共载TFPI-2及顺铂磁性纳米复合物的制备及其对鼻咽癌的治疗

The Preparation of a Novel Magnetic Nanoparticles Carrying TFPI-2 and Cisplatin and Its Evaluation of Inhibitory Effect on Nasopharyngeal Carcinoma In Vitro

构建共载组织途径抑制因子-2(tissue factor pathway inhibitor-2,TFPI-2)及顺铂(cisplatin,CDDP)的磁性纳米(magnetic nanoparticles,MNP)复合物(MNP-CDDP/TFPI-2),研究该复合物对鼻咽癌(nasopharyngeal carcinoma,NPC)的综合抑制效应。将前期制备的载CDDP磁性纳米颗粒(MNP-CDDP)和聚乙二醇单甲醚–聚乙烯亚胺(MPEG-PEI)TFPI-2静电吸附,制备MNP-CDDP/TFPI-2,通过红外光谱、透射电镜、激光粒度仪观察复合物的结构、粒径大小及电位,邻苯二胺(o-phenylenediamine,OPDA)法检测复合物中CDDP含量。通过流式细胞仪检测基因转染效率,分析磁性纳米复合物转染TFPI-2的情况;RT-PCR及Western blot法检测CNE-2细胞转染TFPI-2质粒后TFPI-2 m RNA和蛋白表达情况。采用CCK-8实验、流式细胞术及Matrigel侵袭实验检测MNPCDDP/TFPI-2对CNE-2细胞增殖、凋亡及细胞侵袭能力的影响。研究结果显示,纳米复合物成功合成,平均水动力学粒径151.2 nm,Zeta电位+14.5 m V,复合物中CDDP含量平均为120μg/m L,包封率33.3%。复合物所载TFPI-2质粒转染率22.7%±2.5%。CNE-2转染TFPI-2质粒后,胞内TFPI-2 m RNA和蛋白表达明显增加。MNP-CDDP/TFPI-2组细胞生长抑制率及凋亡率分别为34.8%和42.3%,明显高于MNP-CDDP组(27.1%、38.0%)和(MPEG-PEI)TFPI-2组(18.9%、16.2%)(P<0.05)。Matrigel侵袭实验结果显示,纳米复合药物组穿膜细胞数为28±3个,明显低于对照组(P<0.05),提示MNP-CDDP/TFPI-2组细胞侵袭和迁移能力降低。结果表明,本研究已成功构建携带TFPI-2表达基因及CDDP的MNP-CDDP/TFPI-2;体外实验显示,MNP-CDDP/TFPI-2对NPC细胞具有良好的综合抑制效应。

MNP-CDDP/TFPI-2 was prepared to construct a novel multifunctional magnetic nanocarrier loading tissue factor pathway inhibitor-2（TFPI-2） and cisplatin（CDDP）. We evaluated comprehensive inhibitory effect of this nanocomposites on nasopharyngeal carcinoma（NPC）. MNP-CDDP/TFPI-2 was constructed with magnetic nanoparticles-cisplatin（MNP-CDDP） and（MPEG-PEI）TFPI-2. The CDDP concentration, morphology, particle size and zeta potential of nanocomposites were measured by OPDA（o-phenylenediamine） colourimetry, Fourier transform infrared spectrum（FT-IR）, transmission electron microscope（TEM） and laser particle detector, respectively. Flow cytometry was used to analyze the gene transfection efficiency of magnetic nanocomposites. TFPI-2 m RNA and protein expression levels were evaluated by RT-PCR and Western blot. Proliferation rate, cell apoptosis and invasion ability of CNE-2 cells cultured with MNP-CDDP/TFPI-2 were measured by CCK-8, fl ow cytometry and matrigel invasion test. The results showed that nanocomposites were successfully synthesized, with the average particle size 151.2 nm, Zeta potential ＋14.5 m V. OPDA showed that the CDDP concentration of nanocomposites was 120 μg/m L and the encapsulation efficiency of CDDP was 33.3%. The transfection efficiency of TFPI-2 in the nanocomposites was 22.7%±2.5%. The m RNA and protein expression levels of TFPI-2 in CNE-2 cells of MNP-CDDP/TFPI-2 group were significantly increased compared with those of the control group. The rate of growth inhibition and cell apoptosis were 34.8% and 42.3%, whereas that in MNP-CDDP group were 27.1% and 38.0%, in（MPEG-PEI）TFPI-2 group were 18.9% and 16.2%, respectively（P〈0.05）. Matrigel invasion assay showed that the number of transmembrane cells was 28±3 in MNP-CDDP/TFPI-2 group, which was lower than that in the control group（P〈0.05）. MNP-CDDP/TFPI-2 carrying cisplatin and TFPI-2 was successfully constructed and proved to be significantly inhibitory effect on the growth of CNE-2 cells.