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

Purpose: 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 ZD2GVs had only 267.73 & PLUSMN; 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.

Automated summary

In this study, a nanoscale acoustic probe based on gas vesicles (GVs) was developed by conjugating ZD2 peptides to the biosynthetic GVs. The resulting ZD2-GVs showed excellent binding capability to tumor cells and demonstrated strong penetration ability in tumor tissues. This study suggests that ZD2-GVs can serve as a potential UMI probe for early diagnosis of bladder cancer.