Welz C, Neuhuber W, Schreier H, Metzler M, Repp R, Rascher W, Fahr A
Department of Pharmaceutics and Biopharmacy, Philipps-University Marburg, Germany.
Pharm Res. 2000 Oct;17(10):1206-11. doi: 10.1023/a:1026410612600.
The aim of this study was to characterize the intracellular fate and nuclear uptake kinetics of oligonucleotides (ON) that were complexed with protamine sulfate (PS) and negatively charged liposomes at different ratios of ON to PS.
Double-fluorescence labelling of ON and liposomal lipid was applied to simultaneously monitor the interaction as well as the individual fate of active agent and carrier upon intracellular delivery using confocal laser scanning microscopy (CLSM). A DNA-analogue of a 68-mer intramolecular double-stranded RNA:DNA-hybridoligonucleotide (chimeraplasts) with unmodified phosphate backbone was employed. This construct was condensed with PS and coated with a liposomal formulation (AVE-3 = artificial viral envelope).
PS-ON complexes and AVE -3-coated complexes with a defined composition were very effective in nuclear transport of ON for a ON:PS charge ratio of 1:3. Nucleus:cytosol fluorescence ratios peaked at about 10 hrs and started to decrease again at 21 hrs.
AVE associates with PS-condensed ON, and this complex is able to be taken up by cells and to deliver ON to the nucleus. PS-ON complexes are released from the liposomal formulation, mainly as an extranuclear enzymatic degradation of the liposomal phospholipids. The results of the kinetic analysis can be used to optimize transfection protocols with ON in HepG2 cells.
本研究旨在表征寡核苷酸（ON）与硫酸鱼精蛋白（PS）以及带负电荷脂质体以不同ON与PS比例复合后的细胞内命运和核摄取动力学。
应用ON和脂质体脂质的双荧光标记，通过共聚焦激光扫描显微镜（CLSM）同时监测细胞内递送时活性剂与载体的相互作用以及各自的命运。使用具有未修饰磷酸骨架的68聚体分子内双链RNA：DNA杂交寡核苷酸（嵌合体）的DNA类似物。该构建体与PS凝聚并用脂质体制剂（AVE-3 =人工病毒包膜）包被。
对于ON：PS电荷比为1:3的情况，具有确定组成的PS-ON复合物和AVE-3包被的复合物在ON的核转运中非常有效。核：细胞质荧光比在约10小时达到峰值，并在21小时再次开始下降。
AVE与PS凝聚的ON结合，并且该复合物能够被细胞摄取并将ON递送至细胞核。PS-ON复合物从脂质体制剂中释放，主要是由于脂质体磷脂的核外酶促降解。动力学分析结果可用于优化HepG2细胞中ON的转染方案。
