Stage-Specific Stability of Spray-Freeze-Dried Ovalbumin-Loaded Liposomes: Dissecting the Impact of Atomization, Freezing, and Drying on Cryoprotectant Performance.
Cheng Xi X, Li Mengyuan M, Yan Shen S, Zhang Shengyu S et al.
Objectives: Aqueous liposomal suspensions suffer from inherent physical and chemical instability, motivating the development of solid powder formulations. Spray-freeze-drying is a promising solidification technique; however, systematic investigations that isolate the impact of individual unit operations-atomization, freezing, and drying-on liposomal stability remain scarce. This study aimed to elucidate the stage-specific effects of each unit operation on the critical quality attributes of liposomal powders. Methods: Ovalbumin-loaded liposomes (O@L) prepared from DSPC, HSPC, or DPPC were processed using a self-developed microfluidic jet spray freezing tower coupled to a freeze dryer, with trehalose or mannitol as cryoprotectants. The effects of phospholipid composition, freezing temperature (-30 °C, -50 °C, and -70 °C), and freeze-drying cycle were assessed through the z-average, zeta potential, and encapsulation efficiency (EE) of the reconstituted liposomes, as well as the morphology, crystallinity, residual moisture, and hygroscopicity of the resulting powders. Results: Atomization induced protein leakage from the trehalose formulations, whereas mannitol better preserved EE. During freezing, trehalose provided superior cryoprotection, as mannitol partially crystallized and failed to uniformly coat the vesicles. After drying, however, vesicles frozen at -70 °C exhibited fusion attributable to non-uniform cryoprotectant distribution; a moderate freezing temperature (-50 °C) combined with a stepwise primary drying program proved optimal. Trehalose-based powders displayed higher residual moisture and irreversible crystallization above ~60% RH, whereas crystalline mannitol ensured low, reversible hygroscopicity. Conclusions: The optimal condition for a single unit operation does not guarantee the best overall outcome; rational spray-freeze-drying process design requires balancing stage-specific effects.