Freeman D J, Niven R W
Amgen Inc., Thousand Oaks, California 91320, USA.
Pharm Res. 1996 Feb;13(2):202-9. doi: 10.1023/a:1016078728202.
A plasmid containing the luciferase 'marker' cDNA was constructed to test non viral gene delivery formulations in vivo.
A scale up procedure was devised to produce up to gram quantities of plasmid. Sufficient quantities were generated to process and test the DNA with various additives and to generate a spray-dried powder formulation of the plasmid. Male Sprague-Dawley rats (250 g) were intratracheally instilled with 200-250 microl of solution containing 200 microg plasmid +/- lipid [DC Chol:DOPE 1:1 molar (2mg/kg)] growth factors [KGF (10 mg/kg), EGF (5 mg/kg)], permeation enhancers [sodium glycocholate (0.01 to 10% w/v)), sodium deoxycholate (1% w/v), beta-cyclodextrin (1% w/v)], surfactant [Tween 80 (1% w/v)], a mucolytic [N-acetylcysteine (10% w/v)] and positively charged synthetic polymers [PVAVAM 6 and 14%]. Animals were sacrificed 24 hr post-dose and the lungs were assayed for luciferase using a chemiluminescent assay.
The relative ability of the materials to promote luciferase production in the lungs was permeation enhancer >> DNA alone > or = lipid, mucolytic, surfactant, growth factor > polymer. Protein production in the lungs ranged from 10 times below the DNA control (approximately 16 pg) using the polymers (approximately 1.5 pg) to approximately 125 times greater than the control using the permeation enhancer (approximately 2050 pg). The transfection capabilities of the majority of additives was low. The enhancing effects of sodium glycocholate were dose-dependent and perhaps associated with the critical micelle concentration. Although the bile salt was the most successful of the tested compounds, it resulted in significant mortality when used at concentrations greater than 1% w/v.
The results suggest that transfection can be significantly enhanced by additives such as NaGC but some toxicity may be unavoidable.
构建一种含有荧光素酶“标记”cDNA的质粒，用于在体内测试非病毒基因递送制剂。
设计了一种放大程序以生产多达克级的质粒。生成了足够数量的质粒，用于用各种添加剂处理和测试DNA，并制备质粒的喷雾干燥粉末制剂。将雄性Sprague-Dawley大鼠（250 g）经气管内滴注200 - 250微升含有200微克质粒±脂质[DC Chol:DOPE 1:1摩尔比（2毫克/千克）]、生长因子[KGF（10毫克/千克）、EGF（5毫克/千克）]、渗透增强剂[甘氨胆酸钠（0.01至10% w/v）、脱氧胆酸钠（1% w/v）、β-环糊精（1% w/v）]、表面活性剂[吐温80（1% w/v）]、黏液溶解剂[N-乙酰半胱氨酸（10% w/v）]和带正电荷的合成聚合物[PVAVAM 6%和14%]的溶液。给药后24小时处死动物，使用化学发光测定法测定肺组织中的荧光素酶。
材料促进肺组织中荧光素酶产生的相对能力为：渗透增强剂>>单独的DNA>或=脂质、黏液溶解剂、表面活性剂、生长因子>聚合物。肺组织中的蛋白质产生量范围从使用聚合物时比DNA对照低10倍（约1.5皮克）到使用渗透增强剂时比对照高约125倍（约2050皮克）。大多数添加剂的转染能力较低。甘氨胆酸钠的增强作用呈剂量依赖性，可能与临界胶束浓度有关。尽管胆盐是测试化合物中最成功的，但当浓度大于1% w/v使用时会导致显著的死亡率。
结果表明，NaGC等添加剂可显著增强转染，但一些毒性可能不可避免。
