by Cassio Luiz Coutinho Almeida-da-Silva, Leticia Ferreira Cabido, Wei-Chun Chin, Ge Wang, David M. Ojcius and Changqing Li.
Engineered nanoparticles (NPs) composed of elements such as silica and titanium, smaller than 100 nm in diameter and their aggregates, are found in consumer products such as cosmetics, food, antimicrobials and drug delivery systems, and oral health products such as toothpaste and dental materials. They may also interact accidently with epithelial tissues in the intestines and oral cavity, where they can aggregate into larger particles and induce inflammation through pathways such as inflammasome activation. Persistent inflammation can lead to precancerous lesions. Both the particles and lesions are difficult to detect in biopsies, especially in clinical settings that screen large numbers of patients. As diagnosis of early stages of disease can be lifesaving, there is growing interest in better understanding interactions between NPs and epithelium and developing rapid imaging techniques that could detect foreign particles and markers of inflammation in epithelial tissues. NPs can be labelled with fluorescence or radioactive isotopes, but it is challenging to detect unlabeled NPs with conventional imaging techniques. Different current imaging techniques such as synchrotron radiation X-ray fluorescence spectroscopy are discussed here. Improvements in imaging techniques, coupled with the use of machine learning tools, are needed before diagnosis of particles in biopsies by automated imaging could move usefully into the clinic.
1. Introduction
Engineered nanoparticles (NPs) have been defined as nanomaterials that range in size between 1 and 100 nm (in at least one dimension). NPs are used in the fabrication of numerous consumer goods, including various paints, food, detergents, bactericides, coatings, cosmetics, sunscreens, tires, personal care products, computer construction, and drug and gene delivery [1]. NPs are also present in medical and healthcare goods, sunscreens, toothpaste, and various devices used in dental procedures, such as professional polishing agents and dental restorative materials [[1], [2], [3], [4]]. These NPs can also aggregate into larger particles, which have different effects than the original NPs on biological responses from tissues exposed to the particles.
Given the promising application of NPs, funding for the National Nanotechnology Initiative (NNI) in the USA alone approached $1 billion in 2005. As of 2009, this new technology supported a worldwide market of about a quarter of a trillion dollars, of which about $91 billion was found in U.S. products that include nanomaterials [5]. With the rapid development of nanotechnology and its related commercial products, it is inevitable that NPs have become a common part of our daily lives. The major concern with NPs in terms of their potential health hazards or risks (e.g., the potential for producing reactive oxygen species and associated negative biological consequences) is mainly related to their large surface reactivity due to their unique small dimensions.
Upon interaction with the organism, different NPs have been demonstrated to interact with immune cells and epithelial cells [[6], [7], [8], [9], [10]]. Epithelial cells are particularly important because they are the first cells to encounter foreign particles in the skin and in the oral mucosa and, by secreting cytokines and chemokines, can initiate and orchestrate an immune response. In fact, we and others have described how challenged oral epithelial cells are able to sense and generate immune responses in response to infection [[11], [12], [13]]. Due to the large distribution of NPs in several products used routinely, NPs can be found in human tissue, including oral tissues [6].
Imaging techniques such as light microscopy are the most common imaging methods used by pathologists for detection of tissue anomalies. Even though NPs can be implicated in different pathological conditions, their identification is challenging due to their small size and lack of natural staining. Given that early diagnosis of diseases can improve the quality of life and save people's lives, it is crucial to better understand the relationship between NPs and the epithelium, and to develop efficient and robust imaging techniques that could detect simultaneously inflammatory markers and NPs in epithelial tissues.
The present review will focus on the interactions of silica and titanium nanoparticles with human cells, and their ability to induce immune responses and exacerbate certain pathological conditions. While previous excellent and comprehensive reviews have focused on the factors and cellular mechanisms related to the toxicity of NPs [1,14], and their effects on biological systems [14], the goal of this study is to discuss the interactions of silica and titanium nanoparticles across the continuum of oral and gastrointestinal epithelia. We will also discuss the potential use of innovative imaging techniques and machine learning tools to effectively detect nanoparticles in soft tissue samples for diagnostic purposes.
2. Engineered nanoparticles (NPs)
The following three types of NPs are the most frequent nanomaterials that the public encounters in daily life.
2.1. Titanium dioxide NPs (TiO2 NPs)
Titanium dioxide (TiO2) has traditionally been considered as a biologically inert substance to both animals and humans. As a consequence, TiO2 NPs are manufactured in large quantities for a wide range of applications as one of most common nanomaterials, such as the nanotechnology and biomedical products, paints, cosmetics, textiles, papers, plastics, sunscreens, and food [2,3]. Among household products, including foods and personal care products, toothpastes and chewing gums have some of the highest content of TiO2 NPs, up to around 6 mg Ti/g (roughly 10 mg TiO2/g toothpaste) and 3 mg/g chewing gum. Popular brands like Sensodyne, Aquafresh, Colgate, Mentos, Eclipse, and Trident are among the toothpastes/chewing gums with the highest TiO2 concentrations (for details see (Weir et al., 2012) [2]).
The use of titanium in dental materials and the enormous annual global production and growing commercial popularity of TiO2 raise the risk of occupational and environmental exposures. Nonetheless, TiO2 NPs are also known for their potential hazardous effects by triggering harmful responses (e.g., inflammation) in various tissues and animal models [3]. In particular, the introduction of titanium into gingival tissues can result in foreign body gingivitis, an inflammatory process involving the gingiva [7,15], which will be discussed in the sections below.
2.2. Silicon dioxide NPs (SiO2 NPs)
SiO2 NPs possess many unique physical-chemical characteristics; therefore, they are utilized in many aspects of everyday life and many household products. According to the Consumer Products Inventory (CPI), there are over 100 commercial products that contain SiO2 NPs, including food, toothpastes, cosmetics, paints, electronic devices, and even drugs and dietary supplements. Due to their versatile applications, SiO2 NPs have become the second most-highly produced nanomaterial [4]. The large-scale production and widespread application of SiO2 NPs have increased the risk of human exposure. Many published reports confirmed that SiO2 NPs are capable of inducing proinflammatory responses, NLRP3 inflammasome activation and oxidative stress including reactive oxygen species (ROS) production in various cells and tissues, as reviewed by Dong et al. [4] and Chen et al. [16]. The role of SiO2 NPs in inducing inflammation in different types of epithelial cells will be discussed in the sections below.
2.3. Cerium oxide NPs (CeO2 NPs)
Cerium, which is the first element in the lanthanide group (rare earth element family) with 4f electrons, which give cerium oxide (CeO2) NPs unique catalytic, magnetic and electronic properties, has attracted attention from researchers in physics, chemistry, biotechnology and materials science [17,18]. Cerium oxide is involved in a wide range of applications, including oil refining (cracking catalysts), polishing agents (for glass mirrors, plate glass, television tubes, ophthalmic lenses, precision optics, electronic wafers), sensors, semiconductors, solar cells, thin-film coatings and solid-oxide fuel cells [[17], [18], [19]]. CeO2 NPs can also serve as fuel additives, three-way catalysts for automobile exhaust-gas treatments, oxidative coupling of methane and water-gas shift reactions [18,19]. Recently, CeO2 NPs have been added in many consumer products (cigarette additives, sunscreens, or cosmetics) and as potential pharmacological agents [17,18]. CeO2 possesses its unique properties due to its varying valence electrons that are either +3 or +4, and its large surface-area-to-volume ratio creates oxygen defects [20,21]. Earlier studies have suggested that CeO2 NPs can induce production of ROS [[22], [23], [24], [25]] and can cause immunological responses in rats that included alveolar functional changes, lung tissue inflammation and cytotoxicity [24].
3. Pathogenic effects of NPs on oral and gut epithelia
Because NPs are commonly used as food additives and in dental materials, these particles frequently interact with oral and gut epithelia. To understand the safety of engineered NPs in food, it is important to assess the presence, dissolution, agglomeration state, and release of these materials in the nano-size range from food during human digestion. A study using an in vitro digestion model found that SiO2 NPs (E551) in food additives either disappeared or were only present in low amounts in the gastric stage of digestion; however, SiO2 NPs became bioavailable under conditions found in the gut lumen. Hence, the human intestinal epithelium is likely exposed daily to SiO2 NPs [8]. Accordingly, the potential effects of SiO2 NPs and other NPs on the intestinal mucosa should not be overlooked. The effects of NPs are particularly significant in individuals whose intestinal homeostasis is already disrupted, such as those with inflammatory bowel diseases. Indeed, Ogawa et al. found that the oral administration of small (10 nm) SiO2 NPs exacerbated intestinal inflammation in a dextran-sulfate sodium (DSS) colitis model through activation of the ASC inflammasome in macrophages [26]. Strikingly, a recent study using a small intestinal epithelial cellular model has found that co-exposure to boscalid, a commonly used pesticide, and TiO2 (E171) or SiO2 (E551) downregulated cell junction gene expression, increasing pesticide translocation across the small intestinal epithelium [27].
TiO2 particles are widely used as a food additive (E171), which is a powder composed of particles with an average grain size larger than 100 nm, but also containing a significant amount of nanosized (<100 nm) particles. TiO2 in food has been recently declared unsafe, causing several European nations to ban E171 as food additive in early 2022. According to an updated safety assessment from the European Food Safety Authority (EFSA), E171 is no longer considered a safe food additive, particularly due to concerns regarding potential genotoxicity [28]. In addition, a large body of evidence has found that exposure to TiO2 may cause gut barrier dysfunction and be associated with the etiology and/or exacerbation of inflammatory bowel disease. For instance, a study has reported that orally administered TiO2 NPs worsen acute colitis in mice and, in vitro, TiO2 particles induced ROS generation and increased epithelial permeability in intestinal epithelial cell monolayers. Most notably, increased levels of titanium were found in the blood of patients with active ulcerative colitis [29]. Exposure to TiO2 has been shown to affect the morphology of intestinal epithelium as E171 caused disruption of microvilli organization in Caco-2 BBE1 cells in vitro. Another in vitro study has shown that, in addition to altering tight junctions, acute exposure of Caco-2/HT29-MTX cells to TiO2 NPs reduces absorptive microvilli. Accordingly, both the protective and absorptive functions of the intestinal epithelium are impaired by TiO2 exposure [28].
Importantly, TiO2 is rated by the International Agency for Research on Cancer (IARC) as possibly carcinogenic for humans by inhalation (Group 2B). This fact, when combined with the findings that TiO2 disturbs the intestinal barrier and induces inflammation, raises concerns about a potential role for titanium in intestinal carcinogenesis. Indeed, TiO2 has been shown to enhance the formation of intestinal tumors in mice suffering from colitis-associated cancer. This aggravated tumor formation is associated with premalignant changes in the colonic epithelium, but also with a dramatic decrease in goblet cells leading to intestinal barrier disruption. Tumorigenesis could also be attributed to ROS production as well as DNA damage caused by the interaction between E171 and microtubules, or oxidative DNA damage in cells acutely or repeatedly exposed to E171. Finally, TiO2 NPs have been reported to promote epithelial to mesenchymal transition in colorectal cancer cells. In conclusion, additional studies are needed to better elucidate the potential involvement of TiO2 particles in the development of colorectal cancer [28].
Foreign body gingivitis (FBG) is a term to describe gingival inflammation associated with the iatrogenic implantation of foreign material [7]. These lesions are thought to occur when damage to the mucosal epithelium during dental or oral hygiene procedures allows the introduction of small foreign particles into the gingival tissues. Unfortunately, quite often the individual foreign particles are less than 1 μm in diameter and very easily overlooked by the pathologist who examines FBG biopsies under light microscopy (Fig. 1D) [6]. Free article. Read more at: https://www.sciencedirect.com/science/article/pii/S240584402301229X
Fig. 2. Effects of different NPs on NLRP3 inflammasome activation in oral epithelial cells. On the left side of the oral cell, gold, silver and palladium NPs are shown to induce inflammasome activation in oral cells. On the right of the oral cell, we postulate that silica, aluminum and titanium NPs may be involved in NLRP3 inflammasome activation in oral cells, although this remains to be shown.