Evaluation of cytotoxicity of fullerenol С<sub>60</sub>(OH)<sub>22-24</sub> towards human peripheral blood NK cells <i>in vitro</i>

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 players of the antiviral and antitumor immune response. However, they have been largely understudied as targets for fullerenol nanoparticles. The aim of this work 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 study internalization of these nanoparticles into the cells. The studies were conducted with mononuclear cells from peripheral blood of healthy donors (n = 4). Fullerenol (MST-WS60-Bio, fullerenol C60(OH)24 99.99%, MST-Nano, Russia; 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 the cells were assessed using flow cytometry. We have 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 period. 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 10% of NK cells did adhere/internalize fullerenol particles at a concentration of 100 μg/mL, with about 50% of cells consumed the particles at 200 μg/mL. 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 at both longer cultivation period and higher nanoparticle concentration. Fullerenol didn’t exhibit cytotoxic effects on the studied cell population.