Formation of stable cationic lipid/DNA complexes for gene transfer.

Stable cationic lipid/DNA complexes were formed by solubilizing cationic liposomes with 1% octylglucoside and complexing a DNA plasmid with the lipid in the presence of detergent. Removal of the detergent by dialysis yielded a lipid/DNA suspension that was able to transfect tissue culture cells up to 90 days after formation with no loss in activity. Similar levels of gene transfer were obtained by mixing the cationic lipid in a liposome form with DNA just prior to cell addition. However, expression was completely lost 24 hr after mixing. The transfection efficiency of the stable complex in 15% fetal calf serum was 30% of that obtained in the absence of serum, whereas the transient complex was completely inactivated with 2% fetal calf serum. A 90-day stability study comparing various storage conditions showed that the stable complex could be stored frozen or as a suspension at 4 degrees C with no loss in transfection efficiency. Centrifugation of the stable complex produced a pellet that contained approximately 90% of the DNA and 10% of the lipid. Transfection of cells with the resuspended pellet and the supernatant showed that the majority of the transfection activity was in the pellet and all the toxicity was in the supernatant. Formation of a stable cationic lipid/DNA complex has produced a transfection vehicle that can be stored indefinitely, can be concentrated with no loss in transfection efficiency, and the toxicity levels can be greatly reduced when the active complex is isolated from the uncomplexed lipid.

本研究通过以1%辛基葡萄糖苷（octylglucoside）溶解阳离子脂质体，并在去污剂存在的条件下将质粒DNA与脂质复合，制备得到稳定的阳离子脂质/DNA复合物。通过透析去除去污剂后，可获得脂质/DNA悬液，该悬液在复合物制备完成后长达90天内仍可转染组织培养细胞，且转染活性无任何衰减。若仅在加入细胞前即刻将脂质体形式的阳离子脂质与DNA混合，也可获得相当水平的基因转移效果，但该临时混合体系在制备完成24小时后便完全丧失基因表达活性。稳定复合物在15%胎牛血清（fetal calf serum）中的转染效率仅为无血清环境下的30%，而瞬时复合物（transient complex）在2%胎牛血清中便会完全失活。一项对比多种储存条件的90天稳定性研究结果显示，稳定复合物可通过冷冻保存，或以悬液形式在4℃下储存，且转染效率无任何损失。对稳定复合物进行离心处理后，可得到沉淀组分，该组分中约包含90%的DNA与10%的脂质。分别使用重悬后的沉淀组分与上清液进行细胞转染实验，结果表明绝大多数转染活性存在于沉淀组分中，而全部细胞毒性均集中于上清液。本研究制备的稳定阳离子脂质/DNA复合物作为转染载体，可实现长期无限期储存，可在不损失转染效率的前提下进行浓缩，且当从未结合的游离脂质中分离得到活性复合物后，体系的毒性水平可大幅降低。