A Tumor-Responsive Enzymatic Cascade System Inducing pH-Activable Metabolic Starvation and H₂O₂-Induced Apoptosis

Conventional tumor-targeted therapies primarily focus on drug delivery systems exploiting the tumor microenvironment for controlled drug release. While prodrug strategies activate small-molecule drugs in tumors, they rely on single-step chemical conversions and lack sustained tumor-selective activity. Here, a novel strategy of tumor-responsive, enzymatic cascade therapy is introduced by co-delivering arginine decarboxylase (RDC) and diamine oxidase (DAO) into the tumor site. Unlike traditional arginine depletion strategies, RDC remains inactive at physiological pH and is selectively activated under acidic tumor conditions, ensuring localized metabolic starvation while minimizing systemic toxicity. To overcome a key limitation of RDC-namely, the accumulation of toxic agmatine-RDC is co-delivered with DAO using a chitosan-functionalized Pluronic-based nanocarrier (RDC/DAO@NC), enabling tumor-specific conversion of agmatine into hydrogen peroxide (H₂O₂). This pH-sensitive cascade reaction is expected to limit agmatine accumulation in normal tissues while amplifying RDC-mediated antitumor effects through oxidative-stress-induced apoptosis. In vitro, RDC/DAO@NC suppresses tumor cell proliferation through arginine depletion and induces apoptosis via H₂O₂ generation. In vivo, RDC/DAO@NC substantially enhances tumor accumulation and therapeutic efficacy compared to free enzymes. This study provides the first in vivo validation of RDC-based cancer therapy, suggesting its potential as a tumor-selective strategy integrating enzyme activation in the tumor microenvironment, metabolic starvation, and reactive-oxygen-species-induced apoptosis in cancer treatment.