纳米级液态氟碳靶向超声造影剂的制备及体外寻靶的实验研究

The manufacture of a novel ultrasound contrast agent:hepatocyte targeted perfluorocarbon lipid nanoparticle,and its targeting study in vitro of liver

目的制备一种新型的具备特异性肝细胞靶向性能的超声造影剂,即携半乳糖化多聚赖氨酸(Gal-PLL)的脂质包裹的纳米级液态氟碳微球超声造影剂,并研究其在体外肝细胞的寻靶能力。方法应用还原胺化法优化制备肝脏去唾液酸糖蛋白受体的配体Gal-PLL,将其磷脂膜洗脱下来并逐滴加入液态氟碳,通过超声波细胞粉碎机进行振荡获得目标造影剂,并观察其形态,检测其粒径和电位。将靶向造影剂和非靶向造影剂分别滴入贴壁生长的正常大鼠肝细胞及正常人肝细胞中,比较观察两组造影剂与肝细胞的靶向效果。结果成功制备Gal-PLL,并合成了靶向纳米液态氟碳脂质微球超声造影剂,电镜下其形态圆整、大小均一,性质稳定,平均粒径200 nm,平均电位35 m V。制备的靶向纳米液态氟碳脂质微球超声造影剂能靶向聚集于体外正常大鼠肝细胞和正常人肝细胞周围。结论自制携Gal-PLL的脂质包裹的纳米级液态氟碳微球超声造影剂具有性质稳定、靶向性等优点,为今后实时在体监测并定量评估肝脏损伤程度的超声显影研究提供了前期基础。

Objective To prepare a new type of ultrasound contrast agent,perfluorooctyl bromide lipid nanoparticle with Galactocylated Poly-L-Lysine（Gal-PLL）,and study the ability of this contrast for hepatocyte targeting in vitro.Methods The preparation of high-efficiency Gal-PLL ligand of the asialoglycoprotein receptor in liver was optimized by chemical reactions of reductive amination.Lipid membrane prepared before was eluted with Gal-PLL,adding perfluorocarbon in it,then objective ultrasound contrast agent was prepared by means of ultrasonic cell disruptor agitation,its feature was observed,the grain diameter and electric potential were detected.Targeting contrast agent and non-targeting contrast agent were instilled into normal adherence growth rat hepatocyte BRL and human hepatocyte L02,respectively. The targeting effect of the contrast agent for both rat hepatocyte BRL and human hepatocyte L02 were observed and compared. Results High-efficiency Gal-PLL ligand of the asialoglycoprotein receptor for hepatocyte and novel ultrasound contrast agent-perfluorooctyl bromide lipid nanoparticle were successfully prepared. The contrast agent was round with size stability（the average diameter was 200 nm）,under the electron microscope,meanwhile,with stable property.Its average electric potential was 35 m V.The contrast agent flocked together around the rat hepatocyte BRL and human hepatocyte L02 cells. Conclusion The self-perpared novel ultrasound contrast agent,perfluorooctyl bromide lipid nanoparticle,is stable in property with targeting function for hepatocyte.This novel contrast may be used for evaluation on liver damage degree quantitatiely in real time in future.