Nanobiotechnology for health-care

The effect of cerium dioxide nanoparticles on the oxidative stress parameters in gamma-irradiated skin fibroblasts 3D-culture 

Kot Yu.1, Kot K.1, Filonenko S.1, Polonska A.1, Klochkov V.2, Kavok N.2, Yefymova S.2, Malyukin Yu.2, Perskyi Ye.1 

1 V. N. Karazin Kharkiv National University, biochemistry department, 4 Svobody Sq., Kharkiv, 61022, Ukraine.
E-mail: kot.juriy@gmail.com
2 Institute for Scintillation Materials, Nanocrystal Materials Department, Nauky ave. 60, Kharkiv, 61072, Ukraine.

Cerium dioxide nanocrystals (CeO2) are today one of the most promising classes of antioxidant materials. 
The aim of the study was to substantiate experimentally in vitro the possibility of using CeO2 nanoparticles as an antioxidant for cell therapy in radiation burns.
Spherical CeO2 nanoparticles (D=1-2 nm) were synthesized in the Nanocrystal Materials Department of Institute for Scintillation Materials of National Academy of Sciences of Ukraine.
The work was carried out on 3D-culture of skin fibroblasts of male Wistar rats of 3 months old. The isolated and subcultured until passage 3 cells (DMEM+ 10% FBS, 370С, 5%СО2) with viability of 89% (Invitrogen Countess) were seeded on a three-dimensional matrix (Alvetex Scaffold, 96 well) and cultured for 8 days until the culture density of 90%. 1 hour prior to irradiation, a suspension of nanoparticles was added to the culture medium to a final concentration of 1.72 μg/L (10nM), 3.44 μg/L (20nM), 6.88 μg/L (40nM), and incubated for 1 hour.
After replacing the medium with nanoparticles free one, the cells were irradiated on the gamma complex Rokus AM (Co60). The dose of irradiation was 0.75 Gy (104kV, 9mA, 0.2 Gy/min, duration of irradiation – 225 sec).
Two and six hours after irradiation, the fluorometric determination of the concentration of reactive oxygen species (DCFDA, Ex/Em=495/529 nm), lipid hydroperoxides (MitoPeDPP, Ex/Em=452/470 nm), determination of the degree of DNA fragmentation by comet analysis (CASPlab software), spectrophotometric determination of the enzymatic activity of superoxide dismutase and catalase (Abcam) were carried out in the fibroblasts culture.
It was shown that in the absence of irradiation, nanoparticles at concentrations of 1.72 and 3.44 μg/L do not cause changes in the studied parameters at all investigation times. The concentration of the nanoparticles 6.88 μg/L caused a decrease in cell viability by 4% after 6 hours of cultivation on a background of absence of changes in other parameters. This concentration also proved to be ineffective for maintaining the vitality of cells after irradiation - the number of living cells decreased both in the presence and absence of nanoparticles after 6 hours of cultivation by 31%. Pre-incubation with nanoparticles at a concentration of 1.72 and 3.44 μg/L resulted in an increase in the number of living cells in culture 6 hours after irradiation by 14% and 18%, respectively, compared with irradiated cells. 
In 2 and 6 hours after irradiation, the concentration of ROS and lipid hydroperoxides increased by 5.3 and 7.7 times, respectively, in the culture, and the degree of fragmentation of nuclear DNA by 5.0 times. In this case, the activity of SOD and catalase decreased by 6 hours of observation by 2.8 and 4.7 times, respectively. 
Pre-incubation with CeO2 nanoparticles at all used concentrations resulted in a significant decrease in prooxidant indices in irradiated culture. Herewith, the most corrective effect was provided by the concentration of 3.44 μg/L – for 6th hour of cultivation after irradiation, the concentration of ROS and lipid hydroperoxides was respectively 40% and 27% less, and the activity of SOD and catalase was 64% and 123% higher than in irradiated culture without nanoparticles. At the same time, regardless of the concentration of nanoparticles, the degree of DNA fragmentation remained at the level of irradiated cells, which apparently is either a consequence of long-term effects of ROS overproduction or the inability to fix defects by cell repair systems over the past observation time.
Thus, spherical nanoparticles CeO2 (D=1-2 nm) at a concentration of 3.44 μg/L can be used in cell therapy of skin lesions to normalize the prooxidant-antioxidant balance.
Acknowledgements. The authors express their gratitude to to the chief doctor Vasiliev L.Ya., to the doctor-radiologist Vasilyev L.L., and to the doctor-roentgenologist Trofimov A.V. for the consultations and for the provided technical possibility of irradiation on the basis of Grigoriev Institute for medical Radiology NAMS of Ukraine.