Institute of Biophysics and Cellular Engineering of NASB, Minsk, Belarus.
J Control Release. 2010 Jan 25;141(2):110-27. doi: 10.1016/j.jconrel.2009.09.030. Epub 2009 Oct 6.
This paper reviews different techniques for analyzing the transfection efficiencies and cytotoxicities of dendriplexes-complexes of nucleic acids with dendrimers. Analysis shows that three plasmids are mainly used in transfection experiments: plasmid DNA encoding luciferase from the firefly Photinus pyralis, beta-galactosidase, or green fluorescent protein. The effective charge ratio of transfection does not directly correlate with the charge ratio obtained from gel electrophoresis, zeta-potential or ethidium bromide intercalation data. The most popular cells for transfection studies are human embryonic kidney cells (HEK293), mouse embryonic cells (NIH/3T3), SV40 transformed monkey kidney fibroblasts (COS-7) and human epithelioid cervical carcinoma cells (HeLa). Cellular uptake is estimated using fluorescently-labeled dendrimers or nucleic acids. Transfection efficiency is measured by the luciferase reporter assay for luciferase, X-Gal staining or beta-galactosidase assay for beta-galactosidase, and confocal microscopy for green fluorescent protein. Cytotoxicity is determined by the MTT test and lactate dehydrogenase assays. On the basis of the papers reviewed, a standard essential set of techniques for characterizing dendriplexes was constructed: (1) analysis of size and shape of dendriplexes in dried/frozen state by electron or atomic force microscopy; (2) analysis of charge/molar ratio of complexes by gel electrophoresis or ethidium bromide intercalation assay or zeta-potential measurement; (3) analysis of hydrodynamic diameter of dendriplexes in solution by dynamic light scattering. For the evaluation of transfection efficiency the essential techniques are (4) luciferase reporter assay, beta-galactosidase assay or green fluorescent protein microscopy, and (5) cytotoxicity by the MTT test. All these tests allow the transfection efficiencies and cytotoxicities of different kinds of dendrimers to be compared.
本文综述了分析树枝状聚合物-核酸复合物转染效率和细胞毒性的不同技术。分析表明，转染实验主要使用三种质粒：萤火虫荧光素酶基因的质粒 DNA、β-半乳糖苷酶或绿色荧光蛋白。转染的有效电荷比与凝胶电泳、动电位或溴化乙锭嵌入数据得到的电荷比没有直接关系。转染研究最常用的细胞是人胚肾细胞（HEK293）、小鼠胚胎细胞（NIH/3T3）、SV40 转化的猴肾成纤维细胞（COS-7）和人上皮样宫颈癌细胞（HeLa）。使用荧光标记的树枝状聚合物或核酸来估计细胞摄取。通过荧光素酶报告基因测定、X-Gal 染色或β-半乳糖苷酶测定测定β-半乳糖苷酶的转染效率，通过共聚焦显微镜测定绿色荧光蛋白的转染效率。通过 MTT 试验和乳酸脱氢酶测定来确定细胞毒性。根据综述的论文，构建了用于表征树枝状聚合物的一套标准基本技术：（1）通过电子或原子力显微镜分析干燥/冷冻状态下树枝状聚合物的大小和形状；（2）通过凝胶电泳或溴化乙锭嵌入试验或动电位测量分析复合物的电荷/摩尔比；（3）通过动态光散射分析溶液中树枝状聚合物的水动力直径。为了评估转染效率，基本技术是（4）荧光素酶报告基因测定、β-半乳糖苷酶测定或绿色荧光蛋白显微镜测定，以及（5）通过 MTT 试验测定细胞毒性。所有这些测试都允许比较不同类型的树枝状聚合物的转染效率和细胞毒性。
