Chen Lin,1,2 Xiaoying Li,1 Yingnan Wu,1 Yuanyuan Wang,3 Weijian Song,1 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 Ultrasound Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, 325000, 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

Correspondence: Litao Sun, Email litaosun1971@sina.com Fei Yan, Email fei.yan@siat.ac.cn

Purpose: C-X-C chemokine receptor 4 (CXCR4) mediates the inflammatory response of atherosclerotic vulnerable plaques (ASVP) and is a potential biomarker of atherosclerotic vulnerable plaques. The purpose of this study was to use the imaging ability of a new type of ultrasound contrast agent, nanoscale biosynthetic gas vesicles (GVs), on the vascular wall and to combine the specific ligand of CXCR4 to construct a targeted molecular probe to achieve early identification of atherosclerotic vulnerable plaques and guide clinical treatment decisions.
Materials and Methods: Compared three contrast agents: GVs, the micro-contrast agent SonoVue, and polyethylene glycol (PEG)-modified GVs in the carotid artery. The expression of CXCR4 in atherosclerotic plaques was demonstrated using flow cytometry and immunofluorescence experiments. Cell adhesion and in vivo ultrasound imaging experiments demonstrated their ability to target the nanoscale biosynthetic gas vesicles. The safety of GVs, PEG-GVs, and CXCR4-GVs was tested the CCk8 test, H&E staining, and serum detection.
Results: Strong CXCR4 expression was observed in plaques, whereas little expression was observed in normal vessels. GVs can produce stable contrast signals on the carotid artery walls of rats, whereas PEG-GVs can produce more lasting contrast signals on the carotid artery wall of rats. CXCR4-GVs exhibited excellent binding capability to ox-LDL-induced RAW264.7 cells. Animal experiments showed that compared with Con-GVs, CXCR4-GVs injected plaque imaging signal was stronger and more durable. In vitro scanning of vulnerable plaques in rats injected with fluorescent vesicles demonstrated that CXCR4-GVs oozed through the neovasculars within vulnerable plaques and aggregated in vulnerable plaques. Through the CCK8 test, H&E staining, and serum detection, the safety of CXCR4-GVs was confirmed.
Conclusion: CXCR4-GVs were constructed as targeted molecular probes, which can be proven to have good targeting properties to vulnerable atherosclerotic plaques.

Keywords: ultrasound molecular imaging, vulnerable plaque, CXCR4, nanoscale biosynthetic gas vesicles