叶酸靶向共载TFPI-2及顺铂磁性纳米复合物的制备及其对鼻咽癌HNE-1细胞的靶向性和体外抑制效应

Preparation of folate-targeted magnetic nanocomposites loaded with TFPI-2 plasmid and cisplatin and evaluation of its targeting and inhibitory effect on nasopharyngeal carcinoma HNE-1 cells in vitro

目的 制备叶酸靶向共载组织因子途径抑制物2（TFPI-2）及顺铂磁性纳米复合物,探讨其对鼻咽癌HNE-1细胞的靶向性和体外抑制效应.方法 通过酰胺化反应合成叶酸靶向高聚物叶酸-聚乙二醇-聚乙烯亚胺（FA-PEG-PEI）,采用静电吸附技术,将其与本课题组前期制备的载顺铂磁纳米粒（MNP-CDDP）及TFPI-2质粒混合,得到终产物FA-PEG-PEI/MNP-CDDP/TFPI-2.核磁氢谱验证高分子聚合物的合成.动态光散射（DLS）及透射电镜（TEM）观测其粒径、电荷及形态.邻二氮菲法及邻苯二胺法测定纳米复合物中铁及顺铂（CDDP）的含量.琼脂糖凝胶电泳分析载体FA-PEG-PEI/MNP-CDDP与TFPI-2质粒的结合情况.普鲁士蓝铁染色法及荧光显微镜观察磁性纳米复合物对鼻咽癌（NPC）细胞的分子靶向性.Western印迹检测磁性纳米复合物转染HNE-1细胞后TFPI-2蛋白的表达情况.Cell Counting Kit-8（CCK-8）细胞计数试剂盒、流式细胞技术检测复合物对HNE-1细胞增殖、凋亡的影响.结果 核磁氢谱在FA、PEG和PEI相应的频率上可见特征吸收峰.DLS测得产物FA-PEG-PEI/MNP-CDDP/TFPI-2复合物平均粒径为141.1 nm,平均电荷为21.5 mV.透射电镜下可见纳米微粒有良好的分散性,但粒径大小不太均匀.铁含量与顺铂载药量分别为116.2 μg/ml及92.88μg/ml.琼脂糖凝胶电泳显示,当载体FA-PEG-PEI/MNP-CDDP与TFPI-2质粒质量比≥1时,载体能完全吸附质粒,并且有效保护质粒免受DNA酶的消化作用.铁染色和荧光显微镜观测可见叶酸受体（FR）阳性HNE-1细胞内蓝染磁颗粒及绿色荧光点比FR阴性CNE-2细胞显著增多（P＜0.05）.HNE-1细胞被载TFPI-2质粒的复合物转染后,TFPI-2蛋白的表达量比空白载体组及单纯质粒组明显增高（P＜0.05）.复合物对HNE-1细胞有明显的体外抑制效应,抑制率及凋亡率分别为64.00％及49.61％,明显高于载体组（8.19％,9.26％）、单载质粒组（40.35％,19.85％）、单载顺铂组（56.15％,36.46％）,均P＜0.05.结论 采用酰胺化反应和静电吸附技术成功合成了FA-PEG-PEI/MNP-CDDP/TFPI-2磁性纳米复合物,该复合物对鼻咽癌HNE-1细胞具有良好的靶向性及体外抑制效应.

Objective To prepare a novel folate-targeted magnetic nanocomposites loaded with tissue facor pathway inhibitor 2 （TFPI-2） and cisplatin （CDDP） and to investigate its targeting ability and anti-tumor effect on nasopharyngeal carcinoma HNE-1 cells in vitro.Methods The copolymer folic acidpolyethylene glycol-polyethyleneimine （FA-PEG-PEI） was synthesized through amidation reaction,and then FA-PEG-PEI/ magnetic nanoparticles-CDDP/TFPI-2 （MNP-CDDP/TFPI-2） nanocomposites was obtained by electrostatic adsorption between TFPI-2 plasmid and magnetic nanoparticles loaded with CDDP （MNP-CDDP） with vortex FA-PEG-PEI.1H Nuclear Magnetic Resonance （1H NMR） was used to determine if FA-PEG-PEI was synthesized.The particle size,zeta potential and morphology were detected by dynamic light scattering （DLS） and transmission electron microscope （TEM）.The content of Fe and CDDP was measured by phenanthroline and o-phenylenediamine （OPDA） colourimetry.Agarose gel electrophoresis was used to analyze the binding ability of FA-PEG-PEI/MNP-CDDP to TFPI-2 plasmid.Molecular targeted uptake of FA-PEG-PEI/MNP-CDDP/TFPI-2 coupling with green fluorescent protein （GFP） in NPC cells were observed by Prussian-blue iron staining and fluorescence microscope.The levels of TFPI-2 protein expression after transfection were evaluated by Western blot.The effects of nanocomposites on HNE-1 cells proliferation and apoptosis were measured with Cell Counting Kit-8 （CCK-8） and flow cytometry.Results Special peak value of FA,PEG and PEI were showed on 1H NMR spectrogram.The mean size and zeta potential of FA-PEG-PEI/MNP-CDDP/TFPI-2 were 141.1 nm and 21.5 mV.The nanocomposites showed a good monodispersity and an insufficient size uniformity under TEM.The content of Fe and CDDP were 116.2 μg/ml and 92.88 μg/ml,respectively.Agarose gel electrophoresis showed TFPI-2 could be encapsulated completely and protected from digestion of DNA enzyme as the mass ratio of FA-PEG-PEI/MNP-CDDP and TFPI-2 plasmid was equal or higher than 1：1.More blue-stained magnetic granulars and green fluorescence were seen in folate receptor （FR）-positive HNE-1 cells than in FR-negative CNE-2 （P 〈0.05） under microscope and fluorescence microscope.The level of TFPI-2 protein expression in HNE-1cells increased significantly after transfection by FA-PEG-PEI/MNP-CDDP/TFPI-2,compared with other control groups （FA-PEG-PEI/MNP-CDDP group and TFPI-2 group）,all P 〈 0.05.The nanocomposites inhibitory effect on HNE-1 including cell growth inhibition rate （64.00%） and apoptosis rate （49.61%） were significantly higher than that in FA-PEG-PEI/MNP group （8.19%,9.26%）,FA-PEG-PEI/TFPI-2 group （40.35%,19.85%） and FA-PEG-PEI/MNP-CDDP group（56.15%,36.46%） （P 〈 0.05）.Conclusion FA-PEG-PEI/MNP-CDDP/TFPI-2 nanocomposites was successfully synthesized using amidation and electrostatic adsorption technology and has a good molecular targeting and inhibitory effect on FR-positive HNE-1cells in vitro.