Synthesis of Gold Nanoparticles by Using Green Machinery: Characterization and In Vitro Toxicity

Abstract

Green synthesis of gold nanoparticles (GNPs) with plant extracts has gained considerable interest in the field of biomedicine. Recently, the bioreduction nature of herbal extracts has helped to synthesize spherical GNPs of different potential from gold salt. In this study, a fast ecofriendly method was adopted for the synthesis of GNPs using fresh peel (aqueous) extracts of Benincasa hispida, which acted as reducing and stabilizing agents. The biosynthesized GNPs were characterized by UV-VIS and Fourier transform infrared spectroscopy, transmission electron microscopy (TEM), and dynamic light scattering. In addition, the in vitro antibacterial and anticancer activities of synthesized GNPs were investigated. The formation of gold nanoparticles was confirmed by the existence of a sharp absorption peak at 520 nm, corresponding to the surface plasmon resonance (SPR) band of the GNPs. TEM analysis revealed that the prepared GNPs were spherical in shape and had an average particle size of 22.18 ± 2 nm. Most importantly, the synthesized GNPs exhibited considerable antibacterial activity against different Gram-positive and Gram-negative bacteria. Furthermore, the biosynthesized GNPs exerted remarkable in vitro cytotoxicity against human cervical cancer cell line, while sparing normal human primary osteoblast cells. Such cytotoxic effect was attributed to the increased production of reactive oxygen species (ROS) that contributed to the damage of HeLa cells. Collectively, peel extracts of B. hispida can be efficiently used for the synthesis of GNPs, which can be adopted as a natural source of antimicrobial and anticancer agent.

Keywords: Benincasa hispida; antibacterial; anticancer; auric chloride (gold salt); gold nanoparticles (GNPs).

Figure 1

Characterization of gold nanoparticles (GNPs): (A) UV–VIS spectroscopy, (B) transmission electron microscopy, (C) dynamic light scattering, (D) zeta potential, and (E) Fourier transform infrared (FTIR) spectrum.

Figure 2

Qualitative assessment of antibacterial activity of GNPs. Müeller–Hinton (MH) agar plates were seeded with standardized suspensions (equivalent to the 0.5 McFarland) of (A) Escherichia coli, (B) Staphylococcus aureus, (C) Salmonella abony, and (D) Klebsiella pneumonia. Equal amounts of GNPs and PBS (negative control) were poured in the wells made in MH plates. After overnight incubation at 37 °C, inhibition zones around wells of GNPs (10 µg/mL) against all tested bacterial species, in comparison to control, were observed.

Figure 3

Determination of minimum inhibitory concentration (MIC) of GNPs. Aliquots of GNPs were serially diluted in 96-well microtiter plates in tryptic soy broth (TSB) medium. Aliquots (10 μL) from prepared standard suspensions (equivalent to the 0.5 McFarland) of tested bacterial strains were added to each well. After overnight incubation at 37 °C, the bacterial cells were collected and viably counted. The experiment was repeated in triplicate and the data shown are the means ± standard errors. The MIC was the lowest concentration of GNPs that completely inhibited the bacterial growth, and MIC₅₀ was the GNPs concentration that inhibited 50% of the bacteria.

Figure 4

Assessment of GNPs cytotoxicity and determination GNPs IC₅₀ on cancer cells. Normal human primary osteoblasts and human cervical cancer cell (HeLa cells) were plated overnight at a density of 1 × 10⁴ cell per well in a 96-well plate at 37 °C. The normal or cancer cells were treated with different concentrations of GNPs and the in vitro cytotoxicity was evaluated using MTT assay. The inhibition percentages were calculated relative to negative control and IC₅₀ was the GNPs concentration, which inhibits 50% of HeLa cells. The experiment was conducted in triplicate and the data shown are the means ± standard errors.

Figure 5

Evaluation of anticancer activity of GNPs. (A,B) Changes in cellular morphology: HeLa cells were pretreated with PBS (negative control) or GNPs in IC₅₀ for 48 h at 37 °C in 5% CO₂ atmosphere and the morphological changes were observed. (C,D) Changes in nuclear morphology: the nuclei of untreated control or treated HeLa cell with GNPs at IC₅₀ were stained with fluorescent nuclear dye DAPI. (E) The mean signal intensities and standard deviations for at least 50 DAPI stained treated or untreated HeLa cells with GNPs at IC₅₀ were measured. (F,G) Intracellular reactive oxygen species (ROS) production: untreated (control) or treated HeLa cells with GNPs at IC₅₀ were stained with fluorogenic reagent H2DCFDA. (H) The intensities of fluorescence in GNPs (IC₅₀) treated HeLa cells in comparison to untreated cells were measured; the mean signal intensities and standard deviations for at least 50 treated cells were calculated.