Kim J S, Maruyama A, Akaike T, Kim S W
Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City 84112, USA.
Pharm Res. 1998 Jan;15(1):116-21. doi: 10.1023/a:1011917224044.
To characterize the physical and biochemical properties of the DNA terplex delivery system, which has previously been shown to deliver and express pSV-beta-gal plasmid efficiently in cultured smooth muscle cells (SMC) (1).
Atomic force microscopy (AFM), zeta-potential measurement (ZP), gel electrophoresis (GE), circular dichroism (CD), fluorescence quenching and 1H-NMR spectrometry were used.
AFM showed that the plasmid DNA of about 600 nm long in its extended state was condensed to the size of about 100 nm by terplex formation. The DNA condensing effect of the terplex system was as good as unmodified PLL, as shown by an ethidium bromide displacement assay. Zeta-potential measurement showed that the terplex system exerts a slightly positive surface charge (+2 mV) at a 1:1:1 weight ratio of DNA:LDL:stearyl-PLL, which showed the best transfection efficiency on SMC. GE indicated that electrophoretic mobility of the terplex system decreased with increasing amounts of stearyl-PLL, indicating that the surface charge of the terplex system became more positive as more stearyl-PLL was added. Results from CD showed that there was no significant changes in tertiary structure of plasmid DNA from the terplex formation. Presence of strong hydrophobic interaction between stearyl-PLL and LDL was confirmed by 1H-NMR, where about a 30% decrease in epsilon-methylene peak of PLL backbone was observed when stearyl-PLL was mixed with LDL, but this phenomenon was not observed when unmodified PLL was used.
Our results indicate that the plasmid DNA, when formulated with the stearyl-PLL and LDL, forms a stable and hydrophobicity/charge balanced terplex system of optimal size for efficient cellular uptake and the DNA is still intact after the terplex formation. This information is expected to be utilized for the development of much improved transfection vectors for in vivo gene therapy.
表征DNA三元复合物递送系统的物理和生化特性，该系统先前已被证明能在培养的平滑肌细胞（SMC）中高效递送和表达pSV-β-半乳糖苷酶质粒（1）。
使用原子力显微镜（AFM）、ζ电位测量（ZP）、凝胶电泳（GE）、圆二色性（CD）、荧光猝灭和1H-核磁共振光谱法。
AFM显示，处于伸展状态时长约600 nm的质粒DNA通过形成三元复合物被浓缩至约100 nm大小。溴化乙锭置换试验表明，三元复合物系统的DNA凝聚效果与未修饰的聚赖氨酸（PLL）一样好。ζ电位测量显示，在DNA:低密度脂蛋白（LDL）:硬脂酰-PLL重量比为1:1:1时，三元复合物系统呈现轻微的正表面电荷（+2 mV），这在SMC上显示出最佳转染效率。GE表明，三元复合物系统的电泳迁移率随硬脂酰-PLL量的增加而降低，表明随着添加更多硬脂酰-PLL，三元复合物系统的表面电荷变得更正。CD结果显示，形成三元复合物后质粒DNA的三级结构没有显著变化。1H-核磁共振证实硬脂酰-PLL与LDL之间存在强疏水相互作用，当硬脂酰-PLL与LDL混合时，观察到PLL主链的ε-亚甲基峰下降约30%，但使用未修饰的PLL时未观察到这种现象。
我们的结果表明，质粒DNA与硬脂酰-PLL和LDL配制时，形成了一个稳定且疏水性/电荷平衡的最佳大小的三元复合物系统，有利于细胞有效摄取，并且形成三元复合物后DNA仍然完整。预计该信息将用于开发用于体内基因治疗的大大改进的转染载体。
