Efficient gene transfer using reversibly cross-linked low molecular weight polyethylenimine
Efficient gene transfer using reversibly cross-linked low molecular weight polyethylenimine
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DOI：
10.1021/bc0100455
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发表时间：
2001-11-01
影响因子：
4.7
通讯作者：
Lee, RJ
中科院分区：
文献类型：
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作者：
Gosselin, MA;Guo, WJ;Lee, RJ
聚乙烯亚胺(PEI)是一种具有潜在应用前景的阳离子非病毒载体。在这里，我们证明了在与同双功能胺反应性可还原交联剂偶联后，低分子聚乙烯亚胺有效地介导了体外基因输送到中国仓鼠卵巢(CHO)细胞。根据两种交联剂的反应性和化学性质，选择了二硫代二丁二胺基丙酸酯(DSP)和二甲基-3，3‘-二硫代双丙酰亚胺-2HCI(DTBP)作为交联剂。这两种试剂都与伯胺反应形成可还原的交联键；然而，与DSP不同的是，DTBP交联剂通过酰胺键的形成来保持聚合物的净电荷。将重均相对分子质量(M-w)为800Da的PEI与DSP或DTBP在PEI伯胺：交联反应基团比例分别为1：1和2：1的条件下反应，用荧光素酶报告基因在巨细胞病毒(CMV)启动子的作用下评价其在CHO细胞中的转染率。我们的结果表明，根据交联剂、偶联程度和NIP比率的不同，交联聚合物可以调节不同水平的转染率。总的来说，我们发现共轭大小与基因转移效率成正比。通过凝胶滞留分析，我们还评估了用45 mM二硫苏糖醇(DTT)还原前后的交联物缩合DNA的能力。DTT以1：1的比例与数字信号处理器(DSP)进行交联物交联物的二硫键还原，抑制DNA缩合反应，但对其余聚合物影响不大。用还原型谷胱甘肽进行类似的细胞内还原可以促进PEI与DNA的解偶联，从而增强基因的表达。
Polyethylenimine (PEI) is a polycation with potential application as a nonviral vector for gene delivery. Here we show that after conjugation with homobifunctional amine reactive reducible cross-linking reagents, low molecular weight polyethylenimine efficiently mediates in vitro gene delivery to Chinese hamster ovary (CHO) cells. Two cross-linking reagents, dithiobis(succinimidylpropionate) (DSP) and dimethyl-3,3'-dithiobispropionimidate-2HCI (DTBP), were utilized based on their reactivity and chemical properties. Both reagents react with primary amines to form reducible cross-links; however, unlike DSP, the DTBP cross-linker maintains net polymer charge through amidine bond formation. PEI with a reported weight-average molecular weight (M-w) of 800 Da was reacted with either DSP or DTBP at PEI primary amine:cross-link reactive group ratios of 1:1 and 2:1. The transfection efficiencies of the resulting cross-linked products were evaluated in CHO cells using a luciferase reporter gene under a cytomegalovirus (CMV) promoter. Our results show that cross-linked polymers mediate variable levels of transfection depending on the cross-linking reagent, the extent of conjugation, and the NIP ratio. In general, we found conjugate size to be proportional to gene transfer efficiency. Using gel retardation analysis, we also evaluate the capacity of the cross-linked polymers to condense plasmid DNA before and after reduction with 45 mM dithiothreitol (DTT). DTT mediated reduction of intracross-link disulfide bonds and inhibited condensation of DNA by conjugates cross-linked with DSP at a ratio of 1:1, but had little effect on the remaining polymers. Analogous intracellular reduction of transfection complexes by reduced glutathione could facilitate uncoupling of PEI from DNA to enhance gene expression.