免疫毫微粒的内化作用及其逆转人肝癌细胞多药耐药性的研究

Internalization of Antibody-targeted Immunonanoparticles into Human Hepatoma Cells and Its Reversal Effect on MDR

目的 观察免疫毫微粒能否内化进入人肝癌细胞及其多药耐药性 ( MDR)细胞 ,研究免疫毫微粒能否逆转 MDR。方法 将抗人肝癌阿霉素白蛋白免疫毫微粒 ( HAb18- ADR- HSA- NP)与人肝癌株 SMMC- 772 1细胞及其 MDR细胞 ( SMMC- 772 1/ MDR+ )共同孵育 ,采用扫描电镜 ,激光共聚焦显微镜 ,透射电镜技术观察免疫毫微粒与靶细胞的结合及免疫毫微粒的内化过程 ;采用 MTT比色分析法测定免疫毫微粒对靶细胞的杀伤率 ,计算免疫毫微粒对靶细胞的 IC50 和 MDR细胞的耐药倍数 ( RF)。结果 激光共聚焦显微镜观察 ,SMMC- 772 1细胞表面和胞浆中均有许多荧光毫微粒 ;透射电镜观察 ,免疫毫微粒与 SMMC- 772 1细胞及其 MDR细胞在 3 7℃孵育后 ,胞浆中可见有免疫毫微粒 ,随时间的延长细胞变性损伤越严重 ;当 SMMC- 772 1及其 MDR细胞预先经 HAb18抗体处理 ,再与 HAb18- ADR- HSA- NP孵育 ,则胞浆中未见免疫毫微粒 ;扫描电镜显示 ,多个 HAb18- ADR- HSA- NP紧密结合在 SMMC- 772 1/ MDR+ 表面 ,MDR细胞对于免疫毫微粒的耐药倍数 ( 2 .1)较其对于游离 ADR( 4.4)明显降低。结论 人肝癌特异性阿霉素白蛋白免疫毫微粒通过抗体介导能特异性内化进入人肝癌 SMMC- 772 1细胞及其 MDR细胞 ;该免疫毫微粒能增强

Objective To observe if antibody-targeted immunonanoparticles could internalize into sensitive and multidrug resistance (MDR) cells of human hepatoma; and to study if the immunonanoparticles could reverse the MDR. Methods Human hepatoma-specific adriamycin-loaded human serum albumin immunonanoparticles (HAb18-ADR-HSA-NP) were incubated with human hepatoma sensitive cell line (SMMC-7721) or MDR cell line(SMMC-7721/MDR +) and the internalization of immunonanoparticles were observed by laser confocus microscopy, scanning electron microscopy and transmission electron microscopy; MTT colorimetric assay was used for assaying in vitro cytotoxicities of HAb18-ADR-HSA-NP to the resistant variant cells.Then based on these data,IC 50 value of the immunonanoparticles and RF (Resistant Factor) of MDR cells were calculated. Results Laser confocus microscopy showed that many fluorescent particles (labeled immunonanoparticles) tightly adsorbed to SMMC-7721 cells and were also seen in cytoplasm of SMMC-7721 cells. When incubated with immunonanoparticles at 37℃, many of immunonanoparticles were visualized in cytoplasm of SMCC-7721 or SMCC-7721/MDR +. These immunonanoparticles-contained cells exhibited damaged ultrastructures and the damage degree depended on incubation time.When the human hepatoma cells were pretreated with HAb18 antibody and incubated with immunonanoparticles, few immunonanoparticles were seen in cytoplasm of the cells, suggesting antibody-specific internalization of the immunonanoparticles. Scanning electron microscopy demonstrated specific binding of the immunonanoparticles to the resistant variant cells. That immunonanoparticles exerted enhanced cytotoxicity to the resistant variant cells was demonstrated by a decrease of RF value of MDR cells, compared with free ADR(4.4 vs.2 1). Conclusion Human hepatoma-specific adriamycin-loaded human serum albumin immunonanoparticles could specifically internalize into sensitive or multidrug resistance cells of human hepatoma via antibody direction. The immunonanoparticles could enhance the sensitivity of MDR cells to ADR cytotoxicity, suggesting its reverse effect on MDR.