Pinnaduwage P, Schmitt L, Huang L
Department of Biochemistry, University of Tennessee, Knoxville 37996-0840.
Biochim Biophys Acta. 1989 Oct 2;985(1):33-7. doi: 10.1016/0005-2736(89)90099-0.
Sonicated liposomes composed of dioleoylphosphatidylethanolamine (DOPE) and a quaternary ammonium detergent (dodecyl-, tetradecyl-, or cetyl-trimethylammonium bromide) mediates functional transfer of pSV2 CAT plasmid DNA to mouse L929 fibroblasts. Successful transfection was determined by assaying for chloramphenicol acetyltransferase activity in cell lysates collected 40 h after exposure to the lipid-DNA complexes. Liposomes prepared with the quaternary ammonium detergents were less toxic than the free detergents at the same concentrations and were more efficient in their delivery of the plasmid DNA to the cells. Analysis of the three detergents in combination with the lipid showed that cetyltrimethylammonium bromide was least toxic to the cells. This detergent, at a minimal concentration of 20 mol% in DOPE, allowed for stable liposome preparations and efficient transfection. Optimal efficiency of transfection occurred with 30 micrograms of DNA. Further increases in the DNA concentration caused a decrease in the transfection efficiency, perhaps due to charge repulsions between the liposomes now saturated with negatively charged DNA and the negatively charged cell surface. The transfection activity of the liposome was limited by its cytotoxicity at high liposome concentrations. These results are compared with that of the Lipofectin, another positively charged liposome preparation which is commercially available. Although the overall transfection activity of the liposome containing the quaternary ammonium detergent is somewhat lower than that of the Lipofectin, it may serve as an inexpensive and convenient alternative.
由二油酰磷脂酰乙醇胺（DOPE）和季铵洗涤剂（十二烷基、十四烷基或十六烷基三甲基溴化铵）组成的超声处理脂质体介导了pSV2 CAT质粒DNA向小鼠L929成纤维细胞的功能转移。通过在暴露于脂质-DNA复合物40小时后收集的细胞裂解物中检测氯霉素乙酰转移酶活性来确定成功转染。在相同浓度下，用季铵洗涤剂制备的脂质体比游离洗涤剂毒性更小，并且在将质粒DNA递送至细胞方面更有效。对三种洗涤剂与脂质组合的分析表明，十六烷基三甲基溴化铵对细胞毒性最小。这种洗涤剂在DOPE中的最低浓度为20摩尔％时，可制备稳定的脂质体并实现高效转染。转染的最佳效率出现在DNA浓度为30微克时。DNA浓度进一步增加导致转染效率降低，这可能是由于现在被带负电荷的DNA饱和的脂质体与带负电荷的细胞表面之间的电荷排斥所致。脂质体的转染活性在高脂质体浓度下受到其细胞毒性的限制。将这些结果与另一种市售的带正电荷脂质体制剂Lipofectin的结果进行了比较。尽管含有季铵洗涤剂的脂质体的总体转染活性略低于Lipofectin，但它可能是一种廉价且方便的替代品。
