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Development of new tools to study drug-lipid interactions and their application to investigating amphotericin b's association with model cell membranes

University of British Columbia

The interaction of different formulations of the antifungal drug amphotericin B (AmB) with model cell membranes was studied and new techniques of measuring this interaction using electrochemical and/or spectroscopic methods were developed. Two model cell membrane systems were used: sterol-free lipid monolayers adsorbed to a Hg electrode and sterol-free or sterol-containing floating lipid monolayers on a Langmuir trough. Electrochemical control over the adsorbed monolayer allowed the defectiveness of the layer to be varied and the interaction of AmB with both well-ordered and defective monolayers characterized. Measurements of monolayer capacitance and permeability were used to indicate the nature of the interaction. Capacitance provides a measure of the lipid organization, while permeability was measured via electro reduction of thallium (I)cation. The three AmB formulations and two control samples were examined and showed different interaction behaviour. The disruption of lipid order and permeabilization induced by the two commercial formulations correlated generally with in vivo studies of their toxicity. An experimental and possibly less toxic AmB formulation made monolayer significantly more permeable. In situ fluorescence microscopy of the monolayer on Hg was carried out after introducing a low concentration of fluorophore into the layer. Fluorescence intensity as a function of electrode potential was measured and was used to characterize the lipid on Hg model membrane system before we attempted to measure AmB's influence on the fluorescence. The fluorescence excitation and emission spectra of AmB itself were measured ex situ for two of the formulations. Using added surfactant to control AmB aggregation state, the relationship between AmB aggregation and its fluorescence properties was examined. We discovered AmB to have unusual dual fluorescence properties, the extent of which differed between formulations. We measured AmB's fluorescence in situ as the drug interacted with floating lipid monolayers on the Langmuir trough. Both the variation in fluorescence during compression of a mixed AmB/lipid monolayer and penetration of AmB into a phospholipid monolayer were measured. This experimental setup was configured to collect fluorescence only from AmB at the monolayer, and not from AmB in bulk solution. Fluorescence excitation was made using a laser diode extracted from a consumer electronics device.

Thesis/Dissertation

application/pdf

2008-02-15

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/355

Chemistry

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/