The Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London, UK.
J Biotechnol. 2011 Oct 10;155(4):377-86. doi: 10.1016/j.jbiotec.2011.07.023. Epub 2011 Jul 27.
The effect of DNA vector topology when complexed to poly-l-lysine (PLL) and its quantification in transfection efficiency has not been fully addressed even though it is thought to be of importance from both production and regulatory viewpoints. This study investigates and quantifies cell uptake followed by transfection efficiency of PLL:DNA complexes (polyplexes) in Chinese hamster ovary (CHO) cells and their dependence on DNA topology. PLL is known for its ability to condense DNA and serve as an effective gene delivery vehicle. Characterization of PLL conjugated to a 6.9kb plasmid was carried out. Dual labeling of both the plasmid DNA (pDNA) and PLL enabled quantitative tracking of the complexed as well as dissociated elements, within the cell, and their dependence on DNA topology. Polyplex uptake was quantified by confocal microscopy and image analysis. Supercoiled (SC) pDNA when complexed with PLL, forms a polyplex with a mean diameter of 139.06nm (±0.84% relative standard error [RSE]), whereas open circular (OC) and linear-pDNA counterparts displayed mean diameters of 305.54 (±3.2% RSE) and 841.5nm (±7.2% RSE) respectively. Complexes containing SC-pDNA were also more resistant to nuclease attack than its topological counterparts. Confocal microscope images reveal how the PLL and DNA remain bound post transfection. Quantification studies revealed that by 1h post transfection 61% of SC-pDNA polyplexes were identified to be associated with the nucleus, in comparison to OC- (24.3%) and linear-pDNA polyplexes (3.5%) respectively. SC-pDNA polyplexes displayed the greatest transfection efficiency of 41% which dwarfed that of linear-pDNA polyplexes of 18.6%. Collectively these findings emphasize the importance of pDNA topology when complexed with PLL for gene delivery with the SC-form being a key pre-requisite.
尽管从生产和监管的角度来看，DNA 载体拓扑结构与聚-L-赖氨酸（PLL）复合时的影响及其在转染效率中的定量尚未得到充分解决，但它被认为是很重要的。本研究调查并定量了 PLL：DNA 复合物（多聚物）在中华仓鼠卵巢（CHO）细胞中的细胞摄取及其转染效率，以及它们对 DNA 拓扑结构的依赖性。PLL 以其能够使 DNA 凝聚并作为有效的基因传递载体的能力而闻名。对与 6.9kb 质粒偶联的 PLL 进行了表征。同时对质粒 DNA（pDNA）和 PLL 进行双重标记，能够定量跟踪细胞内复合和游离的元素，以及它们对 DNA 拓扑结构的依赖性。通过共聚焦显微镜和图像分析来定量多聚物摄取。当与 PLL 复合时，超螺旋（SC）pDNA 形成的多聚物平均直径为 139.06nm（±0.84%相对标准误差[RSE]），而开环（OC）和线性-pDNA 对应的平均直径分别为 305.54nm（±3.2%RSE）和 841.5nm（±7.2%RSE）。含有 SC-pDNA 的复合物也比其拓扑结构对应物更能抵抗核酸酶的攻击。共聚焦显微镜图像揭示了 PLL 和 DNA 在转染后是如何保持结合的。定量研究表明，在转染后 1 小时，61%的 SC-pDNA 多聚物被鉴定与核相关，而 OC-（24.3%）和线性-pDNA 多聚物分别为 3.5%。SC-pDNA 多聚物显示出最大的转染效率为 41%，这大大超过了线性-pDNA 多聚物的 18.6%。这些发现共同强调了 DNA 拓扑结构在与 PLL 复合用于基因传递时的重要性，SC 形式是一个关键的先决条件。
