Disruption of intracellular calcium homeostasis drives graphene quantum dots-induced inflammatory response in liver macrophages

Graphene quantum dots (GQDs), with photoluminescent properties, high stability, and excellent biocompatibility, hold tremendous potential in biomedicine. It is urgent to evaluate their safety and potential health risks to promote their clinical application. Kupffer cells (KCs), as primary immune cells encountered by foreign substances entering the liver and integral to liver immunity, have yet to be systematically studied for toxicity responses to different GQDs. This study focused on three widely used GQDs (OH-GQDs, N-GQDs, and NH₂-GQDs), examining their effects on KCs and elucidating the underlying mechanisms. Our findings suggested that the toxicity levels of the three GQDs on KCs are ranked as OH-GQDs > N-GQDs > NH₂-GQDs, with inflammation being the main form of toxic effect, which was a consequence of GQD-induced calcium homeostasis disruption. Specifically, cytoplasmic calcium imbalance caused by GQDs leaded to mitochondrial Ca^2 + overload, mitochondrial dysfunction, and mtROS generation, which subsequently activated the NLRP3 inflammasome-dependent inflammation. Crucially, we identified upstream mechanistic differences in calcium homeostasis disruption induced by each GQDs, with the most toxic OH-GQDs inducing ER stress-mediated Ca²⁺ release, which was closely related to the depletion of GSH caused by the generation of oxygen free radicals (•OH and O₂•−). By tracing Ca²⁺ homeostasis, this work comprehensively mapped the upstream and downstream mechanisms of GQD-induced liver macrophage inflammation, providing new insights into the toxic effects of GQDs. Additionally, linking the intrinsic properties of GQDs, we identified the molecular initiating events of OH-GQDs mediated excessive inflammation in KCs, offering strategies for the de novo safe design of GQDs that target the content of oxygen-containing functional groups and the generation capacity of free radicals, which is of great significance for the development of safe, non-toxic, and efficient GQDs for clinical diagnosis and treatment.