Cytotoxicity and cellular proteome impact of cellulose nanocrystals using simulated digestion and an in vitro small intestinal epithelium cellular model

Cellulose nanocrystals (CNC) (0.75 or 1.5% w/w) in either a fasting food model (FFM) or a standardized food model (SFM) based on the average USA diet, were digested using a three-phase (oral-gastric-small intestinal) digestion simulator, and the resulting small intestinal phase digestas were applied to an in vitro triculture human cellular model of the small intestinal epithelium (SIE). Effects of exposures on SIE monolayer integrity, cytotoxicity, and oxidative stress were evaluated, and a global cellular proteomic analysis of exposed cells was performed to obtain a more comprehensive understanding of the cellular response to ingested CNC in different food models. Other than a slight increase in cytotoxicity (13.2%, p < 0.05) in the presence of 1.5% w/w CNC in FFM, CNC did not significantly impact SIE health. Furthermore, two-way ANOVA revealed a significant interaction between the food matrix and CNC ingestion in cytotoxicity outcomes. No significant perturbations of the cellular proteome were caused by CNC ingestion in either food model. However, 125 significantly differentially expressed proteins were identified only in food model comparisons in the presence of CNC, indicating significant interactions between CNC and food matrix. Gene ontology analysis showed that CNC-food matrix interactions have impacts on multiple biological processes related to nutrient absorption and utilization, such as protein transport, glycolysis, and fatty acid metabolism. These results suggest that CNC has limited cytotoxic potential, but may alter the cellular response to co-ingested nutrients, and underscore the need to include such biological endpoints in risk assessment studies of ingested nanomaterials.