CeO2 nanoparticles improve prooxidant/antioxidant balance, life quality and survival of old male rats.

Due to its unique redox chemistry, nanoceria is considered as potent free radical scavenger and antioxidant. However, their protective capacity in aging organisms remains controversial. To detect the anti-aging effects associated with the redox activity of 2 and 10 nm nano-CeO2, different test systems were used, including in vitro analysis, in situ assay of mitochondria function and in vivo studies of suitable nano-CeO2 on aging of male Wistar rats from 22 months-old to the end of life. The 2 nm nanoparticles exhibited not only antioxidant (·OH scavenging; chemiluminescence assay; decomposition of H2O2, phosphatidylcholine autooxidation) but also prooxidant properties (reduced glutathione and reduced nicotinamide adenine dinucleotide phosphate oxidation) as well as affected mitochondria whereas in most test systems 10 nm nano-CeO2 showed less activity or was inert. Prolonged use of the more redox active 2 nm nano-CeO2 (0.25-0.3 mg/kg/day) in vivo with drinking water resulted in improvement in physiological parameters and normalization of the prooxidant/antioxidant balance in liver and blood of aging animals. Survival analysis using Kaplan-Meier curve and Gehan tests with Yates' correction showed that by the time the prooxidant-antioxidant balance was assessed (32 months), survival rates exceeded the control values most considerably. The apparent median survival for the control rats was 900 days, and for the experimental rats-960 days. In general, the data obtained indicate the ability of extra-small 2 nm nano-CeO2 to improve quality of life and increase the survival rate of an aging organism.

Comparative Studies of Orthovanadate Nanoparticles and Metformin on Life Quality and Survival of Senile Wistar Rats.

Effect of prolong use of orthovanadate nanoparticles (GdVO4/Eu3+ NPs (8 × 25 nm)) on life quality and survival of male Wistar rats on the late stage of ontogenesis (from 23 months to the end of life) has been investigated. Multi-parametric assessment of orthovanadate NPs influences against metformin (Met) which is a well-known calorie restriction mimetic (CR-mimetic) has been completed. The quality of life was assessed by taking into account age-related hallmarks-phenotype and some physiological parameters (condition of the coat, body weight, concentration of thyroxine, rectal temperature) as well as indicators of the pro-oxidant/antioxidant balance of blood and liver (the content of lipid hydroperoxides; aconitase, glutathione peroxidase, glutathione reductase, glutaredoxin activity, and activity of NADP+-dehydrogenases (DG) (glucose-6-phosphate DG, malate DG, and isocitrate DG)) in aging animals. Kaplan-Meier curve and Gehan tests with Yates' correction were performed for the survival analysis. It has been found that long-term use of GdVO4/Eu3+ NPs (0.25-0.30 mg/kg/day), as well as Met (100-110 mg/kg/day) with drinking water led to reliable improvement of physiological parameters and normalization of the pro-oxidant/antioxidant balance in the liver and blood of experimental animals. A significant increase in the survival rate of aging rats was observed; the apparent median survival for control rats was 900 days, while for experimental rats, 1010 and 990 days for GdVO4/Eu3+ NPs and Met, respectively. In general, the data obtained demonstrate the ability of GdVO4/Eu3+ NPs and CR-mimetic-Met to improve the quality of life and increase the survival of an elderly organism.

Cytotoxicity, Retention, and Anti-inflammatory Effects of a CeO2 Nanoparticle-Based Supramolecular Complex in a 3D Liver Cell Culture Model.

Both cerium oxide (CeOx) nanoparticles and mefenamic acid (MFA) are known anti-inflammatory agents with hepatoprotective properties and are therefore prescribed for one of the major diseases in the world, nonalcoholic fatty liver disease (NAFLD). To study the potential cytotoxicity and anti-inflammatory effects as well as drug retention of a potential therapeutic CeOx/MFA supramolecular complex, a well-standardized hepatic (HepG2) spheroid model was used. Results showed that the highest cytotoxicity for the CeOx/MFA supramolecular complex was found at 50 µg/mL, while effective doses of 0.1 and 1 µg/mL yielded a significant decrease of TNF-α and IL-8 secretion. Time-resolved analysis of HepG2 spheroids revealed a spatiotemporal distribution of the supramolecular complex and limited clearance from the internal microtissue over a period of 8 days in cultivation. In summary, our results point at rapid uptake, distribution, and biostability of the supramolecular complex within the HepG2 liver spheroid model as well as a significant anti-inflammatory response at noncytotoxic levels.

Anti-aging Effects of Antioxidant Rare-Earth Orthovanadate Nanoparticles in Wistar Rats.

Biomedical application of rare-earth-based nanoparticles attracts much attention due to their unique optical and redox properties and quite low toxicity. Earlier, we found age-related beneficial effects of rare-earth-based orthovanadate nanoparticles (OV NPs) on the prooxidant/antioxidant balance in liver and blood of Wistar rats, as reported by Nikitchenko et al. (Biol Trace Elem Res (2020). https://doi.org/10.1007/s12011-020-02196-7 ). However, the question remained unclear whether OV NPs' redox activity directly defines the protection ability. In the present work, antiradical, antioxidant, and membrane-protective properties of GdYVO4/Eu3+ NPs (1-2 nm), GdVO4/Eu3+ NPs (8 × 25 nm), LaVO4/Eu3+ (57 × 8 nm) were assayed in a comparative manner in various model systems. All OV NPs demonstrated the protective properties, but extra-small GdYVO4/Eu3+ NPs revealed the weakest antioxidant efficacy. In isolated mitochondria, OV NPs lowered (most evidently-extra-small NPs) respiration and oxidative phosphorylation, as well as ATP concentration. We conclude that not only the direct antioxidant effect but also slight suppression of bioenergetic processes by the OV NPs as well as the triggering of GSH-dependent antioxidant system may represent the principal mechanisms of their beneficial influences in an aged organism. This statement is consistent with improvement of the oxidative balance of 33-month-old rats due to prolonged administration of GdVO4 /Eu3+ NPs (for 11 months) accompanied by retention of the GSH signaling of the old rats at the level of 12 months mature animals. Consequently, an increase of antioxidant defense upon prolonged usage of OV NPs will lead to oxidative balance stabilization increasing the health span and survival of an organism.

Age-Related Effects of Orthovanadate Nanoparticles Involve Activation of GSH-Dependent Antioxidant System in Liver Mitochondria.

Vanadium is an important ultra-trace element nowadays attracting attention with particular emphasis on medical application. But the therapeutic application of vanadium-based drugs is still questionable and restricted due to some toxic side effects. It was found that unique redox properties of vanadium in nanoform provided antioxidant activity and prevented oxidative disturbance in cells in vitro. Though, on the organism level, ambiguous effects of vanadium-based nanoparticles were observed. In this study, the age-related features of prooxidant/antioxidant balance in blood serum and liver mitochondrial and postmitochondrial fractions of 3 and 18-month-old Wistar male rats treated with orthovanadate nanoparticles (GdVO4/Eu3+, 8 × 25 nm) within 2 months have been investigated. Prooxidant potential-related indexes were the content of lipid hydroperoxides as well as aconitase activity. Activity of glutathione peroxidase, glutathione-S-transferase, glutaredoxin, glutathione reductase, glucose-6-phosphate dehydrogenase, and NADPH-dependent isocitrate dehydrogenase designated the tissue antioxidant potential. Based on the obtained values, the integral index of the prooxidant/antioxidant balance-the reliability coefficient (Kr) has been calculated. The data show that due to activation some chain links of GSH-dependent antioxidant system, GdVO4/Eu3+ nanoparticles increase the reliability of the prooxidant-antioxidant balance in tissues and especially in the liver mitochondria of old animals (Kr in mitochondria of young rats was 2.94, and in mitochondria of old ones-9.83 conventional units). Detected in vitro glutathione peroxidase-like activity of the GdVO4/Eu3+ nanoparticles is supposed to be among factors increasing the reliability of the system. So, for the first time, the beneficial effect of the long-term orthovanadate nanoparticle consumption in old males has been discovered.

Experimental Evaluation of the Impact of Gadolinium Orthovanadate GdVO4:Eu3+ Nanoparticles on the Carrageenan-Induced Intestinal Inflammation.

AIM: To evaluate the effects of orally administered gadolinium orthovanadate GdVO4:Eu3+ nanoparticles (VNPs) on the course of chronic carrageenan-induced intestinal inflammation. METHODS: Samples of small intestinal tissue were collected from four groups of rats (intact, after administration of VNPs, with carrageenaninduced intestinal inflammation, with carrageenan-induced intestinal inflammation orally exposed to VNPs) to assess the intestinal morphology and HSP90α expression. Levels of seromucoid, C-reactive protein, TNF-α, IL-1ß and IL-10 were determined in blood serum. RESULTS: Oral exposure to VNPs was associated with neither elevation of inflammation markers in blood serum nor HSP90α overexpression in the small intestine, i.e. no toxic effects of VNPs were observed. Carrageenan-induced intestinal inflammation was accompanied by higher levels of TNF-α and IL-1ß, as well as HSP90α upregulation in the intestinal mucosa, compared with controls. Administration of VNPs to rats with enteritis did not lead to statistically significant changes in concentrations of circulating pro-inflammatory cytokines with the trend towards their increase. CONCLUSION: No adverse effects were observed in rats orally exposed to VNPs at a dose of 20 µg/kg during two weeks. Using the experimental model of carrageenan-induced enteritis, it was demonstrated that VNPs at the dose used in our study did not affect the course of intestinal inflammation.

Orally administered gadolinium orthovanadate GdVO4:Eu3+ nanoparticles do not affect the hydrophobic region of cell membranes of leukocytes.

AIM: To assess the phospholipid bilayer of white blood cells (WBCs) and the ability of leukocytes to generate reactive oxygen species (ROS) in rats orally exposed to GdVO4:Eu3+ nanoparticle (VNP) solution for 2 weeks by fluorescent probes-ortho-hydroxy derivatives of 2,5-diaryl­1,3­oxazole. METHODS: Steady-state fluorescence spectroscopy, i.e., a study by the environment-sensitive fluorescent probes 2­(2'-OH-phenyl)-5-(4'-phenyl-phenyl)-1,3-oxazole (probe O6O) and 2­(2'-OH-phenyl)-phenanthro[9,10]-1,3-oxazole (probe PH7), and flow cytometry, i.e., analysis of 2',7'-dichlorofluorescein (DCF), a product of a dye 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), fluorescence in CD45+/7-aminoactinomycin D (7-AAD)- cells, were used to evaluate the state of cell membranes and reactive oxygen species (ROS) generation in leukocytes of rats orally exposed to gadolinium orthovanadate nanoparticles(VNPs). RESULTS: No significant changes were detected in the spectra of the fluorescent probes bound to the WBCs from the rats orally exposed to nanoparticles in comparison with the corresponding spectra of the probes bound to the cells from the control group of animals. This indicates that in the case of the rats orally exposed to nanoparticles, no noticeable changes in physicochemical properties (i.e., in the polarity and the proton-donor ability) are observed in the lipid membranes of WBCs in the region where the probes locate. There was no statistically significant difference in the amount of ROShigh viable leukocytes in rats treated with VNPs and control samples. CONCLUSION: Neither changes in the physical and chemical properties of the leukocyte membranes nor in ROS generation by WBCs are detected in the rats orally exposed to VNP solution for 2 weeks.

Nanotechniques Inactivate Cancer Stem Cells.

One of the tasks of current oncology is identification of cancer stem cells and search of therapeutic means capable of their specific inhibition. The paper presents the data on phenotype characteristics of Ehrlich carcinoma cells as convenient and easy-to-follow model of tumor growth. The evidence of cancer stem cells as a part of Ehrlich carcinoma and significance of CD44+ and CD44- subpopulations in maintaining the growth of this type of tumor were demonstrated. A high (tenfold) tumorigenic activity of the Ehrlich carcinoma CD44+ cells if compared to CD44- cells was proven. In this pair of comparison, the CD44+ cells had a higher potential of generating in peritoneal cavity of CD44high, CD44+CD24-, CD44+CD24+ cell subpopulations, highlighting the presence of cancer stem cells in a pool of CD44+ cells.In this study, the ability of synthesized hybrid nanocomplexes, comprising the nanoparticles of rare earth orthovanadates GdYVO4:Eu3+ and cholesterol to inhibit the tumor growth and to increase the survival of the animals with tumors was established. A special contribution into tumor-inhibiting effect is made by each of its components. Treatment of Ehrlich carcinoma cells with two-component hybrid complex resulted in maximum reduction in the concentration of the most tumorigenic CD44high cells with simultaneous rise in the number of CD117+ cells that decreased an intensity of tumor growth by 74.70 ± 4.38% if compared with the control.

Interactions of Nanosized Aggregates of Fullerene C60 with Electrolytes in Methanol: Coagulation and Overcharging of Particles.

Contrary to numerous studies on the stability of fullerene aqueous colloidal solutions in the presence of electrolytes, the corresponding issue for the organosols was until recently almost unexplored. In this article, the state of C60 in methyl alcohol and the regularities of the coagulation of colloidal solution in this solvent were examined in the presence of electrolytes. Alcosols with a fullerene concentration of 4 × 10-6 M were prepared by the dilution of the C60 saturated solution in toluene by methanol. The ca. 300 nm-sized aggregates possess a negative electrokinetic potential value, ζ = -37 ± 8 mV. To determine the critical coagulation concentrations, CCC, the size increase of the species was followed up using the dynamic light scattering method. The analysis of the coagulation in terms of the Fuchs function, W, was accompanied by zeta potential monitoring. The consideration of the data for 1:1 electrolytes NaClO4 and N(n-C4H9)4ClO4 allows a rough estimate of the Hamaker constant of fullerene-fullerene attraction. Whereas in the case of these two electrolytes the colloidal species are negatively charged at the CCC, expressed overcharging of up to ζ = +36 mV by H+, Ca2+, Ba2+, and La3+ ions was observed. The action of HClO4 should be attributed to the interfacial acid-base reaction, whereas the excessive attraction of metal cations is caused by poor solvation in methanol; the negative charge is restored when the metal cations are shielded by a macrocyclic ligand.

Towards better understanding of C60 organosols.

It is of common knowledge that fullerenes form colloids in polar solvents. However, the coagulation via electrolytes and the origin of the negative charge of species are still unexplored. Using a 'radical scavenger' and electrospray ionization spectroscopy (ESI), we proved the formation of ion-radical C60˙(-) and its (probable) transformation into C60(2-) or (C60)2(2-). The coagulation of C60 organosols by NaClO4 and other perchlorates and nitrates in acetonitrile and its mixture with benzene obeys the Schulze-Hardy rule. At higher Ca(ClO4)2 and La(ClO4)3 concentrations, instead of coagulation, stable re-charged colloidal particles appeared, up to a zeta-potential of +(20-42) mV, as compared with -(33-35) mV of the initial organosols. The influence of both HClO4 and CF3SO3H was similar. This phenomenon is attributed to poor solvation of inorganic cations in cationo- and protophobic acetonitrile, which was proven using [2.2.2] cryptand. Further increasing the concentration of Ca(ClO4)2 led again to coagulation, thus demonstrating a novel type of 'coagulation zones'.

Mitochondrial potential (ΔΨm) changes in single rat hepatocytes: the effect of orthovanadate nanoparticles doped with rare-earth elements.

Rare-earth-based nanoparticles (NPs) are widely used as fluorescent probes for imaging in vitro and in vivo. One of the challenges that restrain NPs applications in biomedical research is their effect on subcellular structures. In this paper, the ability of lanthanide NPs to affect the cellular oxidative balance and alter the mitochondrial function was analyzed. Since size and shape mutually affect the cellular internalization and intracellular distribution of NPs, the investigations were performed with NPs of spherical (GdYVO4:Eu(3+), spindle-(GdVO4: Eu(3+) and rod-like (LaVO4: Eu(3+) shapes. Quantitative microfluorimetry with JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide) as a mitochondrial probe was used for monitoring of the mitochondrial transmembrane potential (ΔΨ m) in single living cells. Changes in the ratio of the JC-1 probe fluorescence were used to analyze the NPs effect on ΔΨ(m). The fastest suppressive effect (within 1 hour) was found for spherical NPs. Gradual lowering of ΔΨ(m) was observed at the exposure of cells within 24 hours for all types of NPs. Exogenous thiols were required for ΔΨ(m) protection. The protective role of exogenous glutathione (GSH) proves that the increase of reactive oxygen species (ROS) formation with depletion of GSH can mediate NPs toxicity. The dynamics of the shape-dependent effect can be explained by the features of NPs transportation into cells.

Nanostructures of hydrated C60 fullerene (C60HyFn) protect rat brain against alcohol impact and attenuate behavioral impairments of alcoholized animals.

It is well known that chronic ethyl alcohol (EtOH) consumption is capable to injure brain cells and to cause essential abnormalities in behavioral characteristics of animals addicted to alcohol. In this work we for the first time have shown that administration of aqueous solutions of hydrated C60 fullerenes (C60HyFn) with C60 concentration of 30nM as a drinking water during chronic alcoholization of rats (a) protects the tissues of central nervous system (CNS) from damage caused by oxidative stress with high efficacy, (b) prevents the pathological loss of both astrocytes (the main cells of CNS) and astrocytic marker, glial fibrillary acidic proteins (GFAP) and, as consequence, (c) due to their adaptogenic effects, C60HyFn significantly improves behavioral response and eliminates emotional deficits induced by chronic alcohol uptake. The wide range of beneficial biological effects, zero-toxicity, and efficacy even in super-small doses provide a rationale for the possible application of C60HyFn for the treatment of alcohol-induced encephalopathy as well as alcoholism prophylaxis.