Unilamellar liposomes made with the French pressure cell: a simple preparative and semiquantitative technique

Abstract

A simple, rapid, and almost quantitative technique is described for the preparation of 1-40 ml of homogeneous unilamellar liposomes from dilute or concentrated aqueous suspensions of egg phosphatidylcholine. Aqueous suspensions of lipid are placed with the chamber of a French pressure cell at room temperature and rapidly extruded at 20,000 psi through the small orifice. A single pass transforms more than 70% of the extruded lipid into a homogeneous population of single-wall bilayer vesicles; more than 90% is transformed by recycling the lipid through the French pressure cell. About 95% of these liposomes range between 150-300 A in diameter (mean 200 A). The liposomes are stable for days to months when stored under nitrogen at 0.4 degrees C and can be prepared at 0 degrees, 25 degrees, or 37 degrees C. The liposomes appeared unaltered by repeated passages through the French pressure cell and no degradation of the phospholipid was detected after ten consecutive cycles at 20,000 psi in the absence of a nitrogen atmosphere. The method is especially useful for trapping small molecular weight substances because the concentration of both lipid and solute can be made quite high. Cholesterol up to 45 mole % can be incorporated into larger liposomes of egg phosphatidylcholine (mean diameter 315 A). Other phospholipids and different lipid mixtures can also be transformed into unilamellar vesicles with this method which has the advantage that additional steps of ultracentrifugation, column chromatography, dialysis, and concentrating procedures are usually unnecessary. Multilayered liposomes of small size (980 A mean diameter; > 95% between 500-1,500 A) are produced at lower pressure (3,000 psi). The latter are separated by gel permeation chromatography from a second population of homogeneous vesicles of even smaller size (580 A mean diameter; > 95% between 300-900 A) that contain two bilayer shells.