Chen Jiji, Tian Buning, Yin Xiang, Zhang Yanqiong, Hu Duosha, Hu Zhiyuan, Liu Meizhou, Pan Yifeng, Zhao Jinfeng, Li Hao, Hou Can, Wang Jiwei, Zhang Yangde
National Key Laboratory of Nanobiological Technology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, PR China.
J Biotechnol. 2007 Jun 15;130(2):107-13. doi: 10.1016/j.jbiotec.2007.02.007. Epub 2007 Feb 17.
The cationic polylactic acid (PLA) nanoparticle has emerged as a promising non-viral vector for gene delivery because of its biocompatibility and biodegradability. However, they are not capable of prolonging gene transfer and high transfection efficiency. In order to achieve prolonged delivery of cationic PLA/DNA complexes and higher transfection efficiency, in this study, we used copolymer methoxypolyethyleneglycol-PLA (MePEG-PLA), PLA and chitosan (CS) to prepare MePEG-PLA-CS NPs and PLA-CS NPs by a diafiltration method and prepared NPs/DNA complexes through the complex coacervation of nanoparticles with the pDNA. The object of our work is to evaluate the characterization and transfection efficiency of MePEG-PLA-CS versus PLA-CS NPs. The MePEG-PLA-CS NPs have a zeta potential of 15.7 mV at pH 7.4 and size under 100 nm, while the zeta potential of PLA-CS NPs was only 4.5 mV at pH 7.4. Electrophoretic analysis suggested that both MePEG-PLA-CS NPs and PLA-CS NPs with positive charges could protect the DNA from nuclease degradation and cell viability assay showed MePEG-PLA-CS NPs exhibit a low cytotoxicity to normal human liver cells. The potential of PLA-CS NPs and MePEG-PLA-CS NPs as a non-viral gene delivery vector to transfer exogenous gene in vitro and in vivo were examined. The pDNA being carried by MePEG-PLA-CS NPs, PLA-CS NPs and lipofectamine could enter and express in COS7 cells. However, the transfection efficiency of MePEG-PLA-CS/DNA complexes was better than PLA-CS/DNA and lipofectamine/DNA complexes by inversion fluorescence microscope and flow cytometry. It was distinctively to find that the transfection activity of PEGylation of complexes was improved. The nanoparticles were also tested for their ability to transport across the gastrointestinal mucosa in vivo in mice. In vivo experiments showed obviously that MePEG-PLA-CS/DNA complexes mediated higher gene expression in stomach and intestine of BALB/C mice compared to PLA-CS/DNA and lipofectamine/DNA complexes. These results suggested that MePEG-PLA-CS NPs have favorable properties for non-viral gene delivery.
阳离子聚乳酸（PLA）纳米颗粒因其生物相容性和可生物降解性，已成为一种很有前景的非病毒基因递送载体。然而，它们无法延长基因传递时间并实现高转染效率。为了实现阳离子PLA/DNA复合物的长效递送并提高转染效率，在本研究中，我们使用甲氧基聚乙二醇 - 聚乳酸共聚物（MePEG - PLA）、聚乳酸（PLA）和壳聚糖（CS），通过透析过滤法制备了MePEG - PLA - CS纳米颗粒和PLA - CS纳米颗粒，并通过纳米颗粒与质粒DNA（pDNA）的复合凝聚制备了纳米颗粒/DNA复合物。我们工作的目的是评估MePEG - PLA - CS与PLA - CS纳米颗粒的特性及转染效率。MePEG - PLA - CS纳米颗粒在pH 7.4时的zeta电位为15.7 mV，粒径小于100 nm，而PLA - CS纳米颗粒在pH 7.4时的zeta电位仅为4.5 mV。电泳分析表明，带正电荷的MePEG - PLA - CS纳米颗粒和PLA - CS纳米颗粒均可保护DNA免受核酸酶降解，细胞活力测定显示MePEG - PLA - CS纳米颗粒对正常人肝细胞的细胞毒性较低。研究了PLA - CS纳米颗粒和MePEG - PLA - CS纳米颗粒作为非病毒基因递送载体在体外和体内转移外源基因的潜力。由MePEG - PLA - CS纳米颗粒、PLA - CS纳米颗粒和脂质体携带的pDNA可进入COS7细胞并在其中表达。然而，通过倒置荧光显微镜和流式细胞术检测发现，MePEG - PLA - CS/DNA复合物的转染效率优于PLA - CS/DNA和脂质体/DNA复合物。明显可以发现复合物聚乙二醇化后的转染活性得到了提高。还测试了这些纳米颗粒在小鼠体内跨胃肠道黏膜的转运能力。体内实验明显显示，与PLA - CS/DNA和脂质体/DNA复合物相比，MePEG - PLA - CS/DNA复合物在BALB/C小鼠的胃和肠道中介导了更高的基因表达。这些结果表明MePEG - PLA - CS纳米颗粒具有用于非病毒基因递送的良好特性。
