Anita Krokosz

Fullerenols as a New Therapeutic Approach in Nanomedicine

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, todays 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.

Membrane fluidity and activity of membrane ATPases in human erythrocytes under the influence of polyhydroxylated fullerene.

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.

Damage to human erythrocytes by radiation-generated HO* radicals: molecular changes in erythrocyte membranes.

The effectiveness of radiation-generated HO* radicals in initiating erythrocyte hemolysis in the presence of oxygen and under anaerobic conditions and prehemolytic structural changes in the plasma-erythrocyte membrane were studied. Under anaerobic conditions the efficacy of HO* radicals in induction of hemolysis was 16-fold lower than under air. In both conditions, hemolysis was the final consequence of changes of the erythrocyte membrane. Preceding hemolysis, the dominating process under anaerobic conditions was the aggregation of membrane proteins. The aggregates were principally formed by -S-S- bridges. A decrease in spectrin and protein of band 3 content suggests their participation in the formation of the aggregates. These processes were accompanied by changes in protein conformation determined by means of 4-maleimido-2,2,6,6-tetramethylpiperidine-N-oxyl (MSL) spin label attached to membrane proteins. Under anaerobic conditions, in the range of prehemolytical doses, the reaction of HO* with lipids caused a slight (10-16%) increase in fluidity of the lipid bilayer in its hydrophobic region with a lack of lipid peroxidation. However, in the presence of oxygen, hemolysis was preceded by intense lipid peroxidation and by profound changes in the conformation of membrane proteins. At the radiation dose that normally initiates hemolysis a slight aggregation of proteins was observed. Changes were not observed in particular protein fractions. It can be suggested the cross-linking induced by HO* radicals under anaerobic conditions and a lack of lipid peroxidation are the cause of a decrease in erythrocyte sensitivity to hemolysis. Contrary, under aerobic conditions, molecular oxygen suppresses cross-linking, catalysing further steps of protein and lipid oxidation, which accelerate hemolysis.

Can melatonin delay oxidative damage of human erythrocytes during prolonged incubation

PURPOSE Melatonin (MEL) is an effective antioxidant in numerous experimental models, both in vitro and in vivo. However, it should be stressed that there are also papers reporting limited antioxidative activity of MEL or even giving evidence for its pro-oxidative properties. In the present paper we investigated the influence of MEL on the oxidative damage of human erythrocytes during prolonged incubation. MATERIAL/METHODS Human erythrocytes suspended in phosphate-buffered saline (PBS), pH 7.4 were incubated at 37ºC either in absence or presence of melatonin at concentration range 0.02 mM-3 mM for up to 96 hrs. The influence of MEL on erythrocyte damage was assessed on the basis of the intensity of intracellular oxidation processes (the oxidation of HbO₂, GSH, fluorescent label DCFH₂) as well as damage to the plasma membrane (lipid peroxidation, the potassium leakage) and the kinetics of hemolysis. RESULTS The prolonged incubation of erythrocytes induced a progressive destruction of erythrocytes. Melatonin prevented lipid peroxidation and hemolysis whereas the oxidation of HbO₂ and DCFH₂ was enhanced by melatonin at concentrations higher than 0.6 mM. In the case of erythrocytes incubated with 3 mM of MEL, the hemolysis rate constant (0.0498±0.0039 H%•h⁻¹) was 50% lower than that of the control while the HbO₂ oxidation rate constants were about 1.4 and 1.5 times higher for 1.5 and 3 mM of MEL, respectively. Melatonin had no influence on the oxidation of GSH and the potassium leakage. CONCLUSIONS Probably, MEL can stabilize the erythrocyte membrane due to interaction with lipids, thus prolonging the existence of cells. On the contrary, in the presence of MEL the accelerated oxidation of HbO₂ and generally, increased oxidative stress was observed in erythrocytes. Pro- and antioxidative properties of melatonin depend on the type of cells, redox state, as well as experimental conditions.

Effect of dose‐rate and dose fractionation on radiation‐induced hemolysis of human erythrocytes

Human erythrocytes suspended in an isotonic Na‐phosphate buffer, pH 7.4 (hematocrit 2 %) were exposed under air to gamma radiation at a dose rates of 2.2 kGy·h‐1 and 4.2 kGy·h‐1. The dose‐response curves for hemolysis of erythrocytes indicated that the process of hemolysis is inversely related to the dose‐rate. At both dose‐rates we observed a reduced level of hemolysis, when erythrocytes were irradiated with a split dose (0.4 kGy+2.3 kGy with an interval time between the subsequent exposures from 1 to 4 h) in comparison with the same single dose (2.7 kGy). The maximal effect of fractionation was observed when the interfraction time was equal to 3.5 h. The influence of the interfraction temperature on this effect was observed. The results obtained indicate that enucleated human erythrocytes under suitable radiation conditions are capable of repairing radiation damage which leads to hemolysis.

The Effect of Highly Hydroxylated Fullerenol C60(OH)36 on Human Erythrocyte Membrane Organization

The mechanism of the interaction of highly hydroxylated fullerenol C60(OH)36 with 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 the hw/hs parameter, when fullerenol was added before the maleimide spin label (MSL) to the membrane suspension. The increase of the hw/hs parameter 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.

Fullerenol C60(OH)36 protects human erythrocyte membrane against high-energy electrons

Fullerenols (polyhydroxylated fullerene C60) are nanomaterial with potentially broad applicability in biomedical sciences with high antioxidant ability, thus, we investigated the radioprotecting potential of fullerenol C60(OH)36 on human erythrocytes irradiated by high-energy electrons of 6 MeV. The results demonstrate that C60(OH)36 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 C60(OH)36. 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 C60(OH)36 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 C60(OH)36. 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.

Fullerenols in therapy and diagnosis of cancer

Malignant tumors are one of the main causes of death in Poland. One of the objectives of contemporary biomedical research is to maximize the effects of therapeutic strategies. The actions undertaken to improve therapeutic agents are aimed at reducing the side effects of cancer treatments. Another direction of investigations is the search for protective substances that reduce the toxicity of the drug to normal cells. Carbon-based nanomaterials (fullerenes and their derivatives, graphene, carbon nanotubes, nanodiamonds) are a broad class of nanoparticles that have potential biomedical applications in both therapy and diagnostics. The aim of this paper is to review biological properties of fullerenols in the context of their use in various strategies of cancer treatments. The authors also discuss the possibility of simultaneous use of nanoparticles in therapy and diagnosis, that is, in theranostics. Current knowledge indicates that fullerenes and their hydrophilic derivatives, especially fullerenols, show low or no toxicity. They may contribute to the inhibition of tumor growth and protection of normal cells through their antioxidant properties, as well as to the regulation of expression of genes involved in apoptosis and angiogenesis, and stimulation of the immune response. Gadoliniumcontaining endohedral fullerenes are less toxic as a contrast agents in magnetic resonance imaging, and they may also inhibit tumor growth, which is a promising result for theranostics. Med Pr 2016;67(6):817-831.

The role of resveratrol and melatonin in the nitric oxide and its oxidation products mediated functional and structural modifications of two glycolytic enzymes: GAPDH and LDH

Abstract Background Nitric oxide is a well-known gaseous signaling molecule and protein modifying agent. However, at higher concentrations or during oxidative stress nitric oxide may exert some deleterious effects on protein structure and function. Here we investigated the influence of nitric oxide and products of its oxidation on two glycolytic enzymes: GAPDH and LDH under in vitro nitrosative stress conditions. Secondly, we applied natural antioxidants: melatonin and resveratrol to examine their effects on the enzymes under studied conditions. Methods By means of UV–VIS and fluorescence spectroscopy methods we compared nitric oxide mediated changes of enzyme activities, amount of free sulfhydryl groups (-SH) and bis-ANS probe binding. Finally, we predicted potential cysteine residues modified by nitric oxide in studied proteins using GPS-SNO software. Results Our results indicated that nitric oxide reversibly inactivates GAPDH but does not affect the activity of LDH. Nitric oxide dependent GAPDH activity decline was accompanied by the reduction of the amount of free –SH groups and GAPDH-bound bis-ANS fluorescence. Reduction of the number of free –SH groups and protein-bound bis-ANS fluorescence was also observed in LDH treated with NO. Applied antioxidants increased inactivation of GAPDH and structural changes of GAPDH and LDH. Conclusions Nitric oxide modifies function and structure of thiol-dependent enzyme such as GAPDH and structure of LDH which function do not rely on cysteine thiols. Both resveratrol and melatonin exerted prooxidative properties in studied conditions. General significance Extensively studied antioxidants: resveratrol and melatonin may function as a prooxidative species under in vitro nitrosative stress conditions.

Activity of Membrane ATPases in Human Erythrocytes Under the Influence of Highly Hydroxylated Fullerenol

Incubation of erythrocyte membranes with highly hydroxylated fullerenol C60(OH)x, x > 30 led to decreases in Na,K-ATPase, Ca-ATPase and Mg-ATPase activities.