**3. Impact of Dietary Exposure to Silver Nanoparticles in Health: Gut Nanotoxicology E**ff**ects**

As the investigation into the application of nanotechnology in the food sector increases, the potential of nanotechnology in food science/industry also expands and consequently, so does the human exposure to these substances. In the case of antimicrobial silver nanoparticles with application in food industry, the subject of this review, the main human exposure source is through the oral-gastrointestinal tract [68]. The mean dietary exposure level of Ag-NPs is estimated at 70–90 μg/day [69]. After ingestion, the Ag-NPs come in contact with lumen of the oral cavity and esophagus. There is little published information on the absorption rate of particulates through the epithelium of these two compartments, probably due to both a low surface area and a short residence time for most food matrices [68]. After that, during the gastrointestinal digestion process in the stomach and small intestine, the interaction of Ag-NPs with biological fluids can lead to its agglomeration, aggregation, and dissolution [69–73]. In addition, silver nanoparticle absorption (transcellular and paracellular transport and vesicular phagocytosis) through the gastrointestinal tract epithelium could take place. Finally, the nanoparticles that escape the absorption process reach the colon where they could modulate the composition and/or activity of gut microbiota, affecting the production and toxicity of bacterial metabolites [69]. Part of the initial intake of nanoparticles could be extracted in feces. According to the anatomy of the gastrointestinal tract, several environments characterizdc by specific microbiota composition are found. Gut microbiota harbors more than 100,000 billion microorganisms, including bacteria, fungi, viruses, protozoa and archaea, with bacteria representing a majority. The dominant gut bacterial phyla are the *Firmicutes* (including *Clostridium*, *Enterococcus*, *Lactobacillus*, and *Ruminococcus* genera) and

*Bacteroidetes* (including *Bacteroides* and *Prevotella* genera). These bacteria play an important role in the development and conservation of host health. Gut microbes play a role in human physiology through several mechanisms, including their contribution to nutrient and xenobiotic metabolism (e.g., synthesis of vitamins, digestion of oligo, and polysaccharides, drugs, etc.) and to the regulation of immune and neurodendocrine functions. Some of these effects are mediated by products of bacterial metabolism, such as short-chain fatty acids (SCFA), including propionate, butyrate or acetate, which influence the gut barrier, the inflammatory tone and the metabolic homeostatic control in different tissues [74]. To date, little is known about the effect of nanoparticles on the intestinal microbiota, but what is known is that there are numerous factors that can produce an imbalance in the intestinal bacterial populations, like food, triggering certain diseases. That is why the investigation of the NPs-gut microbiota relationship is so important and should continue [68,69].

The physical and chemical transformations of Ag-NPs during the gastrointestinal digestion could involve modifications in their toxic effect. Despite the specific features of these particles and the differences among them, they all display a close relationship between physicochemical reactivity and bioavailability/biopersistence in the gastrointestinal tract. Recently, Mercier-Bonin et al. [68] and Bouwmeester et al. [72] discussed the potential impact of the luminal and gastrointestinal environment on nanomaterial properties and toxicity studies. In this section, with a specific focus on silver nanoparticles, we report *in vitro* and *in vivo* studies considering both local and systemic levels effects, with a particular emphasis on their impact on gut microbiota.

#### *3.1. In Vitro Studies: Static and Dynamic Gut Simulators and Epithelium Cell Models*

Today, several *in vitro* models, from cell models to static and dynamic gastrointestinal models can be used alone or in combination for the study of Ag-NPs toxicity. As mentioned above, concentration/dose is a very important factor for the use of nanoparticles as an antimicrobial agent in the food field. In general, cytotoxicity of Ag-NPs is concentration-dependent. Moreover, depending on the cell type, silver nanoparticles cytotoxicity varies notably, and this should be taken into consideration for their application in consumer products [75]. As said above in relation to their antimicrobial activity, size, shape, charge and surface are also factors that affect the cytotoxicity of these nanoparticles. Ag-NPs' security depends on their state as they can form aggregates during their synthesis and use due to surface charge or they are covered by a high viscosity substance or suspended in a high viscosity environment. It has been shown that coated silver nanoparticles have lower cytotoxic due to the stabilization effect of the coating, which in turn, depends on the coating material and the thickness of the layer [76,77].

Different studies have evaluated the cytotoxic effect of silver nanoparticles in various human cell lines trying to understand the possible risks after exposure or ingestion (Table 2). However, today there are not many studies that evaluated the effect of silver nanoparticles in the oral cavity and the evaluation of the effect of these nanoparticles on oral microbiota is even more limited [68]. In one of these studies, it was found that Ag-NPs increased oxidative stress, inflammation and apoptosis in the human gingival fibroblast cell line (CRL-2014) [78]. Likewise, Niska et al. [79] observed that Ag-NP induced cell death in a concentration-dependent manner, not being toxic until concentrations greater than 40 μg/mL on human gingival fibroblasts (HGF-1). On the other hand, Hernández-Sierra et al. [80] studied the effect of Ag-NPs of different sizes of periodontal fibroblasts extracted from volunteers. They concluded that only nanoparticles with a size smaller than 20 nm increased the cytotoxicity of fibroblasts. Another study with human periodontal fibroblasts, specifically with the cell line HPLF, found that nanoparticles at low concentrations (≤16 μg/mL) had little influence on proliferation and cell cycle, while at high concentrations (32 and 64 μg/mL), they inhibited cell proliferation and significantly changed morphology [81]. The effect of Ag-NPs on oral bacteria has also been evaluated, with bacteria of the genus *Streptococcus* being more sensitive to them [82]. In another work, it was observed how the MIC and MBC of the silver nanoparticles was between 100 and 250 μg/mL for peri-implantitis pathogens [83]. On the other hand, Lu et al. [19] reported a MIC range between 25 and 50 μg/mL and this could be due to the smaller size of the nanoparticles used.


**Table 2.** Studies regarding silver nanoparticles cytotoxicity effects in several cell lines.


**Table 2.** *Cont*.

Unlike what happens with the oral cavity, there are numerous *in vitro* investigations on the effect of silver nanoparticles in the intestine (Table 2). It was observed that the intake of Ag-NPs within a food matrix increased its absorption by colon epithelial cells, the opposite being the case when ingested without food. This shows us the ease with which nanoparticles can reach our intestines due to their consumption along with food [84]. The toxicity difference between digested and undigested silver nanoparticles was also studied. It was possible to verify how the undigested ones were mostly captured by the cellular model Caco-2/HT29-MTX [88]. In the study of Silvan et al. [33], exposure of GSH-Ag NPs to epithelial cells (HT-29, Caco-2 and CCD-18) showed a dose-dependent cytotoxic effect and no significant cytotoxicity occurred until concentrations of 4.93 μg/mL. This is supported by other works in which the toxicity of silver nanoparticles is usually in the range of 10 to 100 μg [93]. It was observed in the work of Vila et al. [87] that the exposure of small-sized Ag-NPs (≈ 8 nm) at a concentration of 100 μg/mL only reached 20% cytotoxicity in Caco-2 cells. It was also shown that cell integrity was not altered using concentrations below 50 μg/mL.

The toxicity of these nanoparticles has not only been studied on oral and intestinal cell lines. There is a study in which non-cytotoxic doses of Ag-NPs were used against the HepG2 cell line. Moreover, at low doses (2 and 4 mg/L), Ag-NPs presented "hormesis" effects by accelerating cell proliferation and an activation of mitogen-activated protein kinase (MAPK) [91]. On the other hand, Khorrami et al. [92] described a cytotoxicity level of 70%, at concentrations between 10 and 60 μg/mL, on the MCF-7 breast cancer cell line, while for the L-929 cell line (non-carcinogenic), it was only 15%. In another study, the toxic effect of Ag-NPs on the MCF-7 cell line was also evaluated. Cellular cytotoxicity was observed from a nanoparticle concentration of 10 μg/mL [90]. This is opening the door to the use of this nanomaterial against cancer cells and therefore, to be a possible cancer therapy, alone or in combination with other existing methods [86,94]. Other studies reported that Ag-NPs may interact with the cerebral microvasculature producing a proinflammatory cascade in rat brain microvessel endothelial cells, as well as that larger NPs were less toxic, and blood–brain barrier (BBB) dysfunction and astrocyte swelling causing neuronal degeneration [89,95].

In reference to static models of gastrointestinal digestion (Table 3), there is one study that showed that Ag-NPs with a size of 60 nm and a concentration of 10 mg/mL (1661 particles/mL) in the presence of proteins survived the extreme conditions of the digestion and reached the intestine [71]. This probably means that epithelial cells of the intestine would be exposed to these nanoparticles, causing cellular damage. On the contrary, in the absence of proteins, the fraction of NPs that reached the intestine was smaller [71]. In other works they also studied the effect of nanoparticles during the passage through the gastrointestinal tract. It was found that by contacting them with synthetic human stomach fluid, the Ag-NPs aggregated significantly and also released ionic silver that was physically associated with the aggregates of particles such as silver chloride. In addition, it was seen that NPs smaller than 10 nm were added to a greater extent than larger one [96]. It was also demonstrated that depending on the composition and pH, the morphology and the size of the Ag-NPs changed when passing through the different fluids (simulated saliva and gastric and intestinal fluids); in addition, there was only a low toxicity in a pilot study of reconstituted human tissues model [85]. When Ag-NPs interact with proteins, a corona is always formed and it decreases the entry of nanoparticles into cells and therefore, cellular toxicity decreases [97]. Gil-Sánchez et al. [74] evaluated the effect of static *in vitro* digestion on silver nanoparticles with two types of coating. It was observed that the glutathione-coated nanoparticles agglomerated less than those that had the polyethylene glycol coating and were less toxic to colon cells. Studying the changes of NPs in dynamic models is more limited. In the work of Cueva et al. [98], the dynamic gastrointestinal simulator simgi® was used to digest Ag-NPs and study their effect on the colonic microbiota (Table 3). They did not observe changes in the bacterial composition or in the production of ammonium ions during the simulations, so it was concluded that Ag-NPs did not alter the composition and metabolic activity of the human intestinal microbiota. Another dynamic study showed that 90% of Ag-NPs were already dissolved by passing through the stomach and that many of the released ions bind to the food matrix. This results in less bioavailable ions and therefore, less toxicity (Table 3) [73].


**Table 3.** Studies in *in vitro* static and gastrointestinal simulation models regarding silver nanoparticles effects at gut level and microbiota.


**Table 3.** *Cont*.

A limited number of studies on the interaction of nanomaterials with the microbiome are available, most of them in rodents. In one *in vitro* study, it was observed that the Ag-NPs modified the *Firmicutes*/*Bacteroidetes* phylum ratio, increasing *Firmicutes* and decreasing *Bacteroidetes*. It was seen that the nanoparticles altered the intestinal microbiota as would a metabolic and inflammatory disease [100]. On the other hand, after exposure of silver nanoparticles (10 nm) to a concentration range of 0–100 μg/mL, a marked decrease in saturated fatty acids was observed, except in palmitic acid, which increased by 26–32%. The observation of these variations led to the sequencing of bacterial DNA. According to the results of Das et al. [99], Ag-NP ingestion, either deliberate or inadvertent, could have negative consequences on our intestinal microbiota, as evidenced by a significant decreasing of *Bacteroidetes* due to both ionic silver (AgCl; 25–200 mg/L) and nanosilver-mediated changes.
