Nitrogen → Cholesterol

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A 3:2:1 molar mixture of Phosphatidylcholine: Triolein: Cholesteryl Oleate (PC:TO:CO) was dissolved in chloroform/methanol 2:1 (V/V) and the solvent removed under a stream of nitrogen. The lipids were then re-dissolved and lyophilised from t-butanol for 24 hours (EF4 Modulyo Freeze Dryer, Edwards High Vacuum, Crawley, United Kingdom), then re-suspended in 0.01M Tris-HCl buffer (pH 8.0) to give a final concentration of 7 to 8 % w/v for extrusion. The lipid dispersions were sonicated under a stream of N2 for two hours using a 250W sonicator, centrifuged at 10,000 rpm for 60 minutes (MSE Superspeed 75 Ultracentrifuge, MSE Ltd, London, United Kingdom) and then transferred to the Extruder vessel (Lipex Biomembranes Inc, Vancouver, Canada) which was maintained at 50-55° C. throughout. The lipid mixture was successively extruded through polycarbonate filters of pore size 0.1 and 0.05 μm using two stacked filters and at least four extrusions under 60 psig pressure provided by a nitrogen source8. Samples of lipid microemulsion were diluted with Tris-HCl buffer to give a cholesterol concentration of approximately 1 mmol/l and heated to 55° C. in a stirring water bath. Aliquots of peptide dissolved in DMSO were added under the surface of the stirring microemulsion, control experiments were performed with DMSO alone. The volume of DMSO added in each case was kept below 20 μl/ml of microemulsion mixture. The peptide-microemulsion complex was incubated at 55° C. for 15 minutes then dialysed overnight against 5 liter PBS. The resulting non-naturally occurring LDL (nLDL) were filter sterilised (0.2 μm) and stored at 4° C. under N2 before use.

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