Rational Design and Preclinical Evaluation of LRP1-Targeted 68Ga-Labeled Angiopep-2 Peptides for PET Imaging of Hepatocellular Carcinoma

Abstract Hepatocellular carcinoma (HCC) is a heterogeneous malignancy lacking effective strategies for subtype-specific molecular imaging. Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional receptor implicated in HCC progression. Herein, we report the rational design and systematic comparison of three Angiopep-2-derived 68Ga-labeled radiotracers with distinct DOTA conjugation positions, aiming to elucidate how chelator placement modulates targeting specificity and in vivo imaging contrast for LRP1-positive HCC. Immunohistochemical analysis of an HCC tissue microarray confirmed LRP1 overexpression in 19.3% of clinical specimens, which correlated with poorer overall survival and defined a high-risk subgroup. All tracers exhibited high radiochemical purity, excellent stability, and favorable pharmacokinetic profiles. In vitro, 68Ga-DOTA-ANG-2 and 68Ga-DOTA-ANG-3 showed robust uptake in LRP1-high Huh-7 cells. Although 68Ga-DOTA-ANG-2 achieved the highest absolute tumor uptake, 68Ga-DOTA-ANG-3 demonstrated markedly reduced nonspecific hepatic accumulation, yielding a superior tumor-to-liver contrast (5.61 ± 1.33 at 120 min). MicroPET/CT imaging in subcutaneous and orthotopic HCC models verified that 68Ga-DOTA-ANG-3 enables sensitive and high-contrast detection of intrahepatic lesions. Collectively, 68Ga-DOTA-ANG-3 is a promising positron emission tomography (PET) agent for subtype-specific HCC imaging, supporting further evaluation of LRP1-targeted PET imaging in a biomarker-defined subset of HCC.