The Potential of a Graphene Monolayer in Macrophage Polarization Using RAW 264.7 Cells

Maintaining an appropriate balance of macrophage subpopulations throughout the wound healing process, using a graphene monolayer as a substrate, may represent a promising therapeutic strategy. In this study, the effect of a graphene monolayer on the polarization of RAW 264.7 macrophages was investigated using flow cytometry, fluorescence microscopy, and ELISA. Analysis of surface M1 (MHC II, CD80, CD86) and M2 (CD163, CD200R, CD206) markers demonstrated generally higher expression of M1 markers in M1-polarized groups (control, CM1; and graphene monolayer, GM1) compared to M2-polarized groups (CM2 and GM2), likely as a result of LPS and IFN-γ stimulation. Culturing macrophages on a graphene monolayer as a substrate for LPS- and IFN-γ-stimulated cells was associated with a trend toward reduced expression of all analyzed M1-associated markers compared with the control M1 group; however, this effect did not reach statistical significance. TNF-α secretion was higher in GM1 compared to CM0, GM0, and CM2. In contrast, surface markers alone were less conclusive for identifying M2 polarization, whereas intracellular markers such as ARG1 provided a more robust indication of the M2 phenotype. ARG1 expression was significantly elevated in CM2 and GM2 groups, with GM2 showing a significant increase relative to the control groups (CM0, CM1) and GM0 and GM1. These findings further support ARG1 and NOS2 as reliable markers of M2 and M1 polarization, respectively. The graphene monolayer did not induce spontaneous macrophage polarization. Only under M1 (LPS and IFN-γ) and M2 (IL-4 and IL-13) stimulation did it show a consistent trend toward modest modulation of macrophage polarization, possibly creating conditions conducive to tissue healing.