Wangerek L A, Dahl H H, Senden T J, Carlin J B, Jans D A, Dunstan D E, Ioannou P A, Williamson R, Forrest S M
Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Australia.
J Gene Med. 2001 Jan-Feb;3(1):72-81. doi: 10.1002/1521-2254(200101/02)3:1<72::AID-JGM157>3.0.CO;2-M.
Cationic liposomes represent an important gene delivery system due to their low immunogenicity, but are relatively inefficient, with optimisation of DNA-liposome complexes (lipoplexes) for transfection necessary for each cell type of interest. There have been few studies examining optimisation in neuronal cell types or determining how the structure of lipoplexes affects transfection efficiency.
Four commercially available cationic liposome formulations were used to optimise transfection efficiency in neuronal cells. The DNA to liposome ratio and the amount of DNA used in transfections were varied. Transfection efficiency was determined by the percentage of cells positive for the micro-galactosidase reporter gene product. The structure of lipoplexes was studied using atomic force microscopy. Lipoplexes were characterised further using dynamic light scattering to determine size and fluorescence techniques to show DNA compaction.
Optimal transfection conditions were found to differ between immortalised cell lines and primary cells. High transfection efficiencies in immortalised cell lines were achieved predominantly with multivalent cationic liposomes while primary neuronal cells showed optimal transfection efficiency with monovalent cationic liposomes. The structure of lipoplexes was observed with atomic force microscopy and showed globular complexes for multivalent cationic liposomes, while monovalent liposomes gave less compact structures. In support of this finding, high levels of DNA compaction with multivalent liposomes were observed using fluorescence quenching measurements for all DNA to liposome ratios tested. One monovalent liposome showed increasing levels of compaction with increasing liposome amount. Dynamic light scattering showed little change in complex size when the different lipoplexes were studied.
Optimisation of transfection efficiency was different for cell lines and primary neurons. Immortalised cells showed optimal transfection with multivalent liposomes while primary neurons showed optimal transfection with monovalent liposomes. The charge ratio of the monovalent liposome was below one, suggesting a different mechanism of lipoplex binding and uptake in primary neurons. The structure of lipoplexes, as
阳离子脂质体因其低免疫原性而成为一种重要的基因递送系统，但效率相对较低，对于每种感兴趣的细胞类型都需要优化用于转染的DNA - 脂质体复合物（脂质体转染复合物）。很少有研究考察在神经元细胞类型中的优化情况或确定脂质体转染复合物的结构如何影响转染效率。
使用四种市售阳离子脂质体制剂来优化神经元细胞中的转染效率。改变转染中DNA与脂质体的比例以及所用DNA的量。通过微半乳糖苷酶报告基因产物阳性细胞的百分比来确定转染效率。使用原子力显微镜研究脂质体转染复合物的结构。进一步使用动态光散射来确定大小以及荧光技术来显示DNA压缩，从而对脂质体转染复合物进行表征。
发现永生化细胞系和原代细胞的最佳转染条件不同。永生化细胞系中的高转染效率主要通过多价阳离子脂质体实现，而原代神经元细胞用单价阳离子脂质体显示出最佳转染效率。用原子力显微镜观察到脂质体转染复合物的结构，多价阳离子脂质体呈现球状复合物，而单价脂质体形成的结构较松散。支持这一发现的是，对于所有测试的DNA与脂质体比例，使用荧光猝灭测量观察到多价脂质体具有高水平的DNA压缩。一种单价脂质体随着脂质体用量增加显示出压缩水平增加。研究不同脂质体转染复合物时，动态光散射显示复合物大小变化不大。
细胞系和原代神经元的转染效率优化不同。永生化细胞用多价脂质体显示出最佳转染效果而原代神经元用单价脂质体显示出最佳转染效果。单价脂质体的电荷比低于1，表明在原代神经元中脂质体转染复合物的结合和摄取机制不同。脂质体转染复合物的结构，如
