Nanochemistry and biotechnology

The kinetics of hepatocytes autofluorescence at interaction with GdVO4:Eu3+ nanoparticles

Kot Yu.1, Kot K.1, Lukan R.1, Yehemberdinov Ye.1, Klochkov V.2, Kavok N.2, 
Yefymova S.2, Malyukin Yu2, 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.

Spherical GdVO4:Eu3+ nanoparticles (diameter 1-2 nm) were synthesized in the Nanocrystal Materials Department of Institute for Scintillation Materials of National Academy of Sciences of Ukraine. The ability to penetrate into cells and autofluorescent properties make them very promising for use in visualization or indications of the structural and functional state of the cell. During the investigation of the fluorescence spectra of cells under penetration of nanoparticles into them, it was found that the autofluorescence of cells changes. The aim of the research was to study the hepatocytes auto-fluorescence kinetics under interaction with nanoparticles.
The study was performed on isolated hepatocytes of female Wistar rats of 3 months old with a viability of 89%. The nanoparticles were introduced into the cell suspension at a concentration of 12.5 μg/104 cells. The cell suspension was analyzed on a Zeiss LSM510 META confocal scanning microscope. The spectral characteristics of the suspension were obtained every 2 minutes for 150 minutes. The data was analyzed using Zeiss LSM Image Examiner and Bitmap Imaris software.
At an excitation wavelength of 405 nm, autofluorescence of hepatocytes in blue (459-470 nm), green (524-545 nm) and red (620-631) channels was observed. A three-dimensional analysis of the fluorescence solocalization in the cell through all three channels showed that the autofluorochromes are not localized in the plasma membrane and nucleus region, but are distributed in the space between them. 
When interacting with nanoparticles, there was an increase in autofluorescence in all three channels, compared with native cells, and after reaching a maximum, a decrease to the control level. However, these changes for each wavelength range have their own characteristics. The maximum of autofluorescence in the "blue" and "red" channel (the 25th minute of observation) occurs much earlier than in the "green" channel (the 33rd minute of observation). Attenuation of fluorescence to the control value for the "blue" and "red" channel occurs earlier (65th minute of observation) than for the "green" channel (73rd minute of observation). For further observation periods, the values of autofluorescence in all three spectral ranges did not differ from native cells. Analysis of the literature made it possible to correlate the spectral characteristics of the observed autofluorescence and intracellular metabolites-autofluorochromes.
The range of the observed autoflorescence of 459-470 nm is comparable with the maximum of the autofluorescence spectrum of reduced pyridine nucleotides NADH and NADPH (455-470 nm), and the range of 524-545 nm – with the maximum of the autofluorescence spectrum of oxidized flavoproteins FMN and FAD (520-530 nm). Considering also that the ratio of the relative fluorescence of these channels varies from 1.0 to 1.7 from the 20th to 33rd minutes of incubation, it can be said that in hepatocytes, when incubated with nanoparticles, the fluorescence intensity of NADH decreases and of FAD increases at this time, which indicates a decrease in energy processes in the system, and a growing high cell requirement in ATP during the observation period. Thus, with the help of GdVO4:Eu3+ nanoparticles (diameter 1-2 nm), which have a highly active surface and redox activity, it is possible to regulate the pathways of energy metabolism of the cell. 
Acknowledgements. The authors express their gratitude to the scientific researcher of The Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine Igor Kovalenko for the opportunity to work on a confocal microscope and for consultations in processing the data obtained on it.