Delivery of Polyethylenimine/DNA Complexes Assembled in a Microfluidics Device
Delivery of Polyethylenimine/DNA Complexes Assembled in a Microfluidics Device
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DOI：
10.1021/mp900016q
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发表时间：
2009-09-01
影响因子：
4.9
通讯作者：
Lee, L. James
中科院分区：
文献类型：
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作者：
Koh, Chee Guan;Kang, Xihai;Lee, L. James
聚乙烯亚胺（PEI）和质粒DNA（pDNA）复合物（PEI/pDNA）是用于基因递送的非病毒载体。用于生产这些复合物的常规方法涉及PEI和DNA的大量混合（BM），然后涡旋，这在低N/P比下导致大的粒度分布、低细胞毒性和差的基因转染，而在高N/P比下，它导致小的粒度和更好的基因转染但高细胞毒性。为了改善尺寸控制、基因转染效率和细胞毒性，在本研究中，我们使用微流体流体动力聚焦（MF）装置来制备N/P = 3.3和6.7的PEI/pDNA复合物。以小鼠NIH 3 T3成纤维细胞和小鼠胚胎干细胞（mES）为模型细胞系，以编码绿色荧光蛋白（pGFP）和分泌型碱性磷酸酶（pSEAP）的质粒为报告基因，以市售Lipofectamine 2000为阳性对照。通过原子力显微镜（AFM）、动态光散射（DLS）和zeta电位（zeta）测量对配合物进行了表征。共聚焦激光扫描显微镜（CLSM）和荧光标记技术被用来可视化复杂的大小分布，络合均匀性，和细胞分布。结果表明，MF产生的复合物更小，更均匀的复合，并具有更高的细胞活力和提高外源基因的表达。
Polyethylenimine (PEI) and plasmid DNA (pDNA) complexes (PEI/pDNA) are nonviral vectors for gene delivery. The conventional method for producing these complexes involves bulk mixing (BM) of PEI and DNA followed by vortexing which at low N/P ratios results in large particle size distribution, low cytotoxicity, and poor gene transfection, while at high N/P ratios it results in small particle size and better gene transfection but high cytotoxicity. To improve size control, gene transfection efficiency, and cytotoxicity, in this study, we used a microfluidic hydrodynamic focusing (MF) device to prepare PEI/pDNA complexes at N/P = 3.3 and 6.7. We used bulk mixing as control, mouse NIH 3T3 fibroblast cells and mouse embryonic stem (mES) cells as model cell lines, plasmid encoding green fluorescent protein (pGFP) and secreted alkaline phosphatase (pSEAP) as the reporter gene, and commercially available Lipofectamine 2000 as a positive control. The complexes were characterized by atomic force microscopy (AFM), dynamic light scattering (DLS), and zeta potential (zeta) measurement. Confocal laser scanning microscopy (CLSM) and fluorescent labeling techniques were used to visualize the complex size distribution, complexation uniformity, and cellular distribution. The results showed that MF produced complexes were smaller and more uniformly complexed and had higher cell viability and improved exogenous gene expression.