State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China
J Biomed Nanotechnol. 2013 May;9(5):833-44. doi: 10.1166/jbn.2013.1587.
Folate receptor alpha (FRalpha) is overexpressed on ovarian cancer cells and is a promising molecular target for ovarian cancer gene therapy, but there was still no related report. In this study, folate modified cationic liposomes (F-PEG-CLPs) for ovarian cancer gene delivery were developed for the first time. Folate-poly(ethylene glycol)-succinate-cholesterol (F-PEG-suc-Chol) was firstly synthesized and then used to prepare folate-targeted cationic liposomes/plasmid DNA complexes (F-targeted lipoplexes). F-targeted lipoplexes were prepared by post-insertion method, and displayed membrane structure by transmission electron microscopy observation with the diameter of 193 nm-200 nm and the zeta potential of 35 mV-38 mV. DNase degradation experiments showed that plasmid DNA could be effectively shielded by F-targeted lipoplexes in vitro. F-targeted lipoplexes could transfer gene into human ovarian carcinoma cell line SKOV-3, and 0.1% F-PEG-CLPs composed by DOTAP/Chol/mPEG-Chol/F-PEG-suc-Chol (50:45:5:0.1, molar ratio) had the highest transfection efficiency. The transfection activity of F-targeted lipoplexes could be competitively inhibited by free folic acid, demonstrating that folate-FRalpha interaction caused high transfection efficiency of F-targeted lipoplexes. The uptake mechanism of F-targeted lipoplexes was further validated on human oral carcinoma cell line KB and human liver carcinoma cell line HepG2. The concentration-dependent and time-dependent cytotoxicity of targeted material F-PEG-suc-Chol was observed by MTT assay on SKOV-3 cell and its application would not increase the cytotoxicity of F-targeted lipoplexes in SKOV-3 cells. All the data indicated that F-PEG-CLPs would be a promising gene vector targeting for ovarian cancer therapy.
叶酸受体 alpha（FRalpha）在卵巢癌细胞上过度表达，是卵巢癌基因治疗的有前途的分子靶标，但仍没有相关报道。本研究首次开发了用于卵巢癌基因传递的叶酸修饰阳离子脂质体（F-PEG-CLPs）。首先合成了叶酸-聚乙二醇-琥珀酸-胆固醇（F-PEG-suc-Chol），然后用其制备叶酸靶向阳离子脂质体/质粒 DNA 复合物（F-靶向脂质体）。F-靶向脂质体通过后插入法制备，通过透射电子显微镜观察显示出膜结构，直径为 193nm-200nm，zeta 电位为 35mV-38mV。DNase 降解实验表明，F-靶向脂质体可以有效地在体外屏蔽质粒 DNA。F-靶向脂质体可以将基因转染入人卵巢癌细胞系 SKOV-3 中，由 DOTAP/Chol/mPEG-Chol/F-PEG-suc-Chol（50:45:5:0.1，摩尔比）组成的 0.1%F-PEG-CLPs 具有最高的转染效率。F-靶向脂质体的转染活性可被游离叶酸竞争性抑制，表明叶酸-FRalpha 相互作用导致 F-靶向脂质体的高转染效率。进一步在人口腔癌细胞系 KB 和人肝癌细胞系 HepG2 上验证了 F-靶向脂质体的摄取机制。通过 MTT 法在 SKOV-3 细胞上观察到靶向材料 F-PEG-suc-Chol 的浓度依赖性和时间依赖性细胞毒性，其应用不会增加 F-靶向脂质体在 SKOV-3 细胞中的细胞毒性。所有数据表明，F-PEG-CLPs 将是一种有前途的卵巢癌治疗靶向基因载体。
