Department of Chemistry and Bioengineering, Tampere University of Technology, Tampere, Finland.
J Phys Chem B. 2011 Mar 3;115(8):1895-902. doi: 10.1021/jp109984c. Epub 2011 Feb 3.
Polyethylenimine (PEI) is a cationic DNA condensing polymer that facilitates gene transfer into the mammalian cells. The highest gene transfer with branched PEI is obtained at high nitrogen/phosphate (N/P) ratios with free PEI present. The small molecular weight PEI alone is not able to mediate DNA transfection. Here, we used recently developed time-resolved fluorescence spectroscopic method to study the mechanism of PEI-DNA complex formation and to investigate how free PEI, mean molecular weight, and branching of PEI affect the complexes. Analysis of fluorescence lifetimes and time-resolved spectra revealed that for both linear and branched high-molecular-weight PEI the complexation takes place in two steps, but the small-molecular-weight branched PEI complexed DNA at a single step. According to the binding constants obtained from time-resolved spectroscopic measurements, the affinity of N/P complexation per nitrogen atom is highest for LPEI and weakest for BPEI, whereas SPEI-DNA complexation showed intermediate values. Thus, the binding constant alone does not give adequate measure for transfection efficiency. On the other hand, the presence of intermediate states during the polyplex formation seems to be favorable for the gene transfection. Free PEI had no impact on the physical state of PEI-DNA complexes, even though it was essential for gene transfection in the cell culture. In conclusion, the molecular size and topology of PEI have direct influence on the DNA complexation but the free PEI does not. Free PEI must facilitate transfection at the cellular level and not via indirect effects on the PEI-DNA complexes.
聚亚乙基亚胺（PEI）是一种阳离子 DNA 凝聚聚合物，可促进基因转染进入哺乳动物细胞。带有支链的 PEI 的最高基因转染效率是在存在游离 PEI 的情况下通过高氮/磷（N/P）比值获得的。单独的小分子量 PEI 本身无法介导 DNA 转染。在这里，我们使用最近开发的时间分辨荧光光谱法来研究 PEI-DNA 复合物形成的机制，并研究游离 PEI、平均分子量和 PEI 的支化如何影响复合物。荧光寿命和时间分辨光谱的分析表明，对于线性和支化的高分子量 PEI，复合物的形成都分两步进行，但小分子量支化的 PEI 则在一步中与 DNA 结合。根据时间分辨光谱测量得到的结合常数，每氮原子的 N/P 络合亲和力对于 LPEI 最高，对于 BPEI 最低，而 SPEI-DNA 络合则表现出中间值。因此，结合常数本身并不能充分衡量转染效率。另一方面，在多聚物形成过程中存在中间态似乎有利于基因转染。游离的 PEI 对 PEI-DNA 复合物的物理状态没有影响，尽管它对细胞培养中的基因转染是必不可少的。总之，PEI 的分子大小和拓扑结构直接影响 DNA 络合，但游离的 PEI 没有影响。游离的 PEI 必须在细胞水平上促进转染，而不是通过对 PEI-DNA 复合物的间接影响。
