Polycation-DNA complexes for gene delivery: a comparison of the biopharmaceutical properties of cationic polypeptides and cationic lipids
Polycation-DNA complexes for gene delivery: a comparison of the biopharmaceutical properties of cationic polypeptides and cationic lipids
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DOI：
10.1016/s0168-3659(98)00015-7
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发表时间：
1998-04-30
影响因子：
10.8
通讯作者：
Moss, SH
中科院分区：
文献类型：
--
作者：
Pouton, CW;Lucas, P;Moss, SH
DNA质粒与多种阳离子聚氨基酸和阳离子脂质形成颗粒复合物，用于转染培养中的哺乳动物细胞。通过使用荧光测定法测定乙锭的排除来研究络合作用，这表明与阳离子聚氨基酸的络合作用利用了大多数带电官能团。测定了一系列配合物的粒径和zeta电位。通常聚氨基酸在水中形成直径为80-120 nm的均匀颗粒，但它们的颗粒尺寸随着颗粒在电解质或细胞培养基中的稀释而增加。使用pRSVlacZ复合物比较转染效率，pRSVlacZ是一种在劳斯肉瘤病毒启动子控制下表达β-半乳糖苷酶的报告构建体。带正电荷的DNA/聚氨基酸复合物被细胞吸收，但需要内体溶解剂，如氯喹，以促进转染。与其他同聚氨基酸相比，聚鸟氨酸复合物导致最高水平的表达（聚鸟氨酸>聚-L-赖氨酸=聚-D-赖氨酸>聚精氨酸）。赖氨酸和丙氨酸的共聚氨基酸缩合DNA，但在转染实验中活性较低。Copoly（L-Lys，L-Ala 1：1）即使在氯喹存在下也无活性。相反，DNA/阳离子脂质复合物自发转染细胞，氯喹并没有提高表达的程度，而它通常降低效率。细胞系之间脂质复合物的比较效率之间几乎没有相关性，这表明细胞膜的性质和内在化机制的差异是效率的决定因素。为了探索用于基因递送的更好的细胞培养模型，在过滤培养中转染单层Caco-2细胞。随着细胞分化并形成极化单层，β-半乳糖苷酶的表达降低，直到第27天表达与基础活性没有显著差异。Caco-2过滤培养模型值得进一步关注，作为基因递送至上皮表面的模型，例如吸入后在肺中会遇到的。(C)1998年Elsevier Science B.V.
DNA plasmids formed particulate complexes with a variety of cationic polyamino acids and cationic lipids, which were used to transfect mammalian cells in culture. Complexation was studied by assaying for exclusion of ethidium using a fluorometric assay, which indicated that complexation with cationic polyamino acids took place with utilisation of the majority of charged functional groups. The particle sizes and zeta potentials of a range of complexes were determined. Generally polyamino acids formed uniform particles 80-120 nm in diameter in water, but their particle size increased on dilution of the particles in electrolytes or cell culture media. The efficiency of transfection was compared using complexes of pRSVlacZ, a reporter construct which expressed beta-galactosidase under the control of the Rous sarcoma virus promoter. Positively charged DNA/polyamino acid complexes were taken up by cells but required an endosomolytic agent, such as chloroquine, to facilitate transfection. Polyornithine complexes resulted in the highest levels of expression, in comparison with other homopolyamino acids (polyornithine>poly-L-lysine=poly-D-lysine>polyarginine). Copolyamino acids of lysine and alanine condensed DNA but were less active in transfection experiments. Copoly(L-Lys, L-Ala 1:1) was inactive even in the presence of chloroquine. In contrast DNA/cationic lipid complexes transfected cells spontaneously, and chloroquine did not improve the extent of expression, rather it usually reduced efficiency. There was little correlation between comparative efficiencies of lipid complexes between cell lines suggesting that the nature of the cell membrane and differences in mechanisms of internalisation were determinants of efficiency. In an effort to explore better cell culture models for gene delivery, monolayers of Caco-2 cells were transfected in filter culture. As the cells differentiated and formed a polarized monolayer, expression of beta-galactosidase was reduced until at day 27 expression was not significantly different from basal activity. The Caco-2 filter culture model merits further attention as a model of gene delivery to epithelial surfaces, such as would be encountered in the lung after inhalation. (C) 1998 Elsevier Science B.V.