title: Cellular uptake of cationic polymer-DNA complexes via caveolae plays a pivotal role in gene transfection in COS-7 cells.
abstract: OBJECTIVE
  Knowledge about the uptake mechanism and subsequent intracellular routing of non-viral gene delivery systems is important for the development of more efficient carriers. In this study we compared two established cationic polymers pDMAEMA and PEI with regard to their transfection efficiency and mechanism of cellular uptake.
  
  
  METHODS
  The effects of several inhibitors of particular cellular uptake routes on the uptake of polyplexes and subsequent gene expression in COS-7 cells were investigated using FACS and transfection. Moreover, cellular localization of fluorescently labeled polyplexes was assessed by spectral fluorescence microscopy.
  
  
  RESULTS
  Both pDMAEMA- and PEI-complexed DNA showed colocalization with fluorescently-labeled transferrin and cholera toxin after internalization by COS-7 cells, which indicates uptake via the clathrin- and caveolae-dependent pathways. Blocking either routes of uptake with specific inhibitors only resulted in a marginal decrease in polyplex uptake, which may suggest that uptake routes of polyplexes are interchangeable. Despite the marginal effect of inhibitors on polyplex internalization, blocking the caveolae-mediated uptake route resulted in an almost complete loss of polyplex-mediated gene expression, whereas gene expression was not negatively affected by blocking the clathrin-dependent route of uptake.
  
  
  CONCLUSIONS
  These results show the importance of caveolae-mediated uptake for successful gene expression and have implications for the rational design of non-viral gene delivery systems.
authors: Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, P.O. Box 80082, 3508 TB, Utrecht, The Netherlands.
authors: M A E M
authors: MA
authors: van der Aa
authors: U S
authors: US
authors: Huth
authors: S Y
authors: SY
authors: Häfele
authors: R
authors: R
authors: Schubert
authors: R S
authors: RS
authors: Oosting
authors: E
authors: E
authors: Mastrobattista
authors: W E
authors: WE
authors: Hennink
authors: R
authors: R
authors: Peschka-Süss
authors: G A
authors: GA
authors: Koning
authors: D J A
authors: DJ
authors: Crommelin
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D019556:COS Cells; D021941:Caveolae / Q000187:drug effects / Q000378:metabolism*; D002522:Chlorocebus aethiops; D002746:Chlorpromazine / Q000494:pharmacology; D002772:Cholera Toxin / Q000378:metabolism / Q000493:pharmacokinetics; D022163:Clathrin-Coated Vesicles / Q000187:drug effects / Q000378:metabolism; D004247:DNA / Q000737:chemistry*; D004705:Endocytosis / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry; D019833:Genistein / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics; D008689:Methacrylates / Q000737:chemistry; D008856:Microscopy, Fluorescence; D015739:Nocodazole / Q000494:pharmacology; D009757:Nylons / Q000737:chemistry; D011073:Polyamines / Q000737:chemistry*; D000071228:Polyelectrolytes; D011094:Polyethyleneimine / Q000737:chemistry; D014162:Transfection / Q000379:methods; D014168:Transferrin / Q000378:metabolism / Q000493:pharmacokinetics; D000077191:Wortmannin; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D000730:Androstadienes; D002746:Chlorpromazine; D002772:Cholera Toxin; D004247:DNA; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D019833:Genistein; D008156:Luciferases; D046911:Macromolecular Substances; D008689:Methacrylates; D015739:Nocodazole; D009757:Nylons; D011073:Polyamines; D000071228:Polyelectrolytes; D011094:Polyethyleneimine; D014168:Transferrin; D000077191:Wortmannin; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C407037:poly(2-(dimethylamino)ethyl methacrylate)