Translocation of positively and negatively charged polystyrene nanoparticles in an in vitro placental model

AbstractTo obtain insight in translocation of nanoparticles across the placental barrier, translocation was studied for one positively and two negatively charged polystyrene nanoparticles (PS-NPs) of similar size in an in vitro model. The model consisted of BeWo b30 cells, derived from a human choriocarcinoma grown on a transwell insert forming a cell layer that separates an apical from a basolateral compartment. PS-NPs were characterized with respect to size, surface charge, morphology and protein corona. Translocation of PS-NPs was not related to PS-NP charge. Two PS-NPs were translocated across the BeWo transwell model to a lower extent than amoxicillin, a model compound known to be translocated over the placental barrier to only a limited extent, whereas one PS-NP showed a slightly higher translocation. Studies on the effect of transporter inhibitors on the translocation of the PS-NPs indicated that their translocation was not mediated by known transporters and mainly dependent on passive diffusion. It is concluded that the BeWo b30 model can be used as an efficient method to get an initial qualitative impression about the capacity of NPs to translocate across the placental barrier and set priorities in further in vivo studies on translocation of NPs to the fetus

In vitro assays for hazard identification of nanoparticles

The production of nanoparticles (NPs) has increased in the last decades and the number of products in which NPs are being incorporated is still growing. The rapid increase of nanotechnology has several benefits for society, yet there is an increasing concern that exposure to NPs may result in significant adverse health effects. Since NPs are incorporated in a variety of consumer products, it is likely that the general population will be exposed to NPs. It would be desirable that the safety and risk assessment of NPs could be largely based on studies using in vitro models instead of in vivo models as this would reduce the use of test animals, costs and time required to test the large numbers of NPs. The aim of the present thesis was to investigate the potential of in vitro testing strategies to detect hazards of NPs, focusing on toxicokinetic as well as toxicodynamic endpoints. Toxicokinetic studies focused on translocation of NPs in in vitro models of the placental barrier, while toxicodynamic studies were directed at two endpoints that represent potential hazards of NPs that have not yet been well characterized including: developmental toxicity and immunotoxicity. In the present thesis different types of NPs were used. Polystyrene nanoparticles (PS-NPs) were selected because of their commercial availability, with high quality and a wide variety of available physicochemical properties like surface charge, and fluorescent labeling enabling easy detection in toxicokinetic (translocation) studies. Several metal (oxide) NPs were selected as well, of which some are possible constituents of food additives like TiO2, Fe2O3, SiO2 and Ag. Other metal oxide NPs that were selected were Mn2O3, CuO, Cr2O3, CoO and NiO to which we may be exposed via products like paints, catalysts, construction materials, coatings and batteries. Placental translocation of NPs was studied as an important toxicokinetic aspect, since part of the toxicodynamic studies of the present thesis were directed at developmental toxicity testing of NPs. In order to obtain insight in toxicity and translocation of NPs across the placental barrier, cytotoxicity and translocation was studied for one positively and two negatively charged PS-NPs of 50 nm in an in vitro model of the placenta. In this study it appeared that in spite of similar size, surface charge and type of proteins in the protein corona, the differently charged NPs displayed a remarkable difference in cytotoxicity, with only the PS-NPs with an original positive charge inducing cytotoxicity. Translocation of PS-NPs appeared not to be related to PS-NP charge alone. A remarkable difference in translocation was found between the two 50 nm negatively charged PS-NPs that were obtained from different manufacturers. Since none of the characterized parameters, including size, surface charge and protein corona revealed remarkable differences between the two negatively charged NPs, the difference may originate from the chemical groups on the surface of the NPs generating the negative charge. The general conclusion from this study was that the in vitro BeWo b30 model can be used as a fast method to get an initial qualitative impression about the capacity of NPs to translocate across the placental barrier and to set priorities for further in vivo studies on translocation of NPs to the fetus. The same PS-NPs as tested for placental translocation were investigated whether they are able to cause in vitro developmental toxicity in the ES-D3 cell differentiation assay of the embryonic stem cell test (EST) focusing also on the effect that charge may have. The study showed that the two negatively charged PS-NPs did not show any effect in the ES-D3 cell differentiation assay up to the highest concentration tested while the positively charged PS-NP showed a concentration-dependent inhibition of ES-D3 cell differentiation. However, effect concentrations in the ES-D3 cell differentiation assay were close to cytotoxic concentrations, which indicated that the inhibition of the ES-D3 cell differentiation may be due to cytotoxic effects of the positively charged PS-NPs. This indicated that the inhibition of the ES-D3 cell differentiation by the positively charged PS-NPs may be caused by non-specific effects. Although the experiments on placental translocation of the present thesis showed that positively charged PS-NPs are more toxic than negatively charged PS-NPs, it appeared that this may not be generalizable to other NPs. This follows from the fact that in SiO2, Ag and TiO2 NPs that were reported in other studies to inhibit ES-D3 cell differentiation were negatively charged, while the negatively charged PS-NPs of the present study did not affect ES-D3 cell differentiation. Although the limited data available indicate that charge, size and coating of NPs may be important characteristics that determine the developmental toxicity potential of NPs, more (systematic) studies are needed to assess how physicochemical characteristics of NPs relate to their developmental toxicity. This information may help to prioritize NPs for in vitro and in vivo developmental toxicity testing. In addition, toxic effects of a series of metal (oxide) NPs were tested in macrophage RAW264.7 cells in order to obtain insight in effect of these NPs on cells of the innate immune response. In these macrophage RAW264.7 cells the effects of the metal (oxide) NPs were characterized on cell viability, TNF-α production and mitochondria-related parameters like production of reactive oxygen species (ROS), mitochondrial permeability transition pore (MPTP) opening, and intracellular ATP levels. Altogether, results obtained showed no or limited effects of the NP formulations of metal (oxide) food additives on cell viability, ROS production, MPTP opening, ATP levels and TNF-α production in RAW264.7 macrophages. Effects were only observed at high concentrations that may not be physiologically relevant, indicating that related adverse effects upon exposure to the respective NPs in vivo may be limited. Taken together, the present thesis provided further evidence of the influence of physicochemical properties of NPs in driving toxicity in in vitro models. However, the determination of the fate and toxicity of NPs using in vitro or in vivo models is a challenge that needs further evaluation. A combination of several factors likely play a role in determining the outcome of exposure including factors like NP core material and presence and type of coating agents resulting in various physicochemical properties (size, charge, etc.). This appears to hamper conclusive evaluation of the role of physicochemical characteristics of NPs in their potential hazards and risks so far. The results obtained do show however that in vitro assays can detect differences in potential hazards posed by NPs. Therefore it is concluded that the results of the work presented in this thesis will contribute to the further development and use of non-animal based testing strategies for safety testing of NPs providing insight into selected potential hazards of the tested NPs

Effects of systematic variation in size and Ssurface coating of silver nanoparticles on their in vitro toxicity to macrophage RAW 264.7 cells

In literature, varying and sometimes conflicting effects of physicochemical properties of nanoparticles (NPs) are reported on their uptake and effects in organisms. To address this, small- and medium-sized (20 and 50 nm) silver nanoparticles (AgNPs) with specified different surface coating/charges were synthesized and used to systematically assess effects of NP-properties on their uptake and effects in vitro. Silver nanoparticles were fully characterized for charge and size distribution in both water and test media. Macrophage cells (RAW 264.7) were exposed to these AgNPs at different concentrations (0-200 µg/ml). Uptake dynamics, cell viability, induction of tumor necrosis factor (TNF)-α, ATP production, and reactive oxygen species (ROS) generation were assessed. Microscopic imaging of living exposed cells showed rapid uptake and subcellular cytoplasmic accumulation of AgNPs. Exposure to the tested AgNPs resulted in reduced overall viability. Influence of both size and surface coating (charge) was demonstrated, with the 20-nm-sized AgNPs and bovine serum albumin (BSA)-coated (negatively charged) AgNPs being slightly more toxic. On specific mechanisms of toxicity (TNF-α and ROS production) however, the AgNPs differed to a larger extent. The highest induction of TNF-α was found in cells exposed to the negatively charged AgNP_BSA, both sizes (80× higher than control). Reactive oxygen species induction was only significant with the 20 nm positively charged AgNP_Chit.This work was financially supported by NanoNextNL, a micro- and nanotechnology consortium of the Government of The Netherlands and 130 partners; funding was also received from Managing Risks of Nanoparticles, MARINA (EU-FP7, contract CP-FP 263215); and the Strategic Research Funds titled Novel technologies by the Ministry of Economic Affairs of The Netherlands. Synthesis and characterization of the AgNPs used in this study received support from the QualityNano Project (http://www.qualitynano.eu/) financed by the European Community Research Infrastructures under the FP7 Capacities Program (Grant Number INFRA-2010-262163).Peer reviewe

Progress and future of in vitro models to study translocation of nanoparticles

The increasing use of nanoparticles in products likely results in increased exposure of both workers and consumers. Because of their small size, there are concerns that nanoparticles unintentionally cross the barriers of the human body. Several in vivo rodent studies show that, dependent on the exposure route, time, and concentration, and their characteristics, nanoparticles can cross the lung, gut, skin, and placental barrier. This review aims to evaluate the performance of in vitro models that mimic the barriers of the human body, with a focus on the lung, gut, skin, and placental barrier. For these barriers, in vitro models of varying complexity are available, ranging from single-cell-type monolayer to multi-cell (3D) models. Only a few studies are available that allow comparison of the in vitro translocation to in vivo data. This situation could change since the availability of analytical detection techniques is no longer a limiting factor for this comparison. We conclude that to further develop in vitro models to be used in risk assessment, the current strategy to improve the models to more closely mimic the human situation by using co-cultures of different cell types and microfluidic approaches to better control the tissue microenvironments are essential. At the current state of the art, the in vitro models do not yet allow prediction of absolute transfer rates but they do support the definition of relative transfer rates and can thus help to reduce animal testing by setting priorities for subsequent in vivo testin

Induction of Peroxisome Proliferator-Activated Receptor γ (PPARγ)-Mediated Gene Expression by Tomato (<i>Solanum lycopersicum</i> L.) Extracts

Since beneficial effects related to tomato consumption partially overlap with those related to peroxisome proliferator-activated receptor γ (PPARγ) activation, our aim was to test extracts of tomato fruits and tomato components, including polyphenols and isoprenoids, for their capacity to activate PPARγ using the PPARγ2 CALUX reporter cell line. Thirty tomato compounds were tested; seven carotenoids and three polyphenols induced PPARγ2-mediated luciferase expression. Two extracts of tomato, one containing deglycosylated phenolic compounds and one containing isoprenoids, also induced PPARγ2-mediated expression at physiologically relevant concentrations. Furthermore, enzymatically hydrolyzed extracts of seven tomato varieties all induced PPARγ-mediated expression, with a 1.6-fold difference between the least potent and the most potent variety. The two most potent varieties had high flavonoid content, while the two least potent varieties had low flavonoid content. These data indicate that extracts of tomato are able to induce PPARγ-mediated gene expression <i>in vitro</i> and that some tomato varieties are more potent than others