高侵袭性乳头状甲状腺癌（PTC）的准确诊断可以帮助避免PTC的过度诊断和过度治疗，但仍缺乏一种方便而准确的诊断方法。定向微泡是一种新兴的超声造影剂，具有准确诊断高侵袭性PTC的潜力。为了设计和制备一种特定对比增强超声（CEUS）成像高侵袭性PTC的定向微泡，本研究以高侵袭性PTC细胞表面过度表达的β-半乳糖苷结合蛋白酵母菌（Gal-3）为靶点，将与Gal-3具有高亲和力和特异性的C12多肽（ANTPCGPYTHDCPVKR）连接到脂质微泡表面，制备出定向微泡（Gal-3-C12@lipo MBs）。使用薄膜水合法和机械振荡法制备定向微泡，并通过荧光显微镜和AccuSizer研究微泡的形态、直径、浓度和稳定性。通过CCK8法研究微泡的生物安全性。应用共聚焦激光扫描显微镜和流式细胞术研究微泡的细胞摄取以研究对高侵袭性PTC的定向能力。最后，通过临床超声成像系统验证了定向微泡在携带皮下BCPAP肿瘤模型的小鼠中，对高侵袭性PTC的特异性对比增强超声成像。 成功制备了Gal-3-C12@lipo MBs，其显示出明确定义的球形形态，平均直径为1.598±0.848μm。经过制备后4小时，Gal-3-C12@lipo MBs表现出良好的稳定性，不会破裂。在细胞水平上，Gal-3-C12@lipo MBs表现出良好的生物安全性和对BCPAP细胞的优越定向能力，其细胞摄取率比非定向脂质微泡（Lipo MBs）高2.8倍。在动物水平上，Gal-3-C12@lipo MBs显着提高了高侵袭性PTC的对比增强超声成像质量，肿瘤的回声强度显着高于Lipo MBs。 我们设计和制备了一种新型的定向微泡，用于高侵袭性PTC的特定超声成像诊断。该定向微泡具有良好的稳定性、优越的生物安全性和高定向特异性，可以显著提高高侵袭性PTC的肿瘤信噪比，有望促进高侵袭性PTC的准确诊断。版权所有©2023 Ma、Wang、Xi、Tang、Wang、Wang、Wang、Liang和Zhang。

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.To design and prepare a targeted microbubble for specific contrast-enhanced ultrasound (CEUS) imaging of highly invasive PTC.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.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.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.Copyright © 2023 Ma, Wang, Xi, Tang, Wang, Wang, Wang, Liang and Zhang.