Gemcitabine release kinetics determines the anticancer and immunomodulatory activity of HPMA copolymer nanomedicines

Gemcitabine (Gem) is an effective anticancer drug used for the treatment of various cancers. Nevertheless, its clinical benefit is limited owing to its very short plasma half-life, rapid enzymatic inactivation, and systemic toxicity. Here, we report the design, synthesis, and comprehensive biological evaluation of four N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-bound Gem nanomedicines differing in their linker structure and thereby drug release kinetics. The appropriate design enabled us to prepare polymer nanomedicines with highly prolonged Gem circulation and tumor accumulation. All conjugates exerted cytostatic and pro-apoptotic activity in multiple mouse and human cancer cell lines in vitro, with efficacy inversely correlated with Gem release rate. The nanomedicine with the slowest drug release in plasma showed remarkably prolonged systemic exposure up to 96 h versus 2 h for free Gem upon intravenous administration. The prolonged systemic exposure showed superior therapeutic efficacy across multiple mouse and human tumor models, including significantly inhibited tumor growth, considerably extended survival with complete long-term tumor remission, and reduced lung metastatic burden. Mechanistic studies revealed sustained pro-apoptotic activity of the nanomedicine with transcriptional reprogramming of tumor cells, and profound immunomodulatory effects. Optimization of Gem release rate improved pharmacokinetics and antitumor efficacy as well as favorably reshaped the tumor immune microenvironment. Our findings establish Gem release kinetics as a key determinant of HPMA copolymer-Gem conjugate performance and highlight the translational potential of these nanomedicines for overcoming limitations of conventional Gem therapy.