Chemically Programmed Albumin–Enzyme Conjugates to Induce Microenvironment-Responsive Metabolic Disruption

Glucose oxidase (GOx)-mediated glucose depletion is an attractive intervention strategy that can address the abnormal tumor metabolism characterized by elevated glycolysis. But its efficacy is limited by inefficient cellular delivery and hypoxia-induced stabilization of HIF-1α, which is associated with adaptive metabolic responses. Here, we report a chemically programmed albumin–GOx conjugate that couples microenvironment-responsive uptake with reductive release of acriflavine (ACF), a small molecule reported to interfere with HIF-1 signaling. Human serum albumin was used as the core carrier platform and engineered to include (1) dimethylmaleic anhydride groups to sense tumor-relevant acidic conditions and induce charge reversal for concomitant cellular delivery of both GOx and ACF payloads and (2) self-immolative disulfide linkage to release ACF under reductive conditions. The resulting GOx–albumin–ACF conjugate preserved GOx-mediated glucose oxidation and attenuated HIF-1α accumulation. Consequently, stronger metabolic disruption including suppressed lactate production and reduced adenosine triphosphate (ATP) levels was observed, together with enhanced cell death in two-dimensional cultures and three-dimensional tumor spheroids. By integrating chemical activation with modulation of the GOx-associated HIF-1α response, this work establishes a biomacromolecular design strategy for a protein-based, microenvironment-responsive therapeutic system that senses, responds, and acts within the tumor microenvironment to achieve enhanced metabolic disruption and cytotoxicity