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 z8.5 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 mg/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

Nanodisks are nanoscale, disk-shaped phospholipid bilayers whose edge is stabilized by association of apolipoprotein molecules. Self assembled ND particles enriched with all-trans-retinoic acid (ATRA) [phospholipid:ATRA molar ratio = 5.5:1] were generated wherein all reaction components were solubilized. ATRA-ND migrated as a single band (Stokes' diameter ∼20 nm) on native gradient polyacrylamide gel electrophoresis. ATRA, phospholipid and apolipoprotein co-eluted from a Sepharose 6B gel filtration column, consistent with stable integration of ATRA into the ND particle milieu. Spectroscopic analysis of ATRA-ND in buffer yielded an absorbance spectrum characteristic of ATRA. ATRA-ND mediated time-dependent inhibition of cultured HepG2 cell growth more effectively than free ATRA. The nanoscale size of the formulation particles and the stable integration of biologically active ATRA suggest ND represent a potentially useful vehicle for solubilization and in vivo delivery of ATRA.

The N‐terminal domain of human apolipoprotein E3 (apoE3‐NT) harbors residues critical for interaction with members of the low‐density lipoprotein receptor (LDLR) family. While lipid free apoE3‐NT adopts a stable four‐helix bundle conformation, a lipid binding induced conformational adaptation is required for LDLR binding. To investigate the structural basis for this change, the short helix connecting helix 1 and 2 in the four helix bundle was replaced by the β‐turn sequence, ‐NPNG‐. Recombinant helix‐to‐turn (HT) variant apoE3‐NT was produced in E. coli, isolated and characterized. Stability studies revealed an unfolding transition midpoint of 1.9 M guanidine HCl for HT apoE3‐NT. Reconstituted HDL prepared with HT apoE3‐NT binds to the LDLR. HT apoE3‐NT was more effective than wild type apoE3‐NT at inducing a decrease in phospholipid vesicle light scattering intensity. In a competition binding experiment, HT apoE3‐NT preferentially associated with phospholipase C treated LDL. Results indicate that a mutation at one end of the apoE3‐NT four‐helix bundle markedly enhances the lipid binding activity of this protein. In full‐length apoE, increased NT domain lipid binding affinity may alter the ratio of receptor‐active and ‐inactive conformational states and influence lipoprotein‐LDLR interactions. Supported by TRDRP and NIH.

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