Peptide stabilized amphotericin B nanodisks

Abstract

Nanometer scale apolipoprotein A-I stabilized phospholipid disk complexes (nanodisks; ND) have been formulated with the polyene antibiotic amphotericin B (AMB). The present studies were designed to evaluate if a peptide can substitute for the function of the apolipoprotein component of ND with respect to particle formation and stability. An 18-residue synthetic amphipathic alpha-helical peptide, termed 4F (Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH(2)), solubilized vesicles comprised of egg phosphatidylcholine (egg PC), dipentadecanoyl PC or dimyristoylphosphatidylcholine (DMPC) at rates greater than or equal to solubilization rates observed with human apolipoprotein A-I (apoA-I; 243 amino acids). Characterization studies revealed that interaction with DMPC induced a near doubling of 4F tryptophan fluorescence emission quantum yield (excitation 280 nm) and a approximately 7 nm blue shift in emission wavelength maximum. Inclusion of AMB in the vesicle substrate resulted in formation of 4F AMB-ND. Spectra of AMB containing particles revealed the antibiotic is a highly effective quencher of 4F tryptophan fluorescence emission, giving rise to a Ksv=7.7 x 10(4). Negative stain electron microscopy revealed that AMB-ND prepared with 4F possessed a disk shaped morphology similar to ND prepared without AMB or prepared with apoA-I. In yeast and pathogenic fungi growth inhibition assays, 4F AMB-ND was as effective as apoA-I AMB-ND. The data indicate that AMB-ND generated using an amphipathic peptide in lieu of apoA-I form a discrete population of particles that possess potent biological activity. Given their intrinsic versatility, peptides may be preferred for scale up and clinical application of AMB-ND.

Figure 1. Helical wheel projection of 4F

The amphipathic nature of 4F is illustrated with the line denoting the polar – nonpolar interface. Bold font indicates hydrophobic residues. The figure was prepared using AutoCAD LT 2007.

Figure 2. Effect of 4F peptide or apoA-I on the light scattering intensity of phospholipid vesicles

Panel A) egg PC vesicles (100 μg) were incubated with a) buffer alone; b) 40 μg apoA-I c) 40 μg 4F peptide or d) 80 μg 4F peptide. Panel B) dipentadecanoyl PC vesicles (100 μg) were incubated in a) buffer alone; b) 40 μg apoA-I or c) 40 μg 4F peptide. Panel C) DMPC vesicles (100 μg) were incubated with a) buffer alone; b) 40 μg apoA-I or c) 40 μg 4F peptide. Arrows indicate the time at which protein was added to the lipid substrate. Right angle light scattering intensity was monitored as a function of time on a Perkin-Elmer LS 50B luminescence spectrometer with excitation and emission monochromaters set at 600 nm (3.6 nm slit width).

Figure 3. Effect of lipid and AMB on 4F Trp fluorescence emission

4F peptide in buffer (b); empty 4F ND (a) and 4F AMB-ND (c). Samples were excited at 280 nm and emission monitored from 300 to 500 nm.

Figure 4. Electron microscopy of ND samples

A sample of 4F-AMB-ND, negatively stained with uranyl acetate, was imaged on a JEOL 1230 transmission electron microscope at 120 keV. Magnification = 40 000x.

Figure 5. Effect of apoA-I and 4F AMB-ND on growth of Saccharomyces cerevisiae

Twenty μl of a saturated overnight culture of S. cerevisiae grown in YEPD media was used to inoculate 5 ml YEPD media in the presence of indicated amounts of specified AMB-ND. Cultures were incubated at 30°C for 16 h at which time the extent of culture growth was determined spectrophotometrically. apoA-I AMB-ND (○); 4F AMB-ND (●). Values reported are the mean ± S.D. (n= 3).