Nanobiotechnology for health-care 

Pre-treatment conditions determine the antitumor activity of nanoparticles
Goltsev A.N.1, Malyukin Y.V.2, Babenko N.N.1, Gaevskaya Y.A. 1, Bondarovich N.A.1, Dubrava T.G.1, Kovalenko I.F.1, Klochkov V.K.2
1 Institute for Problems of Cryobiology & Cryomedicine of the National Academy of Sciences of Ukraine, Pereyaslavska Str. 23, 61016 Kharkiv, Ukraine 
E-mail: cryo@online.kharkov.ua
2 Institutes for Scintillation Materials of the National Academy of Sciences of Ukraine, 60 Nauki ave., 61001 Kharkiv, Ukraine.

Our previous studies have demonstrated the inhibition of the Ehrlich carcinoma (EC) growth by means of nanoparticles (NPs) of orthovanadates of rare earth metals, it has been suggested that the incubation time of cells with nanomaterials in manifestation of this kind of effect is significant. The purpose of this study was to determine the term of incubation for the EC cells with NPs, which allows a maximal inhibition of tumor growth. Aqueous colloidal solutions of the NPs of spherical shape (1.3 g / l) were obtained by the method of V.K. Klochkov, 2015. The EC cells were incubated with NPs for 1, 2, 3 and 4 hours, and their accumulation in cells was estimated using confocal laser microscope LSM 510 META (Carl Zeiss, Germany). Treated with NPs EC cells were intraperitoneally injected to the Balb/C mice and after 7 days the intensity of the EC development by an absolute number of cells in peritoneal cavity (PC) was assessed. The content of tumorigenic subpopulations of CD44hi, CD44+ CD24- was determined with flow cytometer FACS Calibur (BD, USA).
After 1 hour of incubation, a slight increase in fluorescence intensity was detected in all the EC cells, indicating the onset of accumulation of NPs within the cells. By the 3rd hour of incubation the appearance of single cells (not more than 3%) with an ultrahigh level of fluorescence was noted. After a 3-hour incubation of NPs with  the EC cells, the maximal inhibition of tumor growth was found (about 58.2%), which was accompanied  with a 2-fold decrease  of  the  concentration of CD44hi and CD44+CD24- cells in the formed in the PC tumor, if compared to those in the animals with EC. Thus, the importance of the duration of incubating the cells with NPs to manifest their maximal antitumor effect is emphasized, which in this case was revealed at 3 hours pre-treatment of EC.