Poly(ethylenimine)-mediated transfection: a new paradigm for gene delivery.
Poly(ethylenimine) (PEI) is a synthetic polycation that has been used successfully for gene delivery both in vitro and in vivo due, in theory, to a form of protection that is afforded to the carried plasmids. In this study the stability of PEI/DNA complexes was demonstrated using deoxyribonuclease (DNase) 1 and DNase 2, various levels of pH, and increasing exposure times. DNA that was complexed with PEI was not degraded when exposed to at least 25 Units of either enzyme for 24 h while uncomplexed forms of the same plasmid were digested when exposed to 0.010 Units of DNase 1 for 0.05 h or 0. 003 Units of DNase 2 for 1 h. For further comparison, the stability of complexes made with poly(L-lysine) (PLL) and DNA was examined and found to be lower than that of PEI/DNA complexes; PLL-complexed DNA was digested on exposure to 1.25 Units of DNase 1 for 3 min. Cells were transfected with PEI/DNA complexes and, by using a pH indicator and optical recording techniques, it was found that the normal lysosomal pH value of 5.0 was not altered, bringing into question PEI's hypothesized lysosomal entry. Confocal microscopy showed that PEI/DNA complexes and lysosomes do not merge during transfection (although PLL/DNA complexes do). The lack of lysosomal involvement in PEI-mediated transfection is surprising because it goes against the conventional wisdom that has attempted to explain how PEI functions during transfection. PEI forms a stable complex with DNA, which moves from endocytosis to nuclear entry without significant cellular obstacles.
Godbey
W T
WT
Department of Bioengineering, Rice University, P.O. Box 1892, MS 142, Houston, Texas 77251-1892, USA.
Barry
M A
MA
Saggau
P
P
Wu
K K
KK
Mikos
A G
AG
D000818:Animals; D001672:Biocompatible Materials; D002021:Buffers; D002460:Cell Line; D004274:DNA, Recombinant / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D003851:Deoxyribonucleases; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008247:Lysosomes / Q000378:metabolism; D008422:Materials Testing; D011094:Polyethyleneimine; D014162:Transfection / Q000379:methods*
D001672:Biocompatible Materials; D002021:Buffers; D004274:DNA, Recombinant; D011094:Polyethyleneimine; D003851:Deoxyribonucleases