Green Synthesized Silver Nanoparticles: Antibacterial and Anticancer Activities, Biocompatibility, and Analyses of Surface-Attached Proteins

Abstract

The increasing number of multi-drug-resistant bacteria and cancer cases, that are a real threat to humankind, forces research world to develop new weapons to deal with it. Biogenic silver nanoparticles (AgNPs) are considered as a solution to this problem. Biosynthesis of AgNPs is regarded as a green, eco-friendly, low-priced process that provides small and biocompatible nanostructures with antimicrobial and anticancer activities and potential application in medicine. The biocompatibility of these nanoparticles is related to the coating with biomolecules of natural origin. The synthesis of AgNPs from actinobacterial strain was confirmed using UV-Vis spectroscopy while their morphology, crystalline structure, stability, and coating were characterized using, transmission electron microscopy (TEM), X-ray diffraction (XRD), Zeta potential and Fourier transform infrared spectroscopy (FTIR). Antibacterial activity of biogenic AgNPs was evaluated by determination of minimum inhibitory and minimum biocidal concentrations (MIC and MBC) against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. The potential mechanism of antibacterial action of AgNPs was determined by measurement of ATP level. Since the use of AgNPs in biomedical applications depend on their safety, the in vitro cytotoxicity of biosynthesized AgNPs on MCF-7 human breast cancer cell line and murine macrophage cell line RAW 264.7 using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, cell lactate dehydrogenase (LDH) release and measurement of reactive oxygen species (ROS) level were assessed. The nanoparticle protein capping agent that can be involved in reduction of silver ions to AgNPs and their stabilization was identified using LC-MS/MS. Nanoparticles were spherical in shape, small in size (mean 13.2 nm), showed crystalline nature, good stability (-18.7 mV) and presence of capping agents. They exhibited antibacterial activity (MIC of 8-128 μg ml^-1, MBC of 64-256 μg ml^-1) and significantly decreased ATP levels in bacterial cells after treatment with different concentrations of AgNPs. The in vitro analysis showed that the AgNPs demonstrated dose-dependent cytotoxicity against RAW 264.7 macrophages and MCF-7 breast cancer cells but higher against the latter than the former. Cell viability decrease was found to be 42.2-14.2 and 38.0-15.5% while LDH leakage 14.6-42.7% and 19.0-45.0%, respectively. IC₅₀ values calculated for MTT assay was found to be 16.3 and 12.0 μg ml^-1 and for LDH assay 102.3 and 76.2 μg ml^-1, respectively. Moreover, MCF-7 cells released a greater amount of ROS than RAW 264.7 macrophages during stimulation with all tested concentrations of AgNPs (1.47-3.13 and 1.02-2.58 fold increase, respectively). The SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis revealed the presence of five protein bands at a molecular weight between 31.7 and 280.9 kDa. These proteins showed the highest homology to hypothetical proteins and porins from E. coli, Delftia sp. and Pseudomonas rhodesiae. Based on obtained results it can be concluded that biogenic AgNPs were capped with proteins and demonstrated potential as antimicrobial and anticancer agent.

Keywords: MCF-7; RAW 264.7; antibacterial agent; anticancer agent; biogenic AgNPs; capping agents; cytotoxicity; protein molecules.

Figure 1

UV-Vis spectrum of biosynthesized AgNPs.

Figure 2

TEM of biosynthesized AgNPs.

Figure 3

XRD pattern of biosynthesized AgNPs.

Figure 4

Zeta potential graph of biosynthesized AgNPs.

Figure 5

FTIR spectrum of biosynthesized AgNPs. AgNPs (A) and control (B).

Figure 6

Inhibition of ATP synthesis (%) in Escherichia coli (A), Klebsiella pneumoniae (B), Pseudomonas aeruginosa (C), and Staphylococcus aureus (D) cells after treatment with biosynthesized AgNPs at different concentrations (0.125–512 μg ml⁻¹).

Figure 7

Cytotoxic activity of biosynthesized AgNPs toward human breast cancer cell line (MCF-7) and murine macrophage cell line (RAW 264.7) estimated by MTT assay (A), LDH release (B), and ROS production level (C). Asterisks indicate significant differences between the viability of cells, the levels of LDH released from cells, or the ROS production in cells of both cell lines (***p < 0.001; **p < 0.01; *p < 0.1).

Figure 8

SDS-PAGE of proteins associated with biosynthesized AgNPs.