Oral Delivery of Glutathione: the Antioxidant Function, the Barriers and the Strategies

Abstract

Background: Glutathione (GSH) is a tripeptide, which is recognised as a potent antioxidant involving in numerous essential biological processes, and has been used for interventions in various degenerative diseases. However, as with all protein and peptide drugs, its oral delivery remains challenging due to the physical and enzymatical barriers existing in the gastrointestinal (GI) tract, leading to a low oral bioavailability. Although several approaches have been explored in the past to improve the oral bioavailability of GSH, an appropriate formulation with clinical therapeutic effects is still yet to be developed. This study explores an approach to develop an oral niosome-based GSH loaded delivery system that could provide protection against proteolytic degradation in the GI tract and enhance the molecular absorption across the epithelial membrane. Aim: This project aims to improve GSH physical and chemical stability and explore the mechanism of GSH cellular uptake and transport using a cell culture model. Methods: This study re-developed and validated a high-performance liquid chromatography (HPLC) method to qualify and quantify GSH, which was applied for GSH analysis in the entire project. A thin film hydration method was used to fabricate GLNs, whose composition was optimised by applying a 2 level 3 factors factorial design methodology using Design-Expert® software. The optimised formulation was then characterised for its particle size, zeta potential, entrapment efficiency (EE), morphology, physical stability, in vitro drug release and degradation. The mechanism of GLNs cellular uptake and transport was assessed using Caco-2 cells or co-cultured with Ht29 cells for transport study. Results: The re-developed HPLC method was reliable and accurate, with the GSH calibration curve ranging from 1 to 100 μg/mL displaying excellent linearity (R2 = 0.9999). The optimised formulation of GSH exhibited double-layered vesicular structure, with an average particle size at 253.3 ± 0.6 nm, PDI at 0.353 ± 0.028, zeta potential at -65.3 ± 3.5 mV and EE at 31.45 ± 0.46%. The physical stability study suggested that GSH incorporated with niosomes presented a better physical stability compared to pure GSH solution at the same temperature. The latter appeared to be more stable at 4 ℃ than at 25 ℃ or 40 ℃. The release study suggested that GLNs demonstrated a two-phased release profile with sustained release behaviour as opposed to one-phased release of free GSH solution. Additionally, the in vitro degradation study revealed the GLNs showed protective effect on GSH against enzymatic degradation in extracts from all intestinal regions of rats. Images taken by a confocal microscopy illustrated internalisation of fluorescent-labelled niosomes, suggesting that the mechanism of niosomes uptake and transport into Caco-2 cells might go through endocytosis, the common pathway for nanoparticles absorption across biological membrane and this machenism was confirmed from the transport study (1). The transport study was carried out to determine the effect of niosomes on the GSH transport across the monolayer of the Caco-2/HT29 cells, resulting to significant increases in the flux rates of GSH. Conclusion: A reliable HPLC method was re-developed and validated for its accuracy and reliability based on ICH guideline. A niosomal formulation containing GSH was developed, optimised, and characterised, which has shown positive results in all studies. In addition, the degradation of luminal contents in intestinal areas of rats was superior to those of the mucosa. In terms of enzyme inhibitor, this study discovered that EDTA displayed a significant inhibition effect compared to bacitracin. In vitro studies of the formulation in terms of degradation, cellular uptake, and transport, suggest GSH loaded niosome (GLNs) significantly improve GSH’s stability and cellular absorption across the intestinal membrane.