Physicochemical Characterization and In Vitro Evaluation of Novel Liposomal and Proliposomal Doxorubicin Formulations in Accordance with FDA Guidance

Doxorubicin (Dox) is a widely used chemotherapeutic agent, but its clinical application is limited by severe dose-dependent toxicities, particularly cardiotoxicity. To overcome these challenges, two novel formulations-liposomal doxorubicin (Dox-Lip) and proliposomal doxorubicin (Dox-P-Lip) -were developed and systematically compared with the reference listed drug (RLD), focusing on physicochemical comparability and in vitro bioequivalence in accordance with FDA guidance. Dox-Lip was prepared using alternative lipid compositions and a modified pH gradient-based active loading method, while Dox-P-Lip was obtained by lyophilization of the liposomal formulation. Characterization studies included encapsulation efficiency (EE%), lipid content, drug-to-lipid ratio, internal pH, morphology, bilayer phase behavior, PEG surface density, particle size, zeta potential, and PEG layer thickness determined using the Fixed Aqueous Layer Thickness (FALT) model. In vitro drug release was evaluated under simulated plasma, variable pH and temperature, and ultrasound exposure. Both formulations exhibited high encapsulation efficiencies (EE%) exceeding 90%, comparable to those of the RLD. Moreover, these formulations demonstrated approximately 2.1-fold thicker PEG layers, indicating improved steric stabilization. Although these formulations exhibited slightly higher leakage under stress conditions, the encapsulation efficiency (EE%) consistently remained above 90%, confirming their structural integrity. Overall, the novel formulations demonstrated physicochemical similarity and stability relative to the RLD, supporting their potential as bioequivalent and clinically viable liposomal Dox alternatives.