Nanoparticles and chemicals
Negatively charged 20 nm ‘aged’ silver sulfide nanoparticles (Ag2S NPs) suspensions in milli-Q water were obtained from Applied Nanoparticles (Barcelona, Spain) with a mass concentration of 4.7 g/L. Three types of 50 nm silver nanoparticles with different surface modifications were purchased from Nanocomposix Inc. (San Diego, CA, USA); negatively charged lipoic acid BioPure (LA) AgNPs (pH = 6.1) suspended in milli-Q water, negatively charged citrate BioPure (Cit) AgNPs (pH = 7.4) suspended in 2 mM citrate buffer, and positively charged branched polyethylenimine (BPEI) BioPure AgNPs (pH = 7.0) suspended in milli-Q water. The silver mass concentration in the stock suspensions of the three AgNPs was 1 g/L. All AgNPs suspensions were stored at 4 °C in the dark. Silver nitrate (AgNO3) (Sigma Aldrich; St. Louis, MO, USA) was used as a control (source of Ag+) in all experiments. Dilutions of the AgNPs or AgNO3 were freshly prepared for every exposure experiment in complete cell culture media (depending on the cell line used in the experiment). 5-Fluorouracil (5-FU) was purchased from Sigma-Aldrich.
Physicochemical characterization of nanoparticles
Hydrodynamic diameters of the AgNPs were determined using dynamic light scattering (DLS). Measurements were performed on samples containing 10 mg/L AgNPs suspended in nano-pure water using an ALV dynamic light scattering setup (ALV-Laser Vertriebsgesellschaft; Germany), consisting of a Thorn RFIB263KF photomultiplier detector, an ALV-SP/86 goniometer, an ALV 50/100/200/400/600 μm pinhole detection system, an ALV7002 external correlator, and a Cobolt Samba-300 DPSS laser. The measurements were performed immediately after preparation at room temperature. For each condition, samples were analyzed in triplicate; each measurement consisted of 10 technical replicate-measurements of 30 s each, at an angle of 90°. The results are expressed as the average hydrodynamic diameter (nm) ± standard deviation (SD) that was calculated using AfterALV software (AfterALV 1.0d, Dullware; USA).
The total silver content of the AgNPs suspensions and AgNO3 solution was analyzed using a NexION 350D inductively coupled plasma mass spectrometer (ICP-MS) (PerkinElmer, Waltham, MA, USA). Before analysis, samples were digested using an aqua-regia (1,3, 70% HNO3: 37% HCl) acid digestion for 30 min at 60 °C and diluted with nano-pure water. Silver was measured using the selected element-monitoring mode with m/z values of 107 and 109. A calibration curve of an ionic Ag standard (NIST-AgNO3) (Merck; Darmstadt, Germany) ranging from 0.1 to 50 μg/L was included. Rhodium (Merck) was used as an internal standard. The limit of detection (LODconc) and limit of quantification (LOQconc) were estimated to be 2 and 6 ng/L, respectively. The cell culture media (vehicle controls) did not contain detectable levels of Ag. All samples were analyzed in triplicate.
The particle sizes, size distributions, particle mass- and number-based concentrations of the AgNPs in the AgNPs suspensions and AgNO3 solution were quantified using single particle (sp) ICP-MS. The method for the spICP-MS measurements was described previously  . Briefly, the sample flow rate to the nebulizer was determined before the start of each series of measurements. The dwell time was set at 3 milliseconds and the total acquisition time was set at 60 s. A diluted suspension of 60 nm gold (Au) NPs (Nanocomposix) with a mass concentration of 50 ng/L was used before each analysis to verify the performance of the ICP-MS and to determine the transport efficiency. A calibration curve of ionic silver (NIST-AgNO3) with a concentration range of 0.1–20 μg/L was used for particle mass and size determination. The time scan data of the spICP-MS measurements were exported as .csv files, and the particle size, size distribution, and mass- and number-based concentrations were calculated from the spICP-MS data, using a dedicated spreadsheet. Details about the spreadsheet have been described previously . The LODconc and LOQconc were estimated to be 20 and 67 ng/L, respectively. The NP size was calculated based on the particle mass, assuming spherical particles. The size detection limit (LODsize) was 20 nm and accordingly silver particles with sizes below this limit were included in the ionic silver fraction.
ES-D3 adherent mouse embryonic multipotent stem cells (mESCs; ATCC; Wesel, Germany) were used at passage numbers between 4 and 12. The cells were cultured and maintained in 25 cm2 cell culture flasks (Corning; Oneonta, NY, USA) coated with 0.1% gelatin at 37 °C in a humidified 5% CO2 atmosphere in complete cell culture medium (DMEM+). DMEM+ was prepared by supplementing HyClone AdvanceSTEM Low Osmo Dulbecco’s modified Eagle’s medium (DMEM) culture medium (GE Healthcare Life Sciences; USA) with 20% (v/v) heat inactivated Fetal Bovine Serum (FBS) (ATCC; Manassas, VA, USA), 1% (v/v) Penicillin-Streptomycin-Glutamine 10,000 units penicillin, 10 mg streptomycin/mL, and 29.2 mg/mL L-glutamine (Gibco, Life Technologies; Paisley, UK). The cells were sub-cultured every 2–3 days using non-enzymatic cell dissociation solution (Sigma Aldrich) to detach the cells. The cells were maintained in an undifferentiated state by adding mouse leukemia inhibitory factor (mLIF; Sigma-Aldrich).
The adherent placental choriocarcinoma clone b30 (BeWo b30) was kindly provided by the Institute of Public Health of the Faculty of Health Sciences (University of Copenhagen, Denmark) with permission from Dr. Alan Schwartz (Washington University, St. Louis, MO) and confirmed to be mycoplasma free. The cells were used at passage numbers between 14 and 22. The cells were cultured and maintained in 75 cm2 cell culture flasks (Corning) at 37 °C in a humidified 5% CO2 atmosphere in complete cell culture medium (DMEM+). The DMEM+ was prepared by supplementing DMEM culture medium-GlutaMAX supplement-pyruvate (Gibco, Life Technologies) with 10% (v/v) heat inactivated FBS (Gibco, Life Technologies), 1% (v/v) Penicillin-Streptomycin 10,000 units penicillin, and 10 mg streptomycin/mL (Gibco, Life Technologies). The cells were sub-cultured every 3–4 days using trypsin-EDTA (Sigma Aldrich) to detach the cells.
Cytotoxic effects of the AgNPs and AgNO3 were evaluated using the ATPlite luminescence assay system (PerkinElmer; Waltham MA, USA). In 96-well black flat bottom plates (Greiner bio-one; Frickenhausen Germany) each well was seeded with 100 μL of 1 × 105 cells/mL BeWo b30 cell suspension in DMEM+. Plates were incubated at 37 °C and 5% CO2 for 24 h. The attached cells were then washed with 100 μL/well pre-warmed HBSS buffer w/o phenol red and exposed to 100 μL/well of freshly prepared dilutions (0.1–100 mg/L) of AgNPs or AgNO3. After 24 h exposure, the exposure medium was aspirated and 50 μL/well of mammalian cell lysis solution were added and the plates were shaken (700 rpm) for 5 min at room temperature in an orbital shaker (Heidolph-Trimax 1000; Schwabach, Germany). Next, 50 μL/well of substrate solution were added and the plates were shaken (700 rpm) for 5 min at room temperature and then incubated for 10 min in the dark at room temperature. The luminescence was then measured using a plate reader (BioTek Synergy™ HT Multi-Mode Microplate reader; Winooski VT, USA). The cell viability was expressed as percentage of the control. DMEM+ was used as a negative control and Triton-X100 (0.25%) (Sigma-Aldrich) was used as a positive control.
BeWo b30 placental cell layer barrier integrity assessment
BeWo b30 cells were grown at a density of 1 × 104 cells/cm2 on the upper side of transwell polycarbonate inserts (3 μm pore size, 1.12 cm2 surface area) (Corning) for 6 days (based on . The integrity of the cell layer was assessed before exposure by measuring the transepithelial electrical resistance (TEER) using a Millicell ERS-2 Epithelial Volt- Ohm Meter (Millipore; Darmstadt, Germany). On day 6 post-seeding, only inserts with TEER values between 80 and 100 Ω.cm2 were used for further experiments. The TEER was also measured after exposure to confirm the barrier integrity and comparability of the TEER values before and after exposure.
Additionally, the cell layer integrity was evaluated before exposure to AgNPs suspensions and AgNO3 solution by measuring the transport efficacy of three different markers namely; lucifer yellow (LY) (Sigma-Aldrich) and low (4 kDa) and high (10 kDa) molecular weight fluorescein isothiocyanate dextrans (FITC-D) (Sigma-Aldrich). To the apical compartment, 500 μL/insert of 1 mg/mL of each of the integrity markers in DMEM+ were added separately. After 1 h incubation at 37 °C, the basolateral medium was collected, and the transport of the markers was determined by measuring the fluorescence at 485/530 nm using a fluorescence plate reader (BioTek Synergy™ HT Multi-Mode Microplate reader) and expressed as a percentage of the exposure concentration.
BeWo b30 cellular uptake/association and transport of AgNPs and AgNO3
Six-day old BeWo b30 cell layer were exposed apically to 500 μL/insert of 1 mg/L of the AgNPs suspensions or the AgNO3 solution for 4, 6, 18, and 24 h at 37 °C and 5% CO2. Then, the media from the apical and basolateral compartments were collected. The cells were collected by trypsinzation (500 μL) and sonication (40 kHz for 15–20 min) to form cell lysate. The total silver content in all samples (apical, basolateral and cell lysate) was analyzed using ICP-MS. The particle size, size distribution, and mass- and number-based concentration in in all samples (apical, basolateral and cell lysate) after 24 h exposure using spICP-MS. The total Ag mass balance in the placental transport model upon exposure to AgNPs and AgNO3 was > 90%.
In vitro developmental toxicity assessment of AgNPs and AgNO3
For the assessment of cell viability of mESCs used in the EST, the potential cytotoxicity of AgNPs and AgNO3 was evaluated after 24 and 120 h exposure, reflecting the shortest and longest exposure time during the EST. Each well was seeded with 100 μL of a 2 × 104 (for the 24 h exposure) or 1 × 103 cells/mL (for the 120 h exposure) cell suspension in DMEM+ without mLIF in 96-well black flat bottom plates (Greiner bio-one). The viability was assessed as mentioned above.
The potential of the AgNPs and AgNO3 to inhibit mESCs differentiation into beating cardiomyocytes was evaluated using the EST. The cells were exposed from day 3 to 10 of the 10-day mESCs differentiation. The wells of the 96-well plate (Corning) were filled with 200 μL/well PBS. To start the assay hanging droplets (20 μL) of a 3.75 × 104 cells/mL mESCs suspension were placed on the inner side of a lid of a 96-well plate. Sterile lids of Eppendorf tubes were placed on each corner of the 96-well plate lid to avoid contact of the droplets with the plate. Then the plate was sealed with Micropore tape (3 M; Germany) to prevent evaporation of the hanging drops. The plates were incubated for three days at 37 °C and 5% CO2 in a humidified atmosphere to allow the formation of embryoid bodies (EBs). On day 3, the formed EBs were transferred to 6 cm non-treated tissue culture petri dishes (Greiner bio-one) with 5 mL of DMEM+ containing AgNPs or AgNO3 and incubated at 37 °C and 5% CO2 in a humidified atmosphere for 2 days to allow growth of the EBs. A concentration range between 0.1–25 mg/L was used for each type of AgNPs, while for AgNO3 a concentration range between 0.05–5 mg/L was used. DMEM+ was used as a negative control and 1 μM 5-FU was used as a positive control. On day 5, the EBs were transferred to a 24-well plate (Corning) where each well contained one EB in 1 mL of the same concentration of AgNPs or AgNO3. On day 10, using a light microscope, the wells were visually inspected for contracting cardiomyocytes. The number of wells/plate containing contracting cardiomyocytes were recorded where the experiment was considered valid if at least 21 of the 24 wells of the negative control sample contained contracting cardiomyocytes. For each concentration of each treatment, the fraction of successfully differentiated EBs into contracting cardiomyocytes was calculated and expressed as percentages of the number of wells with beating cardiomyocytes from the number of wells initially seeded with EBs for each concentration.
Characterization of AgNPs dissolution in DMEM+
The stability and dissolution properties of the four AgNPs were evaluated in 1 mg/L AgNPs suspensions in DMEM+ upon incubation in the dark at 37 °C. At 0 and 120 h, aliquots from each AgNPs suspension were extracted for analysis. spICP-MS was used to quantify the total Ag content, particle size, size distribution, and mass- and number-based concentration in all samples as described earlier.
Confocal microscopy of differentiated cardiomyocytes
For confocal imaging, the EBs formed in the EST were transferred into 8 - well μ-Slides (Ibidi; Gräfelfing, Germany) for exposure and differentiation into cardiomyocytes. The EBs were exposed to the AgNPs suspensions or AgNO3 solution using similar conditions as used in the EST. After 5 days exposure, the exposure medium was discarded, and the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 15 min at room temperature. The cells were washed 3 times with PBS for 5 min after discarding the fixation solution. The cells were permeabilized with 0.25% Triton X-100/PBS for 15 min at room temperature. The cells were then washed again 3 times with PBS for 5 min before incubating them with the blocking buffer (1%BSA in PBS) for 30 min at room temperature. After discarding the blocking buffer, Phalloidin - Alexa 488 (6 units) (Dyomics; Jena, Germany) was added to stain cellular actin and the cells were incubated for 30 min at room temperature. Cells were washed three times with PBS before incubating the cells for 10 min at room temperature with RedDot-2 (1: 200) (Biotium; Fremont, CA, USA) to stain the nuclei. Finally, the cells were washed with PBS and stored in the dark until analysis. The cells were analyzed using a confocal laser scanning microscope (SP5X-SMD; Leica Microsystems, Wetzlar, Germany). Samples were excited with 665 and 495 nm lasers and backscattered light was used to detect AgNPs using a 543 nm laser.
Each data point represents the average of three replicates (n = 3) and the results are shown as mean ± standard deviation. Prism (v.8.0.1; GraphPad, USA) software was used for statistical analysis using a one-way analysis of variance (ANOVA) with a Bonferroni’s post-test. A p-value < 0.05 was considered significant.