Immune profiling and tracking of two-dimensional transition metal dichalcogenides in cells and tissues

Two-dimensional (2D) transition metal dichalcogenides (TMDs), such as molybdenum disulfide (MoS2) and tungsten disulfide (WS2), have attracted considerable interest in biomedicine due to their unique combination of physicochemical properties. The effect of nanomaterials on immune cells and their biodistribution are critical aspects of their clinical translation. However, understanding the interactions of these emerging 2D nanomaterials with the complex pool of peripheral blood mononuclear cells (PBMCs) at the single-cell level and their in situ localization in the main organs still needs to be discovered, preventing their translation in medical settings. Here, we report in-depth immune profiling of water-based and defect-free 2D formulations of MoS2 and WS2 through the simultaneous label-free tracking of their immune cell interactions, both ex vivo in human PBMCs and in vivo in mice by high-dimensional analytical approaches, as well as their biodistribution. For comparison, we studied graphene, the hitherto most explored 2D material for biomedical applications. First, we assessed the impact at the protein and gene level by multiplex protein arrays and RNA sequencing, demonstrating a very modest effect of MoS2 and WS2 on immune cell functionality compared to graphene. Then, a single-cell view of the effects of MoS2 and WS2 on 16 primary human immune cell types in terms of viability and functionality was obtained by single-cell mass cytometry by time-of-flight (CyTOF). We explored over 30 markers looking at multiple cell parameters. Finally, we present evidence that MoS2 and WS2 are visible, without the need for labeling, at the single-cell and tissue level by CyTOF, imaging mass cytometry, and multiplexed ion beam imaging by time-of -flight (MIBI-TOF). In particular, MoS2 and WS2 could be detected in the molybdenum (95Mo) and tungsten (180-186W) channels, respectively, which are not used for commercial mass cytometry tags, allowing for the simultaneous interrogation of a wide variety of biological parameters ex vivo and in vivo following intravenous administration of the TMDs. Indeed, we demonstrated the accumulation of TMDs in the main organs by MIBI-TOF and they could also be identified in specific immune cell subsets by CyTOF. Among the two TMDs stud-ied, WS2 exhibited the highest brightness and signal intensity in all the cell subpopulations and tissues analyzed. In conclusion, we identified TMDs as immune-compatible nanoplatforms, traceable at the single-cell and tissue (sub-organ) levels, thus opening up new perspectives for their exploration in biomedicine.