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

## TL;DR

This study develops microbubbles with site-specific antibodies for precise ultrasound imaging of liver cancer, enabling noninvasive molecular profiling.

## Contribution

The use of light-activated site-specific conjugation to attach antibodies to microbubbles for targeted ultrasound imaging of hepatocellular carcinoma.

## Key findings

- LASIC chemistry enabled covalent and site-specific antibody attachment to microbubbles.
- Targeted microbubbles showed significantly increased binding to GPC3-positive cells in vitro.
- In vivo imaging revealed rapid and sustained tumor-specific signal enhancement with LASIC-conjugated microbubbles.

## Abstract

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 antibody’s heavy chain 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 seconds, 30 seconds, and 10 minutes 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 seconds of injection, persisting up to 10 minutes. In contrast, control microbubbles showed diminished signal intensity 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.

This study introduces site-specifically labeled microbubbles for targeted CEUS imaging of HCC, thereby enables noninvasive molecular profiling using off-the-shelf antibodies and offers a clinically translatable strategy for personalized liver cancer diagnostics.