Quantitative comparison of intracellular unpacking kinetics of polyplexes by a model constructed from quantum Dot-FRET
Quantitative comparison of intracellular unpacking kinetics of polyplexes by a model constructed from quantum Dot-FRET
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DOI：
10.1038/sj.mt.6300392
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发表时间：
2008-02-01
影响因子：
12.4
通讯作者：
Leong, Kam W.
中科院分区：
文献类型：
--
作者：
Chen, Hunter H.;Ho, Yi-Ping;Leong, Kam W.
非病毒基因传递的一个主要挑战是获得对限速步骤的机械理解。复合物介导的基因递送中的关键障碍是及时解包靶细胞内的复合物以释放DNA用于有效的基因转移。在这项研究中，组分质粒DNA和多聚体基因载体分别用量子点（QD）和Cy 5染料标记，分别作为荧光共振能量转移（FRET）的供体和受体对。QD介导的FRET的高信噪比使得能够灵敏地检测多聚物稳定性的离散变化。通过共聚焦显微镜随时间捕获通过QD-FRET的复合物的细胞内摄取和解离。根据基于定量图像的分析，释放的质粒在内/溶酶体、胞质和核隔室内的分布形成了构建三室一级动力学模型的基础。对壳聚糖、聚乙烯亚胺和聚氨基磷酸酯的复合物解包动力学进行了比较，发现其与转染效率相关。因此，结合基于图像的定量的QD-FRET使能的复合物稳定性检测是用于研究涉及活细胞内的复合物解包和运输的机制的有价值的方法。我们预计，这种方法也将有助于设计更有效的基因载体。
A major challenge for non-viral gene delivery is gaining a mechanistic understanding of the rate-limiting steps. A critical barrier in polyplex-mediated gene delivery is the timely unpacking of polyplexes within the target cell to liberate DNA for efficient gene transfer. In this study, the component plasmid DNA and polymeric gene carrier were individually labeled with quantum dots (QDs) and Cy5 dyes, respectively, as a donor and acceptor pair for fluorescence resonance energy transfer (FRET). The high signal-to-noise ratio in QD-mediated FRET enabled sensitive detection of discrete changes in polyplex stability. The intracellular uptake and dissociation of polyplexes through QD-FRET was captured over time by confocal microscopy. From quantitative image - based analysis, distributions of released plasmid within the endo/ lysosomal, cytosolic, and nuclear compartments formed the basis for constructing a three-compartment first-order kinetics model. Polyplex unpacking kinetics for chitosan, polyethylenimine, and polyphosphoramidate were compared and found to correlate well with transfection efficiencies. Thus, QD-FRET-enabled detection of polyplex stability combined with image-based quantification is a valuable method for studying mechanisms involved in polyplex unpacking and trafficking within live cells. We anticipate that this method will also aid the design of more efficient gene carriers.