Fullerene C₆₀ Penetration into Leukemic Cells and Its Photoinduced Cytotoxic Effects

Fullerene C₆₀ as a representative of carbon nanocompounds is suggested to be promising agent for application in photodynamic therapy due to its unique physicochemical properties. The goal of this study was to estimate the accumulation of fullerene C₆₀ in leukemic cells and to investigate its phototoxic effect on parental and resistant to cisplatin leukemic cells. Stable homogeneous water colloid solution of pristine C₆₀ with average 50-nm diameter of nanoparticles was used in experiments. Fluorescent labeled C₆₀ was synthesized by covalent conjugation of C₆₀ with rhodamine B isothiocyanate. The results of confocal microscopy showed that leukemic Jurkat cells could effectively uptake fullerene C₆₀ from the medium. Light-emitting diode lamp (100 mW cm^-2, λ = 420-700 nm) was used for excitation of accumulated C₆₀. A time-dependent decrease of viability was detected when leukemic Jurkat cells were exposed to combined treatment with C₆₀ and visible light. The cytotoxic effect of photoexcited C₆₀ was comparable with that induced by H₂O₂, as both agents caused 50% decrease of cell viability at 24 h at concentrations about 50 μM. Using immunoblot analysis, protein phosphotyrosine levels in cells were estimated. Combined action of C₆₀ and visible light was followed by decrease of cellular proteins phosphorylation on tyrosine residues though less intensive as compared with that induced by H₂O₂ or protein tyrosine kinase inhibitor staurosporine. All tested agents reduced phosphorylation of 55, 70, and 90 kDa proteins while total suppression of 26 kDa protein phosphorylation was specific only for photoexcited C₆₀.The cytotoxic effect of C₆₀ in combination with visible light irradiation was demonstrated also on leukemic L1210 cells both sensitive and resistant to cisplatin. It was shown that relative value of mitochondrial membrane potential measured with tetramethylrhodamine ethyl ester perchlorate (TMRE) probe was lower in resistant cells in comparison with sensitive cells and the drop of mitochondrial potential corresponded to further decrease of resistant cell viability after C₆₀ photoexcitation. The data obtained allow to suggest that C₆₀-mediated photodynamic treatment is a candidate for restoration of drug-resistant leukemic cell sensitivity to induction of mitochondrial way of apoptosis.