Development of a Gd(III)-Based Receptor-Induced Magnetization Enhancement (RIME) Contrast Agent for β‑Glucuronidase Activity Profiling

β-Glucuronidase is a key lysosomal enzyme and is often overexpressed in necrotic tumor masses. We report here the synthesis of a pro receptor-induced magnetization enhancement (pro-RIME) magnetic resonance imaging (MRI) contrast agent ((Gd(DOTA-FPβGu))) for molecular imaging of β-glucuronidase activity in tumor tissues. The contrast agent consists of two parts, a gadolinium complex and a β-glucuronidase substrate (β-D-glucopyranuronic acid). The binding association constant (KA) of (Gd(DOTA-FPβGu)) is 7.42 × 10 2 , which is significantly lower than that of a commercially available MS-325 (KA = 3.0 × 10 4 ) RIME contrast agent. The low KA value of (Gd(DOTA-FPβGu)) is due to the pendant β-D-glucopyranuronic acid moiety. Therefore, (Gd(DOTA-FPβGu)) can be used for detection of β-glucuronidase through RIME modulation. The detail mechanism of enzymatic activation of (Gd(DOTA-FPβGu)) was elucidated by LC-MS. The kinetics of β-glucuronidase catalyzed hydrolysis of (Eu(DOTA-FPβGu)) at pH 7.4 best fit the Miechalis−Menten kinetic mode with Km = 1.38 mM, kcat = 3.76 × 10 3 , and kcat/Km = 2.72 × 10 3 M −1 s −1 . The low Km value indicates high affinity of β-glucuronidase for (Gd(DOTA- FPβGu)) at physiological pH. Relaxometric studies revealed that T1 relaxivity of (Gd(DOTA-FPβGu)) changes in response to the concentration of β-glucuronidase. Consistent with the relaxometric studies, (Gd(DOTA-FPβGu)) showed significant change in MR image signal in the presence of β-glucuronidase and HSA. In vitro and in vivo MR images demonstrated appreciable differences in signal enhancement in the cell lines and tumor xenografts in accordance to their expression levels of β-glucuronidase.