title: Early entry and deformation of macropinosomes correlates with high efficiency of decaarginine-polyethylene glycol-lipid-mediated gene delivery.
abstract: BACKGROUND
  Decaarginine-polyethylene glycol-conjugated 3,5-bis(dodecyloxy)benzamide/plasmid DNA [Arg10-polyethylene glycol (PEG)-lipid/plasmid DNA (pDNA)] complexes (designated R10B/DNA complexes) are efficient nonviral carriers for pDNA delivery into human cervical carcinoma HeLa cells. Previous reports indicated that these complexes formed at a relatively low R10B/DNA ratio and showed high transgene expression efficiency. However, the intracellular behaviour of the two different nanostructures, which leads to differences in gene delivery, remains to be elucidated.
  
  
  METHODS
  R10B/DNA complexes prepared at a N/P ratio of 8.5/1 or 42.5/1, corresponding to 5 µm or 25 µm R10B, respectively, were added to HeLa cells, and their uptake and subsequent intracellular fate were examined by cell imaging using electron microscopy (EM) and correlative light-electron microscopy (CLEM).
  
  
  RESULTS
  EM and CLEM analyses revealed that R10B/DNA complexes formed at the lower N/P ratio were mainly taken up by the cells through macropinocytosis, whereas R10B/DNA complexes formed at the higher N/P ratio bound to protruding membrane structures or permeated into the cells by a different pathway. In cells expressing the transgene, R10B/DNA complexes were observed both in macropinosomes and in the cytoplasm. In addition, these cells had macropinosomes with disrupted membranes. These results suggest that cellular uptake through macropinocytosis and subsequent disruption of the macropinosome membrane may be a critical step for R10B-mediated gene delivery.
  
  
  CONCLUSIONS
  We have shown that the existence of R10B/DNA complexes in macropinosomes at the early stages of gene delivery correlates with high efficiency R10B-mediated gene delivery. This finding will provide valuable insights for the engineering of more efficient gene delivery systems based on oligoarginine-mediated carriers.
authors.affiliation: Advanced ICT Research Institute Kobe, National Institute of Information and Communications Technology, Iwaoka, Kobe, Japan.
authors.forename: Shouhei
authors.initials: S
authors.lastname: Kobayashi
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Hattori
authors.forename: Hiroko
authors.initials: H
authors.lastname: Osakada
authors.forename: Kazunori
authors.initials: K
authors.lastname: Toma
authors.forename: Yoshie
authors.initials: Y
authors.lastname: Maitani
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Hiraoka
authors.forename: Tokuko
authors.initials: T
authors.lastname: Haraguchi
mesh_terms: D004247:DNA / Q000008:administration & dosage / Q000737:chemistry; D018014:Gene Transfer Techniques; D006367:HeLa Cells; D006801:Humans; D008055:Lipids / Q000737:chemistry; D008081:Liposomes; D010455:Peptides / Q000737:chemistry*; D020959:Polyethylene / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D014162:Transfection
chemical_list: D008055:Lipids; D008081:Liposomes; D010455:Peptides; C015462:polyarginine; D011092:Polyethylene Glycols; D020959:Polyethylene; D004247:DNA