In Situ X-ray Scattering for Doxorubicin-Loaded Liposomes to Elucidate the Morphological Change Mechanism Depending on the Cooling Rate and Cholesterol Content.

Doxorubicin (DOX)-loaded liposomes are one of the most established and clinically relevant nanomedicines in a drug delivery system. In this study, we elucidate the mechanistic relationships among cooling rate, lipid composition, and the resulting morphological evolution of DOX-loaded liposomes using in situ ultrasmall-angle, small-angle, and wide-angle X-ray scatterings (USAXS/SAXS/WAXS). The wide-range X-ray scattering combined with precisely controlled 2 °C stepwise cooling enabled direct observation of two sequential structural events: DOX-sulfate crystallization, followed by the lipid membrane phase transition. These observations clarify the mechanism by which prolate liposomes containing linearly elongated DOX-sulfate fiber bundles are formed during slow cooling. Combined analysis of cryogenic transmission electron microscopy (cryo-TEM) and USAXS allowed accurate determination of the morphology of each DOX-loaded liposome population. Under a slow cooling condition, the hydrogenated soybean phosphatidylcholine (HSPC)/cholesterol (Chol) = 6:4 formulation contained markedly more linearly elongated DOX-sulfate fiber bundles than the 9:1 formulation. Under a rapid-freezing condition, the HSPC/Chol = 6:4 formulation exhibited a higher proportion of liposomes containing DOX-sulfate linear fiber bundles relative to the curved bundles compared with the 9:1 formulation. These findings highlight the strong influence of Chol content and reveal how the interplay between DOX-sulfate crystallization and lipid membrane phase transition behavior during cooling governs liposome morphology. Notably, USAXS sensitively captured structural changes associated with DOX crystallization, lipid membrane phase transitions, and the resulting morphology of DOX-loaded liposomes. The combined use of wide-range X-ray scattering and conventional cryo-TEM provides mechanistic insight essential for rational formulation design, process control, and analytical evaluation of drug-loaded liposomes.