title: Transfection efficiency and uptake process of polyplexes in human lung endothelial cells: a comparative study in non-polarized and polarized cells.
abstract: BACKGROUND
  Following systemic administration, polyplexes must cross the endothelium barrier to deliver genes to the target cells underneath. To design an efficient gene delivery system into lung epithelium, we evaluated capture and transfection efficiencies of DNA complexed with either Jet-PEI (PEI-polyplexes) or histidylated polylysine (His-polyplexes) in human lung microvascular endothelial cells (HLMEC) and tracheal epithelial cells.
  
  
  METHODS
  After optimizing growth conditions to obtain a tight HLMEC monolayer, we characterized uptake of polyplexes by flow cytometry and evaluated their transfection efficiency. Polyplexes were formulated as small particles. YOYO-labelled plasmid fluorescence intensity and luciferase activity were used as readouts for uptake and gene expression, respectively.
  
  
  RESULTS
  PEI-polyplexes were more efficiently taken up than His-polyplexes by both non-polarized (2-fold) and polarized HLMEC (10-fold). They were mainly internalized by a clathrin-dependent pathway whatever the cell state. In non-polarized cells, His-polyplexes entered also mainly via a clathrin-dependent pathway but with an involvement of cholesterol. The cell polarization decreased this way and a clathrin-independent pathway became predominant. PEI-polyplexes transfected more efficiently HLMEC than His-polyplexes (10(7) vs. 10(5) relative light units (RLU)/mg of proteins) with a more pronounced difference in polarized cells. In contrast, no negative effect of the cell polarization was observed with tracheal epithelial cells in which both polyplexes had comparable efficiency.
  
  
  CONCLUSIONS
  We show that the efficiency of polyplex uptake by HLMEC and their internalization mechanism are polymer-dependent. By contrast with His-polyplexes, the HLMEC polarization has little influence on the uptake process and on the transfection efficiency of PEI-polyplexes.
authors.affiliation: Centre de Biophysique Moléculaire UPR4301 CNRS affiliated to the University of Orléans and INSERM, rue Charles Sadron, 45071 Orléans cedex 2, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Mennesson
authors.forename: Patrick
authors.initials: P
authors.lastname: Erbacher
authors.forename: Véronique
authors.initials: V
authors.lastname: Piller
authors.forename: Claudine
authors.initials: C
authors.lastname: Kieda
authors.forename: Patrick
authors.initials: P
authors.lastname: Midoux
authors.forename: Chantal
authors.initials: C
authors.lastname: Pichon
mesh_terms: D001583:Benzoxazoles; D018929:Cell Culture Techniques; D002461:Cell Line, Transformed; D016764:Cell Polarity; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D004705:Endocytosis / Q000187:drug effects; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D018014:Gene Transfer Techniques; D006639:Histidine / Q000737:chemistry; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008168:Lung / Q000098:blood supply*; D009994:Osmolar Concentration; D010957:Plasmids; D011094:Polyethyleneimine / Q000737:chemistry / Q000378:metabolism*; D011107:Polylysine / Q000737:chemistry / Q000378:metabolism; D011806:Quinolinium Compounds; D013696:Temperature; D014132:Trachea / Q000166:cytology; D014162:Transfection / Q000379:methods*
chemical_list: D001583:Benzoxazoles; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D011107:Polylysine; D006639:Histidine; D011094:Polyethyleneimine; D008156:Luciferases