Targeted co-delivery of gemcitabine and atRA via a GSH-responsive mutual prodrug enhances cytotoxicity through redox imbalance and Pin1 inhibition

Combination therapy offers strong potential to enhance anticancer efficacy, yet its success depends on the concurrent and targeted delivery of multiple agents. To address this challenge, we designed a self-assembling trimeric mutual prodrug, RqGem, which covalently links gemcitabine (GEM) and all-trans retinoic acid (atRA) through a glutathione (GSH)-depleting linker. This architecture enables excipients-free nanoassembly, achieving 100% drug loading and ensuring simultaneous delivery of both agents. RqGem nanoassemblies remained stable under physiological conditions and underwent esterase-mediated activation, releasing GEM and atRA along with a quinone methide intermediate that rapidly depletes intracellular GSH. This GSH depletion-driven redox modulation potentiated the cytotoxic mechanisms of both drugs by elevating oxidative stress and sensitizing cells to DNA damage and differentiation cues.The multiple and synergistic therapeutic actions of RqGem nanoassemblies were evaluated using cell culture and mouse models. RqGem nanoassemblies demonstrated significantly greater cytotoxicity, enhanced cellular uptake, and stronger apoptosis induction than the equivalent mixture of free GEM and atRA, reflecting robust synergy between atRA-mediated signaling modulation and GEM-induced DNA damage. The combined actions of self-assembly, 100% drug loading, concurrent drug release, and redox-dependent sensitization collectively contributed to the high potency of RqGem. Given that the role of atRA in targeting Pin1, a peptidyl-prolyl isomerase overexpressed in cancers and associated with poor prognosis, we further evaluated the impact of RqGem on Pin1 expression. Western blot analyses in AsPC-1 and CFPAC-1 pancreatic cancer cells showed marked Pin1 downregulation following RqGem treatment, supporting its dual mechanism of GEM delivery and atRA-mediated oncogenic pathway inhibition. Ongoing studies in orthotopic pancreatic tumor models aim to define the signaling pathways underlying RqGem-driven Pin1 suppression and its contribution to treatment synergy. Collectively, these findings establish RqGem as a rationally engineered, self-synergistic mutual prodrug that integrates drug self-delivery, controlled activation, and redox regulation to achieve potent and targeted anticancer activity. This platform holds strong translational promise for combination therapy in pancreatic cancer and beyond.