Prylutska S. Elaboration of technology to enhance cisplatin antitumor activity by using C60 fullerene

The dissertation is devoted to elaboration of the new biotechnological approaches to traditional antitumor drugs application in combination with C60 fullerene. Stable aqueous colloidal solution of C60 fullerene (0,15 mg/ml, 2×10-4 M), normal cells (Wistar rat thymocytes and erythrocytes), leukemia L1210 and CCRF-CEM cells, lung carcinoma LLC cells and transplanted model of Lewis lung carcinoma were used in the work. It was found that 10 μM C60 fullerene prevented both ROS production in thymocytes caused by 100 μM H2O2 or 3,3 μM Cis-Pt, H2O2-induced decrease of thymocytes viability and Cis-Pt-induced erythrocytes hemolysis. With the use of fluorescent-labeled C60 fullerene (C60-RITC) the accumulation of nanostructure in leukemic L1210 cells was shown. Immunocytochemical staining with the use of FITC-labeled antibodies to C60 fullerene demonstrated accumulation of C60 fullerenes in the mitochondria of CCRF-CEM leukemic cells. Photoexcitation (irradiation with light-emitting diode lamp, 410-700 nm) of C60 fullerene accumulated by sensitive and resistant to cisplatin leukemic L1210 cells was accompanied by significant increase of ROS production, [Ca2+]i increase, dissipation of mitochondrial membrane potential and cell accumulation in proapoptotic SubG1 phase. Combined action of photoexcited C60 fullerene and Cis-Pt in a low 3,3 μM concentration was followed by cisplatin toxic effect intensification in sensitive leukemic L1210 cells and by its restoration in cisplatin-resistant L1210/Cis-Pt cells. The observed effect was followed by the decrease of both sensitive and resistant leukemic cells viability due to the induction of apoptosis. The noncovalent and stable in aqueous medium nanocomplex of C60 fullerene with Cis-Pt (C60-Cis-Pt) was created and characterized for the first time. Complexation between the C60 and Cis-Pt molecules was confirmed by computer simulation, scanning tunneling microscopy and dynamic light scattering. The toxic effect of C60-Cis-Pt nanocomplex against Lewis lung carcinoma LLC cells was shown to be more effective compared to the effect of free cisplatin at equivalent concentration and was confirmed by cells accumulation in proapoptotic SubG1 phase, caspase 3/7 activation, increased content of cells at early and late stages of apoptosis, inhibition of cells migration activity. The acute toxicity of pristine C60 fullerene and C60-Cis-Pt nanocomplex was studied after intraperitoneal (i.p.) administration to mice. The high LD50 value for C60 fullerene (721 mg/kg) confirmed the absence of toxic effects at low dose of carbon nanostructure in the body of animals. The reduced toxicity of Cis-Pt (LD50=15,6 mg/kg) at complexation with C60 fullerene (LD50=36,1 mg/kg) at i.p. administration to mice was shown. C60-Cis-Pt nanocomplex at doses of 7,5 mg/kg (3,75 mg/kg of C60 and 3,75 mg/kg of Cis-Pt) more effectively inhibited the growth of transplanted Lewis lung carcinoma and metastases formation in mice lung tissue compared to free Cis-Pt in equivalent dose. The observed antitumor and antimetastatic effects of the C60-Cis-Pt nanocomplex were accompanied by prolongation of tumor-bearing mice’s life, normalization of body weight and blood parameters. The proposed technologies of C60 fullerene application both in combination with cisplatin after nanostructure’s photoexcitation and as a component of nanocomplex with cisplatin allowed to decrease the effective dose of cytostatic and to enhance its antitumor effect. The data obtained confirm the prospects of C60 fullerene as a photosencitizer in photodynamic therapy and as a targeted nanocarrier of antitumor drugs.