Molecular Imaging Experimental Study Of Liver Ischemia-Reperfusion Injury Using Targeted Lipid Nanobubble

Objective To prepare a new nanoscale ultrasound contrast agents targeting ICAM-l,to examine its targeting characteristics in vitro and its contrast enhancement effects in vivo, and to assess the feasibility and repeatability of ultrasonic molecular imaging technology in early diagnosing liver ischemia reperfusion injury using a homemade targeted lipid nanobubble.Methods The common lipid nanobubbles were prepared using the thinfilm hydration-sonication method, and then using the avidin-biotin binding method, the biotinylated ICAM-1 antibodies were connected with the biotinylated lipid nanobubble.Finally, the targeted lipid nanobubbles containing anti-ICAM-1 antibody were prepared. Human hepatic sinusoidal endothelial cells (HHSECs) were cultured at the circumstances of hypoxia/reoxygenation (H/R) and low temperature. The binding of the targeted nanobubbles to the HHSECs was photographed under a laser confocal microscope.The rabbit liver IRI model (I/R group) was established using the Pringle’s maneuver, and a sham operation(SO) group as the control.After the success of the modeling, the liver CEUS was performed. Using QLAB software, the liver blood flow parameters:PI, time to peak and time of duration were obtained, to evaluate quantitatively the ultrasound enhancement effect and stability of the targeted nanobubbles, and the reliability of the measurement parameters.Results The size of the targeted lipid nanobubbles was 148.15±39.75nm, Zeta potential-28.4±7.9mV, and the concentration was 3.6-7.4×109bubbles/ml.The targeted lipid nanobubbles containing anti-ICAM-1 antibody adhered well to the surface of HHSECs in the H/R group. After liver IRI, cell apoptosis were found. The apoptosis ratio of liver sinusoidal endothelial cell(LSEC) was 17.53±1.19%, and that of liver cell was 5.61±0.85%. After IRI, ICAM-1 had a high expression in LSEC, and there was a high correlation between ICAM-1 and ALT,LDH(r=0.812,r=0.854).The enhancement effect of targeted lipid nanobubbles in vivo have a good stability and repeatability, ICC=0.938.The variability of CEUS parameters measurement is minimal in 3cm under the liver capsule, CV 3.9%.The measurement results of two observers under the same condition have good consistency and reliability(ICC=0.948).Animal CEUS imaging showed that PI and time of duration of the targeted nanobubbles imaging were significantly higher than that of common nanobubbles in the I/R group, and time to peak was in advance significantly. PI and time of duration of the targeted nanobubbles in the I/R group were also significantly higher than that in the SO group. In the I/R group, PI of the targeted nanobubbles were positively correlated with liver Suzuki’s scores,and were linearly correlated with ICAM-1.Conclusion The targeted lipid nanobubbles have small particle size, stable characteristics, and good targeting ability in cells targeted binding experiment. Compared with the common nanobubbles, the targeted lipid nanobubbles have better specificity, stability and repeatability. Therefore, the targeted lipid nanobubbles are available for assessing liver ischemia-reperfusion injury at the molecular level, noninvasively, quantitatively and specifically.