抗体F(ab′)_2片段靶向的载多柔比星免疫毫微粒的构建及其抗肝癌作用  

Preparation and anti-tumor characteristics of ADR-loaded albumin nanoparticles directed by F(ab′)_2 fragment of anti-human hepatoma antibody

作　　者：王霞[1] 刘晓波[2] 蔡美英[1] 黎光[1] 魏大鹏[1] 

机构地区：[1]四川大学基础医学与法医学院免疫学教研室,成都610041 [2]广州中山大学第一附属医院肾病实验室,广州510080

出　　处：《中国抗生素杂志》2003年第3期176-180,共5页Chinese Journal of Antibiotics

基　　金：卫生部科学研究基金资助项目 (98 1 2 2 5);四川省卫生厅基金资助项目

摘　　要：目的　构建抗体F(ab′) 2 片段靶向的载药免疫毫微粒 ;评价F(ab′) 2 片段免疫毫微粒对肿瘤的特异结合性及杀伤作用。方法　采用异型双功能交联剂琥珀酰亚胺基 3 (2 吡啶二硫 )丙酸酯 (SPDP)将抗人肝癌单抗HAb18或对照抗体 4E3的F(ab′) 2 片段与多柔比星 (ADR)人血清白蛋白毫微粒 (ADR HAS NP)共价交联构建抗体F (ab′) 2 片段靶向的载药免疫毫微粒 (HAb18F(ab′) 2 ADR HSA NP ,4E3F (ab′) 2 ADR HSA NP) ;采用玻片凝集试验 ,免疫荧光染色实验确定F(ab′) 2 片段是否结合在毫微粒表面 ;采用花环形成实验 ,花环形成阻断实验 ,扫描电镜分别从光镜、电镜水平观察F(ab′) 2 片段免疫毫微粒是否同人肝癌细胞株SMMC 772 1特异性结合 ;采用MTT比色分析法研究F(ab′) 2 片段免疫毫微粒的体外细胞毒作用 ;采用皮下荷人肝癌裸鼠模型 ,经瘤体注射观察免疫毫微粒的抗人肝癌作用。结果　F (ab′) 2 片段免疫毫微粒圆球状 ,粒径1 2 μm左右 ,免疫荧光染色阳性而ADR HSA NP为阴性 ,提示F (ab′) 2 片段已偶联到ADR HSA NP表面 ;HAb18F(ab′) 2 ADR HSA NP能有效结合于人肝癌细胞株SMMC 772 1,该结合能被HAb18抗体的F(ab′) 2片段阻断 ,未见该免疫毫微粒与对照细胞 (SW 1116)有效结合 ,这些结果提示 ,HAb18F(ab′)Objective To construct antibody F(ab′) 2 fragment targeted immunonanoparticles and study its in vitro specific binding and antitumor features. Methods Hetero bifunctional crosslinker SPDP was used to couple convalently F(ab′) 2 fragment of monoclonal antibody HAb18 (anti human hepatoma) or control McAb 4E3 with doxorubicin (adrimycin) loaded human serum albumin nanoparticles (ADR HSA NP); Slide agglutination, immunofluorescent assay were used for confirming coupling of F(ab′) 2 fragment to nanoparticles; Rosset formation test, rosset formation blocking test and scanning electron microscopy were used to observe specific binding of the immunonanoparticles to human hepatoma cell line SMCC 7721; MTT colorimetric assay was used for determining in vitro cytotoxicities of the F(ab′) 2 ADR HSA NP or ADR HSA NP to tumor cell lines; Hepatoma bearing nude mice mode was established and used for evaluating the antitumor effects of the immunonanoparticles or ADR HSA NP administered by intratumor injection in the mode. Results F(ab′) 2 ADR HSA NP was sphere with 1 2um of average diameter and stained positively by immunofluorescent assay with rabbit anti mouse F(ab′) 2 and sheep anti rabbit IgG FITC, but ADR HSA NP was stained negatively, which confirmed coupling of F(ab′) 2 fragment to ADR HSA NP; HAb18 F(ab′) 2 ADR HSA NP could efficiently bind to SMMC 7721 cells, but failed to bind to human colon carcinoma cell line SW1116 and this binding could be blocked by F(ab′) 2 fragment of HAb18, which confirmed specific binding of the F(ab′) 2 ADR HSA NP with target cells; HAb18 F(ab′) 2 ADR HSA NP could markedly kill SMMC 7721 cells with dose dependence and hadn′t significant cytotoxicity to SW1116 human colon carcinoma cells; HAb18 F(ab′) 2 ADR HSA NP administered by intratumor route could significantily inhibit the hepatoma growth in hepatoma bearing nude mice model and the 20 day inhibitory rate was 67 5%, significant high

关 键 词：抗体F(ab')2片段 多柔比星 ADR 白蛋白毫微粒 肝癌 载体 

分 类 号：R944.9[医药卫生—药剂学] R735.7[医药卫生—药学]