Quantification of extracellular vesicles in vitro and in vivo using sensitive bioluminescence imaging

Abstract

Extracellular vesicles (EVs) are naturally occurring nano-sized carriers that are secreted by cells and facilitate cell-to-cell communication by their unique ability to transfer biologically active cargo. Despite the pronounced increase in our understanding of EVs over the last decade, from disease pathophysiology to therapeutic drug delivery, improved molecular tools to track their therapeutic delivery are still needed. Unfortunately, the present catalogue of tools utilised for EV labelling lacks sensitivity or are not sufficiently specific. Here, we have explored the bioluminescent labelling of EVs using different luciferase enzymes tethered to CD63 to achieve a highly sensitive system for in vitro and in vivo tracking of EVs. Using tetraspanin fusions to either NanoLuc or ThermoLuc permits performing highly sensitive in vivo quantification of EVs or realtime imaging, respectively, at low cost and in a semi-high throughput manner. We find that the in vivo distribution pattern of EVs is determined by the route of injection, but that different EV subpopulations display differences in biodistribution patterns. By applying this technology for real-time non-invasive in vivo imaging of EVs, we show that their distribution to different internal organs occurs just minutes after administration.

Extracellular vesicles from human multipotent stromal cells protect against hearing loss after noise trauma in vivo

“…It would also be interesting to assess the in vivo localization of EVs after administration into the inner ear and their persistence despite challenges in the labeling of EVs. 40,75,76 The present study provides initial proof of the high potential of UC-MSC-derived EVs to support neuronal survival and to repair noise-induced damage in the inner ear. The capacity to manufacture and characterize clinicalgrade EVs under current GMP will support the rapid translation of these research findings into clinical application and may be helpful also for the intervention in other organ systems and disease indications.…”

“…Whether the effects observed in vitro and in vivo in the present study are due to the applied EVs or due to the combined effects of EVs and cytokine or miRNA profiles need further investigation. It would also be interesting to assess the in vivo localization of EVs after administration into the inner ear and their persistence despite challenges in the labeling of EVs 40,75,76 …”

How to Select Firefly Luciferin Analogues for In Vivo Imaging

“…On the other hand, many researchers have synthesized CTZ analogues for application to optical imaging [ 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 ]. For instance, Hall et al developed an artificial bioluminescence pair, the substrate furimazine (FMZ) and the mutant luciferase NanoLuc ® [ 49 , 50 , 51 ].…”

Diagnostic and Therapeutic Applications of Extracellular Vesicles in Interstitial Lung Diseases

“…Furthermore, little is known regarding EV kinetics and biodistribution in animal models, and, more so, in human subjects. EV labeling and tracking technologies still have significant limitations including quantifiable and reliable measures of EV tracking in vivo [ 123 , 124 ]. Finally, EV engineering to improve scalability, drug delivery, and therapeutic relevance and potency remain active areas of research and discovery.…”