Recently, much attention has been paid to the bioactive properties of water-soluble fullerene derivatives: fullerenols, with emphasis on their pro- and antioxidative properties. Due to their hydrophilic properties and the ability to scavenge free radicals, fullerenols may, in the future, provide a serious alternative to the currently used pharmacological methods in chemotherapy, treatment of neurodegenerative diseases, and radiobiology. Some of the most widely used drugs in chemotherapy are anthracycline antibiotics. Anthracycline therapy, in spite of its effective antitumor activity, induces systemic oxidative stress, which interferes with the effectiveness of the treatment and results in serious side effects. Fullerenols may counteract the harmful effects of anthracyclines by scavenging free radicals and thereby improve the effects of chemotherapy. Additionally, due to the hollow spherical shape, fullerenols may be used as drug carriers. Moreover, because of the existence of the currently ineffective ways for neurodegenerative diseases treatment, alternative compounds, which could prevent the negative effects of oxidative stress in the brain, are still sought. In the search of alternative methods of treatment and diagnosis, today's science is increasingly reaching for tools in the field of nanomedicine, for example, fullerenes and their water-soluble derivatives, which is addressed in the present paper.

The present study was aimed at investigating the effect of fullerenol C60(OH)36 on chosen parameters of the human erythrocyte membrane and the preliminary estimation of the properties of fullerenol as a potential linking agent transferring the compounds (e.g., anticancer drugs) into the membrane of erythrocytes. The results obtained in this study confirm the impact of fullerenol on erythrocyte cytoskeletal transmembrane proteins, particularly on the band 3 protein. The presence of fullerenol in each of the concentrations used prevented degradation of the band 3 protein. The results show that changes in the morphology of red blood cells caused by high concentrations of fullerenol (up to 150mg/L) did not lead to increased red blood cell hemolysis or the leakage of potassium. Moreover, fullerenol slightly prevented hemolysis and potassium efflux. The protective effect of fullerenol at the concentration of 150mg/L was 20.3%, and similar results were obtained for the efflux of potassium. The study shows that fullerenol slightly changed the morphology of the cells and, therefore, altered the intracellular organization of erythrocytes through the association with cytoskeletal proteins.

The influence of fullerenol on the activities of human erythrocyte membrane ATPases and the fluidity of the plasma membrane as well as the possibility of fullerenol incorporation into the plasma membrane were investigated. Fullerenol at concentrations up to 150 μg/mL induced statistically significant decreases in the anisotropy of 1-anilino-8-naphthalene sulfonate (ANS) (14%), N,N,N-trimethyl-4-(6-phenyl-1,3,5,-hexatrien-1-yl)phenylammonium p-toluenesulfonate (TMA-DPH) (7.5%) and 1,6-diphenyl-1,3,5-hexatriene (DPH) (9.5%) after a 1-hour incubation at 37°C. The effect disappeared for ANS and TMA-DPH, but not for DPH, after washing out the fullerenol. Incubation of erythrocyte membranes with fullerenol led to decreases in the activities of Na(+),K(+)-ATPase (to 23% of the control value), Ca(2+)-ATPase (to 16% of control) and Mg(2+)-ATPase (to 22% of control). Washing out the fullerenol lessened the inhibition of the Na(+),K(+)-ATPase (37% of control) and Ca(2+)-ATPase (23.5% of control); however, it did not influence Mg(2+)-ATPase activity. Furthermore, fullerenol could associate with erythrocyte plasma membranes. Our results suggest that fullerenol associates primarily with the surface of the plasma membrane; however, it can also migrate deeper inside the membrane. Moreover, fullerenol influences membrane ATPases so that it may modulate ion transport across membranes.

Fullerenols (polyhydroxylated fullerene C) are nanomaterial with potentially broad applicability in biomedical sciences with high antioxidant ability, thus, we investigated the radioprotecting potential of fullerenol C(OH) on human erythrocytes irradiated by high-energy electrons of 6 MeV. The results demonstrate that C(OH) at concentration of 150 μg/mL protects the erythrocytes against the radiation-induced hemolysis (comparing to non-protected cells, we observed 30% and 39% protection for 0.65 and 1.3 kGy irradiation doses, respectively). The protecting effect was confirmed by 32% decreased release of potassium cations comparing to the cells irradiated without C(OH). Measurements of the amount of lactate dehydrogenase (LDH) released from the irradiated erythrocytes showed that the size of the pores formed by irradiation was not sufficient to release LDH across the erythrocyte membranes. We also report a significant decrease of the affinity of acetylcholinesterase (AChE) for the substrate in the presence of fullerenol, indicating the relatively strong adsorption of C(OH) to components of plasma membrane. Changes in membrane fluidity detected by fluorescence spectroscopy and conformational changes in membrane proteins detected by spin labeling suggest the dose-dependent formation of disulfide groups as an effect of oxidation and this process was inhibited by C(OH) We suppose that scavenging the ROS as well as adsorption of fullerenol to membrane proteins and steric protection of -SH groups against oxidation are responsible for the observed effects.

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The mechanism of the interaction of highly hydroxylated fullerenol C60(OH)36with erythrocyte membranes was studied by electron spin resonance spectroscopy (ESR) of stearic acid derivatives labeled with a nitroxyl radical at C-12 or C-16 and with a nitroxyl derivative of maleimide covalently attached to sulfhydryl groups of membrane proteins. A significant increase in membrane fluidity in the hydrophobic region of the lipid bilayer was observed for 12-doxylstearic acid at fullerenol concentrations of 100 mg/L or 150 mg/L, while for 16-doxylstearic acid significant increase in fluidity was only observed at 150 mg/L. Fullerenol at 100 mg/L or 150 mg/L caused conformational changes in membrane proteins, expressed as an increase in thehw/hsparameter, when fullerenol was added before the maleimide spin label (MSL) to the membrane suspension. The increase of thehw/hsparameter may be caused by changes in lipid-protein or protein-protein interactions which increase the mobility of the MSL label and as a result increase the membrane fluidity. Incubation of the membranes with the MSL before the addition of fullerenol blocked the available membrane protein –SH groups and minimized the interaction of fullerenol with them. This confirms that fullerenol interacts with erythrocyte membrane proteins via available protein –SH groups.