Development of a quantitative method to measure EV uptake

Abstract

The outstanding potential of Extracellular Vesicles (EVs) in medicine, deserves a detailed study of the molecular aspects regulating their incorporation into target cells. However, because EV size lies below the limit of resolution of optical techniques, quantification together with discrimination between EV binding to the target cell and uptake is usually not completely achieved with current techniques. Human tetraspanins CD9 and CD63 were fused to a dual EGFP-Renilla-split tag. Subcellular localization and incorporation of these fusion proteins into EVs was assessed by western-blot and fluorescence microscopy. EV binding and uptake was measured using either a classical Renilla substrate or a cytopermeable one. Incubation of target cells expressing DSP2 with EVs containing the complementary DSP1 portion could not recover fluorescence or luciferase activity. However, using EVs carrying the fully reconstituted Dual-EGFP-Renilla protein and the cytopermeable Renilla luciferase substrate, we could distinguish EV binding from uptake. We provide proof of concept of the system by analysing the effect of different chemical inhibitors, demonstrating that this method is highly sensitive and quantitative, allowing a dynamic follow-up in a high-throughput scheme to unravel the molecular mechanisms of EV uptake in different biological systems.

Extracellular vesicle‐mediated endothelial apoptosis and EV‐associated proteins correlate with COVID‐19 disease severity

“…We also determined the effect of plasma on endothelial apoptosis in the presence and absence of two EV uptake inhibitors: heparin and dynasore (Figure 2f). Heparin inhibits the endocytosis of EVs by binding to heparan sulfate proteoglycans (Franzen et al., 2014; Mrowczynski et al., 2018), while dynasore is known to inhibit clathrin‐ and caveolin‐dependent endocytosis of EVs (Chiba et al., 2018; Toribio et al., 2019). The addition of plasma samples derived from the Severe group resulted in significantly increased caspase activity compared to the plasma from uninfected controls.…”

Exploiting the Natural Properties of Extracellular Vesicles in Targeted Delivery towards Specific Cells and Tissues

“…The uptake of EVs is a complex process that involves a combination of different pathways [51], such as caveolae-dependent endocytosis, clathrin-dependent endocytosis, phagocytosis, pinocytosis, receptor-mediated endocytosis and fusion with the plasma membrane [52][53][54][55][56]. This variety of mechanisms provide EVs with some significant advantages in comparison to other synthetic drug delivery systems with respect to their mode of interaction with host cells and their ability to transfer therapeutic molecules [57].…”

Extracellular Vesicle Transportation and Uptake by Recipient Cells: A Critical Process to Regulate Human Diseases

“…Taken together, these results provided insights into how cells may selectively govern EV uptake. With the constant development of novel technological methodologies for studying EV uptake [86,87], detailed mechanisms governing this complex process will no doubt be elucidated in the near future. Nevertheless, it is apparent that a plethora of molecular mechanisms exist to mediate EV-cell communication and different combinations of these mechanisms may be employed by different EV and recipient cell types, depending on inherent properties of EV or dynamic changes in the physiological states of recipient cells.…”