Mechanistic Analysis of Albumin–Ruthenium Conjugates Reveals Redox‐Sensitive Catalytic Persistence for Extracellular Prodrug Uncaging

Selective use of cytotoxic agents remains limited by systemic exposure and heterogeneous delivery in solid tumors. Extracellular catalytic prodrug activation offers a complementary strategy in which a pericellular catalyst activates a diffusible caged payload without requiring carrier internalization. This approach requires catalysts that retain activity under extracellular redox conditions and concentrate near cells through local binding environments. Here, we use albumin–ruthenium conjugates (ARCs) to define how redox environment and secreted protein acidic and rich in cysteine (SPARC), an albumin‐binding extracellular matrix glycoprotein, shape extracellular catalyst persistence and local albumin association. ARCs uncaged alloc‐protected fluorescent and doxorubicin substrates at low micromolar concentrations with limited intrinsic cytotoxicity. Catalytic activity was attenuated under ambient oxygen but preserved under oxygen‐limited or antioxidant conditions, consistent with oxygen/redox‐associated catalyst deactivation. ARC retained uncaging activity after SPARC capture and washing, indicating that SPARC association does not block catalyst accessibility. In cell‐based assays, SPARC‐expressing 4T1 and BT‐549 cells showed rapid albumin association, whereas low‐SPARC MCF10A cells exhibited weaker early association enhanced by exogenous SPARC. Fluorescence imaging, trypan blue quenching, and washed‐cell uncaging assays support a predominantly extracellular/pericellular mechanism that does not require carrier internalization. Together, these results identify redox‐sensitive catalytic persistence and SPARC‐enhanced albumin capture as mechanistic parameters governing extracellular albumin‐based artificial metalloenzyme function.