Identification of novel superior polycationic vectors for gene delivery by high-throughput synthesis and screening of a combinatorial library
Identification of novel superior polycationic vectors for gene delivery by high-throughput synthesis and screening of a combinatorial library
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DOI：
10.1007/s11095-007-9279-3
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发表时间：
2007-08-01
影响因子：
3.7
通讯作者：
Klibanov, Alexander M.
中科院分区：
文献类型：
--
作者：
Thomas, Mini;Lu, James J.;Klibanov, Alexander M.
目的。效率低和毒性是当前非病毒基因递送载体的两个主要缺点。由于将 DNA 递送至哺乳动物细胞是一个多步骤过程，因此采用高通量合成和筛选方法生成和搜索载体组合文库对于开发新的改进载体来说是一种有吸引力的策略，因为它增加了识别最全面优化载体的机会。材料和方法。与较高分子量同系物相比，小 PEIs 是较差的载体，但毒性较小，增加小 PEIs 的有效分子量，由于更好的 DNA 结合而提高其转染效率，基于这一原理，我们从两个小 PEI 和 24 个双丙烯酸酯和低聚丙烯酸酯合成了 144 种可生物降解衍生物的文库。 423-Da 线性 PEI 及其与 1.8-kDa 支化 PEI 的 1:1 (w/w) 混合物与丙烯酸酯以三种摩尔比在 DMSO 中交联。筛选所得聚合物向 COS-7 猴肾细胞递送 β-半乳糖苷酶表达质粒的效率。从初始筛选中选择的最有效的聚合物在 A549 人肺癌细胞中进行了毒性测试，并在体内采用萤火虫荧光素酶表达质粒在小鼠的全身基因传递模型中进行了毒性测试。结果。从文库中鉴定出几种在体外表现出高效力和低毒性的聚阳离子。线性 423-Da PEI 最有效的衍生物是与三环[5.2.1.0]-癸烷-二甲醇二丙烯酸酯（二丙烯酸酯 14）交联，其效率比母体提高了 3,600 倍以上。最有效的混合 PEI 是用乙二醇二丙烯酸酯（二丙烯酸酯 4）交联的，其效率比物理混合母体 PEI 高 850 倍以上。这些聚合物的相对效率甚至高达线性 22-kDa PEI 的两倍多，被认为是体外和全身基因传递的“金标准”。所鉴定的有效交联聚阳离子的毒性也低于 22-kDa PEI。体内最佳载体为丙二醇甘油酯二丙烯酸酯（二丙烯酸酯7）交联的混合PEI；它在肺中介导的基因表达量最高，其次是脾脏，前者的表达量比后者高53倍。相反，亲本PEI在类似条件下根本不介导基因表达，并且注射在相同条件下制备的最佳N/P为10的22-kDa PEI的聚合复合物杀死了一半的注射小鼠。结论。生物可降解 PEI 交联文库的高通量合成和转染测定已被证明可有效识别高转染载体。与它们的亲本相比，所鉴定的载体在体外和体内系统基因传递模型中都表现出显着优越的效率。这些载体中的大多数介导优先将基因递送至肺部，并且它们的体内毒性与体外相似。
Purpose. Low efficiency and toxicity are two major drawbacks of current non-viral gene delivery vectors. Since DNA delivery to mammalian cells is a multi-step process, generating and searching combinatorial libraries of vectors employing high-throughput synthesis and screening methods is an attractive strategy for the development of new improved vectors because it increases the chance of identifying the most overall optimized vectors.Materials and Methods. Based on the rationale that increasing the effective molecular weight of small PEIs, which are poor vectors compared to the higher molecular weight homologues but less toxic, raises their transfection efficiency due to better DNA binding, we synthesized a library of 144 biodegradable derivatives from two small PEIs and 24 bi- and oligo-acrylate esters. A 423-Da linear PEI and its 1: 1 (w/w) mixture with a 1.8-kDa branched PEI were cross-linked with the acrylates at three molar ratios in DMSO. The resulting polymers were screened for their efficiency in delivering a beta-galactosidase expressing plasmid to COS-7 monkey kidney cells. Selected most potent polymers from the initial screen were tested for toxicity in A549 human lung cancer cells, and in vivo in a systemic gene delivery model in mice employing a firefly luciferase expressing plasmid.Results. Several polycations that exhibited high potency and low toxicity in vitro were identified from the library. The most potent derivative of the linear 423-Da PEI was that cross-linked with tricycle[5.2.1.0]-decane- dimethanol diacrylate (diacrylate 14), which exhibited an over 3,600-fold enhancement in efficiency over the parent. The most potent mixed PEI was that cross-linked with ethylene glycol diacrylate (diacrylate 4) which was over 850-fold more efficient than the physically mixed parent PEIs. The relative efficiencies of these polymers were even up to over twice as high as that of the linear 22-kDa PEI, considered the "gold standard" for in vitro and systemic gene delivery. The potent cross-linked polycations identified were also less toxic than the 22-kDa PEI. The optimal vector in vivo was the mixed PEI cross-linked with propylene glycol glycerolate diacrylate ( diacrylate 7); it mediated the highest gene expression in the lungs, followed by the spleen, with the expression in the former being 53-fold higher compared to the latter. In contrast, the parent PEIs mediated no gene expression at all under similar conditions, and injection of the polyplexes of the 22-kDa PEI at its optimal N/P of 10 prepared under identical conditions killed half of the mice injected.Conclusions. High-throughput synthesis and transfection assay of a cross-linked library of biodegradable PEIs was proven effective in identifying highly transfecting vectors. The identified vectors exhibited dramatically superior efficiency compared to their parents both in vitro and in an in vivo systemic gene delivery model. The majority of these vectors mediated preferential gene delivery to the lung, and their in vivo toxicity paralleled that in vitro.