Co-encapsulated liposomal formulations of tubacin and erlotinib.HCl: a comprehensive stability strategy

The co-encapsulation of two anticancer drugs within liposomes is a promising strategy for delivering a synergistic drug ratio to tumors. In this preliminary study, we systematically investigated the formulation stability of liposomes co-encapsulating tubacin and erlotinib.HCl, as well as the development of a freeze-drying (FD) method as a stabilization strategy. Notably, the effect of cryoprotectants on size and drug encapsulation conservation was studied both before and after freezing and thawing (FD). Tubacin was passively loaded into the lipid bilayer, while erlotinib.HCl was remotely loaded via an ammonium sulfate gradient. Lipid composition studies revealed that erlotinib.HCl encapsulation was the highest at pH 3. Additionally, DSPC:Chol ratios significantly impacted drug loading: high cholesterol content improved erlotinib.HCl retention but reduced tubacin encapsulation. Despite optimization, substantial drug losses occurred during storage at 4 deg C, leading to the development of freeze-drying as a stabilization strategy. Sucrose and trehalose were evaluated as cryoprotectants at different concentrations. Sucrose (10% w/v) demonstrated higher cryoprotection, maintaining liposome sizes within a range optimal for the enhanced permeability and retention (EPR) effect. However, the inclusion of internal sucrose did not further improve stability. Process optimization identified 150 mM ammonium sulfate as the optimal concentration for remote loading while preserving formulation stability. Comparative evaluation of two freeze-drying protocols revealed that the controlled stepwise system (Telstar) outperformed the alternative approach, demonstrating superior drug retention and particle size preservation. Asymmetric flow field-flow fractionation coupled with dynamic light scattering and multi-angle light scattering (AF4-DLS-MALS) analysis confirmed minimal particle aggregation post-lyophilization, with preserved monodispersity and spherical morphology. These findings highlight the critical importance of cryoprotectant selection, buffer optimization, and freeze-drying protocol design for developing stable, co-encapsulated liposomal formulations.