Fullerenols are polyhydroxylated derivatives of fullerene (C₆₀(OH)_n) with antioxidant, antiviral, and antibacterial properties and potential biomedical applications due to their solubility and biocompatibility. However, comprehensive assessment of their cytotoxicity is required, particularly regarding their effects on immune system cells. This study investigated the effects of fullerenol C₆₀(OH)₂₄ (MST-Nano, St. Petersburg, Russia) on the viability, apoptosis, and metabolism of THP-1 human monocytic leukemia cells. Cells were treated with concentrations ranging from 0.25 to 1000 µg/mL and incubated for 24, 48, and 72 h. Viability, apoptosis, and nanoparticle association were assessed by flow cytometry; glycolysis and mitochondrial respiration were measured after 24 h on a Seahorse XFe96 analyzer (Agilent Technologies, Santa Clara, CA, USA). Results showed that the effects of fullerenol depend on concentration and exposure time. At 24 h, 750 µg/mL increased viability, while 1000 µg/mL induced apoptosis. After 48 and 72 h, apoptosis increased at concentrations ≥750 µg/mL, with reduced viability. Nanoparticle association correlated with concentration and inversely correlated with viability but was independent of incubation time. Metabolic analysis revealed decreased glycolysis at 750 µg/mL after 24 h, while mitochondrial respiration was unaffected. Thus, our study demonstrated that fullerenol nanoparticles were safe for the THP-1 monocytic cell line up to 500 µg/mL.

Water-soluble fullerene derivatives such as fullerenol C₆₀(OH)₂₄ are promising candidates for nanomedicine applications, yet their effects on innate immune cells remain poorly characterized. We investigated the interaction of fullerenol with human neutrophils isolated from healthy donors, exposed to concentrations of 0.25–200 μg/mL over 24–72 h. Using multi-parameter flow cytometry, we assessed viability, apoptosis, phagocytic activity, and intracellular reactive oxygen species (ROS) production, complemented by cell-free DPPH radical scavenging assays. Fullerenol was taken up by neutrophils in a concentration- and time-dependent manner. No significant cytotoxicity was observed up to 100 μg/mL, while viability declined at 200 μg/mL. Phagocytosis of opsonized E. coli was preserved at lower concentrations, though a statistically significant negative correlation with fullerenol concentration was detected at higher doses. In cell-free assays, fullerenol scavenged DPPH radicals with an EC₅₀ of 48.90 ± 10.02 μg/mL, exhibiting slower kinetics than Trolox or ascorbic acid. Critically, fullerenol suppressed intracellular ROS production by >33% at 50 μg/mL following PMA stimulation of neutrophils. These findings demonstrate that fullerenol C₆₀(OH)₂₄ combines potent intracellular antioxidant activity with a favorable neutrophil safety profile, supporting its potential application in oxidative stress-related conditions.

Introduction: Breast cancer remains a major healthcare challenge due to its high incidence worldwide. Current treatment methods involve invasive surgical procedures, underscoring the urgent need for more effective and less invasive therapies. One promising approach explored in this study is hyperthermia-based treatment of cancer cells using NIR radiation in the presence of PEGylated graphene oxide. This method aims to leverage the photothermal properties of nanoparticles to selectively ablate cancer cells, offering a potential alternative to other therapeutic strategies. Methods: We investigated graphene oxide coated with linear (LP-GO) or branched (BP-GO) polyethylene glycol at concentrations of 5, 25, and 50 μg/mL in combination with NIR radiation. The irradiation was performed twice for 20 minutes. Cell viability, apoptosis, and the number of MCF-7 cells were assessed at 24 and 48 hours post-irradiation using flow cytometry. Results: The research shows that only LP-GO increases the temperature in a dose-dependent manner. The maximum temperature (45.9°C) was recorded at a concentration of 50 μg/mL after the second irradiation. Additionally, LP-GO is sorbed by MCF-7 cells in a dose-dependent manner. Furthermore, when LP-GO was used at concentrations of 25 and 50 μg/mL in combination with NIR irradiation, the maximum antitumor effect was observed after 48 hours, with a general apoptosis rate of 30%. Discussion: We observed that after NIR irradiation of MCF-7 cells, early apoptosis was induced. According to the literature, this effect may be associated with increased levels of pro-apoptotic proteins and pro-inflammatory cytokines. In addition, apoptosis is known to be triggered after photothermal/photodynamic therapy by increasing the amount of ROS, caspase 8/9 activation, as well as caspase 3/7 and cytochrome c protein. At the same time, we did not observe a great increase in apoptosis, which may be due to the fact that hyperthermia is able to increase the expression of heat shock genes, in particular HSP70, HSP90, and HSP27, HSPВ1, which protect the cell from the negative influence of IR rays. Conclusion: This study demonstrates that LP-GO effectively induces hyperthermia in MCF-7 cells in a concentration- dependent manner, resulting in an increased percentage of apoptotic cells. Therefore, the combination of LP-GO with NIR irradiation shows promise as a hyperthermia-based therapeutic approach for breast cancer treatment.

Introduction. Polyhydroxylated fullerenes, commonly referred to as fullerenols, are among the most promising carbon allotropes due to their hydrophilic nature, stability, and low toxicity. Natural Killer (NK) cells, are key components of the antiviral and antitumor immune response. However, they have been largely understudied as targets for fullerenol nanoparticles. The objective of this study was to assess the immunocompatibility of hydroxylated fullerenol C60(OH)22-24 with СD3-CD56+ NK cells from human peripheral blood, as well as to investigate the internalization of these nanoparticles into cells. Materials and methods. The studies were conducted on mononuclear cells from the peripheral blood of healthy donors (n=4). In the experiments, fullerenol (MST-WS60-Bio, fullerenol C60(OH)24, 99.99%, Modern Synthesis Technology, Latvia; cluster size approximately 130 nm) was used at concentrations of 200, 100, 50, 25, 12.5, 5, 2.5, 0.5, and 0.25 μg/mL. Wells without added nanoparticles served as controls. Cells were incubated in the presence of fullerenol for 24, 48, and 72 hours under conditions of 5% CO2 and 37°C. The viability of NK cells (CD3-CD56+) and the adhesion/internalization of fullerenol into cells were assessed using flow cytometry. Results. It was found that fullerenol nanoparticles at concentrations ranging from 0.25 to 200 μg/mL did not exhibit cytotoxicity towards NK cells during the observation periods of 24, 48, and 72 hours. Thus, no statistically significant decrease in the percentage and absolute number of live NK cells was detected in cultures with fullerenol over these time frames. The study also showed that NK cells did not demonstrate adhesion/internalization of fullerenol nanoparticles at low concentrations (0.25-50 μg/mL) during all observation periods. However, high concentrations of fullerenol were detected inside NK cells at 48 and 72 hours of observation. After 72 hours, approximately 20% of NK cells had adhered/internalized fullerenol at a concentration of 100 μg/mL, and about 50% had done so at a concentration of 200 μg/mL. Conclusion. Thus, for the first time, it was demonstrated that NK cells adhere/internalize fullerenol at high concentrations (100 and 200 μg/mL), and the percentage of fullerenol-positive cells increases with both longer cultivation duration and higher nanoparticle concentration. Fullerenol didn’t exhibit a cytotoxic effect on the studied cell subpopulation.

Objective: The aim of the present study was to evaluate the influence of fullerenol C₆₀(OH)₂₄ nanoparticles on differentiation of dendritic cells (DCs) from human peripheral blood monocytes in an in vitro system. Materials and methods: Peripheral blood monocytes from healthy donors were induced to a DC phenotype using GM-CSF (100 ng/mL) and IL-4 (20 ng/mL); lipopolysaccharide (LPS) (1 μg/mL) was also added to the cultures. Fullerenol C₆₀(OH)₂₄ nanoparticles were applied at concentrations of 0.25–200 μg/mL; controls contained no nanoparticles. After incubation for 7 days in complete RPMI-1640 medium (сRPMI) at 37°C and 5% CO₂, cell viability, expression of CD209 and CD83, nanoparticle uptake by cells (assessed by flow cytometry using the autofluorescence of C₆₀(OH)₂₄), and cell morphology by inverted microscopy were evaluated. Results: Cell viability in cultures treated with 12.5–50 μg/mL fullerenol decreased significantly, on average from 95 to 85%, while at high concentrations (100–200 μg/mL) viability was maintained at approximately 90% on average. The absolute cell count per culture volume significantly decreased at fullerenol concentrations of 25, 50, and 200 μg/mL. Expression of CD209, a marker of DCs, was significantly reduced at 100–200 μg/mL fullerenol, whereas the proportion of CD83⁺ cells, reflecting DC maturation, decreased starting at 50 μg/mL. The percentage of mature DCs (CD209⁺CD83⁺) was significantly reduced at ≥50 μg/mL nanoparticles in culture. It was shown that increasing fullerenol concentration was accompanied by increased nanoparticle uptake by cells, reaching a maximum at 100–200 μg/mL. Morphologically, spindle-shaped cells with dendrites predominated in cultures with 25–50 μg/mL nanoparticles. At concentrations of 100–200 μg/mL, cells remained rounded and lacked dendrites, retaining a monocyte-like appearance. Conclusion: These observations indicate that fullerenol, despite its modest effect on cell viability, decreases the absolute viable cell count in the culture and negatively affects their differentiation into DCs. The study results are relevant for assessment of the immunocompatibility of carbon nanomaterials and their prospects for biomedical application.