Density-based separation of liposomes by glycerol gradient centrifugation

Sonicated liposomes of soybean phospholipids (asolectin) distribute nearly throughout a 19-22% (v/v) glycerol gradient when centrifuged to near equilibrium. Upon recentrifugation on an identical gradient, liposomes selected from several positions in such a gradient migrate as narrow bands to positions close to their original positions, indicating that the liposome distribution in the first gradient is the result of a density-based fractionation. Molecular sieve chromatography, turbidity, and trapped volume measurements indicate that the liposome densities are qualitatively related to their size, with the larger liposomes more dense than the smaller ones. Size estimates obtained by electron microscopy of negatively stained preparations indicate that the fractionation is effective for liposomes with diameters ranging from 200 to 600 A, with maximum efficiency in the range 200-300 A where the majority of the liposomes is found. Interestingly, high concentrations of liposomes improve the efficiency of the fractionation procedure. The size dependence of liposome density is shown not to be due to differential glycerol permeability or lipid composition, and is therefore most likely due to variations in the specific volumes of the individual phospholipid molecules owing to the curvature of the liposomes. Finally, freezing of the glycerol gradient fractions in liquid N2 and storage at -70 degrees C does not modify the size of the isolated liposomes. It is suggested that glycerol density gradient fractionation of liposomes could be a useful general method for obtaining liposomes of reasonably uniform size in large quantities and high concentrations.