Development of intracellular drug delivery system utilizing liposomes

脂质体作为药物载体的作用在国际上得到了广泛的研究，特别是对其在肝脏中的摄取机制的研究。然而，关于内化脂质体的细胞内命运的研究很少报道，普遍认为它们被转移到溶酶体并在溶酶体中降解。细胞内命运的控制，如转移到溶酶体的逃逸和降解，从未尝试过。考虑脂质体内化后的细胞内命运对开发有效的脂质体给药系统以及静脉注射后的靶向性是非常重要的。本研究旨在阐明脂质体在细胞内的命运，得到以下结果：(1)大鼠肝脏灌注研究表明，脂质体被血浆蛋白调理的程度取决于其颗粒大小。(2)通过输注研究，发现肝脏摄取的饱和过程不是通过传统的Michaelis-Menten动力学来表达的，而是通过一种新的动力学模型来表达的，该模型通过增加肝内脂质体的积累量而不是通过血药浓度来降低摄取清除率。(3)经十六烷基甘露糖苷修饰的脂质体并没有像通常预期的那样通过细胞表面的甘露糖受体被kupffer细胞摄取，补体受体在摄取过程中起到了一定的作用。(4)提出了定量评价溶酶体降解过程的方法，并对该方法获得的体内和体外实验数据进行药代动力学分析，使定量评价细胞内降解过程成为可能。本研究的结果被认为为脂质体药物载体的开发提供了非常有用的信息。下一步的研究将重点关注脂质体内化过程与细胞内命运的关系。

The usefulness of liposomes as drug carriers has been widely studied in the world, especially on the uptake mechanism by the liver. However,very few studies on the intracellular fate of internalized liposomes have been reported under the common understanding that they are transferred to lysosomes and degradated there. Control of the intracellular fate such as escape of transfer to lysosome and degradation has never been attempted. It is very important to consider the intracellular fate of liposomes after internalization for the development of useful drug delivery system utilizing liposomes as well as the targetability after intravenous injection.This research was carried out to elucidate the intracellular fate of liposomes and following results were obtained: (1)Perfusion study using rat liver revealed that liposomes were opsonized by plasma protein depending on their particle size. (2)By infusion study,it was found that the saturation process of liver uptake was not expressed by conventional Michaelis-Menten kinetics but by novel kinetic model in which the uptake clearance was decreased by increasing accumulated amount of liposomes in the liver,not by blood concentration. (3)It became apparent that liposomes modified with cetylmannoside were not taken up by kupffer cells via mannose receptor on the cells surface as commonly expected,and the contribution of complement receptors was suggested on the uptake. (4)The method for quantitative estimation of degradation process in lysosomes was presented,and the pharmacokinetic analysis of the data obtained from in vivo and in vitro experiments by this methods made it possible to evaluate the degradation process in the cells quantitatively.Findings obtained in this study are considered to give very useful informations for development of liposomal drug carriers. As next step of the research,the relationship between internalization process and intracellular fate of liposomes will be focussed.