具有活性羧基末端的长循环脂质体的制备和分布

目的　研究长循环免疫脂质体(immunoliposomes,IML)的制备方法, 体外靶细胞杀伤活性和在小鼠体内的组织分布。方法　合成和纯化了1个带末端羧基的磷脂酰乙醇胺(PE)的聚乙二醇衍生物(DPPE-PEG3000-COOH),掺入脂质体中制成长循环脂质体；通过羧基活泼酯化，将膀胱癌单克隆抗体BDI-1或小鼠IgG共价连接到该脂质体表面制成免疫脂质体，体外肿瘤细胞杀伤实验检测载阿霉素免疫脂质体(ADM-BDI-1-IML)特异杀伤靶细胞的能力。用同位素氚示踪法测量免疫脂质体在小鼠的组织分布。结果　抗体在脂质体上的结合率可达30％。体外肿瘤细胞杀伤实验证明载阿霉素免疫脂质体有选择性杀伤靶细胞人膀胱癌细胞EJ的能力。和普通脂质体相比，免疫脂质体在血中的滞留时间明显延长，并减少了在肝、脾的聚集。结论　长循环免疫脂质体在血中有较长的滞留时间，在体外有特异寻靶活性，载阿霉素免疫脂质体有选择性杀伤靶细胞的活性，这些性质为其在体内主动寻靶和选择性杀伤靶肿瘤细胞提供了必要条件。

STUDY ON PREPARATION AND BIODISTRIBUTION OF PEG-IMMUNOLIPOSOMES WITH ACTIVE CARBOXYLIC TERMINALS

AIM: In order to accumulate into its target specifically, the immunoliposomes must possess two characteristics: specific target efficiency to its target cells and prolonged circulation in blood. A new type of polyethylene glycol (PEG)-immunoliposomes carrying monoclonal antibodies at the distal end of PEG chains should be developed. METHODS: A dipalmitoylphosphatidylethanolamine (DPPE) derivative of PEG with carboxyl group (DPPE-PEG3000-COOH) was newly synthesized. Small unilamellar liposomes were prepared from egg phosphatidyl choline and cholesterol (5:4, mol/mol) containing 6 mol% DPPE-PEG3000-COOH using reverse-phase evaporation method followed with bath sonication. Monoclonal antibody of human bladder cancer cell (BDI-1), which is highly specific to human bladder cancer cell, was conjugated to PEG-liposomes as well as mouse IgG at the distal end of polyethylene glycol chain. Doxorubicin was entrapped into these immunoliposomes by remote (NH4)2SO4 gradient loading method. The specific targeting efficiency of these immnoliposomes was tested by cytotoxicity test in vitro, enzyme-linked immune sorbent assay (ELISA) and indirect fluorescent immunoassay. Its biodistribution was carried out in mice. RESULTS: The specific targeting efficiency of BDI-1 immunoliposomes (BDI-1-IML) to EJ cells has been demonstrated, in contrast to the nonspecific human colon carcinoma cells (LOVO). PEG-liposomes linked with mouse IgG (mouse-IgG-immunoliposomes, IgG-IML) displayed lower reticulo-endothelial systems (RES) uptake and longer circulation time than liposomes without PEG after intravenous injection. CONCLUSION: The long circulation of these PEG-immunoliposomes in vivo, combined with its specific targeting efficiency demonstrated in vitro, guarantees the positive targeting efficiency of these immunoliposomes to its target carcinoma in vivo.