Dual-Enzyme-Triggered Covalent Oligomerization Reprograms Intracellular Trafficking for Chemosensitization of...

Multidrug resistance (MDR) remains difficult to overcome because effective intracellular drug engagement is often reduced by limited drug availability and active efflux. Here, we develop a tandem enzyme-triggered self-assembly strategy to modulate these intracellular barriers with spatiotemporal control. A dual-enzyme-responsive precursor, pYFcFYp, is first dephosphorylated by extracellular alkaline phosphatase (ALP) and then oxidized by intracellular tyrosinase (Tyr), initiating covalent oligomerization and the formation of β-sheet-rich nanofibers. The staged assembly induces lysosomal membrane permeabilization (LMP) and cytosolic escape, accompanied by cytoskeletal remodeling and mitochondrial dysfunction, collectively contributing to G1/S-phase arrest and apoptosis. Importantly, LMP-associated doxorubicin redistribution was accompanied by increased intracellular retention and nuclear accumulation of doxorubicin; in parallel, decreased P-glycoprotein (P-gp/ABCB1) levels and a reduced drug-efflux phenotype were observed. These effects correlate with improved antitumor efficacy in vitro and in vivo. Overall, these results support tandem enzyme-guided intracellular self-assembly as a supramolecular route to chemosensitization.