Earthworm Coelomocyte Internalization of MoS2 Nanosheets: Multiplexed Imaging, Molecular Profiling, and Computational Modeling

Fully understanding the cellular uptake and intracellular localization of MoS₂ nanosheets (NSMoS₂) is a prerequisite for their safe applications. Here, we characterized the uptake profile of NSMoS₂ by functional coelomocytes of the earthworm Eisenia fetida. Considering that vacancy engineering is widely applied to enhance the NSMoS₂ performance, we assessed the potential role of such atomic vacancies in regulating cellular uptake processes. Coelomocyte internalization and lysosomal accumulation of NSMoS₂ were tracked by fluorescent labeling imaging. Cellular uptake inhibitors, proteomics, and transcriptomics helped to mechanistically distinguish vacancy-mediated endocytosis pathways. Specifically, Mo ions activated transmembrane transporter and ion-binding pathways, entering the coelomocyte through assisted diffusion. Unlike molybdate, pristine NSMoS₂ (P-NSMoS₂) induced protein polymerization and upregulated gene expression related to actin filament binding, which phenotypically initiated actin-mediated endocytosis. Conversely, vacancy-rich NSMoS₂ (V-NSMoS₂) were internalized by coelomocytes through a vesicle-mediated and energy-dependent pathway. Mechanistically, atomic vacancies inhibited mitochondrial transport gene expression and likely induced membrane stress, significantly enhancing endocytosis (20.3%, p < 0.001). Molecular dynamics modeling revealed structural and conformational damage of cytoskeletal protein caused by P-NSMoS₂, as well as the rapid response of transport protein to V-NSMoS₂. These findings demonstrate that earthworm functional coelomocytes can accumulate NSMoS₂ and directly mediate cytotoxicity and that atomic vacancies can alter the endocytic pathway and enhance cellular uptake by reprogramming protein response and gene expression patterns. This study provides an important mechanistic understanding of the ecological risks of NSMoS₂.