Silver nanoparticles (AgNPs) have been proven to have potent antibacterial properties, offering an attractive alternative to antibiotics in the treatment of several infections such as otitis media. Concerns have been raised though regarding their toxicity. There are few data regarding the toxic effects of AgNPs in cochlear cells. The aim of our study was to evaluate the effects of AgNPs of four sizes as a function of their size on HEI-OC1 cochlear cells and on HaCaT keratinocytes. The cells were treated with different concentrations of AgNPs. We evaluated silver uptake by atomic absorption spectroscopy and transmission electron microscopy (TEM), cytotoxicity with the alamarBlue test, ROS production with 2′,7′-dichloro-dihydro-fluorescein diacetate, and genotoxicity with the comet assay. Silver intracellular concentration increased proportionally with the incubation time and the size of the NPs. Silver uptake was higher in HEI-OC1 cells compared to HaCaT. While after 4 h exposure, only the 50 nm NPs were observed in both cell lines and only the 5 nm NPs were observed in the HaCaT cells, after 24 h, nanoparticles of all sizes could be visualized in both cell lines. The cells showed signs of distress: vacuolizations, autophagosomes, signs of apoptosis, or cellular debris. AgNPs of all sizes reduced viability proportionally with the concentration, HEI-OC1 cells being more affected. The toxicity of AgNPs decreased with the nanoparticle size, and ROS production was dose and size dependent, mainly in the cochlear cells. Genotoxicity assessed by comet assay revealed a higher level of DNA lesions in HEI-OC1 cells after treatment with small-sized AgNPs. The perspective of using AgNPs in the treatment of otitis media, although very attractive, must be regarded with caution: cochlear cells proved to be more sensitive to the toxic effect of AgNPs compared to other cell lines. Potential treatments must be tailored specifically, choosing NPs with minimum toxicity towards auditory cells.

The present study reports for the first time the efficacy of bioactive compounds from Ligustrum ovalifolium L. fruit extract as reducing and capping agents of silver nanoparticles (AgNPs), developing a green, zero energetic, cost effective and simple synthesis method of AgNPs. The obtained nanoparticles were characterized by UV-Vis spectroscopy, transmission electron microscopy (TEM), X-ray diffraction (XRD) and Fourier Transform Infrared spectroscopy (FTIR), confirming that nanoparticles were crystalline in nature, spherical in shape, with an average size of 7 nm. The FTIR spectroscopy analysis demonstrated that the AgNPs were capped and stabilized by bioactive molecules from the fruit extract. The cytotoxicity of the biosynthesized AgNPs was in vitro evaluated against ovarian carcinoma cells and there were found to be effective at low concentration levels.