Interaction of Nanoparticles and Zona Pellucida of Early Mammalian Embryo In Vitro
Maria Kormacheva1*, Elena Perevedentseva1, Artashes Karmenyan1, Alexander Krivokharchenko2, Chia-Liang Cheng1
1Physics, National Dong Hwa University, Hualien, Taiwan
2N.N. Semenov Institute of Chemical Physics, RAS, Moscow, Russian Federation
* presenting author:Maria Kormacheva, email:kormacheva.marie@gmail.com
In vitro fertilization (IVF) is grown rapidly with the advantage of opportunity to diagnose some disease on the stage of preimplantation embryo such as genetic abnormalities and genetic predisposition to some heavy disease. There is number of methods to retrieve the potential for proper development, increasing the chance of pregnancy. The unique physicochemical properties of nanoparticles could help to implement drug delivery into this micro system, mammalian embryo, and expand the scopes of IVF.

Over the past years nanoparticles have attracted attention due to their unique physicochemical properties, which are promising for biomedical application [1] as a drug carriers, biosensors, coating for medical implants and more. Today industry invents various surface functionalizations to customize nanomaterials for different application. Despite of all advantages of nanoparticles the biological impact is still not properly studied. Our work aims to understand the fundamental basis of interaction between thick membrane surrounding a mammalian embryo before implantation, called zona pellucida, and different nanoparticles. It includes the test of toxicity, the probable penetration inside the mammalian embryo along with chemical reaction with zona pellucida.

For that purpose we used embryos from mice. The 2-cell stage embryos have been recovered from female mice of C57bl/6 strain. Nanoparticles have been added into culture medium and development of the in vitro cultivated embryos was evaluated during 72 h. The developmental rate has been compared with control embryos. All embryos were cultivated in 4-well plates with culture medium in incubator at 37°C with 5% of carbon dioxide. Some nanoparticles were examined, such as different kinds of nanodiamond (ND), titanium dioxide particles of different structure, etc., with various concentrations. These kinds of nanoparticles have been selected due to previously demonstrated non-toxicity for cellular models and their optical-spectroscopic properties promising for imaging applications and some bio-optical treatments [2, 3]. NP was added into the medium with 2-cell embryos in concentrations, the highest equaled 200 μg/ml.

The interaction of the nanoparticles with the embryos was analyzed via the confocal fluorescence microscopy (with confocal scanning microscope TCS SP5, Leica, Germany). Due to spectroscopic properties of the used nanoparticles their localization on/in the embryo could be analyzed via their fluorescence. No disturbing effect of these nanoparticles in applied concentrations neither well-observable penetration of the nanoparticles into the embryos has been detected. It opens more wide opportunities for nanobioapplications.
Difference in the interaction of embryos with different nanoparticles was observed: some particle barely lay over the zona pellucida, and some completely cover it.

Acknowledgements
The research was supported by Ministry of Science and Technology of Taiwan with grant No. 103-2112-M-259-001-MY3.

References
1. K. Riehemann, S. W. Schneider, T. A. Luger, B. Godin, M. Ferrari, and H. Fuchs, Angew. Chem. Int. Ed., 2009, 48, 872 – 897.
2. E. Perevedentseva, Y.-C. Lin, Mona Jani, and C. - L. Cheng, Future Medicine. Nanomedicine, 2013, 8(12) 2041-2060.
3. E. Perevedentseva, D. Peer, V. Uvarov, B. Zousman, and O. Levinson, J. Nanoscience & Nanotechnology, 2015, 15(2), 1045-1052.


Keywords: mammalian embryo, nanoparticles, zona pellucida, nanodiamond