Novel electrospun chitosan/PEO membranes for more predictive nanoparticle transport studies at biological barriers.

The design of safe and effective nanoparticles (NPs) for commercial and medical applications requires a profound understanding of NP translocation and effects at biological barriers. To gain mechanistic insights, physiologically relevant and accurate human in vitro biobarrier models are indispensable. However, current transfer models largely rely on artificial porous polymer membranes for the cultivation of cells, which do not provide a close mimic of the natural basal membrane and intrinsically provide limited permeability for NPs. In this study, electrospinning is exploited to develop thin chitosan/polyethylene oxide (PEO) membranes with a high porosity and nanofibrous morphology for more predictive NP transfer studies. The nanofiber membranes allow the cultivation of a tight and functional placental monolayer (BeWo trophoblasts). Translocation studies with differently sized molecules and NPs (Na-fluorescein; 40 kDa FITC-Dextran; 25 nm PMMA; 70, 180 and 520 nm polystyrene NPs) across empty and cell containing membranes reveal a considerably enhanced permeability compared to commercial microporous membranes. Importantly, the transfer data of NPs is highly similar to data from ex vivo perfusion studies of intact human placental tissue. Therefore, the newly developed membranes may decisively contribute to establish physiologically relevant in vitro biobarrier transfer models with superior permeability for a wide range of molecules and particles.

Label-free detection of uptake, accumulation, and translocation of diesel exhaust particles in ex vivo perfused human placenta.

BACKGROUND: Pregnant women and developing fetuses comprise a particularly vulnerable population as multiple studies have shown associations between prenatal air pollution exposure and adverse pregnancy outcomes. However, the mechanisms underlying the observed developmental toxicity are mostly unknown, in particular, if pollution particles can cross the human placenta to reach the fetal circulation. RESULTS: Here, we investigated the accumulation and translocation of diesel exhaust particles (DEPs), as a model particle for combustion-derived pollution, in human perfused placentae using label-free detection by femtosecond pulsed laser illumination. The results do not reveal a significant particle transfer across term placentae within 6 h of perfusion. However, DEPs accumulate in placental tissue, especially in the syncytiotrophoblast layer that mediates a wealth of essential functions to support and maintain a successful pregnancy. Furthermore, DEPs are found in placental macrophages and fetal endothelial cells, showing that some particles can overcome the syncytiotrophoblasts to reach the fetal capillaries. Few particles are also observed inside fetal microvessels. CONCLUSIONS: Overall, we show that DEPs accumulate in key cell types of the placental tissue and can cross the human placenta, although in limited amounts. These findings are crucial for risk assessment and protection of pregnant women and highlight the urgent need for further research on the direct and indirect placenta-mediated developmental toxicity of ambient particulates.

Nano-analytical characterization of endogenous minerals in healthy placental tissue: mineral distribution, composition and ultrastructure.

Despite its crucial role, the placenta is the least understood human organ. Recent clinical studies indicate a direct association between placental calcification and maternal and offspring health. This study reveals distinct characteristics of minerals formed during gestational ageing using cutting-edge nano-analytical characterization and paves the way for investigations focused on the identification of potential markers for disease risks in a clinical setting based on atypical placental mineral fingerprints.

Transfer and Metabolism of the Xenoestrogen Zearalenone in Human Perfused Placenta.

BACKGROUND: Pregnancy is a sensitive condition during which adverse environmental exposures should be monitored thoroughly and minimized whenever possible. In particular, the hormone balance during gestation is delicate, and disturbance may cause acute or chronic long-term health effects. A potential endocrine disruption may be provoked by in utero exposure to xenoestrogens mimicking endogenous estrogens. The mycoestrogen zearalenone (ZEN), a toxic fungal secondary metabolite and mycotoxin found frequently in food and feed, constitutes a prominent example. OBJECTIVES: We performed a comprehensive assessment of the transfer as well as phase I and phase II metabolism of ZEN at the human placental barrier. METHODS: Human placentas were perfused with 1µM (318µg/L) ZEN for 6 h. Samples from the maternal and fetal compartment, placental tissue, and fetal plasma were analyzed by a highly sensitive UHPLC-MS/MS assay to detect ZEN as well as nine key metabolites (α-zearalenol, ß-zearalenol, zearalanone, α-zearalanol, ß-zearalanol, ZEN-14-glucuronide, α-zearalenol-14-glucuronide, ß-zearalenol-14-glucuronide, ZEN-14-sulfate). RESULTS: The model revealed a fast maternofetal transfer of ZEN across the human placental barrier. We also unraveled phase I and phase II metabolism of the parent toxin ZEN into the approximately 70-times more estrogenic α-zearalenol and the less active ZEN-14-sulfate conjugate, which are effectively released into the maternal and fetal circulation in considerable amounts. CONCLUSIONS: Our findings suggest that exposure to ZEN (such as through consumption of ZEN-contaminated cereal-based products) during pregnancy may result in in utero exposure of the fetus, not only to ZEN but also some of its highly estrogenically active metabolites. In the light of the known affinity of ZEN and potentially co-occurring xenoestrogens to the estrogen receptor, and our results demonstrating placental transfer of ZEN and its metabolites in an ex vivo model, we recommend further research and more comprehensive assessment of gestational exposures in women. https://doi.org/10.1289/EHP4860.

Investigating the accumulation and translocation of titanium dioxide nanoparticles with different surface modifications in static and dynamic human placental transfer models.

Titanium dioxide nanoparticles (TiO2 NPs) are widely incorporated in various consumer products such as cosmetics and food. Despite known human exposure, the potential risks of TiO2 NPs during pregnancy are not fully understood, but several studies in mice elucidated toxic effects on fetal development. It has also been shown that modifying NPs with positive or negative surface charge alters cellular uptake and abolishes fetotoxicity of silicon dioxide (SiO2) NPs in mice. Here, we investigated accumulation and translocation of positively charged TiO2-NH2 and negatively charged TiO2-COOH NPs at the placental barrier, to clarify whether surface charge provides a means to control TiO2 NP distribution at the placental barrier. To ensure outcome relevant for humans, the recently developed in vitro human placental co-culture model and the gold standard amongst placental translocation models - the ex vivo perfusion of human term placental tissue - were employed during this study. Sector field-ICP-MS analysis of maternal and fetal supernatants as well as placental cells/tissues revealed a substantial accumulation of both TiO2 NP types while no considerable placental translocation was apparent in both models. Characterization of agglomeration behavior demonstrated a strong and fast agglomeration of TiO2-NH2 and TiO2-COOH NPs in the different culture media. Overall, our results indicate that surface charge is not a key factor to steer placental uptake and transfer of TiO2. Moreover, the negligible placental transfer but high accumulation of TiO2 NPs in placental tissue suggests that potential effects on fetal health may occur indirectly, which calls for further studies elucidating the impact of TiO2 NPs on placental tissue functionality and signaling.

Gold nanoparticle distribution in advanced in vitro and ex vivo human placental barrier models.

BACKGROUND: Gold nanoparticles (AuNPs) are promising candidates to design the next generation NP-based drug formulations specifically treating maternal, fetal or placental complications with reduced side effects. Profound knowledge on AuNP distribution and effects at the human placental barrier in dependence on the particle properties and surface modifications, however, is currently lacking. Moreover, the predictive value of human placental transfer models for NP translocation studies is not yet clearly understood, in particular with regards to differences between static and dynamic exposures. To understand if small (3-4 nm) AuNPs with different surface modifications (PEGylated versus carboxylated) are taken up and cross the human placental barrier, we performed translocation studies in a static human in vitro co-culture placenta model and the dynamic human ex vivo placental perfusion model. The samples were analysed using ICP-MS, laser ablation-ICP-MS and TEM analysis for sensitive, label-free detection of AuNPs. RESULTS: After 24 h of exposure, both AuNP types crossed the human placental barrier in vitro, although in low amounts. Even though cellular uptake was higher for carboxylated AuNPs, translocation was slightly increased for PEGylated AuNPs. After 6 h of perfusion, only PEGylated AuNPs were observed in the fetal circulation and tissue accumulation was similar for both AuNP types. While PEGylated AuNPs were highly stable in the biological media and provided consistent results among the two placenta models, carboxylated AuNPs agglomerated and adhered to the perfusion device, resulting in different cellular doses under static and dynamic exposure conditions. CONCLUSIONS: Gold nanoparticles cross the human placental barrier in limited amounts and accumulate in placental tissue, depending on their size- and/or surface modification. However, it is challenging to identify the contribution of individual characteristics since they often affect colloidal particle stability, resulting in different biological interaction in particular under static versus dynamic conditions. This study highlights that human ex vivo and in vitro placenta models can provide valuable mechanistic insights on NP uptake and translocation if accounting for NP stability and non-specific interactions with the test system.

Translocation of silver nanoparticles in the ex vivo human placenta perfusion model characterized by single particle ICP-MS.

With the extensive use of silver nanoparticles (AgNPs) in various consumer products their potential toxicity is of great concern especially for highly sensitive population groups such as pregnant women and even the developing fetus. To understand if AgNPs are taken up and cross the human placenta, we studied their translocation and accumulation in the human ex vivo placenta perfusion model by single particle ICP-MS (spICP-MS). The impact of different surface modifications on placental transfer was assessed by AgNPs with two different modifications: polyethylene glycol (AgPEG NPs) and sodium carboxylate (AgCOONa NPs). AgNPs and ionic Ag were detected in the fetal circulation in low but not negligible amounts. Slightly higher Ag translocation across the placental barrier for perfusion with AgPEG NPs and higher AgNP accumulation in placental tissue for perfusion with AgCOONa NPs were observed. Since these AgNPs are soluble in water, we tried to distinguish between the translocation of dissolved and particulate Ag. Perfusion with AgNO3 revealed the formation of Ag containing NPs in both circulations over time, of which the amount and their size in the fetal circulation were comparable to those from perfusion experiments with both AgNP types. Although we were not able to clarify whether intact AgNPs and/or Ag precipitates from dissolved Ag cross the placental barrier, our study highlights that uptake of Ag ions and/or dissolution of AgNPs in the tissue followed by re-precipitation in the fetal circulation needs to be considered as an important pathway in studies of AgNP translocation across biological barriers.

A 3D co-culture microtissue model of the human placenta for nanotoxicity assessment.

There is increasing evidence that certain nanoparticles (NPs) can overcome the placental barrier, raising concerns on potential adverse effects on the growing fetus. But even in the absence of placental transfer, NPs may pose a risk to proper fetal development if they interfere with the viability and functionality of the placental tissue. The effects of NPs on the human placenta are not well studied or understood, and predictive in vitro placenta models to achieve mechanistic insights on NP-placenta interactions are essentially lacking. Using the scaffold-free hanging drop technology, we developed a well-organized and highly reproducible 3D co-culture microtissue (MT) model consisting of a core of placental fibroblasts surrounded by a trophoblast cell layer, which resembles the structure of the in vivo placental tissue. We could show that secretion levels of human chorionic gonadotropin (hCG) were significantly higher in 3D than in 2D cell cultures, which indicates an enhanced differentiation of trophoblasts grown on 3D MTs. NP toxicity assessment revealed that cadmium telluride (CdTe) and copper oxide (CuO) NPs but not titanium dioxide (TiO2) NPs decreased MT viability and reduced the release of hCG. NP acute toxicity was significantly reduced in 3D co-culture MTs compared to 2D monocultures. Taken together, 3D placental MTs provide a new and promising model for the fast generation of tissue-relevant acute NP toxicity data, which are indispensable for the safe development of NPs for industrial, commercial and medical applications.

Bidirectional Transfer Study of Polystyrene Nanoparticles across the Placental Barrier in an ex Vivo Human Placental Perfusion Model.

BACKGROUND: Nanoparticle exposure in utero might not be a major concern yet, but it could become more important with the increasing application of nanomaterials in consumer and medical products. Several epidemiologic and in vitro studies have shown that nanoparticles can have potential toxic effects. However, nanoparticles also offer the opportunity to develop new therapeutic strategies to treat specifically either the pregnant mother or the fetus. Previous studies mainly addressed whether nanoparticles are able to cross the placental barrier. However, the transport mechanisms underlying nanoparticle translocation across the placenta are still unknown. OBJECTIVES: In this study we examined which transport mechanisms underlie the placental transfer of nanoparticles. METHODS: We used the ex vivo human placental perfusion model to analyze the bidirectional transfer of plain and carboxylate modified polystyrene particles in a size range between 50 and 300 nm. RESULTS: We observed that the transport of polystyrene particles in the fetal to maternal direction was significantly higher than for the maternal to fetal direction. Regardless of their ability to cross the placental barrier and the direction of perfusion, all polystyrene particles accumulated in the syncytiotrophoblast of the placental tissue. CONCLUSIONS: Our results indicate that the syncytiotrophoblast is the key player in regulating nanoparticle transport across the human placenta. The main mechanism underlying this translocation is not based on passive diffusion, but is likely to involve an active, energy-dependent transport pathway. These findings will be important for reproductive toxicology as well as for pharmaceutical engineering of new drug carriers.

Transfer studies of polystyrene nanoparticles in the ex vivo human placenta perfusion model: key sources of artifacts.

Nanotechnology is a rapidly expanding and highly promising new technology with many different fields of application. Consequently, the investigation of engineered nanoparticles in biological systems is steadily increasing. Questions about the safety of such engineered nanoparticles are very important and the most critical subject with regard to the penetration of biological barriers allowing particle distribution throughout the human body. Such translocation studies are technically challenging and many issues have to be considered to obtain meaningful and comparable results. Here we report on the transfer of polystyrene nanoparticles across the human placenta using an ex vivo human placenta perfusion model. We provide an overview of several challenges that can potentially occur in any translocation study in relation to particle size distribution, functionalization and stability of labels. In conclusion, a careful assessment of nanoparticle properties in a physiologically relevant milieu is as challenging and important as the actual study of nanoparticle-cell interactions itself.

Determination of the transport rate of xenobiotics and nanomaterials across the placenta using the ex vivo human placental perfusion model.

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ (1). Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 (2) and continuously modified by Schneider et al. in 1972 (3), can serve as an excellent model to study the transfer of xenobiotics or particles. Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results. The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

A comparison of acute and long-term effects of industrial multiwalled carbon nanotubes on human lung and immune cells in vitro.

The close resemblance of carbon nanotubes to asbestos fibers regarding their high aspect ratio, biopersistence and reactivity increases public concerns on the widespread use of these materials. The purpose of this study was not only to address the acute adverse effects of industrially produced multiwalled carbon nanotubes (MWCNTs) on human lung and immune cells in vitro but also to further understand if their accumulation and biopersistence leads to long-term consequences or induces adaptive changes in these cells. In contrast to asbestos fibers, pristine MWCNTs did not induce overt cell death in A549 lung epithelial cells and Jurkat T lymphocytes after acute exposure to high doses of this material (up to 30 µg/ml). Nevertheless, very high levels of reactive oxygen species (ROS) and decreased metabolic activity were observed which might affect long-term viability of these cells. However, the continuous presence of low amounts of MWCNTs (0.5 µg/ml) for 6 months did not have major adverse long-term effects although large amounts of nanotubes accumulated at least in A549 cells. Moreover, MWCNTs did not appear to induce adaptive mechanisms against particle stress in long-term treated A549 cells. Our study demonstrates that despite the high potential for ROS formation, pristine MWCNTs can accumulate and persist within cells without having major long-term consequences or inducing adaptive mechanisms.

Barrier capacity of human placenta for nanosized materials.

BACKGROUND: Humans have been exposed to fine and ultrafine particles throughout their history. Since the Industrial Revolution, sources, doses, and types of nanoparticles have changed dramatically. In the last decade, the rapidly developing field of nanotechnology has led to an increase of engineered nanoparticles with novel physical and chemical properties. Regardless of whether this exposure is unintended or not, a careful assessment of possible adverse effects is needed. A large number of projects have been carried out to assess the consequences of combustion-derived or engineered nanoparticle exposure on human health. In recent years there has been a growing concern about the possible health influence of exposure to air pollutants during pregnancy, hence an implicit concern about potential risk for nanoparticle exposure in utero. Previous work has not addressed the question of whether nanoparticles may cross the placenta. OBJECTIVE: In this study we investigated whether particles can cross the placental barrier and affect the fetus. METHODS: We used the ex vivo human placental perfusion model to investigate whether nanoparticles can cross this barrier and whether this process is size dependent. Fluorescently labeled polystyrene beads with diameters of 50, 80, 240, and 500 nm were chosen as model particles. RESULTS: We showed that fluorescent polystyrene particles with diameter up to 240 nm were taken up by the placenta and were able to cross the placental barrier without affecting the viability of the placental explant. CONCLUSIONS: The findings suggest that nanomaterials have the potential for transplacental transfer and underscore the need for further nanotoxicologic studies on this important organ system.

Single walled carbon nanotubes (SWCNT) affect cell physiology and cell architecture.

Single walled carbon nanotubes (SWCNT) find their way in various industrial applications. Due to the expected increased production of various carbon nanotubes and nanoparticle containing products, exposure to engineered nanoparticles will also increase dramatically in parallel. In this study the effects of SWCNT raw material and purified SWCNT (SWCNT bundles) on cell behaviour of mesothelioma cells (MSTO-211H) and on epithelial cells (A549) had been investigated. The effect on cell behaviour (cell proliferation, cell activity, cytoskeleton organization, apoptosis and cell adhesion) were dependent on cell type, SWCNT quality (purified or not) and SWCNT concentration.

Exposure of engineered nanoparticles to human lung epithelial cells: influence of chemical composition and catalytic activity on oxidative stress.

The chemical and catalytic activity of nanoparticles has strongly contributed to the current tremendous interest in engineered nanomaterials and often serves as a guiding principle for the design of functional materials. Since it has most recently become evident that such active materials can enter into cells or organisms, the present study investigates the level of intracellular oxidations after exposure to iron-, cobalt-, manganese-, and titania-containing silica nanoparticles and the corresponding pure oxides in vitro. The resulting oxidative stress was quantitatively measured as the release of reactive oxygen species (ROS). The use of thoroughly characterized nanoparticles of the same morphology, comparable size, shape, and degree of agglomeration allowed separation of physical (rate of particle uptake, agglomeration, sedimentation) and chemical effects (oxidations). Three sets of control experiments elucidated the role of nanoparticles as carriers for heavy metal uptake and excluded a potential interference of the biological assay with the nanomaterial. The present results indicate that the particles could efficiently enter the cells by a Trojan-horse type mechanism which provoked an up to eight times higher oxidative stress in the case of cobalt or manganese if compared to reference cultures exposed to aqueous solutions of the same metals. A systematic investigation on iron-containing nanoparticles as used in industrial fine chemical synthesis demonstrated that the presence of catalytic activity could strongly alter the damaging action of a nanomaterial. This indicates that a proactive development of nanomaterials and their risk assessment should consider chemical and catalytic properties of nanomaterials beyond a mere focus on physical properties such as size, shape, and degree of agglomeration.

The degree and kind of agglomeration affect carbon nanotube cytotoxicity.

The urgent need for toxicological studies on carbon nanotubes (CNTs) has arisen from the rapidly emerging applications of CNTs well beyond material science and engineering. In order to provide a basis for comparison to existing epidemiological data, we have investigated CNTs at various degrees of agglomeration using an in vitro cytotoxicity study with human MSTO-211H cells. Non-cytotoxic polyoxyethylene sorbitan monooleate was found to well-disperse CNT. In the present study, the cytotoxic effects of well-dispersed CNT were compared with that of conventionally purified rope-like agglomerated CNTs and asbestos as a reference. While suspended CNT-bundles were less cytotoxic than asbestos, rope-like agglomerates induced more pronounced cytotoxic effects than asbestos fibres at the same concentrations. The study underlines the need for thorough materials characterization prior to toxicological studies and corroborates the role of agglomeration in the cytotoxic effect of nanomaterials.

In vitro cytotoxicity of oxide nanoparticles: comparison to asbestos, silica, and the effect of particle solubility.

Early indicators for nanoparticle-derived adverse health effects should provide a relative measure for cytotoxicity of nanomaterials in comparison to existing toxicological data. We have therefore evaluated a human mesothelioma and a rodent fibroblast cell line for in vitro cytotoxicity tests using seven industrially important nanoparticles. Their response in terms of metabolic activity and cell proliferation of cultures exposed to 0-30 ppm nanoparticles (microg g(-1)) was compared to the effects of nontoxic amorphous silica and toxic crocidolite asbestos. Solubility was found to strongly influence the cytotoxic response. The results further revealed a nanoparticle-specific cytotoxic mechanism for uncoated iron oxide and partial detoxification or recovery after treatment with zirconia, ceria, or titania. While in vitro experiments may never replace in vivo studies, the relatively simple cytotoxic tests provide a readily available pre-screening method.