基于乙肝病毒的嗜肝性原理构建的肝实质细胞主动靶向给药系统的研究

The liver parenchymal cells targeting drug delivery system based on ligands such as galactose had a low targeting efficiency, due largely to the non-specific distribution of corresponding receptors in other cells or the low affinity between ligands and receptors. Hence, it is a big challenge to find ligands with high efficiency for liver parenchymal cells targeting. It is reported that the surface envelope protein of hepatitis B virus (HBV) has a high affinity to liver parenchymal cells. A recent research demonstrated that HBVpreS/2-48consCn, which was a peptid derivative of a 2nd-48th amino acid sequence located in the pre-S1 region of the surface envelope protein, showed a higher affinity to the specific receptor on liver cells than HBV. Therefore, the objective of this present project is to develop a novel liposome with HBVpreS/2-48consCn serving as a ligand, so that the loaded drug can be highly accumulated in the surface of liver parenchymal cells via specific receptor-ligand interactions and thus absorbed in a large amount by endocytosis. In this project, the ligand modification method, pharmaceutical properties of liposome including preparation procedure, quality evaluation, and stability will be studied. Furthermore, the targeting efficiency, cell uptake, uptake mechanism, pharmacokinetics and preliminary pharmacodynamics of the novel targeting liposome formulation will be evaluated in vitro and in vivo. This research will provide a theoretical basis for developing a safe and highly efficient drug delivery system for liver parenchymal cells targeting.

以半乳糖等配体修饰的肝实质细胞靶向给药系统由于受体在非实质细胞也有表达或结合力不强等原因，导致靶向效率低。寻找高效的肝实质细胞靶向配体，成为目前研究的难点之一。乙肝病毒作为一种嗜肝病毒，其表面包膜蛋白对肝实质细胞有高度的亲和性。最新研究表明该蛋白上pre-S1区中第2位至第48位氨基酸序列的短肽衍生物HBVpreS/2-48consCn具有比乙肝病毒更强的结合肝细胞特异性受体的能力。本课题拟以此短肽衍生物为靶向配体，构建一种新型的主动靶向脂质体，以期该脂质体在配体介导下，载带药物与受体作用浓集于肝实质细胞表面，并通过内吞，促进药物吸收。本课题将系统研究配体修饰方法，靶向脂质体的制备工艺、质量评价、稳定性等制剂学性质；在此基础上，进一步研究靶向脂质体在体内外靶向性、肝实质细胞的摄取效率及摄取机制，动物药代动力学及初步的药效学。为研究新型的安全高效的肝实质细胞主动靶向给药系统提供理论基础。

本课题基于乙肝病毒嗜肝性原理，研究以乙肝病毒表面包裹L蛋白的Pre-S1区的一段多肽为配体的肝实质细胞靶向给药系统。首先通过固相合成技术合成了由乙肝病毒L包裹蛋白pre-S1区第2-48位或第21-47位氨基酸组成的、并在N端肉豆蔻酰化（HBVP、HBVpreS/21-47myr）的短肽；然后用该肽对PEG化脂质体进行修饰，构建一种新型肝实质细胞靶向的给药系统，并对该载体的理化性质、急性毒性以及体内外肝实质细胞靶向性等进行了系统研究。所制得的靶向脂质体粒径为150 nm左右，PDI小于0.2, Zeta电位为-20 mV左右。经原代肝实质细胞摄取试验证实，与未修饰的脂质体相比，所制得的靶向脂质体的摄取可提高5倍；体内肝实质细胞分布试验也证实，所制得的靶向脂质体的分布较未修饰的脂质体提高了8倍。药效学实验进一步证实，所制得靶向脂质体显著提高了水飞蓟宾对CCl4造成的急性肝损伤的保护作用和紫杉醇对肝癌的抑制作用。而且该载体没有表现出明显的急性毒性和免疫毒性。这些结果为肝实质细胞靶向的药物传递系统研究提供了新思路，也为以乙肝病毒Pre-S1短肽在肝靶向药物传递系统中的应用提供了理论基础。.该研究已在Biomaterials、Nanoscale等高水平SCI收录杂志上发表论文15篇，申请相关发明专利1项，参加国际学术会议2次、国内学术会议3次。培养博士研究生2名，硕士研究生2名。