Biological Degradation of Graphitic Carbon Nitride Sheets and Autophagy Induction in Macrophages

Although a metal-free graphitic-C3N4 was studied for potential applications in bioimaging, cancer therapy, etc., its biodegradability and impact on immune modulation and autophagy induction have not yet been reported, which are essential for designing clinical applications. Herein, we studied the biodegradability of two types of g-C3N4 nanosheets (exfoliated and porous) using human myeloperoxidase (hMPO) from the primary immune cells (neutrophils), plant enzyme horseradish peroxidase (HRP) and photo-Fenton reaction (PF, generating hydroxy radicals). The biodegradation was followed by electron microscopy, and spectroscopic techniques such as Raman, X-ray photoelectron, UV-vis and fluorescence spectroscopy. All these results confirmed that the g-C3N4 sheets could be degraded better by hMPO over HRP, where porous g-C3N4 showed higher degradability than exfoliated due to the presence of oxygen groups. Next, the PF reaction was applied to interrogate the degradation by-products using mass spectrometry, and the cytotoxicity of degradation products was assessed in comparison with pristine g-C3N4. Further, the impact of g-C3N4 on the induction of autophagy in the RAW264.7 macrophages was confirmed by over-expression of the autophagy marker, LC3 protein, especially significantly observed for the porous sheets. Finally, the immunomodulatory function of the nanosheets and cytokine production was also evaluated in RAW macrophages following exposure to both porous and exfoliated g-C3N4. These findings demonstrated that the porous nanosheets induced a dose-dependent pro-inflammatory response.