AbstractMultiple 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 Award||2017|
|Supervisor||Matteo Santin (Supervisor)|