Purpose To develop L5 peptide-modified gas vesicles (L5-GVs) for US molecular imaging (USMI) of glypican-3 (GPC3) expression in hepatocellular carcinoma (HCC). Materials and Methods This study was conducted from October 2022 to December 2024. L5-GVs were synthesized by conjugating L5 peptides to gas vesicles derived from Halobacterium NRC-1. In vitro binding was evaluated by incubating fluorescein isothiocyanate-labeled L5-GVs or control GVs (con-GVs) with GPC3-positive HepG2 and GPC3-negative A549 cells. In vivo USMI was performed in subcutaneous HepG2 and A549 tumor-bearing BALB/c nude mice (4-6 weeks old; female; 18-22 g) after injection of con-GVs or L5-GVs. The correlation between USMI signal intensity at 10 minutes and tumor GPC3 immunofluorescence staining was calculated. Single-cell suspensions from 10 resected human HCC specimens were incubated with fluorescein isothiocyanate-labeled L5-GVs, and the correlation between L5-GVs adhesion-positive cell rate and immunohistochemical GPC3 expression was calculated. Results L5-GVs (approximately 252.23 nm ± 1.87) produced stronger fluorescence intensity than con-GVs in HepG2 cells (P P = .96). L5-GVs generated stronger contrast signal than con-GVs in HepG2 tumor-bearing mice (P = .004), with no difference observed in A549 tumors (P = .82); the signal intensity at 10 minutes after injection correlated with GPC3 expression (R2 = 0.89). In patient-derived HCC samples, L5-GVs adhesion-positive cell rate strongly correlated with immunohistochemical GPC3 expression (R2 = 0.94). Conclusion GPC3-targeted L5-GVs enabled specific USMI of HCC in preclinical models, with strong correlation to clinical pathology supporting potential translation for early HCC diagnostic imaging. Keywords: Molecular Imaging, Animal Studies, Ultrasound-Contrast, Contrast Agents-Other Supplemental material is available for this article. © RSNA, 2026 See also commentary by Xu in this issue See also editorial by Zhou in this issue.
Liang et al. (Sun,) studied this question.