No abstract

A new method, the supercritical reverse phase evaporation method, for the preparation of liposomes in one step using supercritical carbon dioxide without any organic solvent toxic to the human body has been developed. This method allowed aqueous dispersions of liposomes to be obtained through emulsion formation by introduction of a given amount of water into a homogeneous mixture of supercritical carbon dioxide/ L-R-dipalmitoylphosphatidylcholine/ethanol with sufficient stirring and subsequent pressure reduction. Transmission electron microscopy observations using the freeze-replica method on the obtained vesicles revealed that most of them are large unilamellar liposomes with diameters of 0.1-1.2 µm. The trapping efficiency of the liposomes examined with the D-(+)-glucose indicated more than 5 times higher trapping efficiency for the water-soluble solute than that of multilamellar vesicles prepared by the Bangham method. The trapping efficiency for cholesterol, an oil-soluble substance, of the liposomes was 63%. These results showed that the supercritical reverse phase evaporation method is an excellent technique that permits one-step preparation of large unilamellar liposomes with a high trapping efficiency for both water-soluble and oil-soluble substances.

The search for a novel producer of glycolipid biosurfactants, mannosylerythritol lipids (MEL) was undertaken based on the analysis of ribosomal DNA sequences on the yeast strains of the genus Pseudozyma. Pseudozyma rugulosa NBRC 10877 was found to produce a large amount of glycolipids from soybean oil. Fluorescence microscopic observation also demonstrated that the strain significantly accumulates polar lipids in the cells. The structure of the glycolipids produced by the strain was analyzed by (1)H and (13)C nuclear magnetic resonance and gas chromatography-mass spectrometry methods, and was determined to be the same as MEL produced by Pseudozyma antarctica, a well-known MEL producer. The major fatty acids of the present MEL consisted of C8 and C10 acids. Based on high performance liquid chromatography, the composition of the produced MEL was as follows: MEL-A (68%), MEL-B (12%), and MEL-C (20%). To enhance the production of MEL by the novel strain, factors affecting the production, such as carbon and nitrogen sources, were further examined. Soybean oil and sodium nitrate were the best carbon and nitrogen sources, respectively. The supplementation of a MEL precursor, such as erythritol, drastically enhanced the production yield from soybean oil at a rate of 70 to 90%. Under the optimal conditions in a shake culture, a maximum yield, productivity, and yield coefficient (on a weight basis to soybean oil supplied) of 142 g l(-1), 5.0 g l(-1) day(-1), and 0.5 g g(-1) were achieved by intermittent feeding of soybean oil and erythritol using the yeast.

We describe an approach for engineering peptide–lipid nanoparticles that function similarly to high-density lipoprotein (HDL). Branched, multivalent constructs, bearing multiple 23- or 16-amino-acid peptides, were designed, synthesized and combined with phospholipids to produce nanometer-scale discoidal HDL-like particles. A variety of biophysical techniques were employed to characterize the constructs, including size exclusion chromatography, analytical ultracentrifuge sedimentation, circular dichroism, transmission electron microscopy, and fluorescence spectroscopy. The nanoparticles functioned in vitro (human and mouse plasma) and in vivo (mice) to rapidly remodel large native HDLs into small lipid-poor HDL particles, which are key acceptors of cholesterol in reverse cholesterol transport. Fluorescent labeling studies showed that the constituents of the nanoparticles readily distributed into native HDLs, such that the peptide constructs coexisted with apolipoprotein A-I, the main structural protein in HDLs. Importantly, nanolipid particles containing multivalent peptides promoted efficient cellular cholesterol efflux and were functionally superior to those derived from monomeric peptides. The multivalent peptide-lipid nanoparticles were also remarkably stable toward enzymatic digestion in vitro and displayed long half-lives and desirable pharmacokinetic profiles in mice, providing a real practical advantage over previously studied linear or tandem helical peptides. Encouragingly, a two-week exploratory efficacy study in a widely used animal model for atherosclerosis research (LDLr-null mice) using nanoparticles constructed from a trimeric peptide demonstrated an exceptional 50% reduction in the plasma total cholesterol levels compared to the control group. Altogether, the studies reported here point to an attractive avenue for designing synthetic, HDL-like nanoparticles, with potential for treating atherosclerosis.