Farhood H, Bottega R, Epand R M, Huang L
Cell, Molecular and Developmental Biology Program, University of Tennessee, Knoxville.
Biochim Biophys Acta. 1992 Nov 9;1111(2):239-46. doi: 10.1016/0005-2736(92)90316-e.
Four different cationic derivatives of cholesterol were synthesized which contain either a tertiary or a quaternary amino head group, with and without a succinyl spacer-arm. Their ability to inhibit protein kinase C (PKC) activity was measured in a detergent mixed micellar solution. Derivatives containing a quaternary amino head group were effective inhibitors (Ki approx. 12 and 59 microM) of PKC and derivatives containing a tertiary amino head group were approx. 4-20-fold less inhibitory. Liposomes containing an equimolar mixture of dioleoylphosphatidylethanolamine (DOPE) and a cationic cholesterol derivative were tested for the DNA-mediated transfection activity in mouse L929 cells. Highest activity was found with the derivative with low PKC inhibitory activity and with a succinyl spacer-arm. The transfection activity of this tertiary amine derivative, N,N-dimethylethylenediaminyl succinyl cholesterol was dependent on DOPE as a helper lipid; liposomes containing dioleoylphosphatidylcholine and this derivative had little activity. The transfection protocol of this new cationic liposome reagent was optimized with respect to the ratio of liposome/DNA, dose of the complex and time of incubation with cells. Several adherent cell lines could be efficiently transfected with this liposome reagent without any apparent cytotoxicity. However, the transfection activity was strongly inhibited by the presence of serum components.
合成了四种不同的胆固醇阳离子衍生物，它们含有叔胺或季胺头部基团，有或没有琥珀酰间隔臂。在去污剂混合胶束溶液中测定了它们抑制蛋白激酶C（PKC）活性的能力。含有季胺头部基团的衍生物是PKC的有效抑制剂（Ki约为12和59 microM），而含有叔胺头部基团的衍生物的抑制作用约低4 - 20倍。测试了含有二油酰磷脂酰乙醇胺（DOPE）和阳离子胆固醇衍生物等摩尔混合物的脂质体在小鼠L929细胞中的DNA介导转染活性。发现PKC抑制活性低且带有琥珀酰间隔臂的衍生物具有最高活性。这种叔胺衍生物N，N - 二甲基乙二胺基琥珀酰胆固醇的转染活性依赖于DOPE作为辅助脂质；含有二油酰磷脂酰胆碱和该衍生物的脂质体活性很小。针对脂质体/ DNA比例、复合物剂量和与细胞孵育时间对这种新型阳离子脂质体试剂的转染方案进行了优化。几种贴壁细胞系可以用这种脂质体试剂有效转染，且没有明显的细胞毒性。然而，血清成分的存在强烈抑制了转染活性。
