A Synergistic Approach Using a ROS-Responsive Lipid-Based Prodrug for Liposomal Delivery of Doxorubicin

Abstract Many clinically used chemotherapeutics, including doxorubicin, suffer from dose-limiting toxicities, poor biodistribution, and limited control over intracellular activation. To mitigate these challenges, two primary delivery strategies have been widely explored: prodrug design and liposomal encapsulation. Prodrugs can improve solubility, pharmacokinetics, and site-selective activation, while liposomes enhance circulation time and reduce off-target exposure. However, each approach presents limitations when employed independently. Prodrugs may introduce stability or solubility concerns and can undergo premature activation, whereas liposomal formulations often rely on passive release mechanisms and may exhibit incomplete or non-specific cargo leakage. In the current work, we sought to develop a synergistic delivery approach that integrates a chemically responsive prodrug directly within the liposomal membrane that responds to reactive oxygen species (ROS) highly upregulated within diseased cells. We report a ROS-responsive doxorubicin lipid prodrug conjugate, ROSDoxL, which is incorporated within the liposomal bilayer, enabling membrane-embedded therapeutic loading rather than aqueous core encapsulation. The prodrug design features a boronate caging group that undergoes oxidation in the presence of elevated reactive oxygen species, triggering a self-immolative release of doxorubicin. A non-responsive control doxorubicin lipid, DoxL, lacking the boronate moiety, was used to evaluate the specific ROS-mediated uncaging mechanism. ESI-HRMS studies confirmed ROS-dependent cleavage of ROSDoxL, while liposomal characterization and cellular studies demonstrated functional delivery and concentration-dependent cytotoxicity in A375 melanoma cells comparable to free doxorubicin. Our membrane-integrated liposomal prodrug approach provides a modular strategy for coupling disease-associated biochemical signals to controlled drug activation, advancing liposomal systems beyond passive carriers towards stimulus-responsive therapeutic delivery.