纳米脂质体在肿瘤亚细胞靶器官分布动力学计算模型的设计

The goal of this study is to develop intracellular kinetic models of liposomes with size at 100-200 nm, both at cell level and at sub-cell level, which show a new method for formulation selection. Cell membrane binding (B) is separated into nonsaturable binding (NSB) and saturable binding (SB). Internalization (I) is further described as liposomes in different sub-cellular compartments, with cytosol and lysosome as the most important organelles studied. Doxorubicin and siRNA are selected as model drugs for chemical medicines and gene therapy. Passive-targeting liposomes and active-targeting liposomes are prepared respectively. Two tumor cells (e.g., MCF-7 and HL-60) are tested. Cell-associated liposomal concentration time courses with equal extracellular liposomes concentrations; and curves with different extracellular concentration at equilibrium are measured. Sub-cellular organelles, i.e., early/late endosomes, lysosomes and nuclei, are distinguished with their respective markers or pH-sensitive fluorescent probes. We will visualize the intracellular trafficking of drug-loaded liposomes in live cells by fluorescence microscopy. The observed data will be fitted in the established mathematic models of computer to get different rate constants among cellular and subcellular compartments. Finally, the established computer models are proposed to enable calculation of the concentration-time profiles of nano-liposomal intracellular trafficking at subcellular target organelles.

拟研究粒径为100-200 nm的纳米脂质体在细胞/亚细胞水平的转运动力学，设计计算机模型计算纳米脂质体在亚细胞靶器官的量变曲线，建立一种制剂筛选的新方法。细胞水平的实验研究主要分为脂质体与细胞表面的结合（B）及内吞（I）过程，设计了时间动力学和稳态浓度研究两组实验，将B按照生理学路径拆分为不饱和的非特异性表面结合（NSB）和可饱和的表面结合（SB）两种方式，建立相应的数学模型拟合实验值，得到细胞水平的转运速率常数；进一步将I拆分为不同亚细胞隔室的脂质体量，选择不同荧光探针标记亚细胞隔室（早/晚期内涵体、溶酶体、细胞核等），精密追踪脂质体在不同隔室间的转运时间及量变曲线，建模拟合得到亚细胞水平的转运速率常数。为完善模型的普适性，选择化学药物阿霉素和基因药物siRNA为两种模型药物，分别以被动靶向脂质体和主动靶向脂质体为研究对象，选择2种肿瘤细胞为实验对象，拟合实验值确定模型参数。

研究了粒径为100-200 nm的纳米脂质体在细胞/亚细胞水平的的转运动力学，设计计算机模型计算纳米脂质体在亚细胞靶器官的量变曲线，建立一种制剂筛选的新方法。细胞水平的实验研究主要分为脂质体与细胞表面的结合（B）及内吞（I）过程，设计了时间动力学和稳态浓度研究两组实验，将B按照生理学路径拆分为不饱和的非特异性表面结合（NSB）和可饱和的表面结合（SB）两种方式，建立相应的数学模型拟合实验值，得到细胞水平的转运速率常数；进一步将I拆分为不同亚细胞隔室的脂质体量，选择不同荧光探针标记亚细胞隔室溶酶体和细胞核，精密追踪脂质体在不同隔室间的转运时间及量变曲线，建模拟合得到亚细胞水平的转运速率常数。为完善模型的普适性，选择化学药物阿霉素和荧光标记的siRNA为两种模型药物，脂质体的处方因素中分别考察了阳离子电荷的高低、促进膜融合的DOPE含量高低、PEG化隐形作用以及靶向配体RGD的作用，分别以被动靶向脂质体和主动靶向脂质体为研究对象，选择HepG2和A375细胞2种肿瘤细胞为实验对象，拟合实验值确定模型参数，同时探讨了内吞机理为脂质阀介导的内吞。