The Effect of Pristine and Pegylated Graphene Oxide Nanosheets on the Functions of Human Neutrophils

Graphene oxide (GO) is very useful for biomedicine, due to its physicochemical properties; therefore, its interaction with cells of the immune system has beenextensively studied. Many studies have aimed toreduce the undesirable effects of GO through chemical modification, including through polyethylene glycol (PEG) coating. Neutrophils are the first to respond to foreign object invasion in the body. Their main functions are the uptake and destruction of foreign particles, including with the help of reactive oxygen species (ROS).Our study aimed to investigate theengulfment of unmodified graphene oxide (GO) and graphene oxide coated with polyethylene glycol (GO-PEG) by human neutrophils and the effect of nanosheets on the production of ROS.We used sheets of GO (Ossila, Great Britain, average plate size 1-5 μm) and GO-PEG (569 ± 14 nm, PEG coating≈ 20%) at concentrations of 12.5μg/mL, 25μg/mL, and 50 μg/mL. The uptake of nanosheets was assessed by flow cytometry, taking into account the level of background adhesion of nanoparticles. ROS production was evaluated by luminol-dependent chemiluminescence (LCL).It was found that GO (12.5μg/mL, 25μg/mL, and 50 μg/mL) was actively internalized by neutrophils, while the uptake of GO-PEG was not detected. GO and GO-PEG particles (25 μg/mLand 50 μg/mL) reduced the total production of ROS by human leukocytes.Thus, the modifying of GOnanosheets with PEG resulted in the abolishment of their active uptake by neutrophils but did not affect the GO inhibitory effect on their oxidative activity. Keywords: graphene oxide surface modification, pegylated graphene oxide nanosheets, nanoparticle uptake, human neutrophils, of reactive oxygen specie

The Effect of PEGylated Graphene Oxide Nanoparticles on the Th17-Polarization of Activated T Helpers

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

Interaction of Graphene Oxide Modified with Linear and Branched PEG with Monocytes Isolated from Human Blood

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