Carbon nitride ( C3N4) is a class of nanomaterials that recently became increasingly important in the field of health and biomedicine. However, there is a lack of data regarding its toxicity and how this type of material affects the cells of the innate immune system, such as macrophages. The objective of this study was to evaluate and compare the effect of C3N4 in its pristine and oxidized ( oxC3N4) forms using a monocytic cell line differentiated into macrophages. Human THP-1 cells, differentiated into macrophages, were exposed to increasing concentrations of C3N4 and oxC3N4 (e.g., 10, 50 and 100 μg/mL). The assessment of the overall cytotoxicity, by quantifying intracellular adenosine triphosphate (ATP), apoptosis, change of mitochondrial membrane potential and generation of reactive oxygen species (ROS), was carried out. The results showed a significant decrease in cellular ATP after 48 and 72 h of exposure to both type of nanomaterials. The subsequent staining with annexin V and propidium iodide confirmed a significant increase in cell death induced by oxC3N4, but not by pristine C3N4. A significant decrease in mitochondrial membrane potential was also observed only when macrophages were exposed to C3N4, raising question about the presence of a cell death mechanism not involving mitochondria and masking apoptosis in the case of the oxidized carbon nitride. In addition, in the non-cytotoxic condition, oxC3N4 was able to induce ROS generation. Taken together, our results show that the oxidized form of C3N4 is that affecting more the viability of macrophages.

Evaluation of the cytotoxicity of pristine two-dimensional carbon nitride and its oxidized form on human macrophages
    Abstract
    1 Introduction
    2 Experimental section
        2.1 Synthesis of pristine C3N4 and oxidized C3N4
        2.2 THP-1 culture and differentiation into macrophages
        2.3 Total ATP quantification
        2.4 Cellular viability using annexin V-PI staining
        2.5 Carbon nitride-induced mitochondrial disruption
        2.6 Reactive oxygen species generation
        2.7 Statistical analysis
    3 Results and discussion
        3.1 Material design and characterization
        3.2 Total ATP quantification
        3.3 Cell death by annexin V and PI staining
        3.4 Study of mitochondrial membrane potential
        3.5 Reactive oxygen species generation
    4 Conclusions
    Acknowledgements 
    References

• Ghidaa Badran(CNRS, Immunology, Immunopathology and Therapeutic Chemistry, UPR 3572, University of Strasbourg, ISIS, 67000 Strasbourg, France)
• Tengfei Wang(CNRS, Immunology, Immunopathology and Therapeutic Chemistry, UPR 3572, University of Strasbourg, ISIS, 67000 Strasbourg, France)
• Alberto Bianco(CNRS, Immunology, Immunopathology and Therapeutic Chemistry, UPR 3572, University of Strasbourg, ISIS, 67000 Strasbourg, France) Corresponding author
• Yunseok Shin(Department of Chemistry and Chemical Engineering, Inha University, 100 Inha‑ro, Michuhol‑gu, Incheon 22212, Korea)
• Sungjin Park(Department of Chemistry and Chemical Engineering, Inha University, 100 Inha‑ro, Michuhol‑gu, Incheon 22212, Korea)