In vitro testing strategies for hazard assessment of nanoparticles

Summary

In chapter 1 of this thesis, an overview of the main applications of NPs was provided and the main properties of NPs were briefly introduced. In addition, some of the key methods that are currently used in the toxicological safety assessment of NPs were presented. The aim of the thesis was introduced which was to investigate the potential of different in vitro methods combined with high-end analytical techniques as a testing strategy to study the toxicokinetic and toxicodynamic properties of silver (Ag) and polystyrene (PS) NPs and set priorities in their further safety testing. The current state of the art of the in vitro methods used in the studies in this thesis and the outline of the thesis were also presented.

In chapter 2, the influences of the size and surface chemistry of pristine PSNPs on the protein corona formation and subsequent uptake/association and transport of these NPs through a Caco-2 intestinal cell model were studied. Four negatively charged PSNPs of different sizes (50 and 200 nm) and with different surface chemistries (sulfone or carboxyl groups) were studied. The protein coronas of these PSNPs were analysed by LC-MS/MS which showed subtle differences in the protein composition of especially the two PSNPs with different surface chemistries. In further experiments, the impact of surface chemistry on the cellular uptake/association and transport was characterised using high-content imaging analysis. These experiments demonstrated that the PSNPs with sulfone surface groups were associated with the cells to a significantly higher extent than the PSNPs with carboxyl surface groups. No clear effect of the size of the PSNPs on the cellular uptake/association was noted. Also, the transport of the PSNPs with sulfone surface groups through the monolayer of cells was significantly higher than that of PSNPs with carboxyl surface groups.

The general conclusion was that the composition of the protein corona and the PSNPs surface chemistry influence the cellular NPs uptake/association and transport, with the effect of the NP surface chemistry outweighing the impact of NPs size on the cellular uptake/associations and transport. These results might be predictive of the intestinal transport of NPs. Still, further studies are required to identify which corona proteins affect the uptake and transport of NPs.

Chapter 3 described an investigation on the impact of the biochemical conditions within the human digestive tract on the intestinal fate of AgNPs with different surface chemistries. On top of that, the transport of these AgNPs across an intestinal in vitro model of Caco-2/HT29-MTX cells was evaluated. An in vitro digestion model was used to simulate the human digestion processes. Two 50 nm negatively charged AgNPs with different surface chemistries were used, lipoic acid (LA) AgNPs and citrate (Cit) AgNPs next to silver nitrate (AgNO3) as a source of ionic silver (Ag+). The co-culture model was exposed to different concentrations of pristine and in vitro digested (IVD) AgNPs or AgNO3 for 24 hr. Using ICP-MS and spICP-MS, the size distribution, dissolution, particle concentration (mass- and number-based) and total silver content of the AgNPs were characterized before and after digestion and in the apical, basolateral and cellular compartments of the Caco-2/HT29-MTX intestinal epithelial model. The surface chemistry of AgNPs had a significant influence on their dissolution and on their biological interactions with the Caco-2/HT29-MTX intestinal model. In general, a significant fraction of the AgNPs dissolved during the digestion up to 86 - 92% and 48 – 70% of the original amount of NPs for the (LA) and (Cit) AgNPs, respectively. Exposure of the monolayer of intestinal cells to increasing concentrations of pristine or IVD AgNPs resulted in a concentration dependent increase of total Ag and AgNPs content in the cellular fractions. The cellular concentrations were significantly lower following exposure to IVD AgNPs compared to the pristine AgNPs. The effect of the surface chemistry on the cellular concentration of Ag was only detected upon exposure to pristine AgNPs, while this difference disappeared upon exposure to IVD AgNPs.

Transport of Ag across the intestinal model layer, as either total Ag or AgNPs, was limited (< 0.1%) following exposure to pristine and IVD AgNPs. De novo formation of AgNPs was shown in the exposure suspensions of IVD AgNPs and AgNO3 and in the cellular fractions, upon cellular exposure to pristine and IVD AgNO3. In conclusion, the surface chemistry of AgNPs and the biochemical conditions during in vitro digestion influences the dissolution and also influences the uptake/association within the Caco-2/HT29-MTX monolayer. This highlights the need to take in vitro digestion into account when studying nanoparticle toxicokinetics in an intestinal cellular in vitro model system. The observation that dissolution characteristics of NPs may change upon digestion could be of added value in the safe(r)-by-design NPs development.

Chapter 4 presented the outcomes from combining the BeWo b30 placental transport model and the embryonic stem cell test (EST) to investigate the capability of pristine AgNPs of different surface chemistries and aged AgNPs (Ag2S NPs) to cross the placental barrier and induce in vitro developmental toxicity. AgNO3 was used as a source of Ag+. The pristine negatively charged AgNPs used in this study were similar to the ones used in chapter 3 while in addition also positively charged 50 nm branched polyethylenimine (BPEI) AgNPs were included in the study. The size distribution, dissolution, particle concentration (mass- and number-based) and total Ag content of the AgNPs in the apical, basolateral and cellular compartments of the BeWo b30 placental transport model at different time points was characterized using ICP-MS and spICP-MS. The ability of the AgNPs to cross the BeWo b30 cell layer was limited and dependent on the surface chemistry of these AgNPs. The particles detected in the basolateral compartment could result from transport of the original AgNPs and/or from the de novo formed AgNPs in the basolateral compartment from Ag+ that was transported.

The in vitro developmental toxicity of the AgNPs was investigated by characterizing their potential to inhibit the differentiation of mouse embryonic stem cells (mESCs) into beating cardiomyocytes using the EST. The observed inhibitory effects of the AgNPs on differentiation of mESCs were most likely the result of cytotoxicity rather than specific effects related to developmental toxicity as the effects on differentiation of the mESCs were only detected at cytotoxic concentrations. Compared to the pristine AgNPs, the aged Ag2S NPs were significantly less cytotoxic, transported less across the BeWo cell layer and did not induce in vitro developmental toxicity. In conclusion, the combination of the BeWo placental transport model with the mESCs differentiation assay appeared to provide a valuable alternative in vitro methodology for prenatal developmental toxicity testing and prioritization for further safety testing of AgNPs, with aged Ag2S NPs appearing to present less of a hazard than pristine AgNPs.

In chapter 5, the In-Cell Western (ICW)-γ-H2AX assay was evaluated as an alternative in vitro assay to detect the potential of aged AgNPs and pristine AgNPs to induce phosphorylation of H2AX in HepG2 liver cells. AgNO3 was used as source of Ag+ to test the effects of Ag+ themselves. The γ-H2AX induction detected was higher after 24 hr exposure compared to 4 hr and was accompanied by a significant cytotoxicity in the HepG2 cells. The increased induction of γ-H2AX measured could be due to the cytotoxicity that occurred at the same concentrations which can result in DNA damage resulting in an increased induction of γ-H2AX. This suggests potential false-positive confounders limiting the use of the ICW-γ-H2AX assay, in the form as applied in chapter 5, for evaluation of the genotoxicity of NPs. Additionally, the potential of the AgNPs to induce ROS production, as a potential underlying mechanism of induction of the cytotoxicity and/or DNA-DSBs, was assessed in HepG2 cells. No increase in ROS levels was measured upon exposure of the cells to the AgNPs for 4 or 24 hr and in the absence of cells, while an increase in ROS levels was detected upon AgNO3 exposure of the cells and in the absence of cells.

In conclusion, the surface chemistry of AgNPs has a significant influence on their cytotoxic effects and the accompanying induction of γ-H2AX levels in HepG2 cells. The aged Ag2S NPs were biologically less active in inducing both cytotoxicity and γ-H2AX levels, as these effects were absent in the dose range tested. The absence of cellular ROS generation upon exposure to all AgNPs indicates that the observed effects were not ROS-mediated. Additional tests, to rule out apoptotic mediated false-positive signals, need to be combined with the ICW-γ-H2AX assay, to render this interesting assay into a robust screening method for the potential genotoxicity of NPs.

Chapter 6 of the present thesis included a general discussion of the results of the previous chapters and highlights on future perspectives for research in the field of in vitro nanotoxicology.

Overall, the work presented in this thesis illustrated the role of surface chemistry and the status of the NPs (pristine or aged and/or digested) on the toxicological behaviour of NPs. Besides, the combination of different in vitro models with high-end analytical techniques was shown to;  1) provide in-depth understanding of the biological behaviour of NPs, 2) assure the value of the alternative in vitro models as a testing strategy for potential hazards that could be induced by NPs and 3) assist in setting priorities for in vivo testing and contributing to reduction, refinement and replacement (3Rs) of animal testing required for the safety evaluation of NPs.