Spectrophotometric analysis of organisation of dipalmitoylphosphatidylcholine bilayers containing the polyene antibiotic amphotericin B

Amphotericin B (AmB) is a polyene antibiotic widely used in the treatment of deep-seated fungal infections. The mode of action of AmB is directly related to the effect of the drug on the lipid phase of biomembranes. In the present work the effect of AmB on the properties of lipid bilayers formed with dipalmitoylphosphatidylcholine (DPPC) and the effect of the lipid phase on the molecular organisation of AmB were studied with application of spectrophotometry in the UV-Vis region. The absorption spectra of AmB in lipid membranes display a complex structure with hypsochromically and bathochromically shifted bands indicative of formation of molecular aggregates of the drug. Formation of molecular aggregates was analysed at different concentrations of the drug in the lipid phase in the range 0.05-5 mol% and at different temperatures in the range 5-55 degreesC, The aggregation level of AmB in the ordered phase of DPPC displayed a minimum corresponding to a concentration of 1 mol% with respect to the lipid. An increase in the aggregation level was observed in the temperature region corresponding to the main phase transition. The structure of molecular aggregates of AmB is analysed on the basis of spectroscopic effects in terms of the exciton splitting model. Analysis of the position of the absorption maximum of AmB in the lipid phase of DPPC in terms of the theory of solvatochrome effects makes it possible to ascribe the refractive indices n = 1.40 and n = 1.49 to the hydrophobic core of the membrane in the L-alpha and the P-beta' phase respectively. Analysis of the aggregation of AmB in the lipid phase in relation to the physical state of the membrane reveals that the temperature range of the main phase transition of a lipid cluster in the immediate vicinity of AmB depends on its concentration. The termination of the phase transition temperature, as read from the AmB aggregation, varies between 42 degreesC at 1 mol% AmB in DPPC and 49 degreesC at 5 mol% AmB in DPPC. The exciton splitting theory applied to the analysis of the spectroscopic data makes it possible to calculate the diameter of the AmB pore as 2.8 Angstrom in the gel phase and 3.6 Angstrom in the fluid phase of the DPPC membrane, on the assumption that the pore is formed by nine AmB molecules. (C) 2001 Elsevier Science B.V. All rights reserved.