Caigui Yu,1– 3,* Lu Zhong,1– 3,* Yanxiang Zhou,1– 3,* Nan Jiang,1– 3 Jinling Chen,1– 3 Sheng Cao1– 3 

1Department of Ultrasound Imaging, Renmin Hospital of Wuhan University, Wuhan, 430060, People’s Republic of China; 2Hubei Key Laboratory of Cardiology, Wuhan, 430060, People’s Republic of China; 3Cardiovascular Research Institute, Wuhan University, Wuhan, 430060, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Jinling Chen; Sheng Cao, Department of Ultrasound Imaging, Renmin Hospital of Wuhan University, 238# Jiefang Road, Wuhan, 430060, People’s Republic of China, Email jenny2774@163.com; caosheng209@126.com

Objective: To explore the feasibility of using cRGD-GNR-PFP-NPs to assess plaque vulnerability in an atherosclerotic plaque mouse model by dual-modal photoacoustic/ultrasonic imaging.
Methods: A nanomolecular probe containing gold nanorods (GNRs) and perfluoropentane (PFP) coated with the cyclic Arg-Gly-Asp (cRGD) peptide were prepared by double emulsion solvent evaporation and carbodiimide methods. The morphology, particle size, potential, cRGD conjugation and absorption features of the nanomolecular probe were characterized, along with its in vitro phase transformation and photoacoustic/ultrasonic dual-modal imaging properties. In vivo fluorescence imaging was used to determine the distribution of cRGD-GNR-PFP-NPs in vivo in apolipoprotein E-deficient (ApoE−/−) atherosclerotic plaque model mice, the optimal imaging time was determined, and photoacoustic/ultrasonic dual-modal molecular imaging of integrin αvβ 3 expressed in atherosclerotic plaques was performed. Pathological assessments verified the imaging results in terms of integrin αvβ 3 expression and plaque vulnerability.
Results: cRGD-GNR-PFP-NPs were spherical with an appropriate particle size (average of approximately 258.03± 6.75 nm), a uniform dispersion, and a potential of approximately − 9.36± 0.53 mV. The probe had a characteristic absorption peak at 780~790 nm, and the surface conjugation of the cRGD peptide reached 92.79%. cRGD-GNR-PFP-NPs were very stable in the non-excited state but very easily underwent phase transformation under low-intensity focused ultrasound (LIFU) and had excellent photoacoustic/ultrasonic dual-modal imaging capability. Mice fed a high-fat diet for 20 weeks had obvious hyperlipidemia with larger, more vulnerable plaques. These plaques could be specifically targeted by cRGD-GNR-PFP-NPs as determined by in vivo fluorescence imaging, and the enrichment of nanomolecular probe increased with the increasing of plaque vulnerability; the photoacoustic/ultrasound signals of the plaques in the high-fat group were stronger. The pathological assessments were in good agreement with the cRGD-GNR-PFP-NPs plaque accumulation, integrin αvβ 3 expression and plaque vulnerability results.
Conclusion: A phase variant photoacoustic/ultrasonic dual-modal cRGD nanomolecular probe was successfully prepared and can be used to identify plaque vulnerability safely and effectively.

Keywords: atherosclerosis, vulnerability, photoacoustic, ultrasound, low-intensity focused ultrasound