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
- 6 January 2004
- journal article
- research article
- Published by Wiley in The Journal of Gene Medicine
- Vol. 6 (1), 76-84
- https://doi.org/10.1002/jgm.470
Abstract
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 polyplex structure. 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 © 2003 John Wiley & Sons, Ltd.Keywords
This publication has 26 references indexed in Scilit:
- Measuring the pH environment of DNA delivered using nonviral vectors: Implications for lysosomal traffickingBiotechnology & Bioengineering, 2002
- Different behavior of branched and linear polyethylenimine for gene deliveryin vitro andin vivoThe Journal of Gene Medicine, 2001
- Development of Biomaterials for Gene TherapyMolecular Therapy, 2000
- Ovarian carcinoma cells are effectively transfected by polyethylenimine (PEI) derivativesCancer Gene Therapy, 2000
- Comparison between cationic polymers and lipids in mediating systemic gene delivery to the lungsGene Therapy, 1999
- The size of DNA/transferrin-PEI complexes is an important factor for gene expression in cultured cellsGene Therapy, 1998
- Size, diffusibility and transfection performance of linear PEI/DNA complexes in the mouse central nervous systemGene Therapy, 1998
- ExGen 500 is an efficient vector for gene delivery to lung epithelial cells in vitro and in vivoGene Therapy, 1997
- Water-Soluble Polyion Complex Associates of DNA and Poly(ethylene glycol)−Poly(l-lysine) Block CopolymerBioconjugate Chemistry, 1997
- Coupling of cell-binding ligands to polyethylenimine for targeted gene deliveryGene Therapy, 1997