Synthesis of non-toxic, biocompatible, and colloidal stable silver nanoparticle using egg-white protein as capping and reducing agents for sustainable antibacterial application

Abstract

For nearly a decade, silver nanoparticles (AgNPs) have been the most prevalent commercial nanomaterials products widely used in different biomedical applications due to their broad-spectrum antimicrobial activity. However, their poor long-term stability in different environments, namely, pH, ionic strength, and temperature, and cytotoxicity toward mammalian cells has restricted their more extensive applications. Hence, there is urgent need to develop highly biocompatible, non-toxic, and stable silver nanoparticles for wide-ranging environments and applications. In the present study, a simple, sustainable, cost-effective and green method has been developed to prepare highly stable aqueous colloidal silver nanoparticles (AgNPs-EW) using the ovalbumin, ovotransferrin, and ovomucoid of egg-white as reducing and capping agents accomplished under the irradiation of direct sunlight. Then, we evaluated the effects of freezing-drying (lyophilization) and freeze-thaw cycles on the stability of AgNPs-EW in aqueous solution under visual inspection, transmission electron microscopy, and absorbance spectroscopy. In addition, we studied the antibacterial activity against Salmonella typhimurium and Escherichia coli, carried out biocompatibility studies on chicken blood, and tested acute, chronic toxicity in Drosophila melanogaster. The results suggest that AgNPs-EW did not aggregate upon freeze-thawing and lyophilization, thus exhibiting remarkable stability. The antibacterial activity results showed that the AgNPs-EW had the highest antibacterial activity, and the minimum inhibitory concentration (MIC) of AgNPs-EW for E. Coli and S. typhimurium were 4 and 6 μg ml^-1, respectively. The biocompatibility study revealed that the AgNPs-EW did not induce any hemolytic effect or structural damage to the cell membranes of chicken erythrocytes up to a concentration of 12 μg ml^-1. Similarly, no acute and chronic toxicity was observed on melanization, fecundity, hatchability, viability, and the duration of development in the 1^st generation of Drosophila melanogaster at the concentration range of 10 mg L^-1 to 100 mg L^-1 of AgNPs-EW, and all the flies completed their full developmental cycle. Therefore, the present study successfully demonstrated the green and sustainable preparation of non-toxic AgNPs-EW having good biocompatibility, enhanced colloidal stability, and antibacterial activity. Hence, the synthesized AgNPs-EW could be used for the development of an antimicrobial formulation for controlling microbial infection.

Fig. 1. (A) Vials showing showing nanoparticles formation at different time intervals (1: 5 min, 2: 10 min, 3: 15 min, 4: 30 min, 5: 45 min, 6: 60 min, 7: 90 min, 8: 120 min, 9: 160 min, 10: 190 min). (B) UV-Vis spectra for nanoparticles formation (reaction kinetics); (C and C′) TEM image of AgNPs-EW, rounded signify protein bounded on the surface of nanoparticles. (D) Size distribution of AgNPs-EW estimated by image J software.

Fig. 2. FTIR spectra showing egg-white in red colour and AgNPs-EW in black colour spectral lines.

Fig. 3. (A) Photographs of nanoparticles containing vials: 1: prior to lyophilization, 2: during ice phase 3: after lyophilization, 4: reconstituted AgNPs-EW; (B) UV-Vis spectra of reconstituted AgNPs-EW solution after freeze-drying (UV-Vis spectrum for AgNPs-EW prior to lyophilization is plotted as a reference spectrum); (C and C′) FE-SEM image of AgNPs-EW before and after freezing–thawing; (D) TEM image of AgNPs-EW after freeze-drying (E and F) zeta potential and DLS analysis for reconstituted AgNPs-EW.

Fig. 4. (A) SDS-PAGE gel of egg-white proteins showing bounding of various proteins on the surface of AgNPs-EW: Lane 1: egg-white proteins alone, Lane 2: bound egg-white protein present over synthesized AgNPs, and Lane 3: unbound egg-white proteins present in the reaction mixture. (B) Histogram representing the total band intensity of bound and unbound egg-white proteins over synthesized AgNPs-EW.

Fig. 5. (A) Photograph showing the dose-dependent inhibition of CFU count (colony forming unit), antimicrobial efficiency of AgNPs-EW against Salmonella Typhimurium on BHI, E. coli on LB plates. (B) Quantitative evaluation of the antibacterial activity of AgNPs-EW by counting the colonies grown on agar and BHI plates. Values are expressed as the mean ± SD (n = 3).

Fig. 6. (A) MTT assay for the viability of E. coli and Salmonella typhimurium after treatment with different concentrations of AgNPs-EW (1, 2, 4, 6, 8, 10, 12 μg ml⁻¹). (B) Intracellular ROS production in the bacterial cell suspensions treated with AgNPs-EW. Values are expressed as the mean ± SD (n = 3).

Fig. 7. Confocal images of Salmonella Typhimurium and E. coli after treatment with AgNPs-EW (4 μg ml⁻¹) for 4 h and stained with acridine orange (green, indicator of intact membrane, live cells) and propidium iodide (red, indicator of membrane damage, dead cells) (scale bar 20 μm).

Fig. 8. Morphology of chicken erythrocytes under upright light microscopy (40× magnification), (A) image of chicken erythrocytes before and (B) after the treatment with 12 μg ml⁻¹ of AgNPs-EW ((C) rounded circle indicates the damaged erythrocytes).

Fig. 9. Acute and chronic toxicity effects of AgNPs-EW on Drosophila melanogaster, (A) rate of fecundity (%), (B) hatchability% (C) viability% (D) toxic effects of AgNPs-EW on duration of developmental stage of Drosophila melanogaster (E) abdominal pigmentation. Values are expressed as the mean ± SD (n = 5).

Fig. 10. Impact of freezing–drying on the antibacterial activity of AgNPs-EW: (A) freeze-dried AgNPs-EW used for the antibacterial study (B) photograph of plates showing impact of freezing–drying on the antibacterial efficiency of AgNPs-EW against Salmonella Typhimurium on BHI and E. coli on LB plates. (C) Quantitative evaluation of the antibacterial activity of AgNPs-EW by counting the colonies grown on agar and BHI plates. Values are expressed as the mean ± SD (n = 3).