The Manufacture Of A Novel Ultrasound Contrast Agent: Liver Targeted Perfluorocarbon Lipid Nanoparticle, And Its Targeting Study In Vitro Of Liver

Objective: To optimize the preparation of high-efficiency ligand of the asialoglycoprotein receptor in liver-Galactosylated Poly-L-Lysine(Gal-PLL), to manufacture a new-type ultrasound contrast agent which had the specificity of liver targeting-perfluorooctyl bromide(PFOB) lipid nanoparticle carrying Gal-PLL, and to study its liver targeting potential, providing experimental proof for future targeting ultrasound imaging which can assess injured level of the liver。Methods:First, the high-efficiency ligand of the asialoglycoprotein receptor in liver-Gal-PLL was prepared by chemical reactions of reductive amination. Second, dipalmitoyl phosphatidylcholine 、 DSPE-020 PA and cholesterol were dissolved at a certain percentage in chloroform and evaporated to be lipid membrane by rotary evaporators. Lipid membrane was eluted with Gal-PLL solution, then adding PFOB in it, and objective targeting ultrasound contrast agent was manufactured by means of ultrasonic cell disruptor agitation; its feature was observed and the grain diameter and electric potential were detected; then the Gal-PLL was replaced with phosphate buffer solution when the non-targeting ultrasound contrast agent was prepared by the same way. Last, The two kinds of contrast agent were all instilled into normal rat hepatocyte BRL and normal human hepatocyte L02 which grew with adherence and were in the logarithmic phase, observing the different targeting effect of the two contrast agent under the electron microscope, inverted fluorescence microscope and laser scanning confocal microscope.Results: High-efficiency ligand of the asialoglycoprotein receptor in liver-Gal-PLL was successfully prepared and novel ultrasound contrast agent-PFOB lipid nanoparticle was manufactured. Its shape under the electron microscope was rounded. The size of the agent was almost the same and it had stable nature also. The average grain diameter was about 200 nanometers, electric poential was about positive 35 mV; the targeting contrast agent can flock together around the BRL and LO2 cells because of its targeting effect, but the non-targeting ultrasound contrast agent had no this potential.Conclusion: Manufactured novel ultrasound contrast agent-PFOB lipid nanoparticle had many advantages, such as uniform grain diameter, good shape and stable nature. It also can target the hepatocyte. This study provided a primary foundation for future study of ultrasonic contrast of real time monitoring in vivo and quantitative assessment of the liver damage degree.