An Enzyme-Responsive Gd(III) MRI Probe for Visualizing β-Hexosaminidase A Activity

We report the design, synthesis, and evaluation of HexA-Gd, a bioresponsive magnetic resonance imaging (MRI) contrast agent for the selective detection of β-hexosaminidase A (Hex-A) activity. The probe employs a self-immolative linker strategy that modulates Gd(III) coordination and T₁ relaxivity in response to enzymatic activation. In vitro relaxometric analyses in phosphate-buffered saline (PBS, pH 7.4, 37 °C, 1.4 T) showed that Hex-A-Gd exhibits a low longitudinal relaxivity (r₁ = 3.06 mM^-1 s^-1) in its pristine form, which increases to 3.78 mM^-1 s^-1, following enzymatic conversion by Hex-A. Time-course measurements confirmed rapid activation kinetics, with significant T₁ shortening within 20 min of enzyme exposure. High-field MRI phantom imaging at 7 T further validated the relaxivity enhancement, yielding r₁ = 5.06 mM^-1 s^-1 and r₂ = 7.67 mM^-1 s^-1 at 25 °C. ¹H NMRD profiling revealed the underlying molecular mechanism, highlighting q-modulated relaxivity changes compared to a nonresponsive control probe (NDO3A-Gd). Cell-based studies in SW480 colorectal cancer cells demonstrated low cytotoxicity and significant probe uptake relative to the control, indicating efficient internalization in a disease-relevant context. Collectively, these findings establish HexA-Gd as a promising platform for noninvasive MRI-based imaging of Hex-A activity in cancer and lysosomal storage disorders.