*4.2. Mechanisms of Nanoparticle Cytotoxicity*

The increased use of AgNPs also increases the concentration of silver ions in soil and water that can adversely affect human and animal health or the environment [137,138].

Little is known about the diversified mechanisms of the action of nanoparticle cytotoxicity, as well as their short- or long-term exposure outcomes on human physiology [139,140]. Many authors claim that the silver ions released from AgNPs through the surface oxidation are the main mechanism that induces cytotoxicity, genotoxicity, immunological responses in biological systems and even cell death [141–145].

Several recent studies revealed that cytotoxicity of AgNPs occur due to the minimum release of silver ions [146–148]. Sambale et al. [149] showed that the mechanism of cell death is different when cells are cultivated with silver particles or ions. Cells treated with particles suffered apoptosis while those cultivated with ions die due to necrosis. They also reported that AgNPs that translocate into cytoplasm through diffusion or channel proteins are oxidized by cytoplasmic enzymes, thereby releasing silver ions. These ions may interact with thiol groups of mitochondrial membrane proteins, causing mitochondrial dysfunction and generating reactive oxygen species (ROS) production [149].

#### *4.3. Effect of Nanoparticles on Cell Membranes*

Nanosilver can interact with cellular membranes and cause toxicity. In particular, nanosilver can interact with the bacterial membrane and this is considered the main mechanism for the antimicrobial toxicity of nanosilver. The AgNPs damaged and destroyed bacterial cells by penetrating and accumulating in the bacterial membrane [150–152]. Khan et al. [153] studied the interaction of nanosilver with five types of bacteria. They found that the adsorption of nanosilver on the bacterial surface or interaction with extracellular proteins is dependent on pH, zeta potential, and NaCl concentration. Baruwati et al. [154] studied "green" synthesized nanosilver from Chinese green tea (*Camellia sinensis*) and found that exposure to these particles alters the membrane permeability of barrier cells (intestinal, brain endothelial) and stimulates oxidative stress pathways in neurons [154].

Metal nanoparticles (copper, silver and zinc oxide) from *Fusarium solani* KJ 623702 were tested against multidrug-resistant bacteria (*P. aeruginosa* and *S. aureus*), *E. coli*, *Klebsiella pneumoniae* and *Entreococcus* sp. and mycotoxigenic *Aspergillus awamori, A. fumigatus* and *F. oxysporum*. The antimicrobial activity of metal nanoparticles increased as follows: CuNPs, ZnONPs, and AgNPs. Besides the internalization of AgNPs with the cell wall resulting in the presence of pits, the fragmentation, complete disappearance of cellular contents, disorganization and leakage of internal components were observed after treatment of microorganisms with mycogenic AgNPs. Authors claimed that AgNPs attach to the microbial cell membrane and alter its structure, transport activity, penetrability, prompt

neutralization of the surface electric charge and produce cracks and pits through which internal cell contents are effluxed [44].

Moreover, AgNPs synthesized using *F. oxysporum* filtrate containing reductase enzymes and quinones showed a toxic effect on the human liver cell line (Huh-7). The IC<sup>50</sup> values were found to be 11.12 µM for propidium iodide assay which is an intercalating fluorescent dye and does not permeant to live cells [155]. Similarly, cell viability (hamster lung fibroblasts, V79) after the treatment with AgNPs synthesized from *F. oxysporum* was studied using neutral red uptake (NRU) assay [156]. These small (around 8 nm) nanoparticles showed a non-cytotoxic effect on fibroblasts at concentrations until 16 µM. The IC<sup>50</sup> of mycogenic AgNPs was recorded at a concentration of 22 µM [156].

Vijayan et al. [157] estimated the release of haemoglobin from red blood cells due to the disruption of the cell membrane by the AgNPs from *F. oxysporum*. The percentage of haemolysis increased from 3.8 to 32.0 with an increase in AgNPs concentration from 25 to 150 µg ml−<sup>1</sup> (at 25 µg ml−<sup>1</sup> interval unit). The authors concluded that these biogenic AgNPs are safe to use as a drug because at safe concentrations they showed antibacterial effect against *Escherichia coli*, *Klebsiella pneumoniae*, *Pseudomonas aeruginosa* and *Salmonella typhi* [157].
