Effect of Nanomaterials on Gut Microbiota

Nanomaterials are widely employed in everyday life, including food and engineering. Food additives on a nanoscale can enter the body via the digestive tract. The human gut microbiota is a dynamically balanced ecosystem composed of a multitude of microorganisms that play a crucial role in maintaining the proper physiological function of the digestive tract and the body’s endocrine coordination. While the antibacterial capabilities of nanomaterials have received much interest in recent years, their impacts on gut microbiota ought to be cautioned about and explored. Nanomaterials exhibit good antibacterial capabilities in vitro. Animal studies have revealed that oral exposure to nanomaterials inhibits probiotic reproduction, stimulates the inflammatory response of the gut immune system, increases opportunistic infections, and changes the composition and structure of the gut microbiota. This article provides an overview of the impacts of nanomaterials, particularly titanium dioxide nanoparticles (TiO2 NPs), on the gut microbiota. It advances nanomaterial safety research and offers a scientific foundation for the prevention, control, and treatment of illnesses associated with gut microbiota abnormalities.


Exposure of the Gut Microbiota to Nanomaterials
Nanomaterials (NMs) are materials with unique properties that are made up of nanostructured basic units or at least one dimension at the nanoscale (geometric scales ranging from 1 nm to 100 nm), such as nanopowders, nanofibers, nanofilms, nanoblocks, and nanopores. According to the classification of chemical composition, they can be divided into metal nanomaterials, nanocrystalline materials, inorganic nonmetallic materials, polymer nanomaterials, and nanocomposites. Nanomaterials exhibit characteristics of a small size effect, high specific surface area, and quantum size effect [1]. Nanomaterials have a wide range of uses due to their superior physical and chemical characteristics, such as biomedicine, diagnostic imaging, DNA nanotechnology, biosensing, and drug-loaded treatment [2]. Notably, nanomaterials have several uses in food engineering [3]. They can be utilized as coatings to minimize mechanical damage or microbiological contamination and improve food color and flavor. Nanocapsules can be employed as carriers to enter, protect, and transport active chemicals in food and medications while preserving the product's appearance and taste [4]. Nanofilms are commonly utilized in chocolate, confectionery, baked goods, and other food-related products because they protect food surfaces from moisture, oil, and gas [5]. Currently, whether it is food itself, food packaging, or the entire process of food manufacturing and production, using different nanomaterials is unavoidable, which undoubtedly increases people's intestinal exposure risk.
Despite their importance in medicine, engineering, food processing, and other fields, the safety of nanomaterials remains a major concern. One of the current and future focuses of nanomaterials is the study of their biological effects and toxicity. To date, many in vivo The antibacterial ability of nanomaterials is affected by their size, production process, and crystal form. Moreover, the temperature, pH, and ionic strength of the environment also have an impact on the antibacterial capabilities of nanomaterials. Smaller particle size nano-TiO 2 and the anatase phase have been shown to be more harmful to E. coli; nevertheless, the toxicity of nano-TiO 2 diminishes with increasing pH (5.0-10.0) and ion concentration [34]. It is worth noting, however, that the above characteristics do not apply to fungi associated with plant rhizomes. According to reports, there were no effects of nanomaterial type, concentration, or charge on the community structure of either rhizobia or AM fungi colonizing plant roots [35].
There are several possible hypotheses for the antibacterial mechanism of nanomaterials ( Figure 1). The electronegative complex groups on the bacterial membrane can attract each other with electropositive metal ions, causing metal nanomaterials to accumulate on the bacterial surface and enter the cell, altering the permeability of the bacterial membrane and allowing bacterial contents to leak out [36]. Nanomaterials that enter bacteria can also alter the function of enzymes and proteins, interfering with the bacterium's regular physiological metabolism [37]. Antimicrobial properties in nanomaterials can also be produced through oxidative stress [38]. H + dispersed on the surface of metal nanoparticles can oxidize OH − and H 2 O to OH. As a powerful oxidant, ·OH causes bacterial redox imbalance. Under UV irradiation, this behavior will be more severe [39]. Nanomaterials offer a unique multiple antibacterial mechanism and have a good killing impact on a range of drug-resistant bacteria when compared to typical disinfectants and medicines [40]. As a result, nanomaterials may offer a solution to multidrug-resistant bacteria.

Antimicrobial Properties of Nanomaterials
Most nanomaterials have antibacterial properties that are effective against common bacteria. Metal oxide nanomaterials such as nano-TiO2, ZnO, and Ag2O can inhibit common bacteria such as E. coli, Bacillus subtilis, and Staphylococcus aureus [31]. Nano-TiO2 and ZnO are poisonous to gram-negative, gram-positive, and fungal microorganisms [32]. Even in the absence of UV irradiation, nano-TiO2 retains its antibacterial ability against E. coli [33]. The antibacterial ability of nanomaterials is affected by their size, production process, and crystal form. Moreover, the temperature, pH, and ionic strength of the environment also have an impact on the antibacterial capabilities of nanomaterials. Smaller particle size nano-TiO2 and the anatase phase have been shown to be more harmful to E. coli; nevertheless, the toxicity of nano-TiO2 diminishes with increasing pH (5.0-10.0) and ion concentration [34]. It is worth noting, however, that the above characteristics do not apply to fungi associated with plant rhizomes. According to reports, there were no effects of nanomaterial type, concentration, or charge on the community structure of either rhizobia or AM fungi colonizing plant roots [35].
There are several possible hypotheses for the antibacterial mechanism of nanomaterials ( Figure 1). The electronegative complex groups on the bacterial membrane can attract each other with electropositive metal ions, causing metal nanomaterials to accumulate on the bacterial surface and enter the cell, altering the permeability of the bacterial membrane and allowing bacterial contents to leak out [36]. Nanomaterials that enter bacteria can also alter the function of enzymes and proteins, interfering with the bacterium's regular physiological metabolism [37]. Antimicrobial properties in nanomaterials can also be produced through oxidative stress [38]. H + dispersed on the surface of metal nanoparticles can oxidize OH − and H2O to OH. As a powerful oxidant, ·OH causes bacterial redox imbalance. Under UV irradiation, this behavior will be more severe [39]. Nanomaterials offer a unique multiple antibacterial mechanism and have a good killing impact on a range of drug-resistant bacteria when compared to typical disinfectants and medicines [40]. As a result, nanomaterials may offer a solution to multidrug-resistant bacteria.

Titanium Dioxide Nanoparticles (TiO2 NPs)
TiO2 NPs have limited impacts on gut microbiota, as evidenced by acute or subchronic experiments that have limited influence on gut microbiota diversity but have a

Titanium Dioxide Nanoparticles (TiO 2 NPs)
TiO 2 NPs have limited impacts on gut microbiota, as evidenced by acute or subchronic experiments that have limited influence on gut microbiota diversity but have a greater impact on gut microbiota quantity ( Figure 2, Table 1). Among these, TiO 2 NPs have a significant impact on bacteria, particularly Lactobacillus, Firmicutes, and Proteobacteria [33][34][35].
Subacute or subchronic exposure to TiO 2 NPs had less of an effect on the gut microbiota in typical rodent models. Li et al. [42] treated mice with TiO 2 NPs (100 mg/kg) for 28 days and observed that TiO 2 NPs did not affect the diversity of gut microbiota but modified the composition structure of the microbiota, in which the abundance of Proteus was reduced dramatically. Wei et al. [43] investigated the long-term toxicity of TiO 2 NP exposure. Weaned young mice were given TiO 2 NPs for three months, and their body weight was found to be lower than that of the control group, which intensified the chronic colitis and immunological response generated by dextran sulfate sodium salt (DSS). According to research, TiO 2 NPs have no effect on the diversity of gut microbiota but drastically affect the quantity of probiotics such as Bifidobacteria and Lactobacilli. Chen et al. [44] found that after 30 days of oral treatment (2, 10, and 50 mg/kg) the structure and composition of the rat gut microbiota were altered, resulting in significant increases in L. gasseri and Turicibacter, while Veillonella was dramatically reduced in the exposure group at 14 days. After 28 days, the abundance of L. gasseri continued to increase significantly, as did L.NK4A136_group. Another study demonstrated that TiO 2 NPs (2, 10, and 50 mg/kg) significantly enhanced the abundance of Lactobacillus and Allobaculum and decreased the abundance of Adlercreutzia and unclassified Clostridiaceae in the exposed group following 21 days of subchronic exposure [45]. In the population, the average long-term intake of titanium dioxide is 0.06 mg/kg bw/day for people over 70 years old, 0.17 mg/kg bw/day for people aged 7-69 years, and 0.67 mg/kg bw/day for children aged 2-6 years [46]. The dose settings of the above animal experiments were considered with a safety factor (100×), which well reflects the situation after TiO 2 NPs exposure.
TiO 2 NPs also have an impact on the gut microbiota in other animal or in vitro models. When TiO 2 NPs were coexposed to bisphenol A (BPA), they increased the abundance of Lawsonia in Danio rerio while decreasing the abundance of Hyphomicrobium [47]. Dudefoi et al. [48] used food-grade TiO 2 NPs to imitate human digestive system dosages in an in vitro model. Following two days of bacterial culture, there were very minor impacts on the gut microbiota. Clostridium cocleatum increased in abundance, whereas Bacteroides ovatus decreased. In vitro studies show that TiO 2 NPs can still have antibacterial properties. According to Albukhaty et al. [49], TiO 2 NPs can effectively inhibit Staphylococcus aureus and Escherichia coli activity in vitro.
TiO 2 NPs can disrupt the tight junctions of intestinal epithelial cells, producing a loss of intestinal barrier structure and altering the diversity and composition of gut microbiota communities in organisms. Li et al. [42] examined the two primary TiO 2 NPs crystals, anatase and rutile, and discovered that the latter had a greater influence on the intestinal ecological habitat of mice. Long intestinal villi and an uneven arrangement of villus epithelial cells were observed in mice fed rutile. However, in the Chen experiment, the intestinal shape of rats was changed significantly by anatase, as evidenced by inflammatory infiltration and mitochondrial abnormalities [44]. Obese mice were more susceptible to this. Mice fed a high-fat diet and exposed to TiO 2 NPs experienced goblet cell loss, the structural distortion of crypts, and the infiltration of inflammatory cells around crypts. The number of dendritic cells and macrophages in the colonic mucosa increased significantly, as did the levels of IL-12, IL-17, KC/GRO, and IL-10 [50].
Moreover, TiO 2 NPs may be hazardous to other digestive system organs, which might have an indirect impact on the gut microbiota. Li et al. [42] showed that TiO 2 NPs accumulated in the spleen, lungs, and kidneys affected the shape and organization of intestinal epithelial cells and altered the composition of gut microbiota over time. Chen et al. [51] discovered that the gut-liver axis regulating mechanism may play a significant role in the influence of nanomaterials on gut microbiota. In rats, subchronic oral TiO 2 NP treatment produces hepatotoxicity, including hepatocyte steatosis and mitochondrial dysfunction. Substantial changes in the alanine, aspartate, and glutamate pathways and metabolic pathways may be critical metabolic pathways leading to disruptions in energy metabolism and oxidation/antioxidant imbalances. A significant increase in the synthesis of lipopolysaccharide (LPS) by the gut microbiota in rats might be proof of the connection between liver metabolism disorder and gut microbiota dysregulation. metabolic pathways may be critical metabolic pathways leading to disruptions in energy metabolism and oxidation/antioxidant imbalances. A significant increase in the synthesis of lipopolysaccharide (LPS) by the gut microbiota in rats might be proof of the connection between liver metabolism disorder and gut microbiota dysregulation.

Silver Nanoparticles (Ag NPs)
Silver NPs are one of the most extensively researched antimicrobial noble metal nanoparticles, with strong antibacterial activity against a wide range of diseases, including drug-resistant bacteria ( Figure 3, Table 2). Silver NPs can alter the diversity and composition of gut microbiota, and the effect is relatively consistent across species. Specifically, this increases the amount of gram-negative bacteria in the gut microbiota, primarily affecting Lactobacillus of the Firmicutes phylum and E. coli of the Proteobacteria phylum [58]. Han et al. [59] discovered that the gut microbiota diversity of fruit flies was dramatically reduced after Ag NP exposure, with the abundance of Acetobacter dropping while Levilactobacillus brevis had a stronger advantage.
In vitro, Ag NPs have strong antibacterial capabilities, and the mechanism is assumed to be direct contact and oxidative stress. The former considers that Ag NPs can slowly release silver ions and covalently bind to sulfhydryl groups (-SH) in proteins, rendering them inactive [60]; the latter believes that Ag NPs catalyze the synthesis of huge amounts of reactive oxygen species (ROS) from water and oxygen, damaging cellular genetic material and triggering apoptosis. Studies have revealed that both pathways occur, with oxidative stress being the primary mechanism of Ag NP antibacterial activity, while silver ions have a limited impact [61,62].
In vivo, the antibacterial mechanism is connected to immunological regulation. Williams et al. [58] investigated the effects of different sizes of nanosilver and silver acetate on gut microbiota and mucosal gene expression in SD rats. Low dosages and small sizes of Ag NPs were discovered to change intestinal gene expression, resulting in the reduced expression of critical immunomodulatory genes such as MUC3, TLR2, TLR4, GPR43, and FOXP3.
Oral Ag NPs affect animal growth and development, but their advantages and risks remain unknown. Fondecila et al. [63] discovered that Ag NPs may decrease the abundance of E. coli linearly in vitro. When giving Ag NPs to piglets, their daily feed intake and weight rose linearly with the dosage of Ag NPs. At the same time, the concentration of E. coli in feces was reduced, whereas the concentration of Lactobacilli was unaffected. Silver NPs altered the composition of the piglet gut microbiota, which benefits development and metabolism. Han et al. [59] discovered that the toxicity of Ag NPs was greater than that of microsilver in fruit flies. Although Ag NPs have no effect on adult fruit flies, they do reduce the rate of development and reproduction. In conclusion, the interference of Ag NPs in gut microbiota may be due to their own antibacterial properties, and an imbalanced gut microbiota exacerbates Ag NP toxicity.

Zinc Oxide Nanoparticles (ZnO NPs)
ZnO NPs have a strong antibacterial effect and inhibit a wide range of bacteria in the gut (Table 3). They have the potential to alter the diversity and composition of the gut microbiota; for example, the abundance of gut probiotics such as Lactobacillus was increased. After 28 days of ZnO NP 1000 mg/kg administration to rats, the abundance of several Lactobacillus probiotics in the intestines of female rats increased significantly [73].
There are several hypotheses about the antibacterial mechanism of ZnO NPs (Figure 4). Antimicrobial processes such as oxidative stress, direct interaction with bacteria, and zinc ion release are all considered feasible. Several investigations have suggested that oxidative stress is the primary antibacterial mechanism of ZnO NPs. In an aqueous solution, ZnO NPs may generate •OH, singlet oxygen or superoxide anions (O 2 • − ), and hydrogen peroxide (H 2 O 2 ). The larger the ZnO NP surface area is, the higher the ROS output [74,75]. During the direct interaction of ZnO NPs with E. coli, the ROS generated trigger the oxidation of the lipid membrane in the cell wall, leading to the leakage of cell contents [76]. Zinc ions produced by ZnO NPs are considered to have antibacterial properties. Nevertheless, ZnO NPs have limited solubility and are sensitive to ambient pH. ZnO NPs tend to remain intact at neutral pH, but in acidic conditions ZnO NPs dissolve and release zinc ions that bind to biomolecules (proteins, carbohydrates, etc.) in bacteria and impede their development [77,78].

Carbon-Based Nanomaterials (CNMs)
Carbon-based nanomaterials with at least one dimension less than 100 nm. There are several common types, such as fullerenes, carbon nanotubes (CNTs), carbon dots, and graphene and its derivatives. Carbon-based nanomaterials can exert antibacterial

Carbon-Based Nanomaterials (CNMs)
Carbon-based nanomaterials with at least one dimension less than 100 nm. There are several common types, such as fullerenes, carbon nanotubes (CNTs), carbon dots, and graphene and its derivatives. Carbon-based nanomaterials can exert antibacterial properties via a variety of mechanisms, including physical destruction, the inflammatory immune response, and oxidative stress ( Figure 5, Table 4).
One of the most typical antimicrobial mechanisms in CNMs is the physical destruction of the outer cell membrane or cell wall. Carbon-based nanomaterials bind to peptidogly-can and proteins in the cell membrane, causing cell membrane rupture [87,88]. Carbon nanotubes and graphene, for example, have sharp edges that may puncture bacterial membranes, resulting in the release of bacterial internal components such as RNA [89,90].
The metabolic inflammatory response is linked to changes in the gut microbiota caused by CNMs. Carbon nanotubes boosted the release of inflammatory factors such as IL-1β, IL-6, and TNF-α in the duodenum and colon, as well as the transition of the phylum Firmicutes to Bacteroidetes and the abundance of the pro-inflammatory bacteria Alitipes_uncultured and Lachnospiraceae bacterium A4 [91].
Oxidative stress is another major antibacterial mechanism in CNMs. Carbon quantum dots generate ROS when exposed to blue light, dramatically inhibiting the activity of methicillin-resistant Staphylococcus aureus and Escherichia coli [92]. Graphene oxide and reduced graphene oxide also showed dose-dependent antibacterial action against Pseudomonas aeruginosa by creating ROS, with graphene oxide inducing bacterial DNA fragmentation [93].
properties via a variety of mechanisms, including physical destruction, the inflammatory immune response, and oxidative stress ( Figure 5, Table 4).
One of the most typical antimicrobial mechanisms in CNMs is the physical destruction of the outer cell membrane or cell wall. Carbon-based nanomaterials bind to peptidoglycan and proteins in the cell membrane, causing cell membrane rupture [87,88]. Carbon nanotubes and graphene, for example, have sharp edges that may puncture bacterial membranes, resulting in the release of bacterial internal components such as RNA [89,90].
The metabolic inflammatory response is linked to changes in the gut microbiota caused by CNMs. Carbon nanotubes boosted the release of inflammatory factors such as IL-1β, IL-6, and TNF-α in the duodenum and colon, as well as the transition of the phylum Firmicutes to Bacteroidetes and the abundance of the pro-inflammatory bacteria Alitipes_uncultured and Lachnospiraceae bacterium A4 [91].
Oxidative stress is another major antibacterial mechanism in CNMs. Carbon quantum dots generate ROS when exposed to blue light, dramatically inhibiting the activity of methicillin-resistant Staphylococcus aureus and Escherichia coli [92]. Graphene oxide and reduced graphene oxide also showed dose-dependent antibacterial action against Pseudomonas aeruginosa by creating ROS, with graphene oxide inducing bacterial DNA fragmentation [93].

Effects of Other Nanomaterials on Gut Microbiota
Silica nanoparticles (SiO 2 NPs) can affect the abundance of gut microbiota through inflammatory immune responses (Table 5). Following 7 days of administering 2.5 mg/kg bw/day SiO 2 NPs to mice, pro-inflammatory factors such as IL-1β, IL-6, and TNF-α increased considerably in the small intestine and colon. Meanwhile, the phylotypes responded to the Firmicutes increase (39.9% vs. 26.1% in control mice) and Bacteroidete decline [98].
Copper-loaded chitosan nanoparticles (CNP-Cu) can increase the abundance of Bifidobacterium and Lactobacillus since some microbiota were inhibited by CNP-Cu [99]. Wang et al. fed weaned piglets CNP-Cu to investigate its effects. The results showed that the abundance of E. coli was dramatically reduced but the numbers of Lactobacillus and Bifidobacterium were increased [100].
In vitro investigations revealed that the richness of the microbiota increased dosedependently when exposed to nano-Al 2 O 3 . The structure of the gut microbiota was altered dramatically at high dosages (50 mg/L), with the number of Firmicutes and Proteobacteria increasing and Bacteroidetes decreasing [101].

Summary and Future Outlooks
The structure and abundance of the gut microbiota are dynamic and influenced by dietary properties. According to the accessible data, Firmicutes were discovered to be the most susceptible microbiota. Firmicutes are one of the most numerous bacterial families in the gut. Lactobacilli, which function as a probiotic in Firmicutes, were sensitive to nanoparticles. Another probiotic called Bifidobacterium was another sensitive microbiota, with increased abundance when exposed to Ag NPs and CNP-Cu NPs and reduced abundance when exposed to titanium dioxide. It can be seen that the change of microbiota is material-specific.
Over the past few decades, rapid advancements in nanomaterials have provided intriguing alternatives to antibacterial therapies. Nanomaterials, as opposed to regular antibiotics, are less prone to causing bacterial resistance. They alter the structure of the gut microbiota, influencing host health by triggering the intestinal immune system and oxidative stress. Unfortunately, most research on the impact of nanomaterials on gut microbiota is restricted to animal or in vitro tests, and studying complicated human environments remains difficult. Since there are still few data on the real exposure concentrations of nanomaterials, dose selection in animal studies needs to be carefully considered. In future, experiments should focus on the influence of nanomaterials on the human gut microbiota, bridging the gap between microbiota disorders and host illnesses and supplementing the safe use of nanomaterials.