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.

Biogenic Silver Nanoparticles: What We Know and What Do We Need to Know?

“…Nowadays, the applications of various fields of conventional biological sciences, along with innovative and efficient nanotechnological tools for the production of greensynthesized AgNPs, have provided a new alternative strategy and perspective for the prevention and treatment of various diseases [117]. To date, many reports are available that demonstrate the antibacterial, antiviral, antifungal, antiprotozoal, antiparasitic, and anticancer efficacy of bio-AgNPs [9,[118][119][120][121][122][123][124][125].…”

“…Due to the increasing antibiotic resistance of bacteria and the inef-fectiveness of conventional therapeutics, there is an urgent need to develop new agents to combat existing pathogens in general and new and emerging pathogens in particular. Nanoparticles can be used as alternative and highly effective antimicrobial agents due to their high surface area, chemical reduction properties, and surface reactivity [9,[126][127][128]. There are a number of reports on bio-AgNPs synthesized by bacteria, fungi, and plants that have demonstrated antimicrobial potential against pathogenic bacteria and fungi (Table 3).…”

“…Overall, bio-AgNPs show a stronger inhibitory effect on Gram-negative than Grampositive bacteria. This difference in efficacy may be due to the variance in thickness of the cell wall present in these bacteria [9,141,[144][145][146]. Bio-AgNPs were also found to effectively inhibit biofilm formation in Gram-positive (Stapylococcus aureus) and Gramnegative (Escherichia coli) bacteria [147].…”

“…Biogenic AgNPs may also be an eco-friendly and biocompatible alternative to conventional anticancer drugs [173]. There are various reports concerning the inhibitory effects of bio-AgNPs on many cancer cell lines (e.g., MCF-7 breast cancer, PC-3 prostate cancer, A549 lung cancer, KB oral cancer, PA1 ovarian cancer) [9,123,142,[174][175][176][177][178]. The proposed mechanism for anticancer activity of biogenic AgNPs is apoptosis caused through caspasedependent and mitochondria-dependent pathways induced by nanoparticles [179,180].…”

“…Biosynthesis has several advantages over the physical and chemical processes because it is eco-friendly, cost-effective, and can be executed at ambient temperature and pressure, with enhanced bioactivity, and less toxicity. It was demonstrated that the proteins and enzymes secreted by the above-mentioned bio-systems act as reducing agents that reduce the bulk metal salt into respective nanoparticles and also act as capping agents that provide stability to nanoparticles and make them bio-compatible for various biological applications [8,9]. Among the metal nanoparticles, silver nanoparticles (AgNPs) have been revealed to have an enormous number of applications, particularly in biomedicine due to their broad range of antimicrobial potential, including antibacterial, antifungal, antiviral, and antiprotozoal properties [10,11].…”