Ruozi Barbara, Montanari Monica, Vighi Eleonora, Tosi Giovanni, Tombesi Andrea, Battini Renata, Restani Cinzia, Leo Eliana, Forni Flavio, Vandelli Maria Angela
Department of Pharmaceutical Sciences, University of Modena and Reggio Emilia, Modena Italy.
J Liposome Res. 2009;19(3):241-51. doi: 10.1080/08982100902788416.
In this study, the mechanism of the internalization and the cellular distribution of 59 fluorescein conjugated PS-ODN (FITC-ODN) after transfection with different mixed lipidic vesicles/oligo complexes (lipoplexes) have been investigated. Mixed lipidic vesicles were prepared with one of the most used cationic lipid (DOTAP) and different amounts of a cholic acid (UDCA) to release the oligo into HaCaT cells. Using flow cytometry, the cellular uptake of the oligo was studied with and without different inhibitors able to block selectively the different pathways involved in the internalization mechanism. The intracellular distribution of the oligo was analyzed by confocal laser scanning microscopy (CLSM), treating the cells with the lipoplexes and directly observing without any fixing procedure. To better carry out the colocalization studies, fluorescent-labeled markers, specific for the different cellular compartments, were coincubated with 59 fluorescein-conjugated 29-mer phosphorotioate oligonucleotide (FITC-ODN). The different lipidic vesicles affect the internalization mechanism of FITC-ODN. After using the inhibitors, the uptake of complexes involved a different internalization mechanism. The live CLSM analysis demonstrated that, after 1 hour from the complex incubation, the oligo was transferred into cells and localized into the endosomes; after 24 hours, the oligo was intracellularly localized close to the nuclear structure in a punctuate pattern. However, the results from fusion experiments showed also a binding of a quite low amount of oligo with the cell membranes.
在本研究中，已对59种荧光素共轭的PS - ODN（FITC - ODN）在与不同混合脂质囊泡/寡核苷酸复合物（脂质体）转染后的内化机制和细胞分布进行了研究。使用最常用的阳离子脂质之一（DOTAP）和不同量的胆酸（UDCA）制备混合脂质囊泡，以便将寡核苷酸释放到HaCaT细胞中。使用流式细胞术，在有和没有能够选择性阻断内化机制中不同途径的不同抑制剂的情况下，研究了寡核苷酸的细胞摄取。通过共聚焦激光扫描显微镜（CLSM）分析寡核苷酸的细胞内分布，用脂质体处理细胞并直接观察，无需任何固定程序。为了更好地进行共定位研究，将针对不同细胞区室的荧光标记标记物与59种荧光素共轭的29聚体硫代磷酸酯寡核苷酸（FITC - ODN）共同孵育。不同的脂质囊泡影响FITC - ODN的内化机制。使用抑制剂后，复合物的摄取涉及不同的内化机制。实时CLSM分析表明，在复合物孵育1小时后，寡核苷酸被转移到细胞中并定位在内体中；24小时后，寡核苷酸以点状模式在细胞内定位在靠近核结构的位置。然而，融合实验的结果也显示相当少量的寡核苷酸与细胞膜有结合。
