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. Citation Grafmueller S, Manser P, Diener L, Diener PA, Maeder-Althaus X, Maurizi L, Jochum W, Krug HF, Buerki-Thurnherr T, von Mandach U, Wick P. 2015. Bidirectional transfer study of polystyrene nanoparticles across the placental barrier in an ex vivo human placental perfusion model. Environ Health Perspect 123:1280–1286; http://dx.doi.org/10.1289/ehp.1409271


Materials and Methods
Cell culture   and human chorionic gonadotropin (hCG) and leptin hormone production rate (D) during ex vivo human placental perfusion with or without particles (control). All data is expressed as the mean ± SD of at least 3 independent experiments.

Cell culture
BeWo cells (b30 clone), a cell line derived from human choriocarcinoma, were obtained from Prof. Dr. Ursula Graf-Hausner (Zurich University of Applied Sciences, Waedenswil, Switzerland) with permission of Dr. Alan L. Schwartz (Washington University School of Medicine, MO, USA) and cultured in Ham´s F-12K medium (Gibco, Thermo Fisher Scientific Inc.) supplemented with 1 % penicillin-streptomycin and 10 % fetal calf serum (FCS) at 37°C and 5 % carbon dioxide (CO 2 ).

MTS viability assay
The in vitro cytotoxicity of the different PS beads was tested using the MTS viability assay. 24 hrs before treatment BeWo cells were seeded in a 96-well plate (8000 cells per well). Different concentrations of PS beads were applied. As negative control, cells without treatment were used and as positive control 1, 10, 100 and 1000 µM CdSO 4 was applied. After 3 or 24 hrs of incubation at 37 °C and 5 % CO 2 , an MTS assay (CellTiter96® AQ ueous One Solution Cell Proliferation Assay; Promega) was performed according to the manufacturer's instructions.
Results were presented as mean percentage of the untreated control from three independent experiments.

Ex vivo human placental perfusion model
A fetal artery and vein of an intact cotyledon were cannulated. Afterwards the placenta was fixed in a tissue holder and placed into a perfusion chamber. To connect the maternal side three blunt cannulas were introduced in the intervillous space and a venous drain was introduced to return the fluid to the maternal circuit. Two peristaltic pumps maintained the fetal flow at 3 -4 mL/min and the maternal flow at 12 mL/min. A water bath kept the temperature of both circuits at 37 °C.
For the fetal side one oxygenator with 95 % N 2 and 5 % CO 2 and for the maternal side one oxygenator with 95% synthetic air and 5 % CO 2 were applied. To flush out the blood and allow a recovery of the tissue from the ischemic period after the delivery, the experiment started with an open perfusion (both circuits) for 20 minutes using only perfusion medium. The perfusion medium contained M199 tissue culture medium (Sigma) diluted with Earl's buffer (dilution 1 : 2), 1 g/L glucose (Sigma), 10 g/L bovine serum albumin (AppliChem GmbH), 10 g/L dextran 40 (Sigma), 2500 IU/L sodium heparin (B.Braun Medical AG), 250 mg/L amoxicillin (GlaxoSmithKline AG) and 2.2 g/L sodium bicarbonate (Merck). After closing of the fetal and maternal circulation by leading the venous outflow back to the corresponding reservoir, the PS particles were added either to the maternal (M) or the fetal (F) reservoir at a final concentration of 25 µg/mL. The concentration of the PS beads in the fetal and maternal circuit was determined by fluorescence measurement in a microplate reader (Biotek Synergy HT) after centrifugation at 800 x g to remove residual erythrocytes. Particle concentrations were corrected for the PM volume in the tubes and volume loss due to sampling before placental transfer was calculated as percentage of transferred PS beads compared to the initially added particle amount. As control radiolabeled 14 C-antipyrine (50 nCi/mL, specific activity: 4.7 mCi/mmol; American Radiolabeled Chemicals Inc.) was also added to the maternal or fetal circuit. The criteria for a successful perfusion were visual control (intact membranes, no lesion, no disruption of the placenta), leak from fetal to maternal side < 4 mL/hr (for reverse perfusions < 1 mL/hr), an equilibrium of 14 C-antipyrine between maternal and fetal circuit after 4 -6 hrs, a fetal perfusion pressure < 70 mmHg and fetal pH at a physiological range of 7.2 -7.4.

Antipyrine transfer
Perfusate samples were mixed with scintillation cocktail (Irgasafe Plus Scintillation Cocktail, Zinsser Analytic) and measured in a liquid scintillation analyzer (Packard Tri-Carb 2200; GMI).

Viability and functionality of the placenta
Glucose and lactate concentration in the fetal and maternal circuit were determined with an automated blood gas system (ABL800 FLEX automated benchtop analyzer, Radiometer Medical ApS). The placental hormones human chorionic gonadotropin (hCG) and leptin in the fetal and maternal circuit before and after perfusion were determined by enzyme-linked immunosorbent assay (ELISA) as described previously (Malek et al. 1997;Malek et al. 2001). The hormone production was calculated by dividing the amount released in both circulations after 6 hrs of perfusion by the weight of perfused tissue.

Histopathological evaluation
Tissue samples of non-perfused (negative control) and perfused placentas were fixed in 4 % formaldehyde (Formafix AG). After standardized dehydratation of the tissue overnight in a Medite Tissue Processor TPC 15 the probes were embedded in paraffin blocks with the Medite Tissue Embedding System TES 99. Using a microtome, 4 µm thick sections were obtained, mounted and air dried on glass slides and stained with haematoxylin and eosin in a Tissue-Tek ® Prisma™. The slides were examined at a Leica DMLB microscope.  with or without PS beads. (C + D) Glucose consumption and lactate production (C) and human chorionic gonadotropin (hCG) and leptin hormone production rate (D) during ex vivo human placental perfusion with or without particles (control). All data is expressed as the mean ± SD of at least 3 independent experiments.