The mechanism exploited by GSH-liposomes to cross the blood-brain barrier (BBB) is not well-defined despite their extensively reported efficiency. Transcytosis, involving endocytosis, cellular sorting, and exocytosis by the endothelial cells of the BBB, is proposed as one of the possible mechanisms . However, there is a lack of strong evidence. Hence, in this study, we investigated the exocytosis of GSH-liposomes from human brain microvascular endothelial cells (hBMECs) and characterise the exocytosed vesicles.
First, GSH-liposomes (GSH-npSL) were formulated as previously reported . The fluorescent (lissamine rhodamine B and calcein)-labeled GSH-nPSL in endothelial growth media was incubated with hBMECs for 2 h at 37 ℃ and washed. Then, exocytosis from hBMECs in the liposomes-free medium was observed over 24 h. The exocytosed vesicles were isolated by differential centrifugation method. The isolated small extracellular vesicles (sEV, < 200 nm) were analysed in terms of particle size and surface charge (by Zetasizer), concentration (by NanoSight tracking analysis), protein content (by BCA assay) and fluorescence intensity (FI) using a Microplate reader (BioTek™ Synergy™ 2, Thermo Fisher Scientific, USA).
The particle size of the isolated sEV from GSH-npSL treated hBMECs was different (129 – 160 nm) from those of non-treated cells (control, 119 - 185 nm). Also, the surface charge of the sEV was either negatively charged (for sEV from GSH-npSL treatment > 140 nm size) or neutral (for control). Furthermore, compared to control, there were more sEV for GSH-npSL treatment (7.8 and 23 folds for particles < 140 nm and > 140 nm size, respectively). The total protein content for every 1010 isolated sEV from GSH-nPSL treated hBMECs was higher compared to control (3 vs 1.6 folds for particles < 140 nm and > 140 nm size, respectively). Also, the isolated sEV emitted fluorescence of both lissamine rhodamine B and calcein dyes (representing the liposomes’ membrane and content label, respectively) which was 4 folds higher for particles < 140 nm compared to >140 nm in size.
These findings suggest that the GSH-liposomes are exocytosed from brain endothelial cells intact and may facilitate the production of more extracellular vesicles.