The role of glutathione conjugation on the transcellular transport process of PEGylated liposomes across the blood brain barrier.

Notwithstanding the growing evidence of improved drug delivery efficiency to the brain by ligand modification of PEGylated liposomes, the comprehensive knowledge of their transport processes and payload across the BBB is yet to be revealed. Herein, this study sought to understand the glutathione (GSH) ligand effect on transcellular transport mechanisms of liposomes through the blood-brain barrier (BBB) by comparing PEGylated liposomes (PEG-L) and GSH PEGylated liposomes (GSH-PEG-L). Endocytosis and exocytosis of liposomes including the role of secreted extracellular vesicles (EVs) of brain endothelial cells (BECs) were assessed. Furthermore, pharmacokinetics and brain distribution analysis of gemcitabine loaded liposomes were carried out in healthy rats to ascertain the in vivo applicability. Our findings suggested that the presence of GSH increased the cellular uptake of liposomes by up to 3-fold in human brain microvascular endothelial cells depending on the dose but not in astrocytes. The cell exposure to liposomes particularly GSH-PEG-L dramatically increased the cell secretion of small and microvesicles with liposomal components, though different liposomes preferred different vesicles for exocytosis. This correlated with GSH-PEG-L transport efficiency of 4 % across the in vitro BBB model in 24 h, 1.7-fold higher than that of PEG-L (p < 0.05). In rats, while PEG-L and GSH-PEG-L showed similar pharmacokinetic profiles and prolonged circulation properties, 3.8 % of the total injected dose (ID) of gemcitabine was found in the brain of the GSH-PEG-L group at 8 h post-injection, compared with 2.8 % ID in the PEG-L group. A brain: blood concentration ratio of 1.27 ± 0.12 indicated that an active transport mechanism to cross the BBB for GSH-PEG-L. Overall, this study revealed that GSH augmented the transcellular transport efficiency of liposomes through BBB to improve targeted brain delivery by enhancing cellular uptake and vesicular exocytosis route of BECs.

The involvement of extracellular vesicles in the transcytosis of nanoliposomes through brain endothelial cells, and the impact of liposomal pH-sensitivity.

Despite the demonstrated effectiveness of nano-materials for drug delivery to the brain, a comprehensive understanding of their transport processes across the blood brain barrier (BBB) remains undefined. This multidisciplinary study aimed to gain an insight into the transport processes across BBB, focusing on the transcytosis of liposomes and the impact of liposomal pH-sensitivity. Glutathione-PEGylated pH-sensitive (GSH-PEG-pSL) and non pH-sensitive liposomes (GSH-PEG-L) were fluorescently labelled with rhodamine-DOPE and calcein, both impermeable to biomembranes. Following exposure to brain microvascular endothelial cells (hBMECs), the key functional component of the BBB, intracellular trafficking were evaluated by confocal live-cell imaging. The exocytosed liposomes, including naturally-occurring extracellular vesicles (EVs), were collected using differential centrifugation and and characterised regarding the EV yield, morphology and EVs origin using nanoparticle tracking analysis, transmission electron microscopy and flow cytometry. The transcytosis of liposomes through a verified BBB model comprising of hBMECs monolayer was also quantified. GSH-PEG-L was initially retained in the endo-lysosomes before exocytosed while packed in EVs of different sizes (<100 â€‹nm to >1 â€‹µm) while GSH-PEG-pSL underwent endosome escape with less degree of exocytosis with more fluorescence remaining in the cytoplasm. Compared with the untreated, hBMECs treated with GSH-PEG-L increased the yield of nano-EV and medium-EV by 7.9-fold and 4.6-fold, respectively. Conversely, GSH-pSL-treated cells produced 2.9-fold more nano-EVs but 2-fold less medium-EVs than the control cells. These vesicles were CD144-positive confirming their endothelial cell-origin. GSH-PEG-L demonstrated 2-fold higher efficiencies than GSH-PEG-pSL to cross the in vitro BBB model via exocytosis. Taken together, GSH-PEG-L might utilize EV secretion pathway to achieve transcytosis across brain endothelial cells of the BBB while liposomal pH-sensitivity favors cytoplasmic delivery.

Zebularine suppressed gemcitabine-induced senescence and improved the cellular and plasma pharmacokinetics of gemcitabine, augmented by liposomal co-delivery.

Chemoresistance is a major factor driving cancer recurrence. This study investigated the potential of zebularine, a dual cytidine deaminase (CDA)/epigenetic inhibitor, to circumvent gemcitabine-resistance in pancreatic cancer using a nanomedicine co-delivery approach. The mRNA expression of key metabolic enzymes, including CDA for gemcitabine deactivation in a gemcitabine-resistant cell line Gr2000 and its parental MIA PaCa-2 was compared using quantitative reverse transcription polymerase chain reaction. A highly gemcitabine-resistant population (HRP) in Gr2000 were characterised for their growth pattern, ß-galactosidase activity (a hallmark of senescence) and chemosensitivity to zebularine after isolation. The CDA inhibition effects of zebularine on the intracellular gemcitabine accumulation and pharmacokinetics in rats when co-delivered with pH-sensitive liposomes (pSL) were investigated. Gr2000 had a 3-time upregulated mRNA expression and enzyme activity for CDA. The HRP (28% of bulk Gr2000) were predominately senescent cells which re-proliferated following a growth arrest for a week. Zebularine suppressed the regrowth of senescent cells, meanwhile enhanced cellular gemcitabine concentration by 2-fold. When co-delivered with pSL, zebularine increased cellular gemcitabine concentration by 4-fold, and extended the half-life of gemcitabine in plasma by 22-fold in rats. In conclusion, multiple mechanisms including therapy-induced senescence were identified with gemcitabine-resistance. Co-delivery of zebularine using liposomes could provide multifaceted benefits in gemcitabine therapy for pancreatic cancer treatment.

Optimisation of glutathione conjugation to liposomes quantified with a validated HPLC assay.

Glutathione (GSH) grafted onto nanoliposomes (GSH-liposomes) have the potential to enhance drug delivery into the brain. GSH is known to be an unstable tripeptide, however, despite widespread use to promote active transport its stability has been largely ignored to date. Therefore this study focuses on the optimisation of GSH conjugation with liposomes, supported with a validated HPLC assay for GSH. An isocratic stability-indicating HPLC assay of GSH was developed after derivatisation of GSH with 5,5'-dithio-bis-2-nitrobenzoic acid and applied for efficient conjugation of GSH to DSPE-PEG2000-maleimide lipid, either in solution or in preformed liposomes (4% molar ratio) at pH 7.4. The conjugation rate was monitored by the HPLC assay to optimise the conjugation conditions, including GSH concentration, GSH:lipid ratio, reaction time, temperature and medium. The physiochemical properties of the resulting GSH-liposomes and their GSH densities were characterised. The HPLC method was linear in the range of 0.05-50 µg/ml, highly sensitive (limit of quantification 50 ng/ml), and accurate (98-102% recoveries) with less than 4% intra-day and inter-day variability. Interestingly, enhanced GSH stability was observed at higher GSH concentrations ≥2 mg/ml and mass spectroscopy confirmed that GSH degradation occurred predominantly by oxidation. Both the proton nuclear magnetic resonance (1H NMR) spectra and HPLC analysis of GSH concentrations confirmed the formation of GSH-PEG-DSPE conjugate. Under the optimal conditions, complete conjugation was attained either by post-insertion or direct conjugation methods with the resulting GSH-liposomes attaining a GSH density of 4% with similar size (120 nm) and zeta potential (-26.7 ±â€¯0.9 mV or -29.8 ±â€¯1.5 mV). The study provides useful information on GSH stability for the optimisation of its conjugation in liposomal formulation.