BeWo B30 Cells Research Articles

Endocytosis and Transport of Silica Nanoparticles in BeWo b30 Cells

Endocytosis and Transport of Silica Nanoparticles in BeWo b30 Cells

Folate-mediated Transport of Nanoparticles across the Placenta.

In this study, a prototype of a targeted nanocarrier for drug delivery for prenatal therapy of the developing fetus was developed and examined in vitro and ex vivo. The folate transport mechanism in the human placenta was utilized as a possible pathway for the transplacental delivery of targeted nanoparticles. Several types of folic acid-decorated polymeric nanoparticles were synthesized and characterized. During transport studies of targeted and non-targeted fluorescent nanoparticles across the placental barrier, the apparent permeability values, uptake, transfer indices, and distribution in placental tissue were determined. The nanoparticles had no effect on BeWo b30 cell viability. In vitro, studies showed significantly higher apparent permeability of the targeted nanoparticles across the cell monolayers as compared to the nontargeted nanoparticles (Pe = 5.92 ± 1.44 ×10-6 cm/s for PLGA-PEG-FA vs. 1.26 ± 0.31 ×10-6 cm/s for PLGA-PEG, P < 0.05), and the transport of the targeted nanoparticles was significantly inhibited by excess folate. Ex vivo placental perfusion showed significantly greater accumulation of the targeted nanoparticles in the placental tissue (4.31 ± 0.91%/g for PLGA-PEG-FA vs. 2.07 ± 0.26%/g for PLGA-PEG). The data obtained suggested different mechanisms for the uptake and transplacental transfer of targeted versus nontargeted nanoparticles. This targeted nanoformulation may be a promising strategy for fetal drug therapy.

Cannabidiol Disrupts Mitochondrial Respiration and Metabolism and Dysregulates Trophoblast Cell Differentiation.

Trophoblast differentiation is a crucial process in the formation of the placenta where cytotrophoblasts (CTs) differentiate and fuse to form the syncytiotrophoblast (ST). The bioactive components of cannabis, such as Δ9-THC, are known to disrupt trophoblast differentiation and fusion, as well as mitochondrial dynamics and respiration. However, less is known about the impact of cannabidiol (CBD) on trophoblast differentiation. Due to the central role of mitochondria in stem cell differentiation, we evaluated the impact of CBD on trophoblast mitochondrial function and differentiation. Using BeWo b30 cells, we observed decreased levels of mRNA for markers of syncytialization (GCM1, ERVW1, hCG) following 20 µM CBD treatment during differentiation. In CTs, CBD elevated transcript levels for the mitochondrial and cellular stress markers HSP60 and HSP70, respectively. Furthermore, CBD treatment also increased the lipid peroxidation and oxidative damage marker 4-hydroxynonenal. Mitochondrial membrane potential, basal respiration and ATP production were diminished with the 20 µM CBD treatment in both sub-lineages. mRNA levels for endocannabinoid system (ECS) components (FAAH, NAPEPLD, TRPV1, CB1, CB2, PPARγ) were altered differentially by CBD in CTs and STs. Overall, we demonstrate that CBD impairs trophoblast differentiation and fusion, as well as mitochondrial bioenergetics and redox homeostasis.

MicroRNA miR-27a as a possible regulator of anti-inflammatory macrophage phenotype in preeclamptic placenta

IntroductionThe role of the TGFβ signaling pathway, an important cascade responsible for the anti-inflammatory polarization of macrophages, in the development of both early- and late-onset preeclampsia (eoPE and loPE), remains poorly understood. In this study, we examined the components of the TGFβ signaling cascade and macrophage markers within placental tissue in normal pregnancy and in PE. MethodsPatients with eoPE, loPE, and normal pregnancy were enrolled in the study (n = 10 in each group). Following techniques were used for the investigation: immunohistochemistry analysis, western blotting, qRT-PCR, isolation of monocytes by magnetic sorting, transfection, microRNA sequencing, and bioinformatic analysis. ResultsWe observed a significant decrease in the anti-inflammatory macrophage marker CD206 in the loPE group, alongside with a significant down-regulation of CD206 protein production in both eoPE and loPE groups. The level of CD68-positive cells and relative levels of CD163 and MARCO production were comparable across the groups. However, we identified a significant decrease in the TGFβ receptor 2 production and its gene expression in the PE group. Further analysis revealed a link between TGFBR2 and MRC1 (CD206) genes through a single miRNA, hsa-miR-27a-3p. Transfecting CD14-derived macrophages with the hsa-miR-27a-3p mimic significantly changed TGFBR2 production, indicating the potential role of this miRNA in regulating the TGFβ signaling pathway. We also revealed the up-regulation of hsa-miR-27a-5p and hsa-miR-27a-3p in the trophoblast BeWo b30 cell line under the severe hypoxia condition and the fact that TGFBR2 3′ UTR could serve as a potential target for these miRNAs. DiscussionOur findings uncover a novel potential therapeutic target for managing patients with PE, significantly contributing to a deeper comprehension of the underlying mechanisms involved in the development of this pathology.

Transplacental passage of hyperforin, hypericin, and valerenic acid.

Safe medications for mild mental diseases in pregnancy are needed. Phytomedicines from St. John's wort and valerian are valid candidates, but safety data in pregnancy are lacking. The transplacental transport of hyperforin and hypericin (from St. John's wort), and valerenic acid (from valerian) was evaluated using the ex vivo cotyledon perfusion model (4h perfusions, term placentae) and, in part, the in vitro Transwell assay with BeWo b30 cells. Antipyrine was used for comparison in both models. U(H)PLC-MS/MS bioanalytical methods were developed to quantify the compounds. Perfusion data obtained with term placentae showed that only minor amounts of hyperforin passed into the fetal circuit, while hypericin did not cross the placental barrier and valerenic acid equilibrated between the maternal and fetal compartments. None of the investigated compounds affected metabolic, functional, and histopathological parameters of the placenta during the perfusion experiments. Data from the Transwell model suggested that valerenic acid does not cross the placental cell layer. Taken together, our data suggest that throughout the pregnancy the potential fetal exposure to hypericin and hyperforin - but not to valerenic acid - is likely to be minimal.

Placental cell translocation of folate-conjugated pullulan acetate non-spherical nanoparticles

Due to the adverse effects of free drugs on the fetus, placental-mediated pregnancy complications still lack effective pharmacotherapy. This study aims to construct a non-spherical drug delivery system based on folate-conjugated pullulan acetate (FPA) for placental targeting and translocation. By adjusting the initial solvent system, FPA nanoparticles with different morphologies were prepared using dialysis method without a surfactant. Cytotoxicity and lactate dehydrogenase release assays indicated the good biocompatibility of FPA nanoparticles in BeWo b30 cells. Cellular uptake and in vitro placental barrier transportation studies showed that FPA nanoparticles entered the cells and transported across the cell monolayer, benefiting from the active target effect mediated by the folate receptor. Moreover, non-spherical FPA nanoparticles showed nuclear translocation due to their shape effect. These findings provide a novel aspect in placental-mediated pregnancy treatment and applications in the obstetrics field of drug use during pregnancy.

Medicinal Plants for the Treatment of Mental Diseases in Pregnancy: An In Vitro Safety Assessment.

Pregnancy is a critical period for medical care, during which the well-being of woman and fetus must be considered. This is particularly relevant in managing non-psychotic mental disorders since treatment with central nervous system-active drugs and untreated NMDs may have negative effects. Some well-known herbal preparations (phytopharmaceuticals), including St. Johnʼs wort, California poppy, valerian, lavender, and hops, possess antidepressant, sedative, anxiolytic, or antidepressant properties and could be used to treat mental diseases such as depression, restlessness, and anxiety in pregnancy. Our goal was to assess their safety in vitro , focusing on cytotoxicity, induction of apoptosis, genotoxicity, and effects on metabolic properties and differentiation in cells widely used as a placental cell model (BeWo b30 placenta choriocarcinoma cells). The lavender essential oil was inconspicuous in all experiments and showed no detrimental effects. At low-to-high concentrations, no extract markedly affected the chosen safety parameters. At an artificially high concentration of 100 µg/mL, extracts from St. Johnʼs wort, California poppy, valerian, and hops had minimal cytotoxic effects. None of the extracts resulted in genotoxic effects or altered glucose consumption or lactate production, nor did they induce or inhibit BeWo b30 cell differentiation. This study suggests that all tested preparations from St. Johnʼs wort, California poppy, valerian, lavender, and hops, in concentrations up to 30 µg/mL, do not possess any cytotoxic or genotoxic potential and do not compromise placental cell viability, metabolic activity, and differentiation. Empirical and clinical studies during pregnancy are needed to support these in vitro data.

Уровень микроРНК miR-374 повышается в клетках BeWo b30 при гипоксии

In humans, trophoblast hypoxia during placental development can be a cause of serious pregnancy complications, such as preeclampsia and fetal growth restriction. The pathogenesis of these conditions is not fully clear and may be associated with changed expression of some genes and regulatory molecules, including miRNA, in trophoblast cells. The aim of this study was to analyze miRNA profiles and measure the expression of their target genes in a model of trophoblast hypoxia. Human choriocarcinoma BeWo b30 cells were used as a trophoblast model. Hypoxia was induced by cobalt chloride (CoCl2) and an oxyquinoline derivative. MRNA and miRNA expression profiles were evaluated by means of next generation sequencing (NGS); the expression of individual genes was analyzed by PCR. We studied the secondary structure of mRNAs of target genes for those miRNAs whose expression had changed significantly and analyzed potential competition between these miRNAs for the binding site. The observed changes in the expression of the key genes involved in the response to hypoxia confirmed the feasibility of using CoCl2 and the oxyquinoline derivative as hypoxia inducers. The analysis revealed an increase in miR-374 levels following the activation of the hypoxia pathway in our trophoblast model. The changes were accompanied by a reduction in FOXM1 mRNA expression; this mRNA is a target for hsa-miR-374a-5p and hsa-miR374b-5p, which can compete with hsa-miR-21-5p for the binding sites on FOXM1 mRNA. The involvement of FOXM1 in the regulation of the invasive cell potential suggests the role of miR-374 and FOXM1 in the pathogenesis of disrupted trophoblast invasion during placental development as predisposing for fetal growth restriction and preeclampsia.

Factors Involved in miRNA Processing Change Its Expression Level during Imitation of Hypoxia in BeWo b30 Cells.

One of the main complications of pregnancy and causes of maternal and perinatal mortality is preeclampsia. The pathophysiology of preeclampsia is associated with the development of placenta and fetal hypoxia and secretion of a number of effective molecules. The human choriocarcinoma cell line BeWo b30 is often used as a model of the placental barrier. It was shown that oxyquinoline derivatives can mimic hypoxia by suppressing HIF-prolyl hydroxylases and the accumulation of HIF-1α. This effect also leads to a change in the expression of microRNAs and their target genes. However, with hypoxia in cells, not only the level of individual miRNAs but also the ratio of miRNA isoforms (isomiRs) can change, presumably due to inaccuracies in the work of the Drosha and Dicer enzymes. In this work, we showed a change in the expression of the factors involved in the maturation of miRNAs when simulating hypoxia in BeWo b30 cells with an oxyquinoline derivative, which may be one of the causes for the change in the ratio of miRNA isoforms.

Combination of the BeWo b30 placental transport model and the embryonic stem cell test to assess the potential developmental toxicity of silver nanoparticles

BackgroundSilver nanoparticles (AgNPs) are used extensively in various consumer products because of their antimicrobial potential. This requires insight in their potential hazards and risks including adverse effects during pregnancy on the developing fetus. Using a combination of the BeWo b30 placental transport model and the mouse embryonic stem cell test (EST), we investigated the capability of pristine AgNPs with different surface chemistries and aged AgNPs (silver sulfide (Ag2S) NPs) to cross the placental barrier and induce developmental toxicity. The uptake/association and transport of AgNPs through the BeWo b30 was characterized using ICP-MS and single particle (sp)ICP-MS at different time points. The developmental toxicity of the AgNPs was investigated by characterizing their potential to inhibit the differentiation of mouse embryonic stem cells (mESCs) into beating cardiomyocytes.ResultsThe AgNPs are able to cross the BeWo b30 cell layer to a level that was limited and dependent on their surface chemistry. In the EST, no in vitro developmental toxicity was observed as the effects on differentiation of the mESCs were only detected at cytotoxic concentrations. The aged AgNPs were significantly less cytotoxic, less bioavailable and did not induce developmental toxicity.ConclusionsPristine AgNPs are capable to cross the placental barrier to an extent that is influenced by their surface chemistry and that this transport is likely low but not negligible. Next to that, the tested AgNPs have low intrinsic potencies for developmental toxicity. The combination of the BeWo b30 model with the EST is of added value in developmental toxicity screening and prioritization of AgNPs.

Transport and toxicity of 5-fluorouracil, doxorubicin, and cyclophosphamide in in vitro placental barrier model based on BeWo b30 cells

An in vitro placental barrier model based on human choriocarcinoma BeWo b30 cell line was considered as a method of preclinical study of the transport and toxicity of antitumor agents and other organic compounds. Low permeabilities were found for 5-fluorouracil as an example of hydrophilic compound and for doxorubicin as an example of a lipophilic compound with a high degree of binding to proteins and DNA and a high permeability was found for cyclophosphamide as an example of lipophilic compound with a low degree of binding to proteins. Using impedance spectrometry and cell viability assessment via reduction of resazurin to resorufin, a pronounced cytotoxic effect of doxorubicin and good tolerance of 5-fluorouracil and cyclophosphamide by the cells were shown for drug concentrations equal to the maximum concentrations in the patients’ blood during the treatment of breast cancer.

Impedance Spectroscopy and Transcriptome Analysis of Choriocarcinoma BeWo b30 as a Model of Human Placenta

Placenta is a highly specialized organ that is necessary for successful gestation. Several models of the placental barrier are used to study how it functions, including the transplacental transport of xenobiotics. One of these models, human choriocarcinoma cell line BeWo is widely used in vitro. Notably, cancerous BeWo cells form multilayer structures that normally are not found in the human placenta. Here, we aim to develop techniques suitable for monitoring BeWo b30 cells in culture. To assess the state of BeWo b30 cells growing on a membrane, we use impedance spectroscopy, which allows us to estimate the number of cell layers by the change in the electrical parameters of the biological system. In mature BeWo b30 cell cultures, we also note a significant increase in the expression of genes encoding metallothioneins (particularly, MT1B, MT1F, and MT2A) and syncytins (ERVW-1 and ERVFRD-1), which can be used as biomarkers reflecting the development of mature phenotypic characteristics, namely, trophoblastic invasion and formation of the syncytium.

Regulation of human placental drug transporters in HCV infection and their influence on direct acting antiviral medications

IntroductionThe objectives of this study were to determine how HCV infection affects placental drug transporters, and to determine the role of drug transporters on the cellular accumulation of direct-acting antiviral drugs in human trophoblasts. MethodsEighty-four ABC and SLC transporter genes were first screened in normal and HCV infected pregnant women using PCR profiler array. The changes in expression were confirmed by qPCR and Western blot. The impact of selected drug transporters on the cellular accumulation of radiolabeled antiviral drugs sofosbuvir, entecavir, and tenofovir was measured in primary human trophoblasts (PHT) and BeWo b30 cells in the presence or absence of transporter-specific inhibitors. PHT were then treated with CL097, ssRNA40, and imquimod to determine the impact of Toll-like receptor (TLR) 7/8 activation on drug transporter expression. ResultsThe expression of the ABC efflux transporters ABCB1/P-gp and ABCG2/BCRP was increased in placenta of women with HCV, while the nucleoside transporters SLC29A1/ENT1 and SLC29A2/ENT2 remained unchanged. The accumulation of sofosbuvir and tenofovir was unaffected by inhibition of these transporters in trophoblast cells. Entecavir accumulation was decreased by the inhibition of ENT2. P-gp and BCRP inhibition enhanced entecavir accumulation in BeWo b30, but not PHT. Overall, there was little effect of TLR7/8 activation on these drug transporters, and the accumulation of entecavir in PHT. DiscussionThe data suggest that expression of placental drug transporters and selection of antiviral drug may impact fetal drug exposure in pregnancies complicated by HCV infections.

Uptake and transport of pullulan acetate nanoparticles in the BeWo b30 placental barrier cell model.

IntroductionNanomedicine has shown a great potential in perinatal medicine because of its characteristics of sustained, controlled release and targeting ability; on the other hand, it may also lead to unexpected toxicities such as embryotoxicity and even malformation after crossing the placental barrier, but data concerning transplacental transport are scarce. Pullulan acetate (PA) nanoparticles (NPs) are a promising nanocarrier derived from natural polysaccharide; however, their transplacental transport ability and mechanism are unknown.Materials and methodsIn this study, fluorescein isothiocyanate (FITC) conjugated PA (PA-FITC) was synthesized. PA-FITC NPs were characterized by dynamic light scattering, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The cytotoxicity of PA-FITC NPs at concentrations of 15, 30, 60, 125, 250, 500, 1,000 and 2,000 μg/mL was studied by cell counting kit-8. The human chorionic gonadotrophin (HCG) cytokine assay was conducted to evaluate the biological function of BeWo b30 cells. Endocytic mechanisms of PA-FITC NPs were investigated via fluorescence analysis. The monolayer properties were characterized by TEM, tight junction staining, transepithelial electrical resistance and fluorescein sodium transportation. The transport ability was measured in the cell based transwell model by confocal imaging and SEM.ResultsPA-FITC NPs were almost spherical shape with a size range of 200–300 nm. Cell viability of BeWo b30 cells was up to 100% in all groups. The concentrations of HCG increased with increasing numbers of cells and culture time, which showed the good biological function of BeWo b30 cells. PA-FITC NPs were rapidly endocytosed through caveolae-mediated endocytosis and pinocytosis, with uptake inhibition rates with nystatin (NY) and colchicines (Col) of 55% and 51% respectively. BeWo b30 cell monolayer was formed over 5 days. PA-FITC NPs were found in the cytoplasm of cells on the transwell membranes; while some NPs were found in the basolateral (fetal) compartment over 24 h.ConclusionIn summary, PA-FITC NPs are nontoxic, can cross the blood-placental barrier, and show mainly internalization to BeWo b30 cells through caveolae-mediated endocytosis and pinocytosis pathways, major via the former pathway. The results could benefit the adjustment and control of the transplacental transport of nanomedicines.

Abstract A118: Efflux of paclitaxel nanoformulation in cell culture model of human placental barrier

Abstract Background: Breast cancer affects 1 of every 3000 pregnancies, and is treated with chemotherapeutics including paclitaxel. Chemotherapy administration in pregnancy is associated with fetal morbidity. Purpose: Here, we investigated the transplacental transfer of both paclitaxel and new paclitaxel nanoformulations that are entering clinical studies or are commercially available for use in breast cancer. In addition, we examined the transfer of fluorescent albumin-based and polymeric micellar nanoformulations with similar properties to those paclitaxel nanoformulations. Methods: Transport studies were performed using BeWo b30 cells (human placental trophoblast cells) seeded in TranswellTM plates coated with human placental collagen. Cells were treated with 20 μg/mL of paclitaxel from marketed formulations. Since paclitaxel undergoes efflux by P-glycoprotein (P-gp) substrate, 100 μM of verapamil was used as a competitive P-gp inhibitor to prevent efflux of free paclitaxel released from the nanoformulations. In order to determine the role of nanomaterials in transport and uptake of paclitaxel, we synthesized fluorescent albumin nanoparticles and fluorescent polymeric micelles with similar properties to the paclitaxel nanoformulations for use in transport studies. Samples were analyzed by high-performance liquid chromatography or fluorometer. Uptake measurements were corrected for protein content. Particle size and polydispersity index were determined using dynamic light scattering. Results: There were no formulation-dependent differences in paclitaxel permeability between the formulations of paclitaxel, nanoparticle albumin-bound paclitaxel (nab-paclitaxel), and paclitaxel-loaded polymeric micelles. In the presence of verapamil, nab-paclitaxel showed a twofold increase in permeability, in contrast to the other formulations. The size of fluorescent albumin nanoparticles (130.3nm) was matched with nab-Paclitaxel (156.2 nm), and the fluorescent micelles (27.6 nm) with paclitaxel-loaded polymeric micelles (27.7nm). The apparent permeability of the fluorescent albumin nanoparticles across the BeWo cells was 1.98 x 10-6 ± 0.81 x 10-6 cm/s, whereas the apparent permeability of the fluorescent micelles was 59.5 x 10-6 ± 10.5 x 10-6 cm/s. Conclusion: These data suggest that paclitaxel-encapsulated micelles may evade efflux by P-gp and cross the trophoblast to a significant extent, while paclitaxel albumin nanoparticles behave like free paclitaxel. Therefore, choice of formulation may have a significant impact on fetal exposure to paclitaxel. We anticipate that these findings will have an impact on future pharmaceutical design and rational development of safe treatment strategies for pregnancy-associated breast cancer and other diseases. Citation Format: Shariq Ali, Sanaalarab Alenazy, Mansi Shah, Luke Bourner, Erik Rytting. Efflux of paclitaxel nanoformulation in cell culture model of human placental barrier [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A118.

Transport of digoxin-loaded polymeric nanoparticles across BeWo cells, an in vitro model of human placental trophoblast.

Fetal arrhythmias can lead to fetal congestive heart failure and hydrops fetalis. Digoxin (the first-line treatment) has low transplacental permeability and high risk of maternal side effects. Biodegradable digoxin-loaded PEGylated poly(lactic-co-glycolic acid) nanoparticles may increase digoxin transport across BeWo b30 cell monolayers (an in vitro model of trophoblast in human placenta) by reducing the drug's interaction with P-gp. Results/methodology: The nanoparticles showed high encapsulation efficiency and sustained release over 48 h. Transport studies revealed significantly increased permeability across BeWo cell layers of digoxin-loaded nanoparticles when compared with free digoxin. P-gp inhibition also increased the permeability of digoxin, but not digoxin-loaded nanoparticles. This represents a novel treatment strategy for fetal cardiovascular disease which may improve maternal and fetal outcomes.

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

To 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.

Suitability of human and mammalian cells of different origin for the assessment of genotoxicity of metal and polymeric engineered nanoparticles

Nanogenotoxicity is a crucial endpoint in safety testing of nanomaterials as it addresses potential mutagenicity, which has implications for risks of both genetic disease and carcinogenesis. Within the NanoTEST project, we investigated the genotoxic potential of well-characterised nanoparticles (NPs): titanium dioxide (TiO2) NPs of nominal size 20 nm, iron oxide (8 nm) both uncoated (U-Fe3O4) and oleic acid coated (OC-Fe3O4), rhodamine-labelled amorphous silica 25 (Fl-25 SiO2) and 50 nm (Fl-50 SiO) and polylactic glycolic acid polyethylene oxide polymeric NPs – as well as Endorem® as a negative control for detection of strand breaks and oxidised DNA lesions with the alkaline comet assay. Using primary cells and cell lines derived from blood (human lymphocytes and lymphoblastoid TK6 cells), vascular/central nervous system (human endothelial human cerebral endothelial cells), liver (rat hepatocytes and Kupffer cells), kidney (monkey Cos-1 and human HEK293 cells), lung (human bronchial 16HBE14o cells) and placenta (human BeWo b30), we were interested in which in vitro cell model is sufficient to detect positive (genotoxic) and negative (non-genotoxic) responses. All in vitro studies were harmonized, i.e. NPs from the same batch, and identical dispersion protocols (for TiO2 NPs, two dispersions were used), exposure time, concentration range, culture conditions and time-courses were used. The results from the statistical evaluation show that OC-Fe3O4 and TiO2 NPs are genotoxic in the experimental conditions used. When all NPs were included in the analysis, no differences were seen among cell lines – demonstrating the usefulness of the assay in all cells to identify genotoxic and non-genotoxic NPs. The TK6 cells, human lymphocytes, BeWo b30 and kidney cells seem to be the most reliable for detecting a dose-response.

Oxcarbazepine-loaded polymeric nanoparticles: development and permeability studies across in vitro models of the blood-brain barrier and human placental trophoblast.

Encapsulation of antiepileptic drugs (AEDs) into nanoparticles may offer promise for treating pregnant women with epilepsy by improving brain delivery and limiting the transplacental permeability of AEDs to avoid fetal exposure and its consequent undesirable adverse effects. Oxcarbazepine-loaded nanoparticles were prepared by a modified solvent displacement method from biocompatible polymers (poly(lactic-co-glycolic acid) [PLGA] with or without surfactant and PEGylated PLGA [Resomer® RGPd5055]). The physical properties of the developed nanoparticles were determined with subsequent evaluation of their permeability across in vitro models of the blood–brain barrier (hCMEC/D3 cells) and human placental trophoblast cells (BeWo b30 cells). Oxcarbazepine-loaded nanoparticles with encapsulation efficiency above 69% were prepared with sizes ranging from 140–170 nm, polydispersity indices below 0.3, and zeta potential values below -34 mV. Differential scanning calorimetry and X-ray diffraction studies confirmed the amorphous state of the nanoencapsulated drug. The apparent permeability (Pe) values of the free and nanoencapsulated oxcarbazepine were comparable across both cell types, likely due to rapid drug release kinetics. Transport studies using fluorescently-labeled nanoparticles (loaded with coumarin-6) demonstrated increased permeability of surfactant-coated nanoparticles. Future developments in enzyme-prodrug therapy and targeted delivery are expected to provide improved options for pregnant patients with epilepsy.

Use of the ES-D3 cell differentiation assay, combined with the BeWo transport model, to predict relative in vivo developmental toxicity of antifungal compounds

We investigated the applicability of the ES-D3 cell differentiation assay combined with the in vitro BeWo transport model to predict the relative in vivo developmental toxicity potencies. To this purpose, the in vitro developmental toxicity of five antifungal compounds was investigated by characterizing their inhibitory effect on the differentiation of ES-D3 cells into cardiomyocytes. The BeWo transport model, consisting of BeWo b30 cells grown on transwell inserts and mimicking the placental barrier, was used to determine the relative placental transport velocity. The ES-D3 cell differentiation data were first compared to benchmark doses (BMDs) for in vivo developmental toxicity as derived from data reported in the literature. Correlation between the benchmark concentration for 50% effect (BMCd50) values, obtained in the ES-D3 cell differentiation assay, with in vivo BMD10 values showed a reasonable correlation (R2=0.57). When the ES-D3 cell differentiation data were combined with the relative transport rates obtained from the BeWo model, the correlation with the in vivo data increased (R2=0.95). In conclusion, we show that the ES-D3 cell differentiation assay is able to better predict the in vivo developmental toxicity ranking of antifungal compounds when combined with the BeWo transport model, than as a stand-alone assay.