Cross-linked small polyethylenimines: while still nontoxic, deliver DNA efficiently to mammalian cells in vitro and in vivo.
Cross-linked small polyethylenimines: while still nontoxic, deliver DNA efficiently to mammalian cells in vitro and in vivo.
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交联的小聚乙基亚胺：虽然仍然无毒，但在体外和体内有效地递送DNA。
DOI：
10.1007/s11095-004-1874-y
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发表时间：
2005-03
影响因子：
3.7
通讯作者：
Klibanov AM
中科院分区：
文献类型：
--
作者：
Thomas M;Ge Q;Lu JJ;Chen J;Klibanov AM
聚乙烯亚胺（PEI）是最有效的非病毒基因传递载体之一。其效率和细胞毒性取决于分子量，其中25 kda的PEI效率最高，但具有细胞毒性。较小的PEIs没有细胞毒性，但效率较低。本文探讨了通过潜在可生物降解的键交联小PEIs以最小细胞毒性提高基因传递效率的方法。假设交联会提高多阳离子的有效分子量，从而提高转染效率，而可生物降解的键在DNA传递后会在细胞内分解，因此不会导致细胞毒性。为了实现这一目标，我们通过酯和/或酰胺键将支化的2-kDa PEI及其1:1 （w/w）混合物与线性423-Da PEI进行交联；研究了交联多阳离子在体内和体外的基因传递效率，以及对哺乳动物细胞的毒性。在猴肾细胞（COS-7）中研究了交联PEIs体外传递β-半乳糖苷酶基因质粒的效率及其细胞毒性。采用动态光散射法比较了未修饰和交联pei的相对DNA凝聚效率。通过在小鼠气管内递送荧光素酶编码质粒和PEIs复合物并估计肺中荧光素酶的表达来评估体内基因递送效率。交联可使小型PEIs在体外的基因传递效率提高40- 550倍；最有效的共轭物的效率甚至超过了分支的25 kda PEI的一个数量级。交联PEIs配合物的平均直径约为300 nm，尺寸分布窄，而未修饰的小PEIs配合物的平均尺寸为100 ~ 700 nm，尺寸分布很宽，这表明了DNA的有效凝聚。在25 kda PEI导致95%细胞死亡的浓度下，偶联物提供了几乎完全的细胞活力。交联pei在体内的效率是未修饰pei的17 ~ 80倍；此外，它们的效率高达25 kda PEI的两倍。小的pei与精心设计的酰胺和酯连接体交联可以提高它们在体外和体内的基因传递效率，而不会增加细胞毒性。高效率取决于连杆的性质和所使用的pei。
Polyethylenimine (PEI) is among the most efficient nonviral gene delivery vectors. Its efficiency and cytotoxicity depend on molecular weight, with the 25-kDa PEI being most efficient but cytotoxic. Smaller PEIs are noncytotoxic but less efficient. Enhancement in gene delivery efficiency with minimal cytotoxicity by cross-linking of small PEIs via potentially biodegradable linkages was explored herein. The hypothesis was that cross-linking would raise the polycation’s effective molecular weight and hence the transfection efficiency, while biodegradable linkages would undergo the intracellular breakdown after DNA delivery and hence not lead to cytotoxicity. Toward this goal, we carried out cross-linking of branched 2-kDa PEI and its 1:1 (w/w) mixture with a linear 423-Da PEI via ester- and/or amide-bearing linkages; the in vitro and in vivo gene delivery efficiency, as well as toxicity to mammalian cells, of the resultant cross-linked polycations were investigated. The efficiency of the cross-linked PEIs in delivering in vitro a plasmid containing β-galactosidase gene and their cytotoxicity were investigated in monkey kidney cells (COS-7). Dynamic light scattering was used to compare the relative DNA condensation efficiency of the unmodified and cross-linked PEIs. In vivo gene delivery efficiency was evaluated by intratracheal delivery in mice of the complexes of a luciferase-encoding plasmid and the PEIs and estimating the luciferase expression in the lungs. Cross-linking boosted the gene delivery efficiency of the small PEIs by 40- to 550-fold in vitro; the efficiency of the most potent conjugates even exceeded by an order of magnitude that of the branched 25-kDa PEI. Effective condensation of DNA was evident from the fact that the mean diameter of the complexes of the cross-linked PEIs was some 300 nm with a narrow size distribution, while the complexes of the unmodified small PEIs exhibited a mean size of >700 nm with a very broad size distribution. At concentrations where the 25-kDa PEI resulted in >95% cell death, the conjugates afforded nearly full cell viability. The cross-linked PEIs were 17 to 80 times m ore efficient than the unmodified ones in vivo; furthermore, their efficiencies were up to twice that of the 25-kDa PEI. Cross-linking of small PEIs with judiciously designed amide- and ester-bearing linkers boosts their gene delivery efficiency both in vitro and in vivo without increasing the cytotoxicity. The high efficiency is dependent on the nature of the linkages and the PEIs used.
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