Poster Presentation Australasian Extracellular Vesicles Conference 2020

Isolation and characterization of exocytosed GSH-liposomes from brain endothelial cells (#67)

Joy N Reginald-Opara 1 2 , Justin Dean 1 , Simon O'Carroll 1 , Darren Svirskis 1 , Zimei Wu 1
  1. University of Auckland , Auckland , New Zealand
  2. Department of Pharmaceutics, University of Nigeria , Nsukka, Nigeria


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 [1]. 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 [2]. 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.


  1. Agrawal M, Ajazuddin, Tripathi DK, Saraf S, Saraf S, Antimisiaris SG, et al. Recent advancements in liposomes targeting strategies to cross blood-brain barrier (BBB) for the treatment of Alzheimer's disease. J Control Rel 2017;260:61-77.
  2. Reginald-Opara JN, Svirskis D, O'Carroll SJ, Sreebhavan S, Dean JM, Wu Z. Optimisation of glutathione conjugation to liposomes quantified with a validated HPLC assay. Int J Pharm 2019;567:118451.