Gene expression profiling of human macrophages after graphene oxide and graphene nanoplatelets treatment reveals particle-specific regulation of pathways.

Graphene and its derivatives are attractive materials envisaged to enable a wealth of novel applications in many fields including energy, electronics, composite materials or health. A comprehensive understanding of the potential adverse effects of graphene-related materials (GRM) in humans is a prerequisite to the safe use of these promising materials. Here, we exploited gene expression profiling to identify transcriptional responses and toxicity pathways induced by graphene oxide (GO) and graphene nanoplatelets (GNP) in human macrophages. Primary human monocyte-derived macrophages (MDM) and a human macrophage cell line, i.e. differentiated THP-1 cells, were exposed to 5 or 20 µg/mL GO and GNP for 6 and 24 h to capture early and more persistent acute responses at realistic or slightly overdose concentrations. GO and GNP induced time-, dose- and macrophage type-specific differential expression of a substantial number of genes with some overlap between the two GRM types (up to 384 genes (9.6%) or 447 genes (20.4%) in THP-1 or MDM, respectively) but also a high number of genes exclusively deregulated from each material type. Furthermore, GRM responses on gene expression were highly different from those induced by inflammogenic material crystalline quartz (maximum of 64 (2.3%) or 318 (11.3%) common genes for MDM treated with 20 µg/mL GO and GNP, respectively). Further bioinformatics analysis revealed that GNP predominantly activated genes controlling inflammatory and apoptotic pathways whereas GO showed only limited inflammatory responses. Interestingly, both GRM affected the expression of genes related to antigen processing and presentation and in addition, GO activated pathways of neutrophil activation, degranulation and immunity in MDM. Overall, this study provides an extensive resource of potential toxicity mechanisms for future safety assessment of GRM in more advanced model systems to verify if the observed changes in gene expression in human macrophages could lead to long-term consequences on human health.

Catching Them Early: Framework Parameters and Progress for Prenatal and Childhood Application of Advanced Therapies.

Advanced therapy medicinal products (ATMPs) are medicines for human use based on genes, cells or tissue engineering. After clear successes in adults, the nascent technology now sees increasing pediatric application. For many still untreatable disorders with pre- or perinatal onset, timely intervention is simply indispensable; thus, prenatal and pediatric applications of ATMPs hold great promise for curative treatments. Moreover, for most inherited disorders, early ATMP application may substantially improve efficiency, economy and accessibility compared with application in adults. Vindicating this notion, initial data for cell-based ATMPs show better cell yields, success rates and corrections of disease parameters for younger patients, in addition to reduced overall cell and vector requirements, illustrating that early application may resolve key obstacles to the widespread application of ATMPs for inherited disorders. Here, we provide a selective review of the latest ATMP developments for prenatal, perinatal and pediatric use, with special emphasis on its comparison with ATMPs for adults. Taken together, we provide a perspective on the enormous potential and key framework parameters of clinical prenatal and pediatric ATMP application.

Differences and Interactions in Placental Manganese and Iron Transfer across an In Vitro Model of Human Villous Trophoblasts.

Manganese (Mn) as well as iron (Fe) are essential trace elements (TE) important for the maintenance of physiological functions including fetal development. However, in the case of Mn, evidence suggests that excess levels of intrauterine Mn are associated with adverse pregnancy outcomes. Although Mn is known to cross the placenta, the fundamentals of Mn transfer kinetics and mechanisms are largely unknown. Moreover, exposure to combinations of TEs should be considered in mechanistic transfer studies, in particular for TEs expected to share similar transfer pathways. Here, we performed a mechanistic in vitro study on the placental transfer of Mn across a BeWo b30 trophoblast layer. Our data revealed distinct differences in the placental transfer of Mn and Fe. While placental permeability to Fe showed a clear inverse dose-dependency, Mn transfer was largely independent of the applied doses. Concurrent exposure of Mn and Fe revealed transfer interactions of Fe and Mn, indicating that they share common transfer mechanisms. In general, mRNA and protein expression of discussed transporters like DMT1, TfR, or FPN were only marginally altered in BeWo cells despite the different exposure scenarios highlighting that Mn transfer across the trophoblast layer likely involves a combination of active and passive transport processes.

Release of graphene-related materials from epoxy-based composites: characterization, quantification and hazard assessment in vitro.

Due to their mechanical strength, thermal stability and electrical conductivity, graphene-related materials (GRMs) have been extensively explored for various applications. Moreover, GRMs have been studied and applied as fillers in polymer composite manufacturing to enhance the polymer performance. With the foreseen growth in GRM production, occupational and consumer exposure is inevitable, thus raising concerns for potential health risks. Therefore, this study aims (1) to characterize aerosol particles released after mechanical abrasion on GRM-reinforced epoxy composites, (2) to quantify the amounts of protruding and free-standing GRMs in the abraded particles and (3) to assess the potential effects of the pristine GRMs as well as the abraded particles on human macrophages differentiated from the THP-1 cell line in vitro. GRMs used in this study included graphene nanoplatelets (GNPs), graphene oxide (GO), and reduced graphene oxide (rGO). All types of pristine GRMs tested induced a dose-dependent increase in reactive oxygen species formation, but a decrease in cell viability was only detected for large GNPs at high concentrations (20 and 40 µg mL-1). The particle modes measured using a scanning mobility particle sizer (SMPS) were 300-400 nm and using an aerodynamic particle sizer (APS) were between 2-3 µm, indicating the release of respirable particles. A significant fraction (51% to 92%) of the GRMs embedded in the epoxy composites was released in the form of free-standing or protruding GRMs in the abraded particles. The abraded particles did not induce any acute cytotoxic effects.

Nanostructure generation during milk digestion in presence of a cell culture model simulating the small intestine.

HYPOTHESIS: The development of advanced oral delivery systems for bioactive compounds requires the fundamental understanding of the digestion process within the gastrointestinal tract. Towards this goal, dynamic invitro digestion models, capable of characterising the molecular as well as colloidal aspects of food, together with their biological interactions with relevant invitro cell culture models, are essential. EXPERIMENTS: In this study, we demonstrate a novel digestion model that combines flow-through time resolved small angle X-ray scattering (SAXS) with an invitro Caco-2/HT-29 cell co-culture model that also contained a mucus layer. This set-up allows the dynamic insitu characterisation of colloidal structures and their transport across a viable intestinal cell layer during simulated digestion. FINDINGS: An integrated online SAXS - invitro cell co-culture model was developed and applied to study the digestion of nature's own emulsion, milk. The impact of the invitro cell culture on the digestion-triggered formation and evolution of highly ordered nanostructures in milk is demonstrated. Reported is also the crucial role of the mucus layer on top of the cell layer, protecting the cells from degradation by digestive juice components such as lipase. The novel model can open unique possibilities for the dynamic investigation of colloidal structure formation during lipid digestion and their effect on the uptake of bioactive molecules by the cells.

Tributyltin and triphenyltin induce 11ß-hydroxysteroid dehydrogenase 2 expression and activity through activation of retinoid X receptor α.

Exposure to the environmental pollutants organotins is of toxicological concern for the marine ecosystem and sensitive human populations, including pregnant women and their unborn children. Using a placenta cell model, we investigated whether organotins at nanomolar concentrations affect the expression and activity of 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2). 11ß-HSD2 represents a placental barrier controlling access of maternal glucocorticoids to the fetus. The organotins tributyltin (TBT) and triphenyltin (TPT) induced 11ß-HSD2 expression and activity in JEG-3 placenta cells, an effect confirmed at the mRNA level in primary human trophoblast cells. Inhibition/knock-down of retinoid X receptor alpha (RXRα) in JEG-3 cells reduced the effect of organotins on 11ß-HSD2 activity, mRNA and protein levels, revealing involvement of RXRα. Experiments using RNA and protein synthesis inhibitors indicated that the effect of organotins on 11ß-HSD2 expression was direct and caused by increased transcription. Induction of placental 11ß-HSD2 activity by TBT, TPT and other endocrine disrupting chemicals acting as RXRα agonists may affect placental barrier function by altering the expression of glucocorticoid-dependent genes and resulting in decreased availability of active glucocorticoids for the fetus, disturbing development and increasing the risk for metabolic and cardiovascular complications in later life.

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.

Impact of particle size and surface modification on gold nanoparticle penetration into human placental microtissues.

AIM: Nanoparticle-based drug carriers hold great promise for the development of targeted therapies in pregnancy with reduced off-target effects. Here, we performed a mechanistic in vitro study on placental localization and penetration of gold nanoparticles (AuNPs) in dependence of particle size and surface modification. MATERIALS & METHODS: AuNP uptake and penetration in human placental coculture microtissues was assessed by inductively coupled plasma-mass spectrometry, transmission electron microscopy and laser ablation-inductively coupled plasma-mass spectrometry. RESULTS: Higher uptake and deeper penetration was observed for smaller (3-4 nm) or sodium carboxylate-modified AuNPs than for larger (13-14 nm) or PEGylate AuNPs, which barely passed the trophoblast barrier layer. CONCLUSION: It is possible to steer placental uptake and penetration of AuNPs by tailoring their properties, which is a prerequisite for the development of targeted therapies in pregnancy.

Weathering of a carbon nanotube/epoxy nanocomposite under UV light and in water bath: impact on abraded particles.

Weathering processes can influence the surface properties of composites with incorporated nanoparticles. These changes may affect the release behavior of nanoparticles when an abrasion process is applied. Therefore, the influence of two different weathering processes, immersion in water and exposure to UV light, on the properties of abraded particles from a carbon nanotube (CNT)/epoxy nanocomposite was investigated. The investigation included the measurement of the weathering impact on the surface chemistry of the exposed samples, the particle size of abraded particles, the quantity of exposed CNTs in the respirable part of the abraded particles, and the toxicity of abraded particles, measured by in vitro toxicity tests using the THP-1 monocyte-derived macrophages. The results showed that weathering by immersion in water had no influence on the properties of abraded particles. The exposure to UV light caused a degradation of the epoxy on the surface, followed by delamination of an approx. 2.5 µm thick layer. An increased quantity of exposed CNTs in abraded particles was not found; on the contrary, longer UV exposure times decreased the released fraction of CNTs from 0.6% to 0.4%. The toxicity tests revealed that abraded particles from the nanocomposites did not induce additional acute cytotoxic effects compared to particles from the neat epoxy.

Carbon Nanotubes Released from an Epoxy-Based Nanocomposite: Quantification and Particle Toxicity.

Studies combining both the quantification of free nanoparticle release and the toxicological investigations of the released particles from actual nanoproducts in a real-life exposure scenario are urgently needed, yet very rare. Here, a new measurement method was established to quantify the amount of free-standing and protruding multiwalled carbon nanotubes (MWCNTs) in the respirable fraction of particles abraded from a MWCNT-epoxy nanocomposite. The quantification approach involves the prelabeling of MWCNTs with lead ions, nanocomposite production, abrasion and collection of the inhalable particle fraction, and quantification of free-standing and protruding MWCNTs by measuring the concentration of released lead ions. In vitro toxicity studies for genotoxicity, reactive oxygen species formation, and cell viability were performed using A549 human alveolar epithelial cells and THP-1 monocyte-derived macrophages. The quantification experiment revealed that in the respirable fraction of the abraded particles, approximately 4000 ppm of the MWCNTs were released as exposed MWCNTs (which could contact lung cells upon inhalation) and approximately 40 ppm as free-standing MWCNTs in the worst-case scenario. The release of exposed MWCNTs was lower for nanocomposites containing agglomerated MWCNTs. The toxicity tests revealed that the abraded particles did not induce any acute cytotoxic effects.

Gene expression profiling of immune-competent human cells exposed to engineered zinc oxide or titanium dioxide nanoparticles.

A comprehensive in vitro assessment of two commercial metal oxide nanoparticles, TiO2 and ZnO, was performed using human monocyte-derived macrophages (HMDM), monocyte-derived dendritic cells (MDDC), and Jurkat T cell leukemia-derived cell line. TiO2 nanoparticles were found to be non-toxic whereas ZnO nanoparticles caused dose-dependent cell death. Subsequently, global gene expression profiling was performed to identify transcriptional response underlying the cytotoxicity caused by ZnO nanoparticles. Analysis was done with doses 1 µg/ml and 10 µg/ml after 6 and 24 h of exposure. Interestingly, 2703 genes were significantly differentially expressed in HMDM upon exposure to 10 µg/ml ZnO nanoparticles, while in MDDCs only 12 genes were affected. In Jurkat cells, 980 genes were differentially expressed. It is noteworthy that only the gene expression of metallothioneins was upregulated in all the three cell types and a notable proportion of the genes were regulated in a cell type-specific manner. Gene ontology analysis revealed that the top biological processes disturbed in HMDM and Jurkat cells were regulating cell death and growth. In addition, genes controlling immune system development were affected. Using a panel of modified ZnO nanoparticles, we obtained an additional support that the cellular response to ZnO nanoparticles is largely dependent on particle dissolution and show that the ligand used to modify ZnO nanoparticles modulates Zn(2+) leaching. Overall, the study provides an extensive resource of transcriptional markers for mediating ZnO nanoparticle-induced toxicity for further mechanistic studies, and demonstrates the value of assessing nanoparticle responses through a combined transcriptomics and bioinformatics approach.

Efficient internalization of silica-coated iron oxide nanoparticles of different sizes by primary human macrophages and dendritic cells.

Engineered nanoparticles are being considered for a wide range of biomedical applications, from magnetic resonance imaging to "smart" drug delivery systems. The development of novel nanomaterials for biomedical applications must be accompanied by careful scrutiny of their biocompatibility. In this regard, particular attention should be paid to the possible interactions between nanoparticles and cells of the immune system, our primary defense system against foreign invasion. On the other hand, labeling of immune cells serves as an ideal tool for visualization, diagnosis or treatment of inflammatory processes, which requires the efficient internalization of the nanoparticles into the cells of interest. Here, we compare novel monodispersed silica-coated iron oxide nanoparticles with commercially available dextran-coated iron oxide nanoparticles. The silica-coated iron oxide nanoparticles displayed excellent magnetic properties. Furthermore, they were non-toxic to primary human monocyte-derived macrophages at all doses tested whereas dose-dependent toxicity of the smaller silica-coated nanoparticles (30nm and 50nm) was observed for primary monocyte-derived dendritic cells, but not for the similarly small dextran-coated iron oxide nanoparticles. No macrophage or dendritic cell secretion of pro-inflammatory cytokines was observed upon administration of nanoparticles. The silica-coated iron oxide nanoparticles were taken up to a significantly higher degree when compared to the dextran-coated nanoparticles, irrespective of size. Cellular internalization of the silica-coated nanoparticles was through an active, actin cytoskeleton-dependent process. We conclude that these novel silica-coated iron oxide nanoparticles are promising materials for medical imaging, cell tracking and other biomedical applications.