*3.2. In Vivo Studies: Animal and Human Trials*

When conducting studies *in vivo*, five main types of models have been used: rats, mice, *Caenorhabditis elegans*, fish (zebrafish), *Drosophila melanogaster* and, in a lesser extent, human studies (Table 4). Each model has its advantages and limitations, but all provide a great deal of information that helps us to conclude facts. Within all these models, rats and mice may be the most used, but the one that generates the most interest is the human model, since it provides real data when it comes to human applications.

**Table 4.** In vivo studies regarding silver nanoparticles effects at gut level and microbiota, organs and tissues.



**Table 4.** *Cont*.


**Table 4.** *Cont*.

Regarding the findings with rat and mice, several studies have been carried out to evaluate the effect of these nanoparticles on the gastrointestinal tract. An abnormal mucus composition of the intestines of the animals was observed, as well as pigmentation of the villi and discharge of mucus granules [102,103,109]. In another study, it was discovered that Ag-NP damaged the microvilli of epithelial cells and intestinal glands in rats, thus decreasing the intestinal absorption of nutrients [104]. In the study by van den Brule et al. [111], by using Next Generation Sequencing (NGS), they observed how the intake of dietary doses Ag-NPs during 28 d did not significantly alter, in a dose-dependent manner, either the uniformity of the intestinal microbiota or populations in rats. But they could see an increase in the relationship between *Firmicutes* and *Bacteroidetes* phyla. Human and mouse gut microbiota are very similar at the phylum level, but not at the genera or species level; however, at least at the phylum level, these results could be extrapolated to humans. It was also discovered that the consumption of Ag-NPs modified the values of cholesterol and alkaline phosphatase in rats, which indicated that exposure to these nanoparticles could cause mild liver damage [103]. Silver nanoparticles are also easily able to cross the tight junction of the blood–brain barrier (BBB); therefore, they can be considered as neurotoxic. Rahman et al. [101] showed a neurotoxic effect induced by oxidative stress of Ag-NPs in three regions of the brain, including the caudate nucleus, the frontal cortex and the hippocampus of adult mice. In addition, another study showed that Ag-NPs produced neuronal degeneration and inflammation of astrocytes in the rat brain due to a low dose of exposure by oral and intragastric administration [105,106].

There are studies with other animal models like fishes. After exposure of fish at Ag-NPs concentrations of 2.5, 10, and 25 μg/L for 24 h, it was observed that the accumulation of silver in the brain was greater than in the liver and gills. In addition, fish that were exposed to the highest concentrations showed alterations in markers of oxidative stress [112]. In another study, various sizes of Ag-NPs coated with gum arabic (10, 40 and 100 nm) were used. Zebrafish embryos were exposed to various concentrations of these nanoparticles for 4 days and only an increase in lethality was observed with the 40 nm nanoparticles. This could be because of the retention of silver in the intestine depends on the particle size and the agglomerates [114]. In the same line is the work of Liu et al. [115], in which they demonstrated that the particle size is more influenced by the toxicity of Ag-NPs than the coating. Ag-NPs of small size (20 nm) and with citrate coating were more toxic and the toxic effect was greater in the intestine than in the gills or muscles. Merrifield et al. [113] showed, in adult zebrafish, that exposure to silver nanoparticles (500 mg/kg food) for 14 days had no effect on the richness and diversity of the microbiota. Similarly, Wilding et al. [107] found that the oral administration of silver nanoparticles of two different sizes (20 and 110 nm) and with two different coatings (PVP and citrate) for 28 days (10 mg/kg bw/day) did not change the diversity of the gut microbiome in mice. In another study, the effect of Ag-NPs in mouse models with inflammatory bowel disease was evaluated. A decrease in inflammation and a positive modulation of the gut microbiota could be observed [110]. By contrast, another study on rats fed twice-daily with oral silver nanoparticles for 13 weeks at various doses (9, 18 and 36 mg/kg bw/day) reported a general increase in the levels of Gram negative bacteria, and a decrease in the levels of *Firmicutes* [108]. It is important to note that there are differences between the human and zebrafish and rodent microbiome. Moreover, differences during gut transit and the interactions with the composition of the food matrix between animals and humans can affect nanomaterial properties in a different way during digestive transit and their putative effects.

There are also studies with *Caenorhabditis elegans*. In one of them, it was observed how the reactive oxygen species in the nematode increased when exposed to *E. coli* contaminated with Ag-NPs. They also increased reproductive toxicity and neurotoxicity [117]. Moon et al. [118] showed that the presence of different silver nanomaterials (including nanoparticles) in the soil decreased the growth and reproduction of *C. elegans*. Similarly, in another study, the hereditary reproductive toxicity produced by Ag-NPs in *C. elegans* was demonstrated and it was observed that this toxicity contributed to inducing germline mutations [116,119].

Finally, another of the most used non-human models is *Drosophila melanogaster*. In one of the studies, the larvae were fed with silver nanoparticles, which were able to reach the intestinal barrier. This was demonstrated by analyzing the increase in intracellular ROS [123]. Reproductive toxicity was

also evaluated in this model. It was observed that exposure of adult specimens to Ag-NPs significantly affected the ability to lay eggs along with a deteriorated ovarian growth [121,122]. In a study of acute and chronic exposure, it was observed that the effect of a solution of Ag-NPs at a concentration of 20 ug/mL 50% of the larvae did not end their development cycle. In addition, after chronic exposure to an Ag-Nps solution of 5 ug/mL, it was shown that after three generations, the flies adapted to silver, recovering the fecundity lost in the first three [120].

As can be seen in the aforementioned paragraphs, Ag-NPs have been shown to have toxic effects to both in *in vitro* and *in vivo* models; however, there is a limited number of studies that reported the impacts of Ag-NPs on human health. One of them is the one carried out by Munger et al. [124]. A total of 60 healthy subjects ingested nanoparticles at concentrations of 10 and 32 ppm (Ag-NPs size: 5–10 nm) for 14 days. No significant changes were detected in the morphology of heart, lungs and other organs, nor in the reactive oxygen species or in the generation of proinflammatory cytokines. Nor did significant changes in metabolic measures appear in the conditions studied. The authors stressed the need to evaluate the effects of longer-term exposure.

Because of the increased potential for consumer exposure to Ag NP, it appeared urgent to assess the possible impact on the gut microbiota and on human health. As reviewed, few studies have investigated this issue and none are conclusive. The differences of results between studies could be related to the techniques used to analyze the microbiota. Moreover, it is difficult to make a comparison between studies published today because different sizes, shapes and concentrations of nanoparticles have been used. As a suggestion, future experiments should consider validated standards to ensure more comparable results and thus, make more reliable conclusions. Moreover, the transfer of results from animals to humans could be improved with the use of "humanized" animals by inoculation of human gut microbiota as well as by investigations conducted with longer exposure durations to better mimic human exposure scenarios.
