Green Synthesis of Ag Nanoparticles for Antibacterial Applications

By Megan Craig, M.Sc.Dec 8 2021

Nanoparticles, particularly protein-derived inorganic nanoparticles, are in great demand. Proteins provide a reducing and stabilizing environment during nanoparticle synthesis. Research published in the International Journal of Nanomedicine discusses the synthesis of AgNPs with Benincasa hispida pulp protein extract before exploring its characterization and evaluation of its biological activity.

Image Credit: CI Photos/Shutterstock.com

Recently, the popularity of inorganic nanomaterials has seen a vast increase. This demand is due to their numerous applications in various macroeconomic industries.

Silver nanoparticles (AgNPs) are used in different fields, specifically as a broad-spectrum antimicrobial agent. Their use also extends to many commercial consumer products like cosmetics, wound dressings, antimicrobial socks, and personal care items. With most nanoparticles, both physical and chemical methods are employed for their synthesis. Each has its own set of disadvantages, with biocompatibility tending to top the list. 

Green synthesis, synthesizing nanoparticles from organic products like bacteria, fungi and plants, is a promising area of nanoparticle research. The phytochemicals from plants can stabilize and reduce agents in synthesizing metal nanoparticles.

Benincasa (Cucurbitaceae), a monotypic genus, has a single species named Benincasa hispida. The fruits of B. hispida are commonly used as a diuretic, and the seeds have been shown to have antiangiogenic effects on prostate cells. The mature fruits of B. hispida harbor proteins, dietary fibers, sterols, enzymes, flavonoid C-glycoside, sugars, phenolic acids, and trace minerals.

In this study, researchers devised a route to synthesize AgNPs with B. hispida pulp protein extract, contributing to the field of green nanotechnology. The generated nanoparticles were found to be highly biocompatible and stable. 

Methodology

Extraction of B. hispida fruit protein took place by finely grinding cleaned fruits and later homogenizing the powdered samples. The proteins were then extracted, and the exact amount was calculated. The extracted protein was then centrifuged, the purified protein extract was utilized for synthesizing AgNPs. The protein concentration was determined with Bradford assay and the purified proteins were examined on 7.5% SDS-PAGE.

The silver nanoparticles were then synthesized from the B. hispida fruit proteins of varying concentrations. Silver nitrate salt was added at different temperatures to obtain the desired-sized nanoparticles.

The synthesized nanoparticles were later characterized with UV-Visible Spectroscopy, the optical density was measured, and the average particle size of AgNPs was also evaluated.

Transmission Electron Microscopy (TEM) was used to investigate the size and the zeta-potential was evaluated with a Zetasizer Nano-ZS. Fourier-Transform Infrared Spectroscopy (FTIR) was used to reveal the information on protein–nanoparticle associations.

The antibacterial activity of the synthesized AgNPs was evaluated with the agar well diffusion technique against E. coli, S. aureus, S. enteric, and S. epidermis. From here, the minimum inhibition concentration of AgNPs was determined along with the anti-biofilm potential of AgNPs.

The team also investigated the anticancer potential of AgNPs against A549 and NRK cells, and studied the cytomorphological changes of the treated A549 cells and the variations in the nuclear morphology of cells.

Results and Discussion

The native charge of proteins performs a vital role in nanoemulsion stability (see Figure 1).

Figure 1. Schematic representation of the mechanism of B. hispida fruit proteins mediated synthesis of biogenic AgNPs and their roles in anticancer, antibacterial, and antibiofilm agents. Image Credit: Baker, et al., 2021

Figure 2 reveals the characterization of the prepared AgNPs using different techniques.

Figure 2. Characterization of B. hispida fruit proteins mediated synthesis of AgNPs by physical techniques: (A) UV-visible spectrum, (B) TEM Micrograph with light gray protein Corona mark by an arrow, (C) size distribution by DLS (D) zeta potential (E) surface characterization by FTIR spectrum. Image Credit: Baker, et al., 2021

The average size was found to be 27 ± 1 nm, and further analysis revealed the shape to be almost spherical. The zeta potential value for AgNPs was –19.7 ± 0.2 mV. FT analysis showed peaks that are characteristics of C=O amide groups of the amide I linkage.

The antibacterial activity of AgNPs examined against the four types of bacteria (see Figure 3) showed broad-spectrum activity.

Figure 3. Graph showing the antibacterial potential of silver nanoparticles. All the data were expressed in the mean ± SD of three experiments. Image Credit: Baker, et al., 2021

Antibacterial activity can be due to several mechanisms like ROS generation, enhanced permeability, change in membrane fluidity, and membrane integrity loss, interrupting the actin, binding to the 30S subunit of ribosomes.

The biosynthesized AgNPs can obstruct the biofilm formation of different pathogenic bacteria; a significant reduction of 70-90% was seen in the test conducted (see Figure 4). This antibiofilm formation was dose-dependent and that the increased concentration of AgNPs decreased the biofilm formation.

Figure 4. Image and graph showing inhibition of biofilm formation of silver nanoparticles. All the data were expressed in the mean ± SD of three experiments. Image Credit: Baker, et al., 2021

Anticancer studies revealed that the AgNPs were active against A549 cells (see Figure 5A), however, they did not exhibit any activity against NRK cells up to 200 µM (see Figure 5B).

Figure 5. The cytotoxicity study graph of MTT assay for AgNPs treatment against (A) A549 cells and (B) NRK cells. Image Credit: Baker, et al., 2021

Also, the anticancer effect is dose-dependent, the activity increases with an increase in the concentration of AgNPs. Figure 6 indicates the morphological changes of A549 cells after incubation with various concentrations (IC25, IC50 & IC75) of AgNPs.

Figure 6. The cytomorphological study of AgNPs treated A549 cells (A) control, (B) IC25 (C) IC50, (D) IC75 at 20× magnification. Image Credit: Baker, et al., 2021

Observations show cells having deformed morphologies while some cells had their plasma membrane intact, indicating the start of apoptosis.

The AgNPs' interaction with nuclear materials was analyzed using fluorescent dye. The treated cells showed apoptotic impact (see Figure 7A and B) when compared with untreated cells. It was noted that AgNPs produced more fluorescence compared to untreated cells (Figure 8A).

Figure 7. Images observed for AgNPs treated cells under phase contrast microscope after 48 hours of treatment for DAPI staining (A) control (B) treated cells; for Mito Tracker Red staining (C) control (D) treated cells and for DCFDA staining (E) control (F) treated cells. Image Credit: Baker, et al., 2021

Figure 8. Graphical representation in terms of percent (A) nuclear condensation, (B) mitochondrial content, and (C) intracellular ROS generation. All the data were expressed in the mean ± SD of three experiments. Image Credit: Baker, et al., 2021

It was seen that AgNPs can disrupt the mitochondrial membrane potential (ΔΨm) of A549 cells. ROS generation of A549 cells after interaction with AgNPs was evaluated and the intensity of fluorescence was a direct measure of ROS generated in the cells. A greater intensity of fluorescence was observed while the untreated cells showed no fluorescence.

Conclusion

Researchers detailed a green protocol for producing silver nanoparticles with the fruit protein extract of Benincasa hispida. The synthesized silver nanoparticles enhanced the potential of Benincasa hispida fruit protein, and showed efficient antibacterial, antibiofilm, and anticancer activities. However further studies are required to analyze their toxicity.

Journal Reference

Baker, A., Iram, S., Syed, A., Elgorban, A. M., Bahkali, A. H., Ahmad, K., Khan, M. S., Kim, J. (2021) Fruit Derived Potentially Bioactive Bioengineered Silver Nanoparticles. International Journal of Nanomedicine, 16, pp. 7711–7726. Available online: https://www.dovepress.com/getfile.php?fileID=75944.

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