Studying the intracellular dissociation of polymer-oligonucleotide complexes by dual color fluorescence fluctuation spectroscopy and confocal imaging
Studying the intracellular dissociation of polymer-oligonucleotide complexes by dual color fluorescence fluctuation spectroscopy and confocal imaging
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DOI：
10.1021/bi0476883
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发表时间：
2005-07-26
期刊：
影响因子：
2.9
通讯作者：
Demeester, J
中科院分区：
文献类型：
--
作者：
Lucas, B;Remaut, K;Demeester, J
为了转染细胞，阳离子聚合物和阳离子脂质体作为寡核苷酸和质粒DNA的载体被广泛研究。DNA在细胞内成功传递的一个重要步骤是从其载体中释放出来。本研究利用双色荧光波动光谱（双色FFS）来表征阳离子聚合物/寡核苷酸复合物的细胞内解离。作为模型，罗丹明绿色标记的寡核苷酸（rhgr - on）与高摩尔质量（Cy5-graft-pDMAEMA, 1700 kDa）或低摩尔质量[Cy5-poly(L-lysine), Cy5-pLL， 30 kDa]的cy5标记的聚合物络合。将FFS结果与共聚焦激光扫描显微镜（CLSM）观察结果进行比较。CLSM证明了Cy5-graft-pDMAEMA/RhGr-ON复合物被Vero细胞内吞噬在细胞质中解离：聚合物仅在细胞质中检测到，而（释放的）RhGr-ON在细胞核中积累。然而，用Cy5-pLL/RhGr-ON复合物转染Vero细胞会导致聚合物和寡核苷酸在细胞核中的共定位。在后一种情况下，CLSM无法证明细胞核中是否存在完整的Cy5-pLL/RhGr-ON复合物，或者这两种成分是否一起位于细胞核中而没有关联。双色FFS，监测（双标记）荧光分子的运动，能够回答这个问题。由于Cy5-pLL/RhGr-ON复合物是多分子的，即由许多RhGr-ON与许多Cy5-pLL链结合而成，因此具有高度的绿色和红色荧光。因此，当Cy5-pLL/RhGr-ON配合物穿过激发体积时，FFS仪器的（绿色和红色）检测器同时检测到强烈的绿色和红色荧光峰。用Cy5-pLL/RhGr-ON复合物转染Vero细胞后，FFS确实能够在细胞质中同时检测到绿色和红色荧光峰，但在细胞核中却无法检测到。从这些结果我们得出结论，转染后细胞核中存在的Cy5-pLL和rhgr - on并不相关。
To transfect cells, cationic polymers as well as cationic liposomes are widely investigated as carriers for both oligonucleotides and plasmid DNA. A major step in the successful intracellular delivery of the DNA is the release from its carrier. In this study, dual color fluorescence fluctuation spectroscopy (dual color FFS) was explored in order to characterize the intracellular dissociation of cationic polymer/oligonucleotide complexes. As a model, rhodamine green-labeled oligonucleotides (RhGr-ONs) were complexed with Cy5-labeled polymers of either high molar mass (Cy5-graft-pDMAEMA, 1700 kDa) or low molar mass [Cy5-poly(L-lysine), Cy5-pLL, 30 kDa]. The FFS results were compared with confocal laser scanning microscopy (CLSM) observations. CLSM proved that Cy5-graft-pDMAEMA/RhGr-ON complexes endocytosed by Vero cells dissociate in the cytoplasm: the polymer was only detected in the cytoplasm whereas the (released) RhGr-ONs accumulated in the nucleus. Transfecting Vero cells with Cy5-pLL/RhGr-ON complexes resulted, however, in colocalization of polymer and oligonucleotides in the nucleus. In the latter case, CLSM was not able to prove whether intact Cy5-pLL/RhGr-ON complexes were present in the nucleus or whether both components were located together in the nucleus without being associated. Dual color FFS, which monitors the movement of (dual labeled) fluorescent molecules, was able to answer this question. As a Cy5-pLL/RhGr-ON complex is multimolecular, i.e., it consists of many RhGr-ONs associated with many Cy5-pLL chains, it is both highly green and red fluorescent. Consequently, when Cy5-pLL/RhGr-ON complexes move through the excitation volume, the (green and red) detectors of the FFS instrument detect simultaneously a strong green and red fluorescence peak. Upon transfecting the Vero cells with Cy5-pLL/RhGr-ON complexes, FFS was indeed able to detect simultaneously green and red fluorescence peaks in the cytoplasm but never in the nucleus. From these results we conclude that the Cy5-pLL and RhGr-ONs present in the nucleus after transfection were not associated.