Dual temperature-sensitive liposome-in-liposome structure for advanced drug delivery systems

Hierarchical liposome-in-liposome (LIL) structures enable sophisticated multi-stage drug delivery, their application is however often limited by complex, time-consuming fabrication and the use of organic solvents. Here we report a simple two-step hydration (TSH) method for fabricating stable LIL architectures in aqueous media within 2 h. This process employs ultrasonication for the formation of structurally robust inner liposomes (IL) and gentle hydration for their subsequent encapsulation within outer liposomes (OL), achieving high-quality dual-spacing configurations. Optical microscopy and differential staining verified the structural integrity and physical encapsulation, demonstrating distinct spatial segregation. The functional efficacy and temperature-sensitive release kinetics of the LIL system were validated through the release theory, confirming that the hierarchical membranes confine encapsulated agents and prevent premature leakage below the phase transition temperature (T_phase) of the OL. The programmable stepwise release was demonstrated through a macroscopic hydrogelation model, where crosslinking was triggered only upon the sequential thermal release of the initiator and accelerator. This was further validated by the independent release of commercial drugs, chlorpheniramine maleate and riboflavin sodium phosphate, as confirmed via HPLC analysis. These results validate the TSH-based LIL platform as a versatile and scalable strategy for developing smart, multi-stage delivery vehicles with environment-responsive release profiles.