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 ß-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 (K_A) of [Gd(DOTA-FPβGu)] is 7.42 × 10², which is significantly lower than that of a commercially available MS-325 (K_A = 3.0 × 10⁴) RIME contrast agent. The low K_A 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 K_m = 1.38 mM, k _cat = 3.76 × 10³, and k_cat/K_m = 2.72 × 10³ M^-1 s^-1. The low K _m 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.