Biological degradation of graphitic carbon nitride sheets and autophagy induction in macrophages

Although metal-free graphitic-C₃N₄ was studied for potential applications in bioimaging, cancer therapy, <i>etc</i>., 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) derived from primary immune cells (neutrophils), the plant enzyme horseradish peroxidase (HRP) and the photo-Fenton reaction (PF, generating hydroxy radicals). Biodegradation was followed by analysis using electron microscopy and spectroscopic techniques, including Raman, X-ray photoelectron, UV-vis and fluorescence spectroscopy. The results confirmed that the g-C₃N₄ sheets could be degraded more effectively by hMPO than by HRP, with porous g-C₃N₄ showing higher degradability than exfoliated g-C₃N₄ due to the presence of oxygen groups. Next, the PF reaction was applied to analyse the degradation by-products using mass spectrometry, and the cytotoxicity of degradation products was assessed in comparison with pristine g-C₃N₄. Additionally, the impact of g-C₃N₄ on the autophagy induction in RAW264.7 macrophages were confirmed by the overexpression of the autophagy marker, LC3 protein, particularly in response to porous sheets. Finally, the immunomodulatory function of the nanosheets and cytokine production were evaluated in RAW macrophages following exposure to both porous and exfoliated g-C₃N₄. These findings demonstrated that porous nanosheets induced a dose-dependent pro-inflammatory response.