title: Gene transfer by chemical vectors, and endocytosis routes of polyplexes, lipoplexes and lipopolyplexes in a myoblast cell line.
abstract: Chemical vectors are widely developed for providing safe DNA delivery systems. It is well admitted that their endocytosis and intracellular trafficking are critical for the transfection efficiency. Here, we have compared the endocytic pathways of lipoplexes, polyplexes and lipopolyplexes formed with carriers of various chemical compositions. Engineered C2C12 mouse myoblast cells expressing Rab5-EGFP, Rab7-EGFP or Cav1-GFP were used to monitor the location of the plasmid DNA into the endocytic compartments by real time fluorescence confocal microscopy. We observed that (i) DNA complexes made with dioleyl succinyl paromomycin:O,O-dioleyl-N-histamine phosphoramidate (DOSP/MM27) liposomes or histidinylated lPEI (His-lPEI) allowing the highest transfection efficiency displayed a positive ζ potential and were internalized by clathrin-mediated endocytosis, (ii) DOSP/MM27 lipoplexes were 6-times more internalized than His-lPEI polyplexes, (iii) all negatively charged DNA complexes lead to less efficient transfection and entered the cells via caveolae and (iv) lipopolyplexes allowing high transfection efficiency were weakly internalized via caveolae. Our results indicate that the transfection efficiency is better correlated with the nature of the endocytic pathway than with the uptake efficacy. This study shows also that engineered cells expressing specific fluorescent compartments are convenient tools to monitor endocytosis of a fluorescent plasmid DNA by real time fluorescence confocal microscopy.
authors.affiliation: Centre de Biophysique Moléculaire, CNRS UPR4301, rue Charles Sadron F-45071 Orléans, Cedex 02, Inserm and Université d'Orléans, France.
authors.forename: Ludivine
authors.initials: L
authors.lastname: Billiet
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Gomez
authors.forename: Mathieu
authors.initials: M
authors.lastname: Berchel
authors.forename: Paul-Alain
authors.initials: PA
authors.lastname: Jaffrès
authors.forename: Tony
authors.initials: T
authors.lastname: Le Gall
authors.forename: Tristan
authors.initials: T
authors.lastname: Montier
authors.forename: Emilie
authors.initials: E
authors.lastname: Bertrand
authors.forename: Hervé
authors.initials: H
authors.lastname: Cheradame
authors.forename: Philippe
authors.initials: P
authors.lastname: Guégan
authors.forename: Mathieu
authors.initials: M
authors.lastname: Mével
authors.forename: Bruno
authors.initials: B
authors.lastname: Pitard
authors.forename: Thierry
authors.initials: T
authors.lastname: Benvegnu
authors.forename: Pierre
authors.initials: P
authors.lastname: Lehn
authors.forename: Chantal
authors.initials: C
authors.lastname: Pichon
authors.forename: Patrick
authors.initials: P
authors.lastname: Midoux
mesh_terms: D000818:Animals; D051242:Caveolin 1 / Q000378:metabolism; D016923:Cell Death; D002460:Cell Line; D002470:Cell Survival; D004247:DNA / Q000378:metabolism; D004705:Endocytosis; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry*; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011108:Polymers / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014168:Transferrin / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
chemical_list: D051242:Caveolin 1; D008081:Liposomes; D011108:Polymers; D011993:Recombinant Fusion Proteins; D014168:Transferrin; C485184:enhanced green fluorescent protein; D000090802:rab7 GTP-Binding Proteins; C000716465:rab7 GTP-binding proteins, mouse; D049452:Green Fluorescent Proteins; D004247:DNA; D008156:Luciferases; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins