Sternberg B, Hong K, Zheng W, Papahadjopoulos D
California Pacific Medical Center, Research Institute, 2340 Clay Street, San Francisco, CA 94115, USA.
Biochim Biophys Acta. 1998 Oct 15;1375(1-2):23-35. doi: 10.1016/s0005-2736(98)00129-1.
We have investigated the morphology and transfection activity of cationic liposome-DNA complexes (CLDC) under conditions relevant to both in vivo and in vitro studies. Moreover we have attempted to establish structure-function relationships relevant for high transfection activities under both conditions. CLDC were composed of dimethyldioctadecylammonium bromide with either 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol (Chol) interacting either with pre-condensed DNA or with uncondensed plasmid DNA. Furthermore for steric stabilization 1% poly(ethylene glycol)-phospholipid conjugate was added to CLDC containing Chol and plasmid DNA. The in vivo studies were carried out in mice following i.v. injection, and the in vitro studies were performed on SK-BR-3 human breast cancer cells in the presence of media with serum. The morphology of the CLDC, monitored by freeze-fracture electron microscopy, was investigated after mixing with mouse serum or the medium where the cells were kept. The substitution of DOPE with Chol, and the addition of N-[omega-methoxypoly(oxyethylene)-alpha-oxycarbonyl-DSPE+ ++ are producing CLDC which are stabilized with respect to time and serum, and are relatively small (100-300 nm). These stabilized complexes show high expression of a marker gene in mouse lungs reaching expression values up to 10 ng luciferase per mg tissue protein, but relatively low expression in SK-BR-3 cells in vitro. Additionally, some of the complexes containing pre-condensed DNA look like 'map-pin' structures showing heads of the size of liposomes and short, stiff and tapering tails. The in vivo transfection activity of these preparations is highest. Similar complexes containing DOPE rather than Chol as helper lipid precipitate in the presence of serum and especially of cell medium and convert into hexagonal lipid (HII) phase. Such complexes, despite their high transfection activity in vitro, show very little transfection activity in vivo. These comparisons may help us to understand the fundamental difference between in vitro and in vivo activity of CLDC: high in vitro transfection activity seems to be associated with hexagonal lipid precipitates whereas high in vivo activity seems to be related with small, stabilized complexes, which in our case also exhibit some protrusions (map-pin structures).
我们研究了在与体内和体外研究相关的条件下阳离子脂质体 - DNA复合物（CLDC）的形态和转染活性。此外，我们试图建立在这两种条件下与高转染活性相关的结构 - 功能关系。CLDC由溴化二甲基二辛基铵与1,2 - 二油酰 - sn - 甘油 - 3 - 磷酸乙醇胺（DOPE）或胆固醇（Chol）组成，它们与预凝聚的DNA或未凝聚的质粒DNA相互作用。此外，为了实现空间稳定，将1%的聚乙二醇 - 磷脂共轭物添加到含有Chol和质粒DNA的CLDC中。体内研究在小鼠静脉注射后进行，体外研究在含有血清的培养基存在下对SK - BR - 3人乳腺癌细胞进行。通过冷冻断裂电子显微镜监测CLDC的形态，在与小鼠血清或细胞培养的培养基混合后进行研究。用Chol替代DOPE以及添加N - [ω - 甲氧基聚（氧乙烯） - α - 氧羰基 - DSPE + ++ 正在产生相对于时间和血清稳定的CLDC，并且相对较小（100 - 300 nm）。这些稳定的复合物在小鼠肺中显示出标记基因的高表达，达到每毫克组织蛋白高达10 ng荧光素酶的表达值，但在体外SK - BR - 3细胞中的表达相对较低。此外，一些含有预凝聚DNA的复合物看起来像“图钉”结构，显示出脂质体大小的头部和短而硬且逐渐变细的尾部。这些制剂的体内转染活性最高。含有DOPE而非Chol作为辅助脂质的类似复合物在血清尤其是细胞培养基存在下沉淀，并转化为六方脂质（HII）相。这种复合物尽管在体外具有高转染活性，但在体内显示出非常低的转染活性。这些比较可能有助于我们理解CLDC体外和体内活性之间的根本差异：高体外转染活性似乎与六方脂质沉淀有关，而高体内活性似乎与小的、稳定的复合物有关，在我们的案例中这些复合物还表现出一些突起（图钉结构）。
