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Title: A biophysical investigation of a membrane-active cyclodextrin-inliposome formulation for antibiotic delivery
Author: Vandera, Kalliopi-Kelli Apostolos
ISNI:       0000 0004 7970 0068
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2019
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Gram-negative bacteria possess numerous defence mechanisms against antibiotics, due to the intrinsic permeability barrier afforded by their outer membrane and the various efflux mechanisms which pump out drugs, explaining the recalcitrance of some common and lifethreatening infections. A novel compound, PPA148, was synthesised in-house and showed promising activity against Gram-negative bacteria. Nevertheless, in some clinical bacterial strains, drug efflux resulted in reduced efficacy was observed, which was reversed in the presence of the efflux pump inhibitor phenylalanine-arginine β naphthylamide (PAβN). A formulation of PPA148 consisting of a drug/cyclodextrin (drug/CD) complex encapsulated in liposomes is investigated as a way to increase drug uptake. PPA148 has very low water solubility, which was measured using spectroscopic techniques: photon correlation spectroscopy (PCS) and Ultra-Violet spectroscopy (UV). Its solubility is improved by complexation with βCD derivatives (HPβCD and RAMEB) and the complex is characterized by applying nuclear magnetic resonance (NMR) and fluorescence. Fluidosomes, loaded with the drug/CD complex, are manufactured by applying the thin-film hydration method followed by extrusion for reducing the size of liposomes. PCS is used to size the particles and measure their zeta-potential. A Stewart assay and UV were used to quantify the lipid and drug concentration respectively, in the final formulation. A disk diffusion microbiological assay is used to assess the efficacy of the formulation against E. coli (DH5a). A variety of in vitro biophysical techniques are used to assess the mechanism of the drug uptake. Phospholipids, Re Lipid A extracted from S. Minnesota and Rc J5 LPS extracted from E. coli are used to make a synthetic monolayer and bilayer model of the outer and inner bacterial membranes. They are adapted for use with the Langmuir trough (LT) and neutron reflectivity (NR) techniques to monitor changes occurring upon interaction with the drug alone and the formulated drug. The results revealed an possible increase in drug efficacy with a possible fusion mechanism of uptake. NR provides a new method to examine the fusion mechanism of fluidosomes with bacterial membrane.
Supervisor: Dreiss, Cecile Ayako ; Jones, Stuart Allen ; Harvey, Richard Douglas Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available