超声分子成像（UMI）已被证明在诊断血管生成、炎症和血栓形成等疾病的发生和发展方面具有很大的潜力。然而，基于微泡的声学探针仅限于血管内靶标，因为它们的粒径相对较大，大大降低了UMI的应用价值，特别是对于血管外靶标来说。外域B型纤维连接蛋白（ED-B FN）是一种与肿瘤起源和发展密切相关的重要糖蛋白，在许多类型的肿瘤中高度表达。在这里，我们通过将ZD2肽与生物合成的气囊泡（GVs）上的ED-B FN进行偶联反应，开发出了一种基于GVs的纳米尺度声学探针（ZD2-GVs），该肽可特异性地靶向ED-B FN。用免疫荧光和Western blot评估了正常肝脏和肿瘤组织中ED-B FN的表达情况。通过酰胺反应将ZD2偶联到GVs表面制备了ZD2-GVs。采用倒置显微镜分析了ZD2-GVs与MB49细胞（膀胱癌细胞系）之间的靶向结合能力。在三只MB49肿瘤小鼠中比较了GVs、非靶向对照GVs（CTR-GVs）和靶向GVs（ZD2-GVs）的增强对比成像特征。通过荧光免疫组化方法评估了ZD2-GVs在肿瘤组织中的穿透能力。通过CCK8、血液生化和HE染色评估了GVs的生物安全性。观察到肿瘤组织中ED-B FN的表达强度较高，而在正常肝脏组织中表达较少。结果显示，所得到的ZD2-GVs的粒径仅为267.73 ± 2.86 nm，与MB49肿瘤细胞显示出良好的结合能力。体内UMI实验显示，与非靶向对照CTR-GVs和GVs相比，ZD2-GVs在BC肿瘤中产生了更强的、更持久的保留。荧光免疫组织化学实验证实了ZD2-GVs能够穿透肿瘤血管进入肿瘤间质空间。生物安全性分析表明，对这些受试小鼠没有明显的细胞毒性。因此，ZD2-GVs可以作为一个潜在的UMI探针，用于膀胱癌的早期诊断。© 2023 Feng et al.

Ultrasound molecular imaging (UMI) has proven promising to diagnose the onset and progression of diseases such as angiogenesis, inflammation, and thrombosis. However, microbubble-based acoustic probes are confined to intravascular targets due to their relatively large particle size, greatly reducing the application value of UMI, especially for extravascular targets. Extradomain B fibronectin (ED-B FN) is an important glycoprotein associated with tumor genesis and development and highly expressed in many types of tumors. Here, we developed a gas vesicles (GVs)-based nanoscale acoustic probe (ZD2-GVs) through conjugating ZD2 peptides which can specially target to ED-B FN to the biosynthetic GVs.ED-B FN expression was evaluated in normal liver and tumor tissues with immunofluorescence and Western blot. ZD2-GVs were prepared by conjugating ZD2 to the surface of GVs by amide reaction. The inverted microscope was used to analyze the targeted binding capacity of ZD2-GVs to MB49 cells (bladder cancer cell line). The contrast-enhanced imaging features of GVs, non-targeted control GVs (CTR-GVs), and targeted GVs (ZD2-GVs) were compared in three MB49 tumor mice. The penetration ability of ZD2-GVs in tumor tissues was assessed by fluorescence immunohistochemistry. The biosafety of GVs was evaluated by CCK8, blood biochemistry, and HE staining.Strong ED-B FN expression was observed in tumor tissues while little expression in normal liver tissues. The resulting ZD2-GVs had only 267.73 ± 2.86 nm particle size and exhibited excellent binding capability to the MB49 tumor cells. The in vivo UMI experiments showed that ZD2-GVs produced stronger and longer retention in the BC tumors than that of the non-targeted CTR-GVs and GVs. Fluorescence immunohistochemistry confirmed that ZD2-GVs could penetrate the tumor vascular into the interstitial space of the tumors. Biosafety analysis revealed there was no significant cytotoxicity to these tested mice.Thus, ZD2-GVs can function as a potential UMI probe for the early diagnosis of bladder cancer.© 2023 Feng et al.