A pH-Reversible Fluorescent Probe for in Situ Imaging of Extracellular Vesicles and Their Secretion from Living Cells

Abstract

Our knowledge in how extracellular vesicles (EVs) are secreted from cells remains inadequate due to the limited technologies available for visualizing them in situ. We report a pH-reversible boron dipyrromethene (BODIPY) fluorescent probe for confocal imaging of EVs secreted from living cells without inducing severe cytotoxicity. This probe predominantly assumes a non-fluorescent leuco-BODIPY form under basic conditions, but it gradually switches to its fluorescent parent BODIPY form upon acidification; such pH transition empowers the imaging of acidic EVs (such as CD81-enriched exosomes and extracellular multivesicular bodies) in weakly basic culture medium and intracellular acidic precursor EVs in weakly basic cytoplasm, with minimal false positive signals frequently encountered for “always-on” dyes. Joint application of this probe with plasmid transfection reveals the secretion of some EVs from cellular pseudopodia via microtubule trackways. This probe may provide mechanistic insights into the extracellular transport of EVs and support the development of EV-based nanomedicines.

Mammalian Cells Exocytose Alkylated Gold Nanoparticles via Extracellular Vesicles

“…While these TEM imaging data suggest NP exocytosis, we acknowledge that TEM imaging can only capture still images of fixed cells and may not allow us to conclusively distinguish between endocytosis and exocytosis based on the direction of NP movement. Yet, live imaging of the exocytosis of a single NP and tracking its three-dimensional (3D) movement in culture medium remain challenging . The fluorescently labeled NPs, once released to the extracellular space, become significantly diluted by the culture medium; the low signal-to-noise ratio renders confocal imaging of the exocytosed NPs impractical (see SI Extended Results and Discussion).…”

“…Yet, live imaging of the exocytosis of a single NP and tracking its three-dimensional (3D) movement in culture medium remain challenging. 33 The fluorescently labeled NPs, once released to the extracellular space, become significantly diluted by the culture medium; the low signal-to-noise ratio renders confocal imaging of the exocytosed NPs impractical (see SI Extended Results and Discussion).…”

Unconventional Protein Secretion Dependent on Two Extracellular Vesicles: Exosomes and Ectosomes

“…In response to various types of stimulation MVBs move, approaching the plasma membrane ( Figure 1 ) ( Calvo and Izquierdo, 2020 ). Understanding of these processes has been strengthened by the use of optical reporters associated with ILV markers ( Verweij et al, 2018 ; Liu X.-M. et al, 2021 ). Upon tethering to specific sites ( Davis et al, 2021 ), some heterogeneity in the molecules participating in MVB exocytosis has been reported among various cell types.…”

“…Fluorescent proteins of exosome membranes, such as CD63-pHfluorin, start emitting fluorescence upon exocytosis. The steps revealed by these approaches are numerous, from the efficacy and intracellular signaling of exogenous stimuli to the frequency, localization and machinery of exocytosis, up to the navigation of the released vesicles ( Liu X.-M. et al, 2021 ; Gurung et al, 2021 ). The exosome localization studies have revealed various unexpected results.…”

Micellar Ratiometric Fluorescent Blood pH Probe Based on Triplet-Sensitized Upconversion and Energy-Transfer Behaviors

“…Fluorescent probes have been widely investigated for pH detection in vitro , in vivo , and in situ . − So far, various fluorescent probes, − such as organic fluorescent molecules, fluorescent polymers, fluorescent hydrogels, metal–organic framework materials, and quantum dots, have been designed with downshifted emissions as signals under ultraviolet light excitation, which, however, limits their applications with regard to physiological pH detection. In addition, the shortcomings of autofluorescence interference in organisms, poor penetrability of an ultraviolet (UV) excitation light source, and detection inaccuracies caused by concentration fluctuations of probe molecules cannot be ignored. − …”