*3.2.1* In vivo *studies*

*Novel Nanomaterials*

to be conserved in the interaction of Ag<sup>+</sup>

through the membrane as previously described.

and transport proteins [80, 91].

not validate the adhesion and penetration abilities of negatively charged nanoparticles [104]. The researchers argued that although the particles were negatively charged, interactions between the particles and building elements of the membrane are likely to have occurred causing structural changes and degradation of the membrane. Morones *et al*. proposed that the interaction of AgNPs and bacterial membranes could be attributed to the strong affinity of the particles to sulphur containing proteins present on the membrane [80]. These interactions are thought

Sondi and Salopek-Sondi [104] further reported that damage to *E. coli* cell membranes might also occur due to the incorporation of AgNPs into their membrane structure. Scanning electron microscopy revealed the formation of "pits" on the surface of the membrane [100]. Similar findings were observed by [102]. Amro *et al*. [109] additionally reported the formation of irregularly shaped "pits" on the outer membrane of *E. coli* cells through the progressive release of lipopolysaccharide molecules. This release of LPS molecules was induced by metal depletion in the cells [105]. A membrane with such morphological changes would display a high increase in permeability, rendering the cell incapable of regulating proper transport

Although these studies have been conducted on Gram-negative bacteria, AgNPs have also been reported to exert inhibitory activities against Gram-positive bacteria which differ from their counterparts based on differences in cell wall structure [106]. It can be tentatively suggested that AgNPs may form interactions with Grampositive bacteria through surface proteins present on the cell wall. Once penetrated, the mechanisms of bacterial activity are conserved with that of Gram-negative

A relatively confined amount of literature focuses on the mechanisms of antifungal activity exerted by AgNPs. However, based on the studies that have been reported, it seems that inhibition of fungal growth by AgNPs may be the result of damage to fungal cellular membranes. Kim *et al*. demonstrated the effect of AgNPs on *Candida albicans*. Transmission electron microscopy (TEM) analysis revealed that the treatment of cells with AgNPs lead to the formation of "pits" on the cell membrane which ultimately disrupts membrane potential [107]. A similar finding was made by Nasrollahi *et al*. who reported that AgNP incubation with *C. albicans* led to damage of the cell membrane [108]. Endo *et al*. reported that disruption of membrane integrity inhibits the normal budding process of daughter cells. Therefore, the authors suggested that AgNPs exert their inhibitory activity by inhibiting the budding of daughter cells due to the destruction of the cell

AgNPs may also disrupt antioxidant defences in fungal cells. Eukaryotic cell studies suggest that AgNPs directly interact with gluthathione, gluthathione reductase or enzymes responsible for maintaining proper levels of gluthathione

affect the function of membrane bound enzymes such as those in the respiratory chain. It has also been reported that exposure of fungal cells to Ag<sup>+</sup>

the loss of DNA replication ability. This results in the deactivation of ribosomal subunit protein expression and synthesis of non-functional enzymes and cellular

From these findings it can be tentatively suggested that bactericidal mechanisms of AgNPs are conserved in their inhibition of fungal cells. In summary, AgNPs exert

[110]. With respect to fungal cells, it has been hypothesised that Ag<sup>+</sup>

and thiol groups on respiratory enzymes

largely

, disrupting the cell membrane/wall,

led to

**266**

bacteria.

membrane [109].

proteins [111].

their antimicrobial effects by releasing Ag<sup>+</sup>

generating ROS and inhibiting proper DNA replication.

Human contact with nanoparticles occurs in the form of intravenous injection, oral administration, inhalation and dermal contact [113]. Injection of AgNPs *in vivo* results in short circulation times and broad tissue distribution. Target sites often include the liver (main target), spleen, lungs and kidneys [114]. Inhalation studies suggest that AgNPs become deposited in the olfactory mucosa and olfactory nerves which can potentially induce impairment and dysfunction of brain cells [115] in addition to immunotoxicity [116]. With regard to oral administration, migration of AgNPs to the gastrointestinal tract promotes dissolution of the particles which subsequently releases Ag<sup>+</sup> [117]. A recent study on oral exposure to Ag+ indicated that these ions interact with sulphur leading to the formation of sulphur containing Ag granules in the intestinal epithelium [118]. The authors suggested that during intestinal digestion, Ag+ give rise to particle formation, possibly in the form of Ag2S or AgCl salt. They further added that this formation might influence their uptake and reduce the toxic effects of Ag<sup>+</sup> , however the effects of Ag salts on the intestine are yet to be elucidated [118, 119]. Reports on the exposure of workers to low doses of Ag dust indicated no significant changes in health status.
