149 UNIMPAIRED DEVELOPMENT OF MURINE EMBRYOS AFTER INJECTION OF SILVER NANOPARTICLES

Abstract

Because of their antibacterial properties, silver nanoparticles (AgNP) are used abundantly in medical products such as wound dressing and catheters, but also in consumer items like clothing and lining of food containers. Despite such widespread exposure and the fact that they are considered toxic to eukaryotic cells as well, knowledge concerning their reprotoxic potential is very limited. Therefore, the aim of the present study was to investigate the effects of AgNP on embryo development. The used AgNP were produced by laser ablation in liquids, which provides particles of high purity and stability. In order to ensure direct contact of the particle with the developing organism, 10 pL of an AgNP-dispersion in water (250 µM Ag; average particle diameter 15 nm) was microinjected into one blastomere of a two-cell-stage embryo (n = 91), derived from superovulated NMRI-mice. As controls, embryos were injected with water only (n = 74) or left untreated as a handling control (n = 102). Subsequently, in vitro culture commenced for 72 hours at 37°C and 5% CO₂ in KSOM plus 1% BSA. In order to distinguish whether possible effects are caused by the nanoparticles as such or by Ag⁺-ions released from the nanoparticles, additional embryos were co-incubated with silver nitrate (25 µM) during culture (n = 41). To exclude influence of the NO^–-ions on the embryo development, potassium nitrate controls (25 µM) were run as well (n = 40). The obtained blastocyst rates were compared using one-way ANOVA and a chi-square test. Day 4 blastocysts derived from the injected groups and the handling control were used for a real-time PCR analysis, to investigate gene expression of developmentally important genes (Bax, Bcl2l2, and Tp53 for apoptosis; Oct4 and Nanog for stemness, Dnmt3a for methylation). Embryo development was assessed on a daily basis. In the injected embryos, no abnormal development was observed. After injection with silver nanoparticles, the development rate reached 61.5%, which did not differ from the water-injected embryos, where 66.2% of the embryos developed to blastocysts. The handling controls reached a significantly higher blastocyst rate of 79.4% compared to both injected groups (P < 0.05). Interestingly, gene expression remained unaltered regardless of whether embryos were injected with AgNP, water, or not injected at all. Exposure to Ag⁺-ions resulted in an immediate arrest of development, while co-incubation of embryos with KNO showed no effect, reaching a blastocyst rate of 80.0%. In conclusion, the results do not indicate any negative effect of intracytoplasmatic injection of silver nanoparticles on murine embryos. Since the presence of Ag⁺-ions was shown to have a tremendously detrimental effect on the development, the nanoparticles do not seem to release silver ions in amounts sufficient to disturb this delicate process. Further studies involving gametes and embryos of large animal models are ongoing to intensify our understanding of silver nanoparticle reprotoxicity.

Supported by DFG Grant SPP1313 and REBIRTH.