Self-synergizing mutual prodrug liposomes co-targeting redox homeostasis and PIN1 for cancer therapy

PIN1 is a prolyl isomerase that catalyzes the cis-trans isomerization of phosphorylated Ser/Thr-Pro motifs and is overexpressed in various cancer types, where it functions as a key regulator that amplifies oncogenic signaling. PIN1 promotes cancer cell proliferation, survival, and metastasis by stabilizing diverse oncoproteins and disrupting tumor-suppressive pathways such as p53, and its activity is highly sensitive to the intracellular redox state, becoming inactivated in a redox-dependent manner in response to changes in oxidative stress levels. In this study, we developed a mutual prodrug in which all-trans retinoic acid (atRA) is integrated into a single molecule with a quinone methide (QM) precursor that amplifies oxidative stress via glutathione (GSH) depletion, to simultaneously maximize atRA-mediated PIN1 inhibition and reactive oxygen species (ROS)-mediated anticancer activity. We first evaluated the synergistic cytotoxicity of atRA and the QM precursor in MCF-7 breast cancer cells by calculating the combination index and assessing changes in PIN1 and its downstream effector cyclin D1 by Western blotting. To exploit this synergy, we designed and synthesized a mutual prodrug in which atRA is covalently linked to the QM precursor, and confirmed its structure by 1 H NMR and LC-MS/MS analysis. Owing to its amphiphilic molecular structure, the mutual prodrug could be stably incorporated into the lipid bilayer at a high loading (~40%) together with DPPC, which enabled the formulation of prodrug-based liposomes. The liposomal surface was subsequently coated with gamma-poly(glutamic acid) (gammaPGA) targeting gamma-glutamyl transferase (GGT), yielding particles with a mean diameter of approximately 150 nm. We then examined esterase-triggered release of atRA and QM and evaluated intracellular GSH depletion and ROS amplification, as well as mitochondrial dysfunction, in MCF-7 cells. We further analyzed redox-responsive, PIN1-related, and apoptosis-related signaling by qPCR and Western blotting. Targeting and in vivo antitumor efficacy of the gammaPGA-coated prodrug liposomes were assessed in an MCF-7 xenograft mouse model, and additional biosafety evaluation revealed no apparent systematic toxicity. Collectively, the mutual prodrug liposomal formulation integrating atRA and the QM precursor disrupted intracellular redox homeostasis and suppressed PIN1-dependent oncogenic signaling, resulting in enhanced anticancer efficacy and reduced systematic toxicity. Thus, this platform offers a next-generation precision nanomedicine candidate that unifies high drug loading, tumor-selective delivery, and self-synergistic mechanisms within a single, clinically translatable system.