Towards a better understanding of the dissociation behavior of liposome-oligonucleotide complexes in the cytosol of cells
Towards a better understanding of the dissociation behavior of liposome-oligonucleotide complexes in the cytosol of cells
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DOI：
10.1016/j.jconrel.2004.12.017
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发表时间：
2005-03-21
影响因子：
10.8
通讯作者：
Demeester, J
中科院分区：
文献类型：
--
作者：
Lucas, B;Remaut, K;Demeester, J
为了在反义治疗中获得真实的突破，有必要了解反义传递系统的细胞行为。荧光波动光谱（FFS），它在时间上测量荧光波动的激发体积的显微镜，因此可以应用于细胞规模，显示出潜力，为这一目的。本研究采用双色荧光光谱法研究了Cy 5标记的寡核苷酸（Cy 5-ONs）与FITC标记的阳离子脂质体（FITC-liposomes）分别在缓冲液、细胞裂解液和Vero细胞胞质溶胶中的复合（结合和解离）。在Hepes缓冲液中，Cy 5-ONs与FITC-脂质体的缔合可以从Cy 5-和FITC-荧光的高峰值清楚地观察到，其同时出现在激发体积中。这可以通过以下事实来解释：在复合状态下，许多Cy 5-ON和FITC-脂质体彼此结合，因此一起移动通过激发体积，从而导致高荧光“FITC/Cy 5-峰”。对细胞裂解物中FITC-脂质体/Cy 5-ONs复合物的FFS测量显示，小部分Cy 5-ONs从复合物中释放。大部分Cy 5-ON保留在复合物中，其似乎也在细胞裂解物中聚集。与细胞裂解物中的测量一致，在Vero细胞的胞质溶胶中显微注射FITC-脂质体/Cy 5-ONs复合物后，一部分Cy 5-ONs被释放（因为通过FFS在细胞核中检测到Cy-ONs），而另一部分保持结合（因为在胞质溶胶中经常观察到Cy 5-峰）。如将要解释的，Cy 5-峰可能是由于Cy 5-ON与胞质溶胶组分聚集以及Cy 5-ON仍然与FITC-脂质体复合并具有淬灭的FITC-荧光。(c)2004 Elsevier B. V.保留所有权利。
To obtain real breakthroughs in antisense therapy, it is necessary to understand the cellular behavior of antisense delivery systems. Fluorescence fluctuation spectroscopy (FFS), which measures in time fluorescence fluctuations in the excitation volume of a microscope and which can thus be applied on a cellular scale, shows potential for this purpose. In this study dual color FFS was explored to characterize the complexation (association and dissociation) between Cy5-labeled oligonucleotides (Cy5-ONs) and FITC-labeled cationic liposomes (FITC-liposomes) in respectively buffer, cell lysate and the cytosol of Vero cells. In Hepes buffer the association of the Cy5-ONs to the FITC-liposomes could be clearly observed from the high peaks of Cy5- and FITC-fluorescence, which appeared simultaneously in the excitation volume. This was explained by the fact that in the complexed state many Cy5-ONs and FITC-liposomes are bound to each other and thus move together through the excitation volume thereby resulting in high fluorescence 'FITC/Cy5 -peaks'. FFS measurements on FITC-liposome/Cy5-ONs complexes in cell lysate revealed that a minor part of the Cy5-ONs was released from the complexes. The major part of the Cy5-ONs remained in the complexes, which also seemed to aggregate in cell lysate. In agreement with the measurements in cell lysate, after microinjection of FITC-liposome/Cy5-ONs complexes in the cytosol of Vero cells a part of the Cy5-ONs was released (as Cy-ONs were detected by FFS in the nuclei) while the other part remained bound (as Cy5-peaks were frequently observed in the cytosol). As will be explained, the Cy5-peaks could be due both to Cy5-ONs clustered with cytosol components and Cy5-ONs still complexed to FITC-liposomes with quenched FITC-fluorescence. (c) 2004 Elsevier B.V. All rights reserved.