Green Synthesis of Silver Nanoparticles for Biomedical Applications

By Megan Craig, M.Sc.Nov 17 2021

Antimicrobial resistance and increasing healthcare costs have inspired researchers to create innovative and effective antimicrobial therapies. Much focus has been placed upon 'nanoparticle-based antimicrobials', as nanoparticles offer distinct benefits compared to traditional chemical antimicrobial agents with multidrug resistance.

Image Credit: Parilov/Shutterstock.com

Silver nanoparticles (AgNPs) include amphiphilic hyperbranched macromolecules, making them an effective antimicrobial surface coating agent. These nanoparticles can even be modified to increase efficiency, enabling their use in various fields, particularly healthcare.

Nanoparticles synthesized through green nanotechnology, however, are an area attracting growing interest. 

Green nanotechnology aims to eliminate or reduce the pollution caused by conventional methods used for their synthesis. Furthermore, environmental impacts in the product chain are estimated and mitigated, resulting in this approach becoming highly favorable for a wide range of biomedical and biotechnological applications.

The production of AgNPs through green nanotechnology is a simple, cost-effective, high-yield synthesis and an eco-friendly procedure. It was introduced to the synthesis of nanoparticles without using toxic chemicals and avoids the production of undesirable toxic products.

A recent study illustrates the production of AgNPs through green technology and its coating by a quaternary ammonium salt, benzalkonium chloride (BAC), with synthesized AgNPs characterized by TEM and Dynamic Light Scattering (DLS). 

Antimicrobial screening of AgNPs and BAC-coated AgNPs was performed against a range of gram-positive (gm+) bacteria and gram-negative (gm−) bacteria by colony-forming units, β-glucosidase activity, and a zone of inhibition.

Methodology

AgNPs were synthesized with slight modifications. Firstly, fresh neem leaves were washed using double-distilled water and then air-dried at room temperature. Secondly, 50 gm neem leaves were soaked in 250 mL double-distilled water and boiled for 1 hour. The obtained extract was cooled down and filtered using Whatman filter paper No. 1.

Then, Azadirachta indica leaf extract and 1 mM AgNO₃ were mixed in a 1:10 ratio. The solution was placed on a magnetic stirrer in an ice bath for 30 minutes at room temperature in a dark chamber to inhibit the photo-activation of silver nitrate.

Ag⁺ reduced to Ag⁰ with the solution turning brown, implying the formation of AgNPs. Benzalkonium chloride (0.1% w/v) was coated on the surface of the resulting AgNPs in an Erlenmeyer glass at room temperature in a dark chamber on a magnetic stirrer.

The morphology, dimensions, and size of AgNPs were obtained. TEM analysis samples were prepared through drop-coating of diluted NP solution on carbon-coated copper grids at standard atmospheric conditions before characterizing AgNPs. 

The bactericidal effect of AgNPs against four different clinical pathogenic bacteria—Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Streptococcus pneumoniae—was analyzed.

Through ultrasonication, AgNPs were dispersed in pre-sterilized millipore water. Desired concentrations (0.0625, 0.1250, 0.250, 0.500, and 1.0 mM) for bacterial effects analysis were formulated with the aqueous dispersions of AgNPs

Results

Figure 1 displays the TEM image of AgNPs, representing the fineness of the particles with a mean diameter of 30 nm. Dynamic light scattering was employed to identify the size distribution profile of AgNPs. From Figure 2, it can be seen that the average mean size of AgNPs was 30 nm.

Figure 1. TEM of the synthesized AgNPs. Image Credit: Ansari & Alshanberi, 2021

Figure 2. DLS of the synthesized AgNPs. Image Credit: Ansari & Alshanberi, 2021

Antibacterial assays found that increased concentration of AgNPs led to a considerable reduction in CFU numbers. For instance, at 0.25 mM AgNPs, 22 × 109 CFU were formed. However, for benzalkonium chloride-coated AgNPs, this figure was reduced to 14 × 109 CFU under similar experimental conditions (see Figure 3).

Figure 3. CFU by the synthesized AgNPs (blue) and BAC-coated AgNPs (orange). Image Credit: Ansari & Alshanberi, 2021

β-glucosidase activity was observed under different AgNPs concentrations. Researchers found a substantial increase in the enzymatic activity of β-glucosidase beyond 0.0625 mM and up to 0.5 mM concentration of AgNPs and BAC–AgNPs (see Figure 4)

Figure 4. Activity of β-glucosidase with the synthesized AgNPs (blue) and BAC-coated AgNPs (red). Image Credit: Ansari & Alshanberi, 2021

No additional changes in activity were observed beyond this concentration.

Table 1 indicates the zone of inhibition of AgNPs and BAC–AgNPs against E. coli, P. aeruginosa, B. subtilis, and S. pneumoniae at 0.25 and 0.50 mM concentration of these bioactive agents, respectively.

Table 1. Zone of inhibition (diameter, cm) of antibacterial test of AgNPs and Benzalkonium chloride (BAC)-coated AgNPs. Source: Ansari & Alshanberi, 2021

An increase in the concentration of these bioactive agents to 0.5 mM led to a significant decrease in the ZOI for BAC–AgNPs than AgNPs. E. coli ZOI was found to be 4.28 and 4.40 cm, while S. pneumoniae ZOI was 3.78 and 3.91 cm for AgNPs and BAC–AgNPs, respectively under similar incubation conditions.

Conclusion

In this study, the synthesis of AgNPs using neem leaf meets all the conditions of a 100% green chemical process. This includes an environmentally friendly, fast, and green approach that is economical and does not use reducing agents or external stabilizers.

Researchers found that the use of benzalkonium chloride as a quaternary ammonium compound led to major improvements in the antibacterial activity of the silver nanoparticles produced from the neem leaves.

Superior antimicrobial efficacy was achieved against bacterial strains like E. coli, P. aeruginosa, B. subtilis, and S. pneumoniae. The persistent antibacterial activity of such nanoparticles against other microorganisms may be studied to extend their use to biomedical, environmental, and biotechnological sectors. This study will also have major implications for the development of other nanoparticles by clean and green technologies. 

Continue reading: An Overview of the Synthesis and Application of Green Nanoparticles.

Journal Reference:

Ansari, S A & Alshanberi, A M (2021) Clinical Application of Silver Nanoparticles Coated by Benzalkonium Chloride. Coatings, 11(11), p. 1382. Available at: https://doi.org/10.3390/coatings11111382.

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