Applications of Novel Tetranuclear Polypyridylruthenium(II) Complexes as Highly Selective Phosphorescent Stains for Extracellular Vesicles

Abstract

It was hypothesised that novel selective phosphorescent RNA stains that consisted of polynuclear polypyridylruthenium(II) complexes could distinguish intact extracellular vesicles (EVs) from cellular debris and other non-EV components. Tetranuclear polypyridylruthenium(II) complexes-based stains, Rubb7-TNL and Rubb7-TL, were confirmed as superior phosphorescent stains for cell biology experiments associated with monocytic and cancer cell EVs, mainly microvesicles (MVs). These novel tetranuclear polypyridylruthenium(II) complexes are remarkably stable in biological media, and act as selective nucleic acid stains for intact EVs. In flow cytometric analysis of EVs released from human monocytes (THP-1 cell line), these stains retain native physical properties of EVs, which is a crucial advantage over many other standard staining technologies. The phosphorescent stains were excellent probes to study interactions of monocytic EVs with an in vitro blood-brain barrier (BBB) model of endothelial cells with tight junctions. Flow cytometry demonstrated the transport of a sub-population of phosphorescently labelled monocytic EVs through a monolayer of brain endothelial cells, which can be used as a model for understanding brain pathologies induced by the immune system response. Phosphorescent EV staining combined with IncuCyte live-cell imaging and live-cell confocal microscopy imaging techniques demonstrated the release and uptake of monocytic EVs by other cell types, A549 lung and MDA-MB-231 breast cancer cells. This enables unprecedented opportunities to study EV biology with these selective nucleic acid stains, and provides new insights into pathways of intercellular communication by which immune-system EVs interact with and release their biochemical cargoes to target cancer cells, to enable potential clinical applications for cancer theranostics.