Site-Specific Native Antibody-Conjugated Microbubbles for Molecular Ultrasound Imaging of Hepatocellular Carcinoma

To develop a microbubble-based contrast agent using light-activated site-specific conjugation (LASIC) for targeted molecular ultrasound imaging of glypican-3 (GPC3) positive hepatocellular carcinoma (HCC).

Azide-functionalized microbubbles were conjugated with commercially available anti-GPC3 antibodies using a LASIC DBCO adapter, which enables precise site-specific labeling at the heavy chain of the antibody without compromising antigen-binding affinity. Binding specificity was confirmed through pre-blocking experiments with free anti-GPC3 antibodies in HCC cell lines. Fluorescence microscopy and quantitative image analysis were employed to evaluate cell-binding efficiency. For in vivo validation, conjugated microbubbles were intravenously administered into mice bearing orthotopic HepG2 xenografts, followed by contrast-enhanced ultrasound (CEUS) imaging at 0 s, 30 s, and 10 min post-injection to assess tumor signal enhancement and persistence.

LASIC chemistry enabled covalent and site-specific antibody attachment to microbubbles. In vitro assays demonstrated significantly increased binding of targeted microbubbles to GPC3-positive cells compared to antigen-blocked controls. In vivo CEUS imaging revealed rapid and sustained tumor-specific signal enhancement within 30 s of injection, persisting up to 10 min. In contrast, control microbubbles showed diminished signal intensity and rapid wash out post-injection.

LASIC-conjugated anti-GPC3 microbubbles exhibit efficient, specific, and durable binding to GPC3-expressing HCC cells both in vitro and in vivo. This strategy enables real-time, image-guided molecular profiling via CEUS and holds promise for precision imaging and theranostic applications in liver cancer management.