Injection of ligand-free gold and silver nanoparticles into murine embryos does not impact pre-implantation development

Abstract

Intended exposure to gold and silver nanoparticles has increased exponentially over the last decade and will continue to rise due to their use in biomedical applications. In particular, reprotoxicological aspects of these particles still need to be addressed so that the potential impacts of this development on human health can be reliably estimated. Therefore, in this study the toxicity of gold and silver nanoparticles on mammalian preimplantation development was assessed by injecting nanoparticles into one blastomere of murine 2 cell-embryos, while the sister blastomere served as an internal control. After treatment, embryos were cultured and embryo development up to the blastocyst stage was assessed. Development rates did not differ between microinjected and control groups (gold nanoparticles: 67.3%, silver nanoparticles: 61.5%, sham: 66.2%, handling control: 79.4%). Real-time PCR analysis of six developmentally important genes (BAX, BCL2L2, TP53, OCT4, NANOG, DNMT3A) did not reveal an influence on gene expression in blastocysts. Contrary to silver nanoparticles, exposure to comparable Ag(+)-ion concentrations resulted in an immediate arrest of embryo development. In conclusion, the results do not indicate any detrimental effect of colloidal gold or silver nanoparticles on the development of murine embryos.

Keywords: biomedical application; confocal microscopy; gene expression; protein corona; toxicity.

Figure 1

Number distribution of nanoparticle size including corresponding TEM micrographs as inserts of (A) gold nanoparticles and (B) silver nanoparticles.

Figure 2

Representative laser scanning microscope images of murine embryos (projections of 10 optical sections (1 µm each)) after the injection of gold nanoparticles (10 pL of a 50 µg/mL nanoparticle dispersion, equal to 1000 nanoparticles/embryo, mean primary particle diameter as determined by TEM: 11 nm). An overlay of the differential interference contrast (DIC) merged with the gold nanoparticle detection channel is shown. Upper panels – Two-cell-stage embryos: (A) handling control, (B) embryo shortly after AuNP injection, one nanoparticle is located in the zona pellucida, highlighting the injection canal, (C) z-axis of projection (B); Lower panels – Day-four-blastocysts: (D) handling control, (E) 3 days after AuNP injection, (F) z-axis of (E). AuNP appear in red, some of which are exemplarily pointed out with arrows.

Figure 3

Representative stereo microscope images of murine blastocysts (A) after silver nanoparticle-injection (10 pL of a 50 µg/mL nanoparticle dispersion, equal to 3300 nanoparticles/embryo, mean primary particle diameter as determined by TEM: 21 nm), (B) untreated handling control, (C) deteriorated 2-cell-stage embryos after co-incubation with silver ions (25 µM AgNO₃).

Figure 4

Gene expression after normalization based on globin/beta-actin transcript abundance. Values are mean ± SD.

Figure 5

Beta actin expression after normalization with globin. Values are mean ± SD.