POLYAMIDOAMINE CASCADE POLYMERS MEDIATE EFFICIENT TRANSFECTION OF CELLS IN CULTURE
POLYAMIDOAMINE CASCADE POLYMERS MEDIATE EFFICIENT TRANSFECTION OF CELLS IN CULTURE
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DOI：
10.1021/bc00023a012
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发表时间：
1993-09-01
影响因子：
4.7
通讯作者：
SZOKA, FC
中科院分区：
文献类型：
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作者：
HAENSLER, J;SZOKA, FC
级联聚合物也称为星爆树枝状聚合物，是球形聚阳离子，可以合成为每个树枝状聚合物具有明确的直径和精确数量的末端胺。我们证明，使用含有荧光素酶和β-半乳糖苷酶的质粒，树枝状聚合物介导多种悬浮和贴壁培养的哺乳动物细胞的高效转染。树枝状聚合物介导的转染是树枝状聚合物/DNA比率和树枝状聚合物直径的函数。使用 68 A 的直径和 6/1 的树枝状聚合物与 DNA 电荷比（末端胺与磷酸盐）获得荧光素酶的最大转染。表达不受氯喹等溶血营养剂的影响，并且仅受培养基中存在 10% 血清的适度影响（降低 2 倍）。通过染料还原测定评估，在不存在 DNA 的情况下，在 150 杯树枝状聚合物/mL 时，细胞活力仅降低 30%，而在存在 DNA 的情况下，细胞活力降低约 75%。在类似的条件下，聚赖氨酸会导致活力完全丧失。当电荷比降低至1:1时，基因表达量下降3个数量级。 当 GALA（一种水溶性膜不稳定肽）通过二硫键共价连接到树枝状聚合物时，1:1 复合物的转染效率提高了 2-3 个数量级。树枝状聚合物的高转染效率可能不仅归因于它们的直径和形状，还可能归因于聚合物中胺的 pK(a)（3.9 和 6.9）。低 pK(a) 允许树枝状聚合物缓冲内体室中的 pH 变化。结构精确控制、良好的pK(a)和低毒性等特点使得树枝状聚合物适合用作基因转移载体。
Cascade polymers also known as Starburst dendrimers are spheroidal polycations that can be synthesized with a well-defined diameter and a precise number of terminal amines per dendrimer. We show, using luciferase and beta-galactosidase containing plasmids, that dendrimers mediate high efficiency transfection of a variety of suspension and adherent cultured mammalian cells. Dendrimer-mediated transfection is a function both of the dendrimer/DNA ratio and the diameter of the dendrimer. Maximal transfection of luciferase are obtained using a diameter of 68 A and a dendrimer to DNA charge ratio of 6/1 (terminal amine to phosphate). Expression is unaffected by lysomotrophic agents such as chloroquine and only modestly affected (2-fold decrease) by the presence of 10% serum in the medium. Cell viability, as assessed by dye reduction assays, decreases by only 30% at 150 mug dendrimer/mL in the absence of DNA and about 75% in the presence of DNA. Under similar conditions polylysine causes a complete loss of viability. Gene expression decreased by 3 orders of magnitude when the charge ratio is reduced to 1:1. When GALA, a water soluble, membrane-destabilizing peptide, is covalently attached to the dendrimer via a disulfide linkage, transfection efficiency of the 1:1 complex is increased by 2-3 orders of magnitude. The high transfection efficiency of the dendrimers may not only be due to their diameter and shape but may also be caused by the pK(a)'s (3.9 and 6.9) of the amines in the polymer. The low pK(a)'s permit the dendrimer to buffer the pH change in the endosomal compartment. The characteristics of precise control of structure, favorable pK(a)'s, and low toxicity make the dendrimers suitable for gene-transfer vehicles.