Size distribution of liposomes by flow field-flow fractionation

The applicability of field-low fractionation (FFF) to the characterization of liposomes is discussed and theoretically described. Because of fundamental differences in their driving forces, sedimentation FFF and flow FFF measure different vesicle properties. Sedimentation FFF, although used previously to measure vesicle sizes and size distributions, is fundamentally a technique that measures the effective mass and mass distribution of particles. It is sensitive to small changes in the effective mass of either the biomembrane or its encapsulated load and thus is likely to be useful in characterizing such properties as drug loading, biomembrane volumes and areas, and distributions of these properties. Size characterization by sedimentation FFF can only be done by deducing size from effective mass. Flow FFF, by contrast, provides a direct measurement of vesicle size and size distribution. After demonstrating the high resolution and relative accuracy of size measurement of flow FFF by the separation of polystyrene latex standards, flow FFF was applied to two preparations of DSPC-DSPA liposomes that were sonicated under different temperature conditions. Fractograms and size distributions are reported as a function of sonication time. The rapid elimination of a large diameter tail to the distribution is shown to constitute a major mechanism for distribution narrowing. Finally, results are provided bearing on the reproducibility of size distribution measurements by flow FFF.