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

Abstract

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 (<i>K</i>_A) of [Gd­(DOTA-FPβGu)] is 7.42 × 10², which is significantly lower than that of a commercially available MS-325 (<i>K</i>_A = 3.0 × 10⁴) RIME contrast agent. The low <i>K</i>_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 <i>K</i>_m = 1.38 mM, <i>k</i>_cat = 3.76 × 10³, and <i>k</i>_cat/<i>K</i>_m = 2.72 × 10³ M^–1 s^–1. The low <i>K</i>_m value indicates high affinity of β-glucuronidase for [Gd­(DOTA-FPβGu)] at physiological pH. Relaxometric studies revealed that <i>T</i>₁ 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. <i>In vitro</i> and <i>in vivo</i> MR images demonstrated appreciable differences in signal enhancement in the cell lines and tumor xenografts in accordance to their expression levels of β-glucuronidase