Spectroscopic studies of amphotericin B solubilized in nanoscale bilayer membranes.

Nanodisks (ND) are discrete nanometer scale phospholipid bilayers whose perimeter is circumscribed by amphipathic apolipoproteins. The membranous environment of ND serves as a matrix for solubilizing the polyene antibiotic amphotericin B (AMB). The spectral properties of AMB in ND are dependent upon AMB concentration. Whereas AMB-ND prepared at a concentration of 2.5 mg AMB per 10 mg phospholipid are consistent with AMB self association in the ND membrane environment, AMB-ND prepared at 0.25 or 0.025 mg AMB per 10 mg phospholipid give rise to spectra reminiscent of AMB in organic solvent. Incubation of ND prepared at a phospholipid/AMB ratio of 400:1 (w/w) at 37 degrees C for 1 h induced a shift in absorbance and near UV circular dichroism spectra consistent with antibiotic self-association. The kinetics of this spectral transition were investigated as a function of incubation temperature. While no change in A388 nm occurred in incubations at 20 degrees C, a time-dependent decrease in A388 nm was observed at 25, 30 and 37 degrees C. Inclusion of ergosterol in the ND membrane attenuated temperature-induced AMB spectral changes. In Saccharomyces cerevisiae growth inhibition assays, ND containing self associated AMB were somewhat less effective than ND possessing a greater proportion of monomeric AMB. On the other hand, inclusion of ergosterol or cholesterol in the ND particle did not alter the growth inhibition properties of AMB-ND. The miniature membrane environment of ND provides a novel milieu for solubilization and characterization of lipophilic biomolecules.

Reconstituted high density lipoprotein enriched with the polyene antibiotic amphotericin B.

The polyene antibiotic amphotericin B (AMB) is an effective antifungal agent whose therapeutic potential is limited by poor aqueous solubility and toxicity toward host tissues. Addition of apolipoprotein A-I to a multilamellar phospholipid vesicle dispersion containing 20% (w/w) AMB induces the formation of reconstituted high density lipoprotein (rHDL), with solubilization of the antibiotic. Density gradient ultracentrifugation resulted in flotation of the complexes to a density of 1.16 g/ml, and negative stain electron microscopy revealed a population of disk-shaped particles. Native gradient polyacrylamide gel electrophoresis indicated a particle diameter of approximately 8.5 nm. Absorbance spectroscopy provided evidence for AMB integration into the lipid milieu. AMB-rHDLs were potent inhibitors of Saccharomyces cerevisiae growth, yielding 90% growth inhibition at <1 microg/ml yeast culture. In studies with pathogenic fungal species, similar growth inhibition characteristics were observed. Compared with AMB-deoxycholate micelles, AMB-rHDL displayed greatly attenuated red blood cell hemolytic activity and decreased toxicity toward cultured hepatoma cells. In in vivo studies in immunocompetent mice, AMB-rHDLs were nontoxic at 10 mg/kg, and they showed efficacy in a mouse model of candidiasis at concentrations as low as 0.25 mg/kg. These results indicate that AMB-rHDLs constitute a novel formulation that effectively solubilizes the antibiotic and elicits strong in vitro and in vivo antifungal activity with no observed toxicity at therapeutic doses.