Extracellular vesicles (EVs) are biologically-derived nanovectors important for intercellular communication and trafficking. As such, EVs show great promise as disease biomarkers and therapeutic drug delivery vehicles. However, despite the rapidly growing interest in EVs, understanding of the biological mechanisms that govern their biogenesis, secretion, and uptake remains poor. Advances in this field have been hampered by both the complex biological origins of EVs, which make them difficult to isolate and identify, and a lack of suitable imaging techniques to properly study their diverse biological roles.
Imperial College London researchers present a new strategy for simultaneous quantitative in vitro imaging and molecular characterisation of EVs in 2D and 3D based on Raman spectroscopy and metabolic labelling. Deuterium, in the form of deuterium oxide (D2O), deuterated choline chloride (d-Chol), or deuterated D-glucose (d-Gluc), is metabolically incorporated into EVs through the growth of parent cells on medium containing one of these compounds. Isolated EVs are thus labelled with deuterium, which acts as a bio-orthogonal Raman-active tag for direct Raman identification of EVs when introduced to unlabelled cell cultures. Metabolic deuterium incorporation demonstrates no apparent adverse effects on EV secretion, marker expression, morphology, or global composition, indicating its capacity for minimally obstructive EV labelling. As such, this metabolic labelling strategy could provide integral insights into EV biocomposition and trafficking. This approach has the potential to enable a deeper understanding of many of the biological mechanisms underpinning EVs, with profound implications for the design of EVs as therapeutic delivery vectors and applications as disease biomarkers.
Raman spectroscopic imaging and analysis of metabolically labelled EVs
in solution and in vitro
Illustration of metabolic labelling and Raman spectroscopic analysis of cells and EVs using the confocal Raman microspectroscopy system; (i) cells cultured on MgF2 windows are imaged via Raman microspectroscopy (control cells) or (ii) are incubated further with deuterium-containing (D2O, d-Chol, or d-Gluc) cell culture medium and imaged via Raman microspectroscopy (deuterium-treated cells) or (iii) the EVs are isolated from the cells and assessed via Raman trapping analysis or (iv) the metabolically deuterium-labelled EVs are incubated with unlabelled cells before cell imaging via Raman microspectroscopy (deuterium EV-treated cells).