Contrast-enhanced ultrasound combined targeted microbubbles for diagnosis of highly aggressive papillary thyroid carcinoma

Background: Accurate diagnosis of highly aggressive papillary thyroid cancer (PTC) may greatly help avoid overdiagnosis and overtreatment of PTC. However, there is still a lack of a convenient and accurate method. Targeted microbubbles, an emerging ultrasound contrast agent, have the potential to accurately diagnose highly aggressive PTC.

Purpose: To design and prepare a targeted microbubble for specific contrast-enhanced ultrasound (CEUS) imaging of highly invasive PTC.

Methods: Using β-galactoside-binding protein galectin-3 (Gal-3) overexpressed on the surface of highly invasive PTC cells as a target, C12 polypeptide (ANTPCGPYTHDCPVKR) with high affinity and specificity for Gal-3 was coupled to the surface of lipid microbubbles to prepare targeted microbubbles (Gal-3-C12@lipo MBs). The targeted microbubbles were prepared by thin-film hydration method and mechanical shaking method. The morphology, diameter, concentration and stability of microbubbles were investigated by fluorescence microscopy and an AccuSizer. The biosafety of microbubbles was studied using BCPAP cells through CCK8 assay. Confocal laser scanning microscope and flow cytometry were applied to research the cellular uptake of microbubbles to investigate the targeting ability to highly aggressive PTC. Finally, the specific contrast-enhanced ultrasound imaging of microbubbles in highly invasive PTC was validated on the mice bearing subcutaneous BCPAP tumor model via a clinically ultrasound imaging system.

Results: Gal-3-C12@lipo MBs were successfully prepared which showed a well-defined spherical morphology with an average diameter of 1.598 ± 0.848 μm. Gal-3-C12@lipo MBs showed good stability without rupture within 4 hours after preparation. At the cellular level, Gal-3-C12@lipo MBs exhibited favorable biosafety and superior targeting ability to BCPAP cells, with 2.8-fold higher cellular uptake than non-targeted lipid microbubbles (Lipo MBs). At the animal level, Gal-3-C12@lipo MBs significantly improved the quality of contrast-enhanced ultrasound imaging in highly invasive PTC, with an echo intensity of tumor significantly higher than that of Lipo MBs.

Conclusion: We designed and fabricated a novel targeted microbubble for the specific ultrasound imaging diagnosis of highly aggressive PTC. The targeted microbubbles have good stability, superior biosafety and high targeting specificity, which can significantly improve the tumor signal-to-noise ratio of highly invasive PTC, and have the potential to facilitate and accurately diagnose highly invasive PTC.