To validate the effectiveness of a bi-specific probe in vivo for enhancing T1-weighted positive contrast to diagnose the early-stage HCC.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality. HCC-targeted magnetic resonance imaging (MRI) is an effective noninvasive diagnostic method that involves targeting clinically-related HCC biomarkers, such as alpha-fetoprotein (AFP) or glypican-3 (GPC3), with iron oxide nanoparticles. However, in vivo studies of HCC-targeted MRI utilize single-target iron oxide nanoprobes as negative (T2) contrast agents, which might weaken their future clinical applications due to tumor heterogeneity and negative MRI contrast. Ultra-small superparamagnetic iron oxide (USPIO) nanoparticles (approximately 5 nm) are potential optimal positive (T1) contrast agents. We previously verified the efficiency of AFP/GPC3-double-antibody-labeled iron oxide MR molecular probe in vitro.

The bi-specific T1-positive contrast-enhanced MRI probe (UAG) for HCC demonstrated increased specificity and sensitivity to diagnose early-stage HCC irrespective of tumor size and/or heterogeneity.

The single- and double-antibody-conjugated 5-nm USPIO probes, including anti-AFP-USPIO (UA), anti-GPC3-USPIO (UG), and anti-AFP-USPIO-anti-GPC3 (UAG), were synthesized. T1- and T2-weighted MRI were performed on day 10 after establishment of the orthotopic HCC mouse model. Following intravenous injection of U, UA, UG, and UAG probes, T1- and T2-weighted images were obtained at 12, 12, and 32 h post-injection. At the end of scanning, mice were euthanized, and a histologic analysis was performed on tumor samples.

T1- and T2-weighted MRI showed that absolute tumor-to-background ratios in UAG-treated HCC mice peaked at 24 h post-injection, with the T1- and T2-weighted signals increasing by 46.7% and decreasing by 11.1%, respectively, relative to pre-injection levels. Additionally, T1-weighted contrast in the UAG-treated group at 24 h post-injection was enhanced 1.52-, 2.64-, and 4.38-fold compared to those observed for single-targeted anti-GPC3-USPIO, anti-AFP-USPIO, and non-targeted USPIO probes, respectively. Comparison of U-, UA-, UG-, and UAG-treated tumor sections revealed that UAG-treated mice exhibited increased stained regions compared to those observed in UG- or UA-treated mice.