title: 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.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113- 8656, Japan.
authors.forename: Keiji
authors.initials: K
authors.lastname: Itaka
authors.forename: Atsushi
authors.initials: A
authors.lastname: Harada
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Yamasaki
authors.forename: Kozo
authors.initials: K
authors.lastname: Nakamura
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kawaguchi
authors.forename: Kazunori
authors.initials: K
authors.lastname: Kataoka
mesh_terms: D018929:Cell Culture Techniques; D003593:Cytoplasm; D004247:DNA / Q000032:analysis*; D005434:Flow Cytometry; D005453:Fluorescence; D020869:Gene Expression Profiling; D018014:Gene Transfer Techniques; D006801:Humans; D018625:Microscopy, Atomic Force; D018613:Microscopy, Confocal; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics*; D011094:Polyethyleneimine; D014162:Transfection
chemical_list: D011094:Polyethyleneimine; D004247:DNA