Intracellular DNA release and elimination correlate poorly with transgene expression after non-viral transfection.
The intracellular limiting steps in non-viral gene delivery are still unclear. The purpose of this study was to quantize intracellular DNA release and elimination kinetics after transfection with various non-viral carriers (calcium phosphate precipitates, branched poly(ethyleneimine), poly-L-lysine, DOTAP, DOTAP/DOPE) and to correlate these factors with transgene expression. Intracellular kinetics of DNA was determined by novel quantitative method based on qRT-PCR and DNase treatment. Intracellular elimination of DNA after calcium phosphate transfection was rapid (half-life of 0.37 h) whereas the amount of DNA in the cells was stable for at least 136 h after poly(ethyleneimine) mediated transfection. Intracellular elimination half-lives for DNA delivered by other carrier systems ranged from 9 to 27 h. Calcium phosphate precipitates are not able to protect DNA, which explains the short elimination half-life. In the case of other carriers DNA is after complex removal mostly carrier bound but after 24 h the major fraction of DNA is in the released or loosened state. Overall, neither total nor released amount of intracellular DNA correlates with the transgene expression.
Ruponen
Marika
M
Department of Pharmaceutics, Faculty of Pharmacy, University of Kuopio, P. O. Box 1627, 70211 Kuopio, Finland. marika.ruponen@uku.fi
Arkko
Satu
S
Urtti
Arto
A
Reinisalo
Mika
M
Ranta
Veli-Pekka
VP
D000818:Animals; D002130:Calcium Phosphates / Q000737:chemistry; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004247:DNA / Q000008:administration & dosage / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D008081:Liposomes / Q000737:chemistry; D008156:Luciferases / Q000235:genetics; D010714:Phosphatidylethanolamines / Q000737:chemistry; D011094:Polyethyleneimine / Q000737:chemistry; D011107:Polylysine / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors; D014162:Transfection; D019076:Transgenes
D002130:Calcium Phosphates; D005229:Fatty Acids, Monounsaturated; D008081:Liposomes; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; D011107:Polylysine; D011094:Polyethyleneimine; D004247:DNA; C020243:calcium phosphate; D008156:Luciferases; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane