Development and Evaluation of an Albumin-Binding GPC3-Targeting Peptide for PET Imaging of Hepatocellular Carcinoma

Accurate imaging-based diagnosis of hepatocellular carcinoma (HCC) remains clinically challenging due to the limited specificity of conventional modalities. Glypican-3 (GPC3), which is highly expressed in most HCCs but absent in normal liver and benign lesions, represents a promising target for precision imaging. In this study, we designed and synthesized two GPC3-targeted 68 Ga-labeled peptide PET tracers: a direct-targeting probe, [ 68 Ga]Ga-HD3, and an albumin-binding–enhanced probe, [ 68 Ga]Ga-HD4. Both probes were obtained in high radiochemical purity and demonstrated good in vitro stability. Their imaging performance was systematically evaluated in GPC3-positive and GPC3-negative xenograft models, as well as in an orthotopic HCC model. While both probes enabled GPC3-specific tumor visualization, [ 68 Ga]Ga-HD4 showed significantly higher tumor uptake and prolonged tumor retention, leading to superior tumor-to-liver (T/L) and tumor-to-muscle (T/M) ratios, particularly at delayed imaging time points. Histological analysis confirmed that tumor uptake was GPC3-specific. In line with its albumin-binding design, [ 68 Ga]Ga-HD4 showed elevated blood-pool activity and extended circulation time. Together, these results demonstrate that the incorporation of an albumin-affinity moiety effectively improves the pharmacokinetics and imaging performance of GPC3-targeted tracers. This work provides a solid practical foundation for the translational development of HCC imaging agents, with [ 68 Ga]Ga-HD4 emerging as the more promising candidate owing to its improved tumor retention and imaging contrast.