In an attempt to develop a quantitative assay for supravesicular structures (SVS)
- such as aggregates, fused liposomes or solid lipid particles - in liposome preparations,
forward vs. side scattering of liposomal doxorubicin (Doxil/Caelyx) was analyzed by
flow cytometry. Based on calibration with fluorescent latex beads, the size resolution
was between about 500 and 1000nm. Caelyx, just as structurally matched empty liposomes
(Doxebo) produced dot plots clearly distinguishable from background, suggesting the
presence of SVS in the above size region. A comparison of gated areas on the scattergrams
obtained for different Caelyx preparations showed differences between current and
expired samples, implying that SVS formation may be storage-time-dependent. Incubation
of doxorubicin with Doxebo in a free drug and lipid concentration range that corresponds
to that in Caelyx also led to varying SVS patterns, raising the possibility that free
doxorubicin in Caelyx might contribute to SVS formation. Dynamic light scattering
and transmission electron microscopic analysis of liposomes following gaiting and
sorting of >500nm particles from Caelyx confirmed the presence of SVS, providing independent
evidence for their stable existence. Based on a rough estimation, the amount of SVS
in Caelyx is some 60 billionth part of all liposomes. These observations raise the
possibility that the presence of an exceedingly small fraction of >500nm particles
may be an intrinsic property of PEGylated small unilamellar liposomes, and that the
described FACS analysis may be developed further as a quality assay for liposomal
homogeneity.
