Ultrasound/Optical Dual Modality Imaging for Evaluation of Atherosclerosis Plaque by OPN Targeted Nanoparticles

Objectives: Intense macrophage infiltration, phenotype transformation of vascular smooth muscle cells (VSMCs) and spotty calcification play vital roles in progressed atherosclerotic plaques. Osteopontin(OPN) is the marker for phenotypic conversion of VSMCs from contractile phenotype to synthetic phenotype. Significant higher OPN expression was found in foam cells along with the aggravating capacity of macrophage recruitment due to its RGD sequence and interaction with CD44. Among all the diagnostic imaging techniques, ultrasound imaging has a unique advantage on account of real time, low cost and high safety. Herein, OPN targeted nanoparticles (Cy5.5-anti-OPN-PEG-PLA-PFOB, denoted as PLA NPs) are constructed to identify the molecular characteristics of high-risk atherosclerosis by virtue of ultrasound and optical imaging. Methods: Osteopontin(OPN)was illuminated as a desired target of atherosclerotic plaques using Western blot, Real-Time PCR, immunofluorescence. Imaging agent was constructed based on previous studies. Polylactic acid-bromoperfluorooctane nanoparticles (PLA-PFOB NPs) were constructed by oil-in-water emulsion method, followed by the hydrophilic modification of amino polyethylene glycol maleimide (NH2-PEG-mal) attached on the surface. OPN antibody tagged with NHS-Cy5.5 fluorescent dye by amide bonds then conjugated with PEG-PLA-PFOB NPs using click reaction. Characterization, biocompatibility, sensibility and specificity were evaluated in vitro. For in vivo study, ApoE-/- mice were fed with high fat diet (containing 15% fat and 0.25% cholesterol) for 16-24 weeks while even-aged C57BL/6 mice were used as control group. Contrast-enhanced ultrasonography and fluorescence imaging were applied to collect signals. Results: In vitro cell study showed that OPN was highly expressed in raw 264.7 cells after incubation with ox-LDL in both RNA and protein levels. The average hydrodynamic size of PLA NPs is 332±40nm and the zeta potential is -20.07±0.75 mV. Good binding sensibility and specificity is confirmed in vitro according to cellular uptake analysis. CCK-8 assay and H&E staining showed excellent biocompatibility. Ultrasound and optical imaging revealed that the nanoparticles were accumulated in the atherosclerotic plaques. Conclusion: OPN targeted PLA nanoparticles(Cy5.5-anti-OPN-PEG-PLA-PFOB) were demonstrated to be a good contrast agent in molecular imaging of synthetic VSMCs and foam cells, which could be a promising tool to identify the vulnera