BIOLOGICAL RESPONSE AND MORPHOLOGICAL ASSESSMENT OF HUMAN THP-1 MACROPHAGES TO RIGID MULTIWALLED CARBON NANOTUBES TRAPPED IN ECM-MIMETIC GELATIN

Carbon nanotubes (CNTs) are nanomaterials of particular interest due to their distinctive physicochemical properties that make them promising for technological and industrial development. Their application in industry is continuously growing, which could lead to accumulation in the environment and a consequent impact on both humans and ecosystems. Exposure to CNTs, especially in the occupational context, occurs mainly by inhalation. However, little is known about the possible effects of CNTs on lung diseases and cancer. Since the extracellular matrix (ECM) modulates the behavior of nanomaterials entrapped in it and the immune response, the study of interactions between immune cell and carbon nanotubes should take into account the extracellular microenvironment in which these interactions occur. In the present study, funded by the PRIN 2022 PNRR program, we evaluated the impact of multiwalled carbon nanotubes (MWCNTs) embedded in gelatin as ECM mimic, on THP-1 macrophages; mainly, studies focused on morphological characterization and biological responses. Macrophage-like cells derived from the human monocytic cell line THP-1 were cultured on ECM-like substrates for up to 5 days. Preliminary experiments demonstrated that macrophages adhered to the substrates and that CNT treatments did not affect cell viability 24 h after cell seeding. However, cell morphology changed at higher CNT concentrations and culture times, showing an increase in filopodia and a decrease in the spreading area that assumed a round shape. Macrophages remodelled ECM-like substrates, making CNTs bioavailable for internalization as indicated by confocal and electron imaging. Interestingly, 24 h after macrophage seeding, ROS generation increased in the presence of CNTs. Since among the numerous immunosuppressive cells in the tumor microenvironment, macrophages play an important role in tumor development, the ability of MWCNT-stimulated macrophages to shape the tumor microenvironment was evaluated. Surprisingly, it was observed that conditioned media of THP-1 cells cultured in the presence of CNTs for 24 h facilitated the migration of endothelial cells in the wound healing assay and reduced the growth of 3D glioblastoma spheroids independently from conditioned media of control cells. Overall, MWCNTs, although entrapped in ECM-like gelatin, are able to stimulate macrophage activity, in a concentration-dependent manner, making them in turn able to remodel the tumor microenvironment.