Assessing regulated cell death modalities as an efficient tool for in vitro nanotoxicity screening: a review.

Nanomedicine is a fast-growing field of nanotechnology. One of the major obstacles for a wider use of nanomaterials for medical application is the lack of standardized toxicity screening protocols for assessing the safety of newly synthesized nanomaterials. In this review, we focus on less frequently studied nanomaterials-induced regulated cell death (RCD) modalities, including eryptosis, necroptosis, pyroptosis, and ferroptosis, as a tool for in vitro nanomaterials safety evaluation. We summarize the latest insights into the mechanisms that mediate these RCDs in response to nanomaterials exposure. Comprehensive data from reviewed studies suggest that ROS (reactive oxygen species) overproduction and ROS-mediated pathways play a central role in nanomaterials-induced RCDs activation. On the other hand, studies also suggest that individual properties of nanomaterials, including size, shape, or surface charge, could determine specific toxicity pathways with consequent RCD induction as well. We anticipate that the evaluation of RCDs can become one of the mechanism-based screening methods in nanotoxicology. In addition to the toxicity assessment, evaluation of necroptosis-, pyroptosis-, and ferroptosis-promoting capacity of nanomaterials could simultaneously provide useful information for specific medical applications as could be their anti-tumor potential. Moreover, a detailed understanding of molecular mechanisms driving nanomaterials-mediated induction of immunogenic RCDs will substantially aid novel anti-tumor nanodrugs development.

Rare-earth orthovanadate nanoparticles trigger Ca2+-dependent eryptosis.

Introduction. Rare-earth orthovanadate nanoparticles (ReVO4:Eu3+, Re = Gd, Y or La) are promising agents for photodynamic therapy of cancer due to their modifiable redox properties. However, their toxicity limits their application.Objective. The aim of this research was to elucidate pro-eryptotic effects of GdVO4:Eu3+and LaVO4:Eu3+nanoparticles with identification of underlying mechanisms of eryptosis induction and to determine their pharmacological potential in eryptosis-related diseases.Methods. Blood samples (n= 9) were incubated for 24 h with 0-10-20-40-80 mg l-1GdVO4:Eu3+or LaVO4:Eu3+nanoparticles, washed and used to prepare erythrocyte suspensions to analyze the cell membrane scrambling (annexin-V-FITC staining), cell shrinkage (forward scatter signaling), reactive oxygen species (ROS) generation through 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) staining and intracellular Ca2+levels via FLUO4 AM staining by flow cytometry. Internalization of europium-enabled luminescent GdVO4:Eu3+and LaVO4:Eu3+nanoparticles was assessed by confocal laser scanning microscopy.Results.Both nanoparticles triggered eryptosis at concentrations of 80 mg l-1. ROS-mediated mechanisms were not involved in rare-earth orthovanadate nanoparticles-induced eryptosis. Elevated cytosolic Ca2+concentrations were revealed even at subtoxic concentrations of nanoparticles. LaVO4:Eu3+nanoparticles increased intracellular calcium levels in a more pronounced way compared with GdVO4:Eu3+nanoparticles. Our data disclose that the small-sized (15 nm) GdVO4:Eu3+nanoparticles were internalized after a 24 h incubation, while the large-sized (∼30 nm) LaVO4:Eu3+nanoparticles were localized preferentially around erythrocytes.Conclusions.Both internalized GdVO4:Eu3+and non-internalized LaVO4:Eu3+nanoparticles (80 mg l-1) promote eryptosis of erythrocytes after a 24 h exposurein vitrovia Ca2+signaling without involvement of oxidative stress. Eryptosis is a promising model for assessing nanotoxicity.

Assessing the Cytotoxicity of TiO2-x Nanoparticles with a Different Ti3+(Ti2+)/Ti4+ Ratio.

Titanium dioxide (TiO2) nanoparticles are promising biomedical agents characterized by good biocompatibility. In this study, we explored the cytotoxicity of TiO2-x nanoparticles with a different Ti3+(Ti2+)/Ti4+ ratio and analyzed the efficiency of eryptosis indices as a tool in nanotoxicology. Two types of TiO2-x nanoparticles (NPs) were synthesized by the hydrolysis of titanium alkoxide varying the nitric acid content in the hydrolysis mixture. Transmission electron microscopy (TEM) images show that 1-TiO2-x and 2-TiO2-x NPs are 5 nm in size, whereas X-ray photoelectron spectroscopy (XPS) reveals different Ti3+ (Ti2+)/Ti4+ ratios in the crystal lattices of synthesized NPs. 1-TiO2-x nanoparticles contained 54% Ti4+, 38% Ti3+, and 8% Ti2+, while the relative amount of Ti4+ and Ti3+ in the crystal lattice of 2-TiO2-x nanoparticles was 63% and 37%, respectively. Cell viability and cell motility induced by TiO2-x nanoparticles were investigated on primary fibroblast cultures. Eryptosis modulation by the nanoparticles along with cell death mechanisms was studied on rat erythrocytes. We report that both TiO2-x nanoparticles do not decrease the viability of fibroblasts simultaneously stimulating cell migration. Data from in vitro studies on erythrocytes indicate that TiO2-x nanoparticles trigger eryptosis via ROS- (1-TiO2-x) and Ca2+-mediated mechanisms (both TiO2-x nanoparticles) suggesting that evaluation of eryptosis parameters is a more sensitive nanotoxicological approach for TiO2-x nanoparticles than cultured fibroblast assays. TiO2-x nanoparticles are characterized by low toxicity against fibroblasts, but they induce eryptosis, which is shown to be a promising tool for nanotoxicity screening. The Ti3+ (Ti2+)/Ti4+ ratio at least partly determines the cytotoxicity mechanisms for TiO2-x nanoparticles.

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.

Antimicrobial activity and cytotoxicity study of cerium oxide nanoparticles with two different sizes.

The control over bacterial diseases requires the development of novel antibacterial agents. The use of antibacterial nanomedicines is one of the strategies to tackle antibiotic resistance. The study was designed to assess the antimicrobial activity of cerium oxide (CeO2 ) nanoparticles (NP) of two different sizes (CeO2 NP1 [1-2 nm] and CeO2 NP2 [10-12 nm]) and their cytotoxicity towards eukaryotic cells. The antimicrobial activity, effects of nanoparticles on DNA cleavage, microbial cell viability, and biofilm formation inhibition were analyzed. The impact of cerium oxide nanoparticles on eryptosis of erythrocytes was estimated using annexin V staining by flow cytometry. The newly synthesized CeO2 NP1 and CeO2 NP2 displayed moderate antimicrobial activities. CeO2 NP1 and CeO2 NP2 exhibited single-strand DNA cleavage ability. CeO2 NPs were found to show 100% microbial cell viability inhibition at a concentration of 500 mg/L. In addition, CeO2 NP1 and CeO2 NP2 inhibited the biofilm formation of S. aureus and P. aeruginosa. Larger cerium oxide nanoparticles were found to be less toxic against erythrocytes compared with the smaller ones. CeO2 nanoparticles demonstrate moderate antimicrobial activity and low cytotoxicity towards erythrocytes, which make them promising antibacterial agents.

Antimicrobial Effects of Nanostructured Rare-Earth-Based Orthovanadates.

The search for novel antimicrobial agents is of huge importance. Nanomaterials can come to the rescue in this case. The aim of this study was to assess the cytotoxicity and antimicrobial effects of rare-earth-based orthovanadate nanoparticles. The cytotoxicity against host cells and antimicrobial activity of LaVO4:Eu3+ and GdVO4:Eu3+ nanoparticles were analyzed. Effects of nanomaterials on fibroblasts were assessed by MTT, neutral red uptake and scratch assays. The antimicrobial effects were evaluated by the micro-dilution method estimating the minimum inhibitory concentration (MIC) of nanoparticles against various strains of microorganisms, DNA cleavage and biofilm inhibition. GdVO4:Eu3+ nanoparticles were found to be less toxic against eukaryotic cells compared with LaVO4:Eu3+. Both nanoparticles exhibited antimicrobial activity and the highest MIC values were 64 mg/L for E. hirae, E. faecalis and S. aureus shown by GdVO4:Eu3+ nanoparticles. Nanoparticles demonstrated good DNA cleavage activity and induction of double-strand breaks in supercoiled plasmid DNA even at the lowest concentrations used. Both nanoparticles showed the biofilm inhibition activity against S. aureus at 500 mg/L and reduced the microbial cell viability. Taken the results of host toxicity and antimicrobial activity studies, it can be assumed that GdVO4:Eu3+ nanoparticles are more promising antibacterial agents compared with LaVO4:Eu3+ nanoparticles.

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.

UV Light-Activated GdYVO4:Eu3+ Nanoparticles Induce Reactive Oxygen Species Generation in Leukocytes Without Affecting Erythrocytes In Vitro.

Nanoparticles (NPs) have been reported to be promising enhancement agents for radiation therapy. The aim of the study was to assess the cytotoxicity of UV non-treated and UV pretreated GdYVO4:Eu3+ nanoparticles against erythrocytes and leukocytes by detecting eryptosis and reactive oxygen species (ROS) generation. Levels of intracellular ROS in erythrocytes and leukocytes using a ROS-sensitive dye 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), as well as eryptosis rate utilizing annexin V staining, following direct exposure to UV-activated and nonactivated NPs were detected by flow cytometry. Blood cells were collected from 9 intact WAG rats. Neither the UV light-untreated GdYVO4:Eu3+ NPs nor the treated ones promoted eryptosis and ROS generation in erythrocytes. Low concentrations of UV light-untreated NPs did not induce oxidative stress in leukocytes, evidenced by unaffected intracellular ROS levels. UV light treatment grants prooxidant properties to NPs, confirmed by NP-induced ROS overproduction in leukocytes. High concentrations of both UV light-treated and untreated NPs altered the redox state of leukocytes. UV light treatment imparts prooxidant properties to GdYVO4:Eu3+ NPs, making them promising radiosensitizing agents in cancer radiation therapy.

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.

Experimental confirmation of antimicrobial effects of GdYVO4:Eu3+ nanoparticles.

Nanotechnology can be applied to design antibacterial agents to combat antibiotic resistance. The aim of the present study was to assess the antimicrobial effects and cytotoxicity of GdYVO4:Eu3+ nanoparticles (NPs). Biofilm inhibition activity, antimicrobial activity, bacterial viability inhibition and DNA cleavage activity of GdYVO4:Eu3+ NPs were studied. In addition, the impact of GdYVO4:Eu3+ NPs on the mitochondrial membrane potential (ΔΨM) of host immune cells and, hence, their apoptosis was analyzed by JC-1 staining using flow cytometry. GdYVO4:Eu3+ NPs demonstrated good antimicrobial, cell viability inhibition and DNA cleavage activities. In addition, GdYVO4:Eu3+ NPs showed good biofilm inhibition activity against S. aureus and P. aeruginosa and inhibition percentages were 89.15% and 79.54%, respectively. However, GdYVO4:Eu3+ NPs promoted mitochondrial depolarization and apoptosis of leukocytes at high concentrations. GdYVO4:Eu3+ nanoparticles are promising antibacterial agents. However, more efforts should be exerted to ensure their safety.

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.

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.

Reactive oxygen species generation in aqueous solutions containing GdVO4:Eu3+ nanoparticles and their complexes with methylene blue.

It this letter, we report the study of free radicals and reactive oxygen species (ROS) generation in water solutions containing gadolinium orthovanadate GdVO4:Eu3+ nanoparticles (VNPs) and their complexes with methylene blue (MB) photosensitizer. The catalytic activity was studied under UV-Vis and X-ray irradiation by three methods (conjugated dienes test, OH· radical, and singlet oxygen detection). It has been shown that the VNPs-MB complexes reveal high efficiency of ROS generation under UV-Vis irradiation associated with both high efficiency of OH· radicals generation by VNPs and singlet oxygen generation by MB due to nonradiative excitation energy transfer from VNPs to MB molecules. Contrary to that under X-ray irradiation, the strong OH . radicals scavenging by VNPs has been observed.

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.

Microspectroscopic Study of Liposome-to-cell Interaction Revealed by Förster Resonance Energy Transfer.

We report the Förster resonance energy transfer (FRET)-labeling of liposomal vesicles as an effective approach to study in dynamics the interaction of liposomes with living cells of different types (rat hepatocytes, rat bone marrow, mouse fibroblast-like cells and human breast cancer cells) and cell organelles (hepatocyte nuclei). The in vitro experiments were performed using fluorescent microspectroscopic technique. Two fluorescent dyes (DiO as the energy donor and DiI as an acceptor) were preloaded in lipid bilayers of phosphatidylcholine liposomes that ensures the necessary distance between the dyes for effective FRET. The change in time of the donor and acceptor relative fluorescence intensities was used to visualize and trace the liposome-to-cell interaction. We show that FRET-labeling of liposome vesicles allows one to reveal the differences in efficiency and dynamics of these interactions, which are associated with composition, fluidity, and metabolic activity of cell plasma membranes.

Pseudoisocyanine J-aggregate to optical waveguiding crystallite transition: microscopic and microspectroscopic exploration.

Using fluorescent microscopy and microspectroscopy, optical properties and morphology transformations in individual pseudoisocyanine (PIC) J-aggregates in aqueous electrolyte solutions have been explored. A stringlike structure of J-aggregates with a string diameter much less than 1 microm has been observed. Photodestruction of the strings under short-wavelength excitation has been revealed. Rodlike PIC crystallites, about 1 microm in diameter, have been observed with time. The fluorescence spectrum of rodlike crystallites has been found to differ from that of stringlike J-aggregate and from PIC crystal powder spectra. The crystallites are very stable, and their photodestruction has not been observed under any excitation conditions. It has been found that rodlike crystallites in contrast to stringlike J-aggregates possess optical waveguide properties. The luminescence of crystallites can be observed only at the excitation spot and at butt ends located up to hundreds of micrometers from the excitation spot.