HABILITATIONSSCHRIFT

 


Tierärztliche Hochschule Hannover / Bibliothek – University of Veterinary Medicine Hannover – Foundation / Library

 

Ulrike Taylor

 

Effects of metal nanoparticle exposure on

somatic and reproductive cells

 

NBN-Prüfziffer

urn:nbn:de:gbv:95-h2911

publication

Hannover, Tierärztliche Hochschule, Habilitationsschrift, 2018

text

http://elib.tiho-hannover.de/dissertations/h_taylor18.pdf

Summary

As their use in technical as well as biomedical applications surges, nanoparticle exposure, intended as well as unintended, has become a common occurrence. The purpose of the here presented work was to study the effects of gold and silver nanoparticles on various aspects of reproduction.

The particles used throughout this study were synthesized by laser ablation in water of a solid metal target. This method provides particles of highest purity, thus avoiding obtaining results biased by the remains of substances used in nanoparticle production.  Prior to investigations using reproductive cells and tissues, gold particles were tested using bovine immortalized endothelial cells, i.e. a somatic cell line, to gather first experiences in the handling, detection but also the toxic potential of gold nanoparticles. Regarding detection, the aim was to establish a tool, which allowed the differentiation of gold nanoparticles with routine laboratory equipment. Trials using a laser scanning confocal microscope proved that this method reliably detected single nanoparticles or nanoparticles agglomerates of 60 nm and larger outside, but also inside cells. Detection was facilitated by the surface plasmon resonance based light scattering abilities of the tested particles. The results obtained by this novel technique showed that the gold nanoparticles were readily incorporated into the somatic cells. In case of ligand-free AuNP, within 48 hours of exposure 75% of all cells were detected to have internalized the particles. Conjugation of the cell penetrating peptide penetratin sped up this process considerably, showing internalization of AuNP in 100% of cells within 2 hours. While uptake of ligand-free nanoparticles seemed to be driven by diffusion, penetratin conjugated particles were incorporated by endocytosis. Toxicological investigations indicated that a detrimental effect of gold nanoparticles on somatic cells is only to be expected if using a very high nanoparticle-to-cell-ratio.

In the following the impact of gold and silver nanoparticles on spermatozoa and oocytes was explored. Nanoparticle incorporation into spermatozoa was investigated using transmission electron microscopy, as sperm cells do not possess sufficient depth, i.e. are to flat, to employ a scanning confocal microscope for the differentiation of intra- and extracellular particles. Interestingly, regardless of surface modification, gold nanoparticles were found unable to penetrate membrane intact spermatozoa prior to acrosome reaction. However, ligand-free as well oligonucleotide-conjugated AuNP could be found attached to the outside of the sperm plasma membrane. Particles conjugated to only peptides or proteins were never found associated to the sperm surface. Yet, membrane association alone seemed to suffice to exert toxicologically relevant effects. Ligand-free as well as oligonucleotide conjugated gold nanoparticles led in a concentration of 10 µg/ml to a drop in sperm motility, probably via complexation of free thiols on the sperm surface. Furthermore, in vitro fertilization trials showed that ligand-free AuNP even caused a decrease in sperm fertilizing ability, most likely due to spacial interference of sperm-oocyte-interactions by nanoparticle aggregates, which unstabilized, ligand-free AuNP are inclined to form in saline solutions. Gold, silver and gold-silver-alloy nanoparticles conjugated to bovine serum albumin showed no toxic effect on spermatozoa.  Oocytes were tested regarding the uptake behavior and toxicity of gold, silver and gold-silver-alloy nanoparticles in conjunction with their cumulus cells, a type of somatic cells, which are closely associated to the oocyte until the end of oocyte maturation. Interestingly, pure gold nanoparticles were internalized exclusively by the oocytes, while even the slightest addition of silver led to a preferential incorporation of the particles into the cumulus cells. Regarding toxicological aspects AuNP remained inert, while silver containing particles led to an arrest in oocyte maturation in a concentration dependent manner.

The final aspect to be tested in the here presented work was the effect of gold and silver nanoparticles on early embryo development. For this purpose ligand-free particles were microinjected into one blastomere of a two-cell-stage murine embryo. This method ensured that only a minimum of variation occurred regarding the applied particle dose. It also allowed for an untreated internal control. Interestingly, the injected nanoparticles had no adverse affect on embryo development up to blastocyst stage. This observation was supported by the finding that developmentally relevant genes remained equally unaffected. While in case of AuNP this result was not unexpected, it was surprising with regard to silver nanoparticles. The reason might be the immediate formation of a protein corona around the silver nanoparticles after injection into the blastomere, preventing the leakage of toxic Ag+-ions.

In conclusion, we could show, that the reprotoxic potential of nanoparticles depends to a large part on chemical composition as well as surface modification. Especially regarding spermatozoa the conjugation to proteins to the particles led via the prevention of sperm surface association to a complete lack of toxicity even by the toxicologically potent silver nanoparticles. While embryos seemed to be very robust towards a challenge with gold and silver nanoparticles, oocytes reacted in a very sensitive manner. This indicates for once that this aspect of reproduction might be especially in danger of being disturbed by nanoparticle contact. However, due to its sensitivity, the fact that it operates with easily obtainable primary cells as well as the possibility to assess functional aspects, rather then mere vitality, it also provides an excellent tool to investigate the toxicology of any given nanoparticle.

keywords

Nanopartikel, Toxizität, Reproduktion, nanoparticle, toxicity, reproduction

kb

4.654