Toxicity of pristine graphene in experiments in a chicken embryo model

Ewa Sawosz,1 Slawomir Jaworski,1 Marta Kutwin,1 Anna Hotowy,1 Mateusz Wierzbicki,1 Marta Grodzik,1 Natalia Kurantowicz,1 Barbara Strojny,1 Ludwika Lipinska,2 André Chwalibog3 1Division of Nanobiotechnology, Warsaw University of Life Sciences, Warsaw, Poland; 2Institute of Electronic Materials Technology, Warsaw, Poland; 3Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Frederiksberg, Denmark Abstract: Evaluation of the potential cytotoxicity of graphene is a key factor for medical applications, where flakes or a surface of graphene may be used as bioactive molecules, drug carriers, or biosensors. In the present work, effects of pristine graphene (pG) on the development of a living organism, with an emphasis on morphological and molecular states of the brain, were investigated using a chicken embryo model. Fertilized chicken eggs were divided into the control group and groups administered with pG suspended in milli-Q water at concentrations of 50 µg/L, 100 µg/L, 500 µg/L, 1,000 µg/L, 5,000 µg/L, and 10,000 µg/L (n=30 per group). The experimental solutions were injected in ovo into the albumin and then the eggs were incubated. After 19 days of incubation, the survival, weight of the body and organs, and blood serum biochemical indices were measured. The brain samples were collected for microscopic examination of brain ultrastructure and measurements of gene and protein expression. Survival of embryos was significantly decreased after treatment with pG, but the body and organ weights as well as biochemical indices were not affected. In all treatment groups, some atypical ultrastructures of the brain were observed, but they were not enhanced by the increasing concentrations of pG. Expression of proliferating cell nuclear antigen at the messenger ribonucleic acid level was downregulated, and the number of proliferating cell nuclear antigen-positive nuc