Ultrasound Molecular Imaging of Bladder Cancer via Extradomain B Fibronectin-Targeted Biosynthetic GVs

Abstract

Yanan Feng,1,2,* Yongsheng Hao,3,* Yuanyuan Wang,3 Weijian Song,1,4 Shanxin Zhang,1 Dong Ni,5 Fei Yan,3 Litao Sun1 1Cancer Center, Department of Ultrasound Medicine, Zhejiang Provincial People’s Hospital (Affiliated People’s Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, 310014, People’s Republic of China; 2Department of Abdominal Ultrasound, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, 266000, People’s Republic of China; 3Center for Cell and Gene Circuit Design, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, People’s Republic of China; 4Bengbu Medical College, Bengbu, 233030, People’s Republic of China; 5Medical Ultrasound Image Computing (MUSIC) Laboratory, Shenzhen University, Shenzhen, 518055, People’s Republic of China*These authors contributed equally to this workCorrespondence: Litao Sun; Fei Yan, Tel [phone], Email [email protected]; [email protected]: 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.Materials and Methods: 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.Results: 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.Conclusion: Thus, ZD2-GVs can function as a potential UMI probe for the early diagnosis of bladder cancer.Graphical Abstract: Keywords: ultrasound contrast agents, gas vesicles, ultrasound molecular imaging, tumor diagnosis

Keywords

• ultrasound contrast agents
• gas vesicles
• ultrasound molecular imaging
• tumor diagnosis