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.