Oxidative stress mediated cytotoxicity of biologically synthesized silver nanoparticles in human lung epithelial adenocarcinoma cell line

Abstract

The goal of the present study was to investigate the toxicity of biologically prepared small size of silver nanoparticles in human lung epithelial adenocarcinoma cells A549. Herein, we describe a facile method for the synthesis of silver nanoparticles by treating the supernatant from a culture of Escherichia coli with silver nitrate. The formation of silver nanoparticles was characterized using various analytical techniques. The results from UV-visible (UV-vis) spectroscopy and X-ray diffraction analysis show a characteristic strong resonance centered at 420 nm and a single crystalline nature, respectively. Fourier transform infrared spectroscopy confirmed the possible bio-molecules responsible for the reduction of silver from silver nitrate into nanoparticles. The particle size analyzer and transmission electron microscopy results suggest that silver nanoparticles are spherical in shape with an average diameter of 15 nm. The results derived from in vitro studies showed a concentration-dependent decrease in cell viability when A549 cells were exposed to silver nanoparticles. This decrease in cell viability corresponded to increased leakage of lactate dehydrogenase (LDH), increased intracellular reactive oxygen species generation (ROS), and decreased mitochondrial transmembrane potential (MTP). Furthermore, uptake and intracellular localization of silver nanoparticles were observed and were accompanied by accumulation of autophagosomes and autolysosomes in A549 cells. The results indicate that silver nanoparticles play a significant role in apoptosis. Interestingly, biologically synthesized silver nanoparticles showed more potent cytotoxicity at the concentrations tested compared to that shown by chemically synthesized silver nanoparticles. Therefore, our results demonstrated that human lung epithelial A549 cells could provide a valuable model to assess the cytotoxicity of silver nanoparticles.

Keywords: Adenocarcinoma cells A549; Lactate dehydrogenase (LDH); Mitochondrial transmembrane potential (MTP); Reactive oxygen species generation (ROS); Silver nanoparticles (AgNP).

Figure 1

Synthesis and characterization of bio-AgNPs using culture supernatant from E. coli. The inset shows tubes containing samples of silver nitrate (AgNO₃) after exposure to 5 h (1), AgNO₃ with the extracellular culture supernatant of E. coli (2), and AgNO₃ plus supernatant of E. coli (3). The color of the solution turned from pale yellow to brown after 5 h of incubation, indicating the formation of silver nanoparticles. The absorption spectrum of AgNPs synthesized by E. coli culture supernatant exhibited a strong broad peak at 420 nm and observation of such a band is assigned to surface plasmon resonance of the particles.

Figure 2

XRD pattern of AgNPs. A representative X-ray diffraction (XRD) pattern of silver nanoparticles formed after reaction of culture supernatant of E. coli with 5 mM of silver nitrate (AgNO₃) for 5 h at 50°C. The XRD pattern shows four intense peaks in the whole spectrum of 2θ values ranging from 20 to 70. The intense peaks were observed at 2θ values of 23.6°, 29.5°, 33.7°, and 46.7°, corresponding to 111, 200, 220, and 311 planes for silver, respectively.

Figure 3

FT-IR spectrum of biologically synthesized silver nanoparticles.

Figure 4

Size and morphology of AgNPs analysis by TEM. (A). Several fields were photographed and used to determine the diameter of silver nanoparticles (AgNPs). (B). Particle size distributions from transmission electron microscopy images. The average range of observed diameter was 15 nm.

Figure 5

Size distribution analysis by dynamic light scattering (DLS). Biologically synthesized silver nanoparticles (bio-AgNPs) and chemically synthesized silver nanoparticles (chem-AgNPs) were dispersed in deionized water and DMEM media with and without serum. The particles were mixed thoroughly via sonication and vortexing, and samples were measured at 25 μg/ml.

Figure 6

Effect of AgNPs on cell viability of A549 human lung epithelial adenocarcinoma cells. Cells were treated with silver nanoparticles (AgNPs) at several concentrations for 24 h and cytotoxicity was determined by the MTT method. The results are expressed as the mean ± SD of three separate experiments each of which contained three replicates. Treated groups showed statistically significant differences from the control group by the Student's t test (p < 0.05).

Figure 7

Effect of AgNPs on LDH release from A549 human lung epithelial adenocarcinoma cells. Lactate dehydrogenase (LDH) was measured by changes in optical density due to NAD⁺ reduction monitored at 490 nm, as described in the ‘Methods’ section. The results are expressed as the mean ± SD of three separate experiments each of which contained three replicates. Treated groups showed statistically significant differences from the control group by the Student's t test (p < 0.05).

Figure 8

ROS generation in AgNP-treated A549 human lung epithelial adenocarcinoma cells. Fluorescence images of A549 cells without silver nanoparticles (AgNPs) (0) and cells treated with biologically synthesized AgNPs (bio-AgNPs) (25 μg/ml) and chemically synthesized AgNPs (chem-AgNPs) (70 μg/ml) and incubated at different time points. Both bio-AgNPs and chem-AgNPs support the formation of hydrogen peroxide inside the A549 cells.

Figure 9

AgNPs modulates mitochondrial transmembrane potential. Changes in mitochondrial transmembrane potential (MTP) was determined using the cationic fluorescent indicator, JC-1. Fluorescence images of control A549 cells (without silver nanoparticles (AgNPs)) and cells treated with biologically synthesized AgNPs (bio-AgNPs) (25 μg/ml) and chemically synthesized AgNPs (chem-AgNPs) (70 μg/ml). The changes of mitochondrial membrane potential by AgNPs were obtained using fluorescence microscopy. JC-1 formed red-fluorescent J-aggregates in healthy A549 cells with high MTP, whereas A549 cells exposed to AgNPs had low MTP and, JC-1 existed as a monomer, showing green fluorescence.

Figure 10

Intracellular localization of AgNPs and accumulation of autophagosomes and autolysosomes. A549 cells were treated with silver nanoparticles (AgNPs) for 24 h and then processed for transmission electron microscopy (TEM) sections. TEM images of ultramicrotome slices of A549 cells without AgNPs (A), internalization of biologically synthesized AgNPs (bio-AgNPs) (B), and internalization of chemically synthesized AgNPs (chem-AgNPs) within the cells (C). Bio-AgNPs induces accumulation of autophagosomes (black arrow) and autolysosomes (white arrow) in cells treated with bio-AgNPs for 24 h (D). Autolysosomes and vesicular structures consistent with autophagy were detected in cells treated with bio-AgNPs (E) and chem-AgNPs (F).