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

Abstract

Background: Silver 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.

Results: The 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.

Conclusions: Pristine 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.

Keywords: Embryotoxicity; Placental transport; Silver nanoparticles; Single particle-ICP-MS; Surface chemistry.

Fig. 1

Concentration dependent effect of (LA), (Cit), (BPEI) AgNPs, Ag₂S NPs, and AgNO₃ on the viability of BeWo b30 cells after 24 h exposure quantified using the ATPlite viability assay. Viability is given as a percentage of the control (mean ± SD; n = 3)

Fig. 2

Total silver content in the a apical, b cell lysate, and c basolateral compartments of the BeWo b30 placental transfer model after 4, 6, 18 and 24 h exposure to 1 mg/L of (LA), (Cit), (BPEI) AgNPs, Ag₂S NPs, or AgNO_3, measured using ICPMS. Concentrations are given as the mean ± SD (n = 3). Values with different letters are significantly different within the same treatment (p ≤ 0.05)

Fig. 3

Total silver content (on the left axis) versus the AgNPs content (mass-based on the right axis) in the a apical, b cell lysate, and c basolateral compartments of the BeWo b30 placental transfer model after 24 h exposure to 1 mg/L of (lA), (Cit), (BPEI) AgNPs, Ag₂S NPs, or AgNO₃, measured using spICP-MS. Concentrations are given as the mean ± SD (n = 3). Values with different letters are significantly different within the same treatment (p ≤ 0.05)

Fig. 4

Number-weighted size distributions of AgNPs generated by spICP-MS measurements of the suspensions (1 mg/L) of: a (LA) AgNPs, b (Cit) AgNPs, c (BPEI) AgNPs, d Ag₂S NPs, and e AgNO₃. Number-weighted size distributions of AgNPs in the apical compartments of the BeWo b30 monolayer model upon 24 h exposure to 1 mg/L of: f (LA) AgNPs, g (Cit) AgNPs, h (BPEI) AgNPs, i Ag₂S NPs, and j AgNO₃. Number-weighted size distributions of AgNPs in the cellular compartments of the BeWo b30 monolayer model upon 24 h exposure to 1 mg/L of: k (LA) AgNPs, l (Cit) AgNPs, m (BPEI) AgNPs, n Ag₂S NPs, and o AgNO₃. Number-weighted size distributions of AgNPs in the basolateral compartments of the BeWo b30 monolayer model upon 24 h to 1 mg/L of: p (LA) AgNPs, q (Cit) AgNPs, r (BPEI) AgNPs, s Ag₂S NPs, and t AgNO₃

Fig. 5

Concentration–response curves for cytotoxicity towards mESCs and for the effect on differentiation into contracting cardiomyocytes of: a (LA) AgNPs, b (Cit) AgNPs, c (BEPI) AgNPs, d (Ag₂S) NPs, and e AgNO_3. The viability of mESCs (right y-axis) was assessed using the ATPlite assay after 24 h and 120 h of exposure. The differentiation of mESCs into contracting cardiomyocytes (left y-axis) was scored after microscopical evaluation. Values are given as a percentage of the control (mean ± SD; n = 3)

Fig. 6

Confocal microscopic images of differentiated cardiomyocytes after 10 days of differentiation. a Negative control, b cells exposed to 1 mg/L (LA) AgNPs, c (Cit) AgNPs, d (BEPI) AgNPs and e Ag₂S NPs. Nuclei were stained in red (RedDot-2), actin was stained in green (Alexa − 488 Phalloidin), and AgNPs are shown in white (back scatter)