Development of an Integrated in Vitro Model for the Prediction of Oral Bioavailability of Nanoparticles

• Main Author: Walczak, Agata Paulina
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2015
• Subjects: Bioavailability; Biodistribution; Cadmium telluride; Chemicals; Enzymes; Food Science; Gene expression; Hydration; Influence; Microscopy; Nanomaterials; Nanoparticles; Peptides; Proteins; Risk assessment; Silver; Small intestine; Stomach; Titanium; Zinc oxides
• ISBN: 9798708785664

Nanotechnology developed in the past decades and resulted in many innovative products. A branch of nanotechnology focuses on the manufacturing and use of nanoparticles (NPs). Currently, we find many products containing NPs on the market (Wijnhoven et al. 2010; Chaudhry et al. 2008; Woodrow Wilson institute). Because many of these products are food or food-related products (Bouwmeester et al. 2014), human oral exposure to NPs is very likely. Before entering the EU market, a risk assessment of NPs applied in these products is required. Many of the products are also available via internet. At the moment, the risk assessment of chemicals and NPs relies heavily on animal studies. For scientific, ethical and economical reasons, there is a demand to refine, reduce and replace animal testing by developing in vitro alternatives for hazard characterization. In vitro methods are ideally incorporated in a tiered testing approach and for oral exposure the focus is on the effect of passing the gastrointestinal tract on the physicochemical properties of the NPs and on the subsequent passage of the gut wall. This can be followed by in vitro effect screening, if indications of systemic bioavailability is obtained. Thus, the aim of the present thesis was to develop an integrated in vitro approach based on in vitro models (i.e. in vitro digestion model and in vitro gut epithelium model) to evaluate the uptake of NPs following ingestion for the prediction of their oral bioavailability in the human body.Chapter 1 of the thesis gives an introduction to the topic, highlights the importance of careful characterization of NPs tested in the various assays and presents existing in vitro models of gastrointestinal digestion and human intestinal epithelium used for toxicity testing of NPs. Some of these in vitro models were selected and evaluated for testing NPs in the next (experimental) chapters. In order to study the fate of NPs after ingestion, the first step was to study the suitability of an in vitro digestion model to assess what happens with NPs during the passage through the human gastrointestinal tract. For this, in chapter 2 of this thesis the effects of the digestion on the fate and physicochemical properties of 60 nm silver nanoparticles (AgNPs) and Ag+ ions were studied. Samples were incubated in a static in vitro human digestion model composed of subsequent oral, gastric and intestinal digestion phases. It was demonstrated that gastrointestinal digestion impacted AgNPs and Ag+ ions in such a way that they changed during gastrointestinal digestion. As measured with (single particle) inductively coupled plasma-mass spectroscopy (SP-ICPMS), AgNPs agglomerated to a high extent during gastric passage, a process facilitated by chlorine interparticle bridges as shown by scanning electron microscopy with energy dispersive X-ray (SEM-EDX) analysis. These clusters were shown to break down during subsequent intestinal incubation, releasing the original AgNPs from the clusters. Strikingly, the NPs retained their original size in the intestinal juice. Thus, it was concluded that orally ingested 60 nm AgNPs, digested under physiologically relevant conditions, ultimately can reach the intestinal wall in their initial size and composition. Interestingly, the results of this study also revealed that intestinal digestion of Ag+ ions resulted in formation of 20-30 nm particles composed of silver, sulphur and chlorine. Therefore, we concluded also that ingestion of Ag+ ions ultimately leads to intestinal exposure to NPs, albeit with a complex chemical composition. In order to reach the systemic circulation, NPs need to pass the gut wall. Therefore, in order to assess the potential bioavailability of NPs, measuring their intestinal translocation is essential. Given the aim to select and validate in vitro alternative testing strategies, selection of an in vitro model was undertaken in Chapter 3 of the thesis. Chapter 3 presents a comparison of three cell culture models: a mono-culture (Caco-2 cells), a co-culture with mucus secreting HT29-MTX cells, and a tri-culture with M-cells. Differently sized (50 and 100 nm) and charged (neutral, positively and negatively) polystyrene nanoparticles (PS-NPs) were used to challenge these models. PS-NPs were shown to translocate across the in vitro intestinal barriers, depending on their physicochemical properties. Size and charge were shown to strongly affect the translocation of NPs, but also surface chemistry was shown to be important, because the two types of negatively charged NPs with different surface modifications had an over 30-fold difference in translocation. This result indicates that the chemical composition at the surface of the NPs is more important than the zeta potential. The relative pattern of NP translocation in all three used intestinal models was similar, but the absolute amounts of translocated NPs differed per model. Therefore, we concluded that for comparing the relative translocation of different NPs, using one intestinal model would be sufficient. Preferably, a model with mucus should be chosen, as mucus creates more realistic exposure conditions, also protecting cells from toxic effect of chyme. However, for screening studies in a tiered risk assessment approach, when absolute translocation values are needed, it should be kept in mind that depending on the chosen model, the outcomes will differ.n chapter 2 in vitro digestion was shown to influence the physicochemical properties of AgNPs. To test if this would affect the translocation efficiency of PS-NPs, we integrated the in vitro digestion model with the in vitro intestinal co-culture model to test the translocation of digested PS-NPs. In chapter 4 the translocation results from this integrated in vitro model are presented. It demonstrates that in vitro digestion of differently charged 50 nm PS-NPs influenced both their translocation behaviour and their protein corona. Translocation of all digested PS-NPs was clearly increased compared to the translocation of their pristine equivalent PS-NPs and was ranging from 1.6% to 12.3%, depending on NP type. One type of negatively charged NP was the NP translocating to the highest extent. The protein corona was affected in both the amount of proteins and their composition. Digested PS-NPs contained less protein in their corona than their equivalent pristine PS-NPs. The composition of the protein corona of PS-NPs was changed, which resulted in a shift from larger proteins (present in coronas of pristine NPs) towards low molecular weight proteins. This study clearly illustrated that gastrointestinal digestion affects in vitro translocation behaviour of different types of PS-NPs. These findings stress the importance of including the in vitro digestion in future in vitro intestinal translocation screening studies for risk assessment of orally taken NPs.To assess the utility of the developed integrated in vitro model for assessing the bioavailability, it required validation by an in vivo study. Chapter 5 presents the estimated bioavailability resulting from an oral exposure of rats to a single dose of the same PS-NPs as tested in the integrated in vitro model. Similar to the in vitro results, the surface charge and surface chemistry affected the uptake and biodistribution of PS-NPs in rats after oral exposure. Also in line with the in vitro results it appeared that one type of negatively charged NP was taken up to a larger extent than the other NPs. The highest amounts of NPs were detected in kidney, heart, stomach wall, and small intestinal wall. This partly confirmed our in vitro findings, where the same NPs translocated to the highest extent among all tested NPs. However, the relative order of uptake for the other NPs differed from the in vitro findings. The estimated bioavailability ranged from 0.2% to 1.7% in vivo, which was much lower than that in vitro (1.6% to 12.3%). These results show that the predicted uptake of PS-NPs from our integrated in vitro model appears to overestimate the actual uptake occurring in the rat in vivo . In conclusion, the integrated in vitro model cannot be directly used for a quantitative prediction of the bioavailability of orally administered NPs. However, the integrated in vitro model can be used for prioritizing NPs, based on their translocation rate, for further in vivo testing for risk assessment.Chapter 6 presents a discussion on the in vitro and in vivo findings of the present thesis and some future perspectives for research on issues raised in this thesis.Overall, the present thesis presents the development of an integrated in vitro strategy for studying the fate and bioavailability of NPs after ingestion. The developed integrated in vitro model combines an in vitro gastrointestinal digestion model with an in vitro intestinal model. The translocation of the NPs, as predicted from the integrated in vitro model, overestimated the translocation across the rat intestine in vivo . Therefore, the integrated in vitro model cannot be directly used for a quantitative prediction of the bioavailability of orally administered NPs. However, the model can be used for screening and prioritizing NPs before further in vivo testing for risk assessment.