Department of Chemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong.
J Control Release. 2011 May 30;152(1):143-51. doi: 10.1016/j.jconrel.2011.03.020. Epub 2011 Mar 30.
Our revisit of the complexation between DNA and polyethylenimine (PEI) by using a combination of laser light scattering and gel electrophoresis confirms that nearly all the DNA chains are complexed with PEI to form polyplexes when the molar ratio of nitrogen from PEI to phosphate from DNA (N:P) reaches 3, irrespective of the PEI chain length and solvent. Each solution mixture with N:P>3 contains two kinds of PEI chains: bound to DNA and free in the solution. It has been shown that it is those free PEI chains that play a vital role in promoting the gene transfection. The effects of the length of the bound and free chains on the gene transfection were respectively studied. Both short and long PEI chains are capable of condensing DNA completely at N:P3 but long ones are ~10²-fold more effective in the gene transfection, apparently due to their fast endocytosis and intracellular trafficking. The cellular uptake kinetics studied by flow cytometry reveals that long free chains increase the uptake rate constant of the DNA/PEI complexes. In the intracellular pathway, they are able to prevent the development of the later endolysosomes, and facilitate the subsequent release of the polyplexes from the endosomes. Our result shows that the "proton sponge" effect is not dominant because the shut-down of the proton pump only partially attenuates the transfection efficiency. A possible mechanism is speculated and presented.
我们通过激光光散射和凝胶电泳的组合，重新研究了 DNA 与聚乙烯亚胺 (PEI) 的复合物形成，结果证实，当 PEI 的氮与 DNA 的磷酸酯摩尔比（N:P）达到3 时，几乎所有的 DNA 链都与 PEI 复合形成聚阳离子复合物，而与 PEI 链长和溶剂无关。N:P>3 的每个溶液混合物都包含两种 PEI 链：与 DNA 结合的和游离在溶液中的。已经表明，正是这些游离的 PEI 链在促进基因转染中起着至关重要的作用。分别研究了结合链和游离链的长度对基因转染的影响。短链和长链 PEI 都能在 N:P3 时完全缩合 DNA，但长链在基因转染中的效率高约 10²倍，显然是由于它们的快速内吞作用和细胞内转运。通过流式细胞术研究细胞摄取动力学表明，长的游离链增加了 DNA/PEI 复合物的摄取速率常数。在细胞内途径中，它们能够防止晚期内溶酶体的形成，并促进随后从内体中释放出多聚物。我们的结果表明，“质子海绵”效应并不占主导地位，因为质子泵的关闭只是部分削弱了转染效率。推测并提出了一种可能的机制。
