Effective polyethylenimine-mediated gene transfer into human endothelial cells.
BACKGROUND
The major advantage in choosing non-viral vectors such as cationic polymers for in vitro and in vivo transfection is their higher biosafety than viral ones. Among the cationic polymers, polyethylenimines (PEIs) are promising molecules for gene delivery to a variety of cells. Efficient transfection of primary endothelial cells using PEIs could be regarded as an interesting strategy of treatment in some ischemic cardiovascular diseases.


METHODS
Efficacies of a 22-kDa linear PEI (L-PEI) and its glucose-grafted derivative (L-PEI-Glc(4)) were compared for gene transfer into human umbilical vein endothelial cells (HUVEC) using the reporter gene luciferase. Cells were incubated for 2, 4 and 24 h with PEI/DNA complexes made in 150 mM sodium chloride (NaCl) or in 5% glucose solution. Luciferase activity was measured 24 h after the onset of transfection. The effects of low (2%) and high (30%) concentrations of serum on transfection efficacy were assessed as well. We then studied the intracellular fate of the PEI/DNA complexes labelled with the DNA intercalator YOYO-1 using flow cytometry analysis (FACS) and confocal microscopy.


RESULTS
PEI/DNA complexes formed in NaCl led to a higher transfection efficacy than those made in glucose. The optimal formulation, depending on the incubation time and the presence of serum in the medium, was obtained using DNA complexed to L-PEI-Glc(4) and incubated for 4 h with the cells. This condition led to 50% fluorescent cells after GFP transfection. A high serum concentration diminished the L-PEI associated toxicity but decreased L-PEI-Glc(4) transfection efficiency. FACS analysis using both vectors showed that almost 90% of the cells had internalized the DNA complexes. Confocal microscopic observations showed a fast attachment of the complexes to the cell surface followed by inclusion into vesicles that migrated to the perinuclear region.


CONCLUSIONS
In this work, we defined the optimal conditions for gene delivery in HUVEC. These conditions were obtained when using derivatives L-PEI and L-PEI-Glc(4) complexed with DNA in 150 mM NaCl and added to cells for 2 and 4 h, respectively. Cellular trafficking of the complexes suggested that cell entry was not a limiting factor for gene delivery using PEI. This study underlined the interest in PEIs as efficient vectors for gene transfer into human endothelial cells.
Laboratoire de Recherche sur l'Angiogenèse, Faculté de Médecine, Strasbourg, France.
Violeta
V
Zaric
Denis
D
Weltin
Patrick
P
Erbacher
Jean-Serge
JS
Remy
Jean-Paul
JP
Behr
Dominique
D
Stephan
D004247:DNA / Q000378:metabolism; D042783:Endothelial Cells; D005434:Flow Cytometry; D006801:Humans; D018613:Microscopy, Confocal; D010957:Plasmids / Q000378:metabolism; D011094:Polyethyleneimine / Q000378:metabolism / Q000633:toxicity; D044967:Serum; D014162:Transfection; D014471:Umbilical Veins
D011094:Polyethyleneimine; D004247:DNA