Enhanced antibacterial activity of hemocyanin purified from Portunus pelagicus hemolymph combined with silver nanoparticles – Intracellular uptake and mode of action.

• Nanosilver (AgNPs) was fabricated using hemocyanin (Hc, 78 kDa) purified from of Portunus pelagigus hemolymph. • Hc-AgNPs were more efficient than purified Hc against bacteria. • Hc-fabricated AgNPs led to higher membrane permeability and ROS formation in bacterial pathogens. • Hc-fabricated AgNPs affected bacterial replication, leading to protein leakage and cell death. Recently, biogenic nanoparticles have been considered promising candidates for manufacturing antibacterial nanodrugs. Here, we synthesized AgNPs using the crab-borne antibacterial agent hemocyanin and assessed the antibacterial action against several pathogenic bacteria. In this study, the crustacean immune protein hemocyanin (Pp- Hc, 78 kDa) purified from Portunus pelagicus hemolymph was used to fabricate silver nanoparticles. Characterization of hemocyanin-fabricated AgNPs (Pp -Hc AgNPs) were achieved using ultraviolet-visible spectrophotometer, X-ray powder diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), high-resolution-transmission electron microscopy (HR-TEM), and energy-dispersive X-ray spectroscopy. The antibacterial efficacy of AgNO 3, Pp -Hc and Pp -Hc AgNPs was compared by growth inhibition, antibiofilm and live and dead analyses. Based on the results, Pp- Hc AgNPs was more efficient than Pp -Hc and AgNO 3 against pathogenic bacteria. Mechanistic analysis revealed membrane damage and reactive oxygen species (ROS) generation, suggesting that Pp -Hc and Pp -Hc AgNPs rely to similar modes of action. Intracellular protein molecules and nucleic acid leakage confirmed that Pp -Hc AgNPs increase membrane permeability, leading to cell death. Based on our results, capping of the exterior surface of nanoparticles with antimicrobial crab-borne peptides, such as Pp- Hc, improves their functions as potential agents against bacterial diseases, which may be useful in clinical applications. [ABSTRACT FROM AUTHOR]

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