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Title: Glutathione-decorated dendrons as potential drug carrier systems in Multiple Sclerosis
Author: Masheta, Dhafir
ISNI:       0000 0004 7427 0660
Awarding Body: University of Brighton
Current Institution: University of Brighton
Date of Award: 2017
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Multiple sclerosis (MS) is a chronic progressive demyelinating disorder of the central nervous system. It is an autoimmune neurodegenerative disease associated with inflammation in the brain white matter mediated by autoreactive T-cells. MS is not curable and the treatment is only aimed at reducing the frequency, limiting the lasting effect of relapses, relief of symptoms, preventing disability arising from disease progression and promoting nerve repair. Glucocorticoid, typically methylprednisolone (MP) is given to reduce the duration of MS relapses. However, due to the presence of the blood-brain barrier (BBB) which impedes the effective delivery of MP to the brain, high doses of MP are given to the patients to reach the minimum therapeutic concentration. Consequently, such elevated doses of MP results in an increase in the adverse effects of the drug. To overcome MP limited permeability, it must be delivered using specialised strategies to avoid high doses administration. This study aims to improve MP cell membrane penetration employing dendrons as drug carriers to achieve higher loading capacities and utilising glutathione molecule as a ligand to be recognised by glutathione receptors in the brain. The aim was achieved by design and characterisation of dendron-drug conjugates, assessment of their cytotoxicity, validation of an in vitro b.End3 cells brain model, penetration studies through this model and biochemical investigation of the anti-inflammatory activity of the final molecule. The successful synthesis of the dendrimers, dendrimer-MP conjugates and attachment with glutathione were achieved using automated solid phase peptide synthesis and characterised by high performance liquid chromatograph, mass spectrometry, nuclear magnetic resonance and Fourier transform infra-red spectroscopy. The cytotoxicity of the drug loaded and unloaded was assessed using lactate dehydrogenase, MTT and calcein/ethidium cytotoxicity assays. Under the conditions used, the assembled drug conjugates’ toxicity levels were within the acceptable range. Transwell inserts were used to support mice immortalised brain endothelial cells, b.End3, to form an in vitro model of the BBB model. The model was validated by using transepithelial electrical resistance measurements, morphological examination, and permeability to paracellular marker (horseradish peroxidase). The data collected revealed that the b.End3 cell line is able to express several important barrier features of the in vivo BBB and can be used as in vitro BBB model for penetration studies. The cells exerted their maximum barrier functions at Day 7 of culturing. Fluorescent staining images confirmed the uptake of the synthesised molecules by b.End3 cells. Quantitative measurements based on high performance liquid chromatography of penetration through the b.End3 cultured cells-barrier indicated improvement in the permeability of MP conjugated to glutathione by almost 3.5 fold compared to free MP reaching 16.8% and 40.9% after 1 and 3 hours of sample introduction, respectively. Biochemical investigations revealed that MP in its attached form retained its anti-inflammatory activity based on the reduction in lactate dehydrogenase and inflammatory cytokines release levels from C6 glial cells treated with tumour necrotic factor- and showed greater antiinflammatory activity compared to unconjugated MP. It can be concluded that the ability of MP to cross an in vitro BBB model can be improved by using glutathione-dendronised carrier system and could provide a suitable base for other poorly penetrating medications intended for the treatment of other neurodegenerative diseases.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available