Experimental Study Of Applying Targeted Nano-Lipid Ultrasound Contrast Agent To Evaluate The Liver Functional Reserve

The liver is the body's vital organ of metabolism. Currently, liver diseases endanger people's health seriously. The liver has a great Compensatory ability, early clinical manifestations of liver damage is not obvious. However, once the liver dysfunction is severe, that often harm to people's lives. Therefore, seek a non-invasive means of monitoring liver function in medical is imminent.ASGPR is a specific receptor on the liver cells membrane, it can conduct receptor-ligand reaction with galactose in the end of material, and the expression of ASGPR is related closely with liver function, research shows that when the liver function reduction, ASGPR content is also reduced correspondingly. Scholars had made tremendous progress using the features of the ASGPR. In recent years, with rapid development of medical molecular imaging, imaging experts hope to use the imaging technology and the feature of ASGPR, to assess the liver functional reserve, which is important not only to estimate risk suffering from liver disease patients who underwent surgery, but also control timely and convenient for the postoperative recovery of patients.With the continuous development of ultrasound contrast agents and ultrasound molecular imaging, we look forward to through the preparation of liver targeted ultrasound contrast agents, use non-invasive which is the unique advantages of ultrasound, to achieve real-time monitoring for the liver reserve function in vivo. For the past, ultrasonic detection of liver function often including to evaluate the regional perfusion of liver blood flow use of ultrasound contrast and detect the dynamics of vascular changes using Doppler ultrasound as the micro-bubble contrast agents trigger rupture, investigating the relationship between the blood hemodynamic parameters and liver function. Some abroad scholars using Doppler ultrasound technique to analyze hepatic vein and portal vein blood flow Doppler waveform to evaluate the liver function. But there had little research in using ASGPR receptor-mediated targeted ultrasound imaging to the liver.Therefore, this subject was studied mainly from the following three parts:The first part, we prepared the GaL-PLL which is the targeting ligand of liver cell membrane receptors ASGPR, and prepared separately the targeting lipid ultrasound contrast agent and the nano-liquid fluorocarbon ultrasound contrast agent, to verify the two targeted ultrasound contrast agents in vitro targeted ability. The second part, we studied the self-made targeted liquid perfluorocarbon nano-particles ultrasound contrast agent in normal rat liver targeting ultrasound imaging characteristics, the same time, established different degrees of acute liver injury model, examined the relationships of the peak echo intensity changes and ASGPR content and serology.The third part, in order to further explore the application of targeted nano-lipid ultrasound contrast agent in the field of liver tumors, we studied the enhanced characteristics of the normal lipid ultrasound contrast agent and the self-made targeted nano-lipid ultrasound contrast agent in the rabbit VX2 tumor model. Objective ( 1 )To compound thetargeting ligands of asialoglycoprotein receptor (ASGPR) that is particularly suitable for liver-specific delivery due to its exclusive expression by parenchymal hepatocytes. To investigate the targeted capacity of ultrasound contrast agent it has a galactose-poly-L-lysine as a ligand,lay the foundation for targeted imaging in vitro. (2)To observe the targeting combined effects of the human liver cells L02 and the targeted ultrasound contrast agent;To evaluate the gathering imaging effects of the targeted ultrasound contrast agent.Methods (1)Galactosylated polylysine was prepared by reductive amination method,identification of galactosylated polylysine synthesis was detected by SDS-PAGE protein electrophoresis, The connected ratio of the amount of the polylysine and the galactose was calculated. The galactosylated polylysine was purificated and collected by Sephadex G-15 column. Prepare targeted lipid microbubbles at room temperature and observate targeting effect in vitro with fluorescence microscopy. (2)A certain proportion of DSPE-020PA and DPPC were dissolved in chloroform,after they were stirred uniform by the magnetic stirrer, they were rinsed and incubated at room temperature for 1 hour with the prepared GaL-PLL solution. Finally,they were oscillated for three minutes by acoustic oscillations. L02 cells were grown in incubator,according to the reacting time of the cells and the targeted nano-lipid ultrasound contrast agent,to observe the combined effects. The nano-lipid ultrasound contrast agent and the degassed water were loaded into cysts and their ultrasound imaging effects were observed by ltrasound diagnostic apparatus Philips iU 22.Results ( 1 ) Galactose and poly-L-lysine connected to high-performance in the molar ratio of 1:100, 24h reaction at room temperature,lipid microbubbles average particle size is 2 micron, size uniformity; see the targeted ultrasound contrast agent targeted combined with liver cancer cells.(2)the particle size of the liquid fluorocarbon nano-targeted lipid ultrasound contrast agent was extremely small, uniform, cylindrical and spherical. 1 hour later, the number of the targeted liquid perfluorocarbon nano-lipid ultrasound contrast agent around the human liver cells membrane was the maximum, and a litter of the targeted liquid perfluorocarbon nano-lipid ultrasound contrast agent was visible in cytoplasm of the L02 cells. The cysts in vitro showed: the side of the targeted liquid perfluorocarbon nano-lipid ultrasound contrast agent showed high echo.Conclusions (1) The targeting lipid microbubbles can be connected to liver cancer cells for its Endocytosis. ( 2 ) The targeted liquid perfluorocarbon nano-lipid ultrasound contrast agent can be effectively targeted to the human liver cells L02 due to carrying home-made GaL-PLL. The targeted ultrasound contrast agents can be imaging by ultrasound and be confirmed in vitro. The size of the contrast agent was small, therefore, it can be considered an ideal ultrasonic molecular probe and achieve the ultrasound molecular imaging in cell level. Objective (1) To research ultrasound imaging characteristics of targeted liquid perfluorocarbon nanoparticles ultrasound contrast agent in normal rat liver ,and provide experimental evidence of targeting ultrasound molecular imaging for the quantitative evaluation of liver function. (2) To analyze comparatively the Relationship of peak echo intensity in acute liver injury at all levels and ASGPR content and serological examination with the different level of acute liver injury model by targeted ultrasound molecular imaging.Methods (1) The standards of the liver function tests (total protein, albumin, alanine aminotransferase, aspartate aminotransferase) were established. 10 healthy male SD rats, they were fasted at the day before the experimental day, 2ml venous blood were placed in the extraction tube with 10ml syringe, labeled and placed at 4℃overnight, until after the natural precipitation of serum constituents and were detected.10 healthy male SD rats, the liquid perfluorocarbon nanoparticles targeted ultrasound contrast agents were injected in tail vein with bolus injection, from the beginning to 0.5h,the ultrasonic imaging were observed and recorded continuously; 0.5h to 1h,the ultrasonic imaging were observed and recorded in each 10min; 1h to the fading of contrast agents,the ultrasonic imaging of the liver parenchyma were observed and recorded in each 30min. finally,the ultrasonic imaging at the different time points of the liver parenchyma of were analysed by DFY diagnostic ultrasound image analysis system.(2) 25 healthy male SD rats were divided into 5 groups randomly, according to different doses of analytical grade in intraperitoneal carbon tetrachloride (CCl4), they were prepared to the CCl4 solution with edible vegetable oil at 10%, 20%, 30%, 40% and 50%, each group was injected 1ml CCl4 solution corresponding to the preparative Concentration, 1 d after,2ml venous blood were placed in the extraction tube with 10ml syringe in each group,labeled and placed at 4℃overnight, until after the natural precipitation of serum constituents and were detected the contents of the total protein and albumin and alanine aminotransferase and aspartate aminotransferase. Until the dose was determined, 20 models were builded with the same method, 4 rats in each group, 1 d after, the content of ASGPR in each group of the liver was detected respectively, and the changes of the liver sinusoidal endothelial cell gap or"fenestrae"were observed by scanning electron microscopy, apoptosis of hepatocytes was analyzed by TUNEL.25 healthy male SD rats were divided into 5 groups randomly,5 rats in each group, the acute liver injury model in different levels were established according to the aforementioned method, they were anesthetized generally for our experiment, thoracic and abdominal skin was prepared, the liquid perfluorocarbon nanoparticles targeted ultrasound contrast agents were injected in tail vein with bolus injection, from the beginning to 0.5h, the ultrasonic imaging were observed and recorded continuously; 0.5h to 1h, the ultrasonic imaging were observed and recorded in each 10min; 1h to the fading of contrast agents,the ultrasonic imaging of the liver parenchyma were observed and recorded in each 30min. Finally, the ultrasonic imagings at the different time points of the liver parenchyma of were analyzed by DFY diagnostic ultrasound image analysis system.Results (1) The normal serum total protein content is 60.0 ~ 80.0g / L, albumin concentration is 30.0 ~ 50.0g / L, aspartate aminotransferase concentration is 0 ~ 200U / L, alanine aminotransferase concentration is 0 ~ 50 U / L. the peak time of the liquid perfluorocarbon nanoparticles targeted ultrasound contrast agents went into the normal rat liver is 61.20±4.83min, the peak echo intensity is 88.20±0.08dB, targeted contrast agents were faded slowly, and the clear time is 310.00±17.80min .(2)The gap of liver sinusoidal endothelial cells was increases after acute liver injury from electron microscope; TUNEL staining, liver cell apoptosis rate increased with the concentration of CCl4 increase which the experimental rats were injected in, analysis of variance in each group ,the differences has statistical significance; With the liver damage increase in, the ASGPR content of liver tissue was decreased,analysis of variance in each group ,P <0.05; with the degree of liver injury increase,the peak echo intensity of liver targeted ultrasound contrast imaging was decreased, the every two groups were P <0.05 except the normal control group and 10% concentration of CCl4 group , while P> 0.05 that was the peak time and return time, hepatic parenchymal echo intensity before imaging and the echo intensity after the contrast agent was returned; total protein and albumin in each group showed no significant change in serology ,while alanine aminotransferase, aspartate aminotransferase were increased at first and then decreased following the degree of liver injury increased.Conclusions The imaging of the liquid perfluorocarbon nanoparticles targeted ultrasound contrast agents Mediated to the liver cell membrane receptors ASGPR is excellent, and benefit to the quantitative evaluation of liver reserve function by this targeted ultrasonic imaging. Object To investigate the ultrasound imaging characteristics of the self-made lipid microbubbles targeted ultrasound contrast agents in normal rabbit liver and VX2 rabbit tumor model, as well as its similarities and differences.Methods Inject common lipid bolus microbubble ultrasound contrast agent and self-made lipid microbubbles targeted ultrasound contrast agents into rabbit ear vein. Use Philips iU22 ultrasound imaging diagnostic mode, real-time monitor two different ultrasound contrast agent imaging characteristics of their own. Analyze the peak time, peak echo intensity and clear time with DFY. The parameters of each group were compared.Results In the normal rabbit liver parenchyma, normal lipid ultrasound contrast agent (MB) time to peak was significantly earlier than the targeted self-made lipid ultrasound contrast agent microbubbles (MB'). Peak intensity of the former high-echo was higher than that of the latter. Removal time of the former was significantly shorter than that of the latter. In the VX2 tumor, MB peak time was significantly earlier than that of the MB'. The peak echo intensity of the former was significantly higher than that of the latter. Removal time of the former was significantly shorter than that of the latter.Conclusions Self-made lipid microbubbles targeted ultrasound contrast agents have their own unique processes and characteristics in ultrasound imaging, which was "negative image" mode.