Microbubbles-Assisted Ultrasound Triggers the Release of Extracellular Vesicles

Abstract

Microbubbles-assisted ultrasound (USMB) has shown promise in improving local drug delivery. The formation of transient membrane pores and endocytosis are reported to be enhanced by USMB, and they contribute to cellular drug uptake. Exocytosis also seems to be linked to endocytosis upon USMB treatment. Based on this rationale, we investigated whether USMB triggers exocytosis resulting in the release of extracellular vesicles (EVs). USMB was performed on a monolayer of head-and-neck cancer cells (FaDu) with clinically approved microbubbles and commonly used ultrasound parameters. At 2, 4, and 24 h, cells and EV-containing conditioned media from USMB and control conditions (untreated cells, cells treated with microbubbles and ultrasound only) were harvested. EVs were measured using flow cytometric immuno-magnetic bead capture assay, immunogold electron microscopy, and western blotting. After USMB, levels of CD9 exposing-EVs significantly increased at 2 and 4 h, whereas levels of CD63 exposing-EVs increased at 2 h. At 24 h, EV levels were comparable to control levels. EVs released after USMB displayed a heterogeneous size distribution profile (30–1200 nm). Typical EV markers CD9, CD63, and alix were enriched in EVs released from USMB-treated FaDu cells. In conclusion, USMB treatment triggers exocytosis leading to the release of EVs from FaDu cells.

Potential Use of Extracellular Vesicles Generated by Microbubble-Assisted Ultrasound as Drug Nanocarriers for Cancer Treatment

“…Our previous findings indicated that levels of EVs exposing CD9 or CD63 significantly increased at 2 and 4 h after USMB [14]. Therefore, we measured EVs containing CTG or BSA FITC in the cell supernatant 2 h after USMB treatment.…”

“…For USMB experiments, cells were prepared according to our previously reported method [14]. Briefly, cells were seeded in cell culture chambers (treated CLINIcell ® 175 µm thick polycarbonate walls, 25 cm 2 ) (Mabio, Tourcoing, France) 1 day prior to USMB.…”

“…The piezoelectric unfocused single element transducer with a diameter of 20 mm (Precision Acoustics) operating at 1.5 MHz was placed at the bottom of a water tank, 8 cm below a cell culture chamber frame. The water surface was another 12 cm above the frame and heated to 37 • C. When exposing cells to US, the cell culture chamber was mounted in the frame and moved over the transducer for 80 s to expose the whole cell culture chamber surface, which resulted in a total exposure of approximately 5 s for each area of the cell culture chamber [14,25]. For USMB treatment control, untreated cells were used.…”

“…For EV measurement using flow cytometry, conditioned medium containing EVs was thawed quickly at 37 • C. We captured EVs using immuno-magnetic beads and measured these EV-bead complexes using BD FACScanto II according to our previously reported method [14]. Using 96-well round-bottom plate 70 µL conditioned medium containing EVs was mixed with 10 µL ExoCap magnetic capture beads CD9 or CD63 (JSR Life Sciences, Leuven, Belgium) to obtain end concentration of 6 × 10 4 beads per sample.…”

“…To investigate whether BSA FITC was carried by EVs after USMB, immunogold electron microscopy was performed as described previously [14,27]. EVs from conditioned medium collected from USMB-treated HUVEC in the presence of BSA FITC or non-conjugated BSA were measured.…”