Mahato R I, Anwer K, Tagliaferri F, Meaney C, Leonard P, Wadhwa M S, Logan M, French M, Rolland A
GeneMedicine, Inc., The Woodlands, TX 77381-4248, USA.
Hum Gene Ther. 1998 Sep 20;9(14):2083-99. doi: 10.1089/hum.1998.9.14-2083.
The objectives of this study were to investigate the influence of physicochemical properties of lipid/plasmid complexes on in vivo gene transfer and biodistribution characteristics. Formulations based on 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and novel biodegradable cationic lipids, such as ethyl dioleoyl phosphatidylcholine (EDOPC), ethyl palmitoyl myristyl phosphatidylcholine (EPMPC), myristyl myristoyl carnitine ester (MMCE), and oleyl oleoyl L-carnitine ester (DOLCE), were assessed for gene expression after tail vein injection of lipid/plasmid complexes in mice. Gene expression was influenced by cationic lipid structure, cationic lipid-to-colipid molar ratios, plasmid-to-lipid charge ratios, and precondensation liposome size. Detectable levels of human growth hormone (hGH) in serum, human factor IX (hFIX) in plasma, and chloramphenicol acetyltransferase (CAT) in the lung and liver were observed with positively charged lipid/plasmid complexes prepared from 400-nm extruded liposomes with a cationic lipid-to-colipid ratio of 4:1 (mol/mol). Intravenous administration of lipid/CAT plasmid complexes resulted in distribution of plasmid DNA mainly to the lung at 15 min after injection. Plasmid DNA accumulation in the liver increased with time up to 24 hr postinjection. There was a 10-fold decrease in the amount of plasmid DNA in the lung at 15 min after injection, when the lipid/plasmid complex charge ratio was decreased from 3:1 to 0.5:1 (+/-). Bright fluorescent aggregates were evident in in vivo-transfected lung with the positively charged pCMV-CAT/DOLCE:dioleyl phosphatidylethanolamine (DOPE) (1:1, mol/mol) complexes, while more discrete punctate fluorescence was observed with a 4:1 molar ratio of cationic lipid:colipid formulations. Preinjection of polyanions such as plasmid, dextran sulfate, polycytidic acid, and polyinosinic acid decreased hGH expression, whereas the preinjection of both positively charged and neutral liposomes had no effect on hGH serum levels. Of the cationic lipids tested, DOLCE was found to be the most effective potentially biodegradable cationic lipid. A correlation between gene expression and cationic lipid:colipid ratios and lipid-to-plasmid charge ratio was also observed for DOTMA- and DOLCE-based formulations.
本研究的目的是调查脂质/质粒复合物的物理化学性质对体内基因转移和生物分布特征的影响。基于1,2-二油酰基-3-三甲基铵丙烷（DOTMA）和新型可生物降解阳离子脂质（如二油酰基磷脂酰胆碱乙酯（EDOPC）、棕榈酰肉豆蔻酰磷脂酰胆碱乙酯（EPMPC）、肉豆蔻酰肉豆蔻酰肉碱酯（MMCE）和油酰油酰L-肉碱酯（DOLCE））的制剂，在小鼠尾静脉注射脂质/质粒复合物后，评估其基因表达情况。基因表达受阳离子脂质结构、阳离子脂质与共脂质的摩尔比、质粒与脂质的电荷比以及预凝聚脂质体大小的影响。用由400 nm挤压脂质体制备的阳离子脂质与共脂质比为4:1（摩尔/摩尔）的带正电荷脂质/质粒复合物，可观察到血清中可检测水平的人生长激素（hGH）、血浆中的人凝血因子IX（hFIX）以及肺和肝脏中的氯霉素乙酰转移酶（CAT）。静脉注射脂质/CAT质粒复合物后，在注射后15分钟时，质粒DNA主要分布到肺。注射后24小时内，肝脏中质粒DNA的积累随时间增加。当脂质/质粒复合物电荷比从3:1降至0.5:1（±）时，注射后15分钟肺中质粒DNA的量减少了10倍。用带正电荷的pCMV-CAT/DOLCE:二油酰基磷脂酰乙醇胺（DOPE）（1:1，摩尔/摩尔）复合物在体内转染的肺中可见明亮的荧光聚集体，而阳离子脂质:共脂质制剂摩尔比为4:1时观察到更离散的点状荧光。预先注射多阴离子如质粒、硫酸葡聚糖、聚胞苷酸和聚肌苷酸会降低hGH表达，而预先注射带正电荷和中性脂质体对hGH血清水平无影响。在所测试的阳离子脂质中，发现DOLCE是最有效的潜在可生物降解阳离子脂质。基于DOTMA和DOLCE的制剂也观察到基因表达与阳离子脂质:共脂质比以及脂质与质粒电荷比之间的相关性。
