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

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 AgNO3,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 AgNO3 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.