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.