Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer
Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer
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DOI：
10.1002/jgm.598
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发表时间：
2004-10-01
影响因子：
3.5
通讯作者：
Ogris, M
中科院分区：
文献类型：
--
作者：
Boeckle, S;von Gersdorff, K;Ogris, M
基于聚乙烯亚胺（PEI）的非病毒载体通常含有过量的不与DNA络合的PEI。由于未结合PEI有助于细胞和系统毒性，从未结合PEI中纯化多聚物是可取的。方法采用粒径排除色谱法（SEC）纯化游离PEI多聚体。在小鼠体内系统应用后，研究了纯化多聚体的转染特性和游离PEI对基因传递的影响。结果SEC未改变复合体的大小和ζ电位。无论用于络合物形成的PEI用量如何，纯化的PEI多聚物最终PEI氮/DNA磷酸盐比均为2.5。值得注意的是，纯化的PEI多聚物显示出较低的细胞和全身毒性。纯化的多聚体在高DNA浓度（8-15马克杯/毫升）下获得了高转染效率。在低DNA浓度（2-4杯/毫升）下，纯化颗粒的基因转移效率低于含有游离PEI的多聚体。机制研究表明，游离PEI部分阻断了DNA复合物的细胞结合，但对随后的细胞内基因传递至关重要。将游离PEI添加到经过纯化颗粒处理的细胞中，延迟达4小时，与未纯化颗粒或没有游离PEI的纯化颗粒相比，转染效率显著提高。本研究提出了一种通过SEC从PEI多丛中去除游离PEI的有效方法。我们的转染实验结果表明，游离PEI在很大程度上有助于高效的基因表达，但也以剂量依赖的方式介导毒性作用。没有游离PEI的纯化多聚物必须在增加的浓度下应用以达到高转染水平，但表现出极大改善的毒性特征。版权所有：John Wiley Sons， Ltd. 2004
Background Nonviral vectors based on polyethylenimine (PEI) usually contain an excess of PEI that is not complexed to DNA. Since unbound PEI contributes to cellular and systemic toxicity, purification of polyplexes from unbound PEI is desirable.Methods Size exclusion chromatography (SEC) was used to purify PEI polyplexes of free PEI. Transfection properties of purified polyplexes and the effect of free PEI on gene delivery were studied in vitro and in vivo after systemic application into mice.Results SEC did not change the size and zeta-potential of polyplexes. Independent of the amount of PEI used for complex formation, purified PEI polyplexes had the same final PEI nitrogen/DNA phosphate ratio of 2.5. Notably, purified PEI polyplexes demonstrated low cellular and systemic toxicity. High transfection efficiency was achieved with purified polyplexes at high DNA concentrations (8-15 mug/ml). At low DNA concentrations (2-4 mug/ml) gene transfer with purified particles was less efficient than with polyplexes containing free PEI both in vitro and in vivo. Mechanistic studies showed that free PEI partly blocked cellular association of DNA complexes but was essential for the following intracellular gene delivery. Adding free PEI to cells treated with purified particles with a delay of up to 4 h resulted in significantly enhanced transfection efficiency compared with non-purified particles or purified particles without free PEI.Conclusions This study presents an efficient method to remove free PEI from PEI polyplexes by SEC. Our results from transfection experiments demonstrate that free PEI substantially contributes to efficient gene expression but also mediates toxic effects in a dose-dependent manner. Purified polyplexes without free PEI have to be applied at increased concentrations to achieve high transfection levels, but exhibit a greatly improved toxicity profile. Copyright (C) 2004 John Wiley Sons, Ltd.