In situ single cell observation by fluorescence resonance energy transfer reveals fast intra-cytoplasmic delivery and easy release of plasmid DNA complexed with linear polyethylenimine
In situ single cell observation by fluorescence resonance energy transfer reveals fast intra-cytoplasmic delivery and easy release of plasmid DNA complexed with linear polyethylenimine
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DOI：
10.1002/jgm.470
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发表时间：
2004-01-01
影响因子：
3.5
通讯作者：
Kataoka, K
中科院分区：
文献类型：
--
作者：
Itaka, K;Harada, A;Kataoka, K
方法利用荧光共振能量转移(FRET)技术，在共聚焦显微镜下研究线状和支化的聚乙烯亚胺(LPEI)、支链聚乙烯亚胺(BPEI)和多聚L赖氨酸(PLL)在细胞内的转运和释放。结果与LPEI形成复合体的PDNA在细胞内的转运和释放发生了明显的变化。由于LPEI/PDNA多链结构的解体，FRET效率显著下降，均匀地扩散到细胞质中。与BPEI络合的PDNA也实现了对内体的快速逃逸。然而，即使在24小时后，PDNA仍然保持着高的FRET效率，这表明BPEI/PDNA复合体具有相当的稳定性，使PDNA保持在凝聚状态。在PLL/PDNA复合体中，既没有观察到内体逃逸，也没有观察到PDNA解聚。这些复合体的细胞内特性与其基因转染率有明显的相关性：与BPEI/PDNA相比，LPEI/PDNA的基因表达更高、更快。原子力显微镜显示BPEI比LPEI更有效地诱导PDNA的缩合，这与复杂的pDNA在细胞质内的有限释放一致。结论LPEI/PDNA在细胞质内的快速逃逸和随后的平稳崩解可能是该复合系统具有良好的转染率的决定因素。这些特性可能特别有利于以迅速的方式实现相当高的基因表达。版权所有(C)2004 John Wiley Sons，Ltd.
Background The investigation into the intracellular mechanisms for gene expression has acquired great impetus for the improvement of the transfection efficiency by a non-viral gene delivery system.Methods Intracellular trafficking as well as release of plasmid DNA (pDNA) complexed with polycations, including linear and branched polyethylenimine (LPEI, BPEI) and poly(L-lysine) (PLL), were explored under confocal microscopy using fluorescence resonance energy transfer (FRET) between a pair of donor-acceptor fluorescent dyes (fluorescein and Cy3) tagged on a single pDNA molecule.Results pDNA complexed with LPEI underwent a rapid escape from the endosomes, spreading uniformly into the cytoplasm with a substantial decrease in FRET efficiency due to the disintegration of LPEI/pDNA polyplexstructure. pDNA complexed with BPEI also achieved a rapid escape from the endosomes. Nevertheless, the pDNA retained high FRET efficiency even after 24 h, indicating an appreciable stability of the BPEI/pDNA polyplex to keep pDNA in a condensed state. In the PLL/pDNA polyplexes, neither endosome escape nor pDNA decondensation was observed. These intracellular characteristics of polyplexes showed a clear correlation to their gene transfection efficiency: The LPEI/pDNA revealed a considerably higher and faster gene expression compared with BPEI/pDNA. Atomic force microscopy revealed that BPEI induced more effective condensation of pDNA than LPEI, being consistent with restricted cytoplasmic release of complexed pDNA.Conclusion Fast endosomal escape and subsequent smooth disintegration of LPEI/pDNA in the cytoplasm are likely to be determining factors for the excellent transfection efficiency of this polyplex system. These properties may be particularly beneficial to achieve appreciably high gene expression in a prompt manner. Copyright (C) 2004 John Wiley Sons, Ltd.