Multiple graphene-based therapeutics have recently been developed, however potential risks related to the interaction between nanomaterials and immune cells are still poorly understood. Therefore, studying the impact of graphene oxide on various populations of immune cells is of importance. In this work, we aimed to investigate the effects of PEGylated graphene oxide on monocytes isolated from human peripheral blood. Graphene oxide nanoparticles with lateral sizes of 100–200 nm and 1–5 μm were modified with linear and branched PEG (GO-PEG). Size, elemental composition, and structure of the resulting nanoparticles were characterized. We confirmed that PEG was successfully attached to the graphene oxide surface. The influence of GO-PEG on the production of reactive oxygen species (ROS), cytokines, phagocytosis, and viability of monocytes was studied. Uptake of GO-PEG by monocytes depends on PEG structure (linear or branched). Branched PEG decreased the number of GO-PEG nanoparticles per monocyte. The viability of monocytes was not altered by co-cultivation with GO-PEG. GO-PEG decreased the phagocytosis of Escherichia coli in a concentration-dependent manner. ROS formation by monocytes was determined by measuring luminol-, lucigenin-, and dichlorodihydrofluorescein-dependent luminescence. GO-PEG decreased luminescent signal probably due to inactivation of ROS, such as hydroxyl and superoxide radicals. Some types of GO-PEG stimulated secretion of IL-10 by monocytes, but this effect did not correlate with their size or PEG structure.

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We investigated the direct effect of PEGylated graphene oxide (P-GO) nanoparticles on the differentiation, viability, and cytokine profile of activated T helper type 17 (Th17) in vitro. The subject of the study were cultures of “naive” T-helpers (CD4+) isolated by immunomagnetic separation and polarized into the Th17 phenotype with a TCR activator and cytokines. It was found that P-GO at low concentrations (5 µg/mL) had no effect on the parameters studied. The presence of high concentrations of P-GO in T-helper cultures (25 μg/mL) did not affect the number and viability of these cells. However, the percentage of proliferating T-helpers in these cultures was reduced. GO nanoparticles modified with linear polyethylene glycol (PEG) significantly increased the percentage of Th17/22 cells in cultures of Th17-polarized T helpers and the production of IFN-γ, whereas those modified with branched PEG suppressed the synthesis of IL-17. Thus, a low concentration of PEGylated GO nanoparticles (5 μg/mL), in contrast to a concentration of 25 μg/mL, has no effect on the Th17-polarization of T helpers, allowing their further use for in-depth studies of the functions of T lymphocytes and other immune cells. Overall, we have studied for the first time the direct effect of P-GO nanoparticles on the conversion of T helper cells to the Th17 phenotype.

Graphene-based materials have an opportunity for use in biomedicine, thanks to their properties. Nevertheless, due to its cytotoxic effects, the use of graphene-based drugs is problematic. However, the surface modification of graphene oxide (GO) nanoparticles with a polyethyleneglycol (PEG) is one way to reduce the harmful effects of graphene on cells. Applying nanoparticles implies their interaction with the immune system, which protects the body. Monocytes are innate immunity cells and the first line of defenсe of the human organism from microorganisms and other alien objects. One of the monocytes’ reactions to a stimulus of any nature is to produce reactive oxygen species (ROS). Published data shows an incomplete picture of modified graphene oxide nanoparticles’ effects on ROS formation by human monocytes. Thus, it was essential to evaluate the pegylated graphene oxide (GO-PEG and GO-8armedPEG) effect on ROS production by human monocytes, assessed by the luminol-dependent chemiluminescence (LCL). The objects of the study were CD14+-cells isolated from mononuclear cells of healthy donors. ROS production was stimulated by opsonized zymosan (OZ), spontaneous LCL was used as a control. PEG-modified (GO-PEG and GO-8armedPEG) GO nanoparticles with sizes of 100-200 nm (“small”) and 1-5 μm (“big”) with PEG covering ~ 20% were used at concentrations of 5 and 25 μg/ml. The study showed that small size nanoparticles at a low concentration of 5 μg/ml and big nanoparticles coated with 8-armed PEG at both concentrations have a significant suppressive effect on spontaneous ROS production. In the stimulated LCL reaction variant, it was found that small nanoparticles (25 μg/ml) also have a suppressive effect on ROS production, such as big-sized particles coated with linear PEG at the same concentration. Thus, we have established for the first time that graphene oxide nanoparticles functionalized with PEG are capable of inhibiting the ROS production by human monocytes, and therefore, we can speak of the antioxidant activity of GO-PEG.

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