Glutathione-decorated dendrons as potential drug carrier systems in Multiple Sclerosis

  • Dhafir Masheta

    Student thesis: Doctoral Thesis


    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
    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
    Date of Award2017
    Original languageEnglish
    Awarding Institution
    • University of Brighton
    SupervisorMatteo Santin (Supervisor)

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