Polystyrene Nanobeads: Stock solutions of commercially available 20 nm rhodamine-labeled polystyrene beads (8.8 × 1014 particles/mL; PS20-RB-2; NanoCS, New York, NY) were suspended in distilled water and 0.01% surfactant and sonicated for 15 minutes prior to measurement. The size of the nanoparticles was measured with Non-Invasive Backscatter optics (NIBS) using a 4 mW, 633 nm laser. The ENM ζ-potential was also measured via Zetasizer Nano ZS. Independent verification of particle size revealed an average particle agglomerate size of 21.86 nm ± 0.026 and a zeta potential of -0.0874 ± 0.195.
Animals: Time-pregnant Sprague Dawley rats were ordered from Charles River Laboratories (Kingston, NY). Animals were delivered on gestational day (GD) 15 and allowed to acclimate within an AAALAC accredited vivarium at Rutgers University for at least 72 hours. Animals had access to food and water ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee of Rutgers University.
Exposure: Rhodamine-labeled nanopolystyrene particles were prepared by vortexing 300 µl of manufacturer’s 1.0% aqueous suspension for 2 minutes, followed by ultra-sonication on ice, for 5 minutes as previously described [21, 22]. Rats were anesthetized with isoflurane gas (5.0% induction). Animals were placed on an angled board by suspending the upper incisor teeth on an incisor loop at a 45° angle. The tongue was retracted using forceps and a cotton-tipped applicator. Using a veterinary operating otoscope fitted with a speculum, the epiglottis was visualized, and a 20 gauge, 4-inch stainless steel ball-tipped oral gavage needle was inserted via the mouth to the trachea. The rats received intratracheal instillation of 300 µL (2.64 × 1014 particles) of nanopolystyrene suspension or vehicle (0.9% NaCl). Rats were monitored after instillation until they regained consciousness and normal physiological activity (e.g., walking, eating, drinking, grooming, and resting).
To extrapolate nanopolystyrene particle dosage, we considered an average 1 mm3 atmospheric microparticle and mathematically converted this to nanosized particles representative of our spherical 20 nm polystyrene beads. Therefore, a single microparticle represents 2.39 × 1014 nanopolystyrene particles (Fig. 1a). Cox et al. reported that the average women inhales 132 microplastic particles per day . Given that maternal minute ventilation, or the volume of gas inhaled during pregnancy, increases by up to 48% while the respiration rate remains unchanged , it is likely that daily total exposure is closer to the upper bound of 279 microplastics identified in the study . These data suggest that the average pregnant woman could be exposed to 6.67 × 1016 nanoplastic particles per day (Fig. 1b). When the surface area of the lung between human (62.7 m2) and our laboratory rat (0.409 m2) model  is considered, the appropriate experimental exposure amounts to 4.34 × 1014 nanoplastic particles (Fig. 1c). This value is much greater than the exposure dose of 2.64 × 1014 nanoplastic particles and therefore, the exposure dose used in this study is within real-world considerations.
Fluorescent Optical Imaging: Twenty-four hours after exposure, dams were fully anesthetized with 3–5% isoflurane in oxygen and Nair was applied to the abdominal region to remove hair prior to imaging. The animal was transferred into the Bruker In-Vivo Multispectral (MS) FX PRO Imager (Bruker, Billerica, MA, USA) imaging chamber with nose cone attached to the manifold and placed in the prone position. The MSFX Pro Bruker detects bioluminescence, fluorescence, radio isotope, and X-ray.
A brightfield was taken to confirm positioning and provide a snapshot/photo of the scan. The primary scans consisted of an excitation of 480 nm with an emission of 535 nm for a 1-minute exposure. Later scans consisted of an excitation of 550 nm with an emission of 600 nm. For these scans, the detectable light refracting off the contrast was recorded. The final scan in this series was an X-ray of the sample that assisted with co-registration of the signal with organ tissues. Following live imaging, animals were sacrificed by removal of the heart according to the Rutgers IACUC approval. Maternal tissues, fetal pups, and fetal tissues were harvested and placed on a polycarbonate tray. After tissue scans were complete, the regions of interest were measured using Bruker MSFX PRO Image software.
Hyperspectral-enhanced darkfield microscopy: Formalin fixed fetal tissues were processed, embedded in paraffin, and sectioned to 4 microns. Slides were visualized via transmitted darkfield hyperspectral images and data captured using CytoViva optics at 60x magnification with oil objective. Dual Mode Fluorescence (DMF) and full fluorescence images were captured with Texas Red excitation filter and triple pass emission filter for further particle confirmation. Data was processed using ENVI 4.8 (CytoViva, Inc., Auburn, AL).
Placental Isolation and Perfusion: A separate cohort of naïve gravid rats were anesthetized with isoflurane (5% induction and 3% maintenance) on GD 20. The right uterine horn was isolated, removed, and placed into a dish of cold (4 °C) physiological salt solution (PSS). Briefly, the uterine horn was dissected, placental unit was identified, amniotic sac opened, fetal pup removed, and umbilical vessels were ligated and unraveled as previously described [25, 26]. The placental unit was removed and placed into a modified isolated vessel chamber (Living Systems Instrumentation, Burlington, VT) filled with warmed (37 °C), oxygenated (21% O2 – 5% CO2 – 74% N2), circulating PSS. The placental vasculature (uterine artery and umbilical artery and vein) were secured to glass pipettes or 26 gauge, 4-inch stainless steel blunt needles, respectively. The uterine artery was perfused with a peristaltic pump at 80 mmHg and the umbilical artery was perfused at 50 mmHg. After a 30-minute equilibration and 10-minute baseline, a bolus of 900 µL (7.92 × 1014 particles/mL) of nanopolystyrene particles were slowly injected into the uterine artery. Effluents were collected and weighed from the distal uterine artery and umbilical vein cannula at 10-minute intervals for a total of 180 minutes. The remaining fluid within the stainless-steel needle cannulating the umbilical vein was collected.
Quantification of Nanopolystyrene particles: 25 µL of effluent from each sampling time point was pipetted in duplicate on a 96-well clear bottom plate. Positive control was identified as 25 µL of stock solution and negative control as PSS only. All samples were diluted by adding 100 µL of PSS into each well. Fluorescence was measured by a spectrophotometer at 546/575 nm (excitation/emission) using a SpectraMax M3 fluorescent microplate reader (Molecular Devices, Sunnyvale, CA). Data were collected using SoftMax Pro 6.3 software.
To confirm that the rhodamine tag remained attached to the polystyrene beads throughout the perfusion, maternal and fetal effluents were pooled together for 4 representative experiments. The samples were centrifuged at 100,000 x g for 1 hour in an ultracentrifuge (Beckman Coulter Max-XP tabletop Ultracentrifuge) to pellet polystyrene ENM. 25 µL of supernatant was removed from each sample and placed in a 96-well clear bottom plate and read at 546/575 nm (excitation/emission) using a SpectraMax M3 fluorescent microplate reader (Molecular Devices, Sunnyvale, CA). Data were collected using SoftMax Pro 6.3 software.
Histology: Representative placentas from the perfusion experiments were fixed in 10% neutral buffered formalin, processed and sectioned to 4 µm. Hematoxylin and eosin (H&E) stained slides were assessed by an ACVP board-certified veterinary pathologist.
Statistics: Outliers were identified as above or below 2 standard deviations from the mean and removed. All data were analyzed by Student’s T-test. Statistical significance was set to p < 0.05 and is indicated with an asterisk (*). Trends were identified as p < 0.10 and are indicated with a (T).