A NAM-based approach for addressing key knowledge gaps in the assessment of potential hazards associated to nanocellulose oral exposure

Nanocellulose (NC) is an emerging material in the pharmaceutical and food sector with several prospective application areas entailing ingestion of NC crystals or fibers. Three main types of NC exist, i.e. cellulose nanocrystals (CNC), nanofibrillated cellulose (NFC), and bacterial NC (BNC). The biological sources and processing conditions affect several physicochemical parameters of NC. Although all NC materials typically have a high aspect ratio, CNC usually consists in rod-shaped crystals, whereas BNC and NFC consists in fibrils composed of fibres with a length up to 2-3 μm. In the present research, a New approach methodology (NAM)-based approach for addressing key knowledge gaps in the assessment of potential hazards associated to NC oral exposure was used. One NC material belonging to each of the three NC types was selected as test item. For the three test materials a thorough physicochemical characterisation was available, which included TEM measurement of median diameters in the range 5-9 nm, along with a material-specific dispersion protocol. The first gap addressed was NC potential to be taken up by intestinal cells and cross the intestinal barrier using Caco-2 monocultures as a model. Two alternative fluorescent agents were tested for NC staining and a novel approach for characterizing cell uptake via Confocal Laser Scanning Microscopy (CLSM) detection was developed. Cell uptake was then studied with a Caco-2/HT29/Raji-B co-culture, which better simulates the barrier properties of the human intestinal epithelium as compared to Caco-2 monolayers. The presence in the model of mucus-secreting goblet cells and M-cells is particularly important (i) as any particle reaching the intestinal epithelium has to cross the mucus layer first and (ii) because particle translocation may largely take place via M-cells. Cell uptake in this model was greater than that observed with the Caco-2 monoculture with CNC, and slightly lower with the nanofibre materials. Importantly, in both models CNC showed the highest internalisation rate, which led to its selection for further studies with the triculture model in repeated exposure conditions. In such conditions (i) an increase in the internalisation rate (with intracellular localization in the lysosomes) was detected and (ii) translocation across the model barrier was demonstrated. The second main pillar of the present research was the assessment of any potential functional impairment of the intestinal barrier after interaction with NC. The triculture cell model was used and Transepithelial Electrical Resistance (TEER) was measured to monitor the cell monolayer integrity, whereas paracellular permeability was assessed by Lucifer Yellow (LY) translocation. TEER decrease and LY translocation increase after NC exposure, both in single and in repeated exposure conditions, showed the potential of NC for functional impairment of the intestinal barrier, at least transiently. Finally, NC uptake and pro-inflammatory responses by macrophage differentiated THP-1 cells were investigated. The three NC materials were massively taken up and did elicit release of Interleukin-8 (IL-8), an important cytokine in inflammation and innate immune system response, with the highest response obtained after exposure to CNC.