An integrated methodology for assessing the impact of food matrix and gastrointestinal effects on the biokinetics and cellular toxicity of ingested engineered nanomaterials

Abstract

BackgroundEngineered nanomaterials (ENMs) are increasingly added to foods to improve their quality, sensory appeal, safety and shelf-life. Human exposure to these ingested ENMs (iENMS) is inevitable, yet little is known of their hazards. To assess potential hazards, efficient in vitro methodologies are needed to evaluate particle biokinetics and toxicity. These methodologies must account for interactions and transformations of iENMs in foods (food matrix effect) and in the gastrointestinal tract (GIT) that are likely to determine nano-biointeractions. Here we report the development and application of an integrated methodology consisting of three interconnected stages: 1) assessment of iENM-food interactions (food matrix effect) using model foods; 2) assessment of gastrointestinal transformations of the nano-enabled model foods using a three-stage GIT simulator; 3) assessment of iENMs biokinetics and cellular toxicity after exposure to simulated GIT conditions using a triculture cell model. As a case study, a model food (corn oil-in-water emulsion) was infused with Fe2O3 (Iron(III) oxide or ferric oxide) ENMs and processed using this three-stage integrated platform to study the impact of food matrix and GIT effects on nanoparticle biokinetics and cytotoxicity .MethodsA corn oil in phosphate buffer emulsion was prepared using a high speed blender and high pressure homogenizer. Iron oxide ENM was dispersed in water by sonication and combined with the food model. The resulting nano-enabled food was passed through a three stage (mouth, stomach and small intestine) GIT simulator. Size distributions of nano-enabled food model and digestae at each stage were analyzed by DLS and laser diffraction. TEM and confocal imaging were used to assess morphology of digestae at each phase. Dissolution of Fe2O3 ENM along the GIT was assessed by ICP-MS analysis of supernatants and pellets following centrifugation of digestae. An in vitro transwell triculture epithelial model was used to assess biokinetics and toxicity of ingested Fe2O3 ENM. Translocation of Fe2O3 ENM was determined by ICP-MS analysis of cell lysates and basolateral compartment fluid over time.ResultsIt was demonstrated that the interactions of iENMs with food and GIT components influenced nanoparticle fate and transport, biokinetics and toxicological profile. Large differences in particle size, charge, and morphology were observed in the model food with and without Fe2O3 and among digestae from different stages of the simulated GIT (mouth, stomach, and small intestine). Immunoflorescence and TEM imaging of the cell culture model revealed markers and morphology of small intestinal epithelium including enterocytes, goblet cells and M cells. Fe2O3 was not toxic at concentrations tested in the digesta. In biokinetics studies, translocation of Fe2O3 after 4 h was <1% and ~2% for digesta with and without serum, respectively, suggesting that use of serum proteins alters iENMs biokinetics and raises concerns about commonly-used approaches that neglect iENM – food-GIT interact...

Synthesis and Physicochemical Transformations of Size‐Sorted Graphene Oxide during Simulated Digestion and Its Toxicological Assessment against an In Vitro Model of the Human Intestinal Epithelium

“…The in vitro simulated digestion of the above samples was performed using a three-phase (oral, gastric, small intestinal) gastrointestinal tract simulator, as previously described in detail by the authors. [36] Briefly, in the oral phase, food inputs were brought to 37 °C, mixed with simulated saliva fluid prewarmed at 37 °C, and inverted by hand for 10 s to mimic agitation in the mouth. The resulting oral phase digestas were then combined with prewarmed simulated gastric fluid and incubated in a shaking incubator at 37 °C for 2 h at 200 rpm to complete the stomach phase.…”

“…A tri-culture, small intestinal epithelial model as previously described and characterized in detail by the authors was employed in this study. [36] In summary, 1.5 mL of Caco-2 cells and HT29-MTX cells were first co-cultured at a ratio of 3:1 (Caco-2:HT29-MTX) for 2 weeks on a 0.4 µm pore membrane transwell inserts (apical compartment) in a 6-well cell culture plate. In the basolateral compartment, 2.5 mL of complete DMEM media was added.…”

“…The synthesized sub-micrometer and a micrometer-sized GO variants were incorporated in a fasting food model (FFM) and submitted to a three-stage (oral, gastric, small intestinal), simulated gastrointestinal digestion according to a methodology previously developed by our group. [36] The physical and chemical transformations of GO were analyzed by employing multiple techniques. Particularly, real-time, ensemble size measurements of GO were performed using multi-angle laser diffraction to follow its agglomeration along the GIT.…”

“…Finally, we performed the toxicological assessment of GO digesta using a physiologically relevant, tri-culture in vitro model of the human small intestinal epithelium consisting of differentiated small intestinal enterocytes (Caco-2 cells), mucus-secreting goblet cell-like cells (HT29), and specialized microfold cells, naturally associated with the lymphatic system. [36] Figure 1 summarizes the study design.…”

Inhalation of printer-emitted particles impairs cardiac conduction, hemodynamics, and autonomic regulation and induces arrhythmia and electrical remodeling in rats

“…Exposures to ENMs released across the lifecycle of nano-enabled products have become inevitable. Due to their size, ENMs can bypass biological barriers, become systemic, interfere with cellular processes, and induce adverse health effects [5][6][7][8][9][10][11][12][13][14][15]. Despite that numerous studies have linked exposure to ambient ultrafine particles to cardiovascular dysfunction, autonomic dysregulation, and heart disease [16][17][18][19], there are only limited investigations into the cardiovascular effects of ENMs [20].…”