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.
Hargreaves
Peter L
PL
Lipid Biology in Health and Disease Research Group, Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, USA.
Nguyen
Thanh-Son
TS
Ryan
Robert O
RO
D000666:Amphotericin B / Q000737:chemistry* / Q000494:pharmacology; D000935:Antifungal Agents / Q000737:chemistry / Q000494:pharmacology; D002784:Cholesterol / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004337:Drug Carriers / Q000737:chemistry; D004875:Ergosterol / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry*; D049329:Nanostructures / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000187:drug effects; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013696:Temperature
D000935:Antifungal Agents; D004337:Drug Carriers; D008051:Lipid Bilayers; D000666:Amphotericin B; D002784:Cholesterol; D004875:Ergosterol