1 dye. (ii) Cells treated with AgNPs showed decreased ψ _m with green florescence (530 nm) after staining with JC1 dye, (b) Ratio of fluorescence at 590–530 nm was plotted against A549 cell types (untreated or treated with AgNPs for 24 h), showing the clear decrease of mitochondrial membrane potential of treated cells. Data is representative of three experiments. *p value <0.05