Mechanistic insights into linear polyethylenimine-mediated gene transfer.
We recently debuted a variety of linear polyethylenimines (LPEIs) with low molecular weight as carriers for gene delivery. The highest transfection efficiency (approximately 44%) was obtained with LPEI 6.6 kDa, while the cytotoxicity remained low (approximately 90% of CHO-K1 cells survived the transfection procedure). Here, we investigated various steps during the transfection process using LPEI 8.1, 5.0 and 1.8 kDa, in order to gain a more complete insight into LPEI-mediated gene transfer and to explore conceptual aspects for further optimization. The cellular uptake characterized by flow cytometry was similar for LPEI 8.1 and 5.0 kDa, while it was significantly lower for LPEI 1.8 kDa. The transfection efficacy in contrast was at NP 24 20.07% for LPEI 8.1 kDa and 39.71% for LPEI 5.0 kDa. This suggests that the endocytosis seems not to be a decisive parameter that determines the efficacy of a polymer in the transfection process. Real-time PCR investigations revealed that LPEI 1.8 kDa likewise or even better protected plasmid from degradation compared to LPEI 5.0 or 8.1 kDa. Furthermore, we found that 1/6 to 1/3 intact plasmid DNA reached the intracellular compartments after complexation with LPEI 1.8 kDa. Therefore, the amount of plasmid DNA available in the cytoplasm seems not to be a limiting factor in the transfection process. That LPEI 8.1-polyplexes built at NP 12 in glucose and transfected in serum-free culture conditions were superior to those built in sodium chloride or transfected in serum-containing conditions points at the structure as a decisive parameter deserving more attention in future studies.
Breunig
Miriam
M
Department of Pharmaceutical Technology, University of Regensburg, Universitaetsstrasse 31, 93040 Regensburg, Germany.
Lungwitz
Uta
U
Liebl
Renate
R
Klar
Juergen
J
Obermayer
Birgit
B
Blunk
Torsten
T
Goepferich
Achim
A
D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D018613:Microscopy, Confocal; D010957:Plasmids; D011094:Polyethyleneimine / Q000493:pharmacokinetics / Q000494:pharmacology*; D014162:Transfection / Q000379:methods*
D049452:Green Fluorescent Proteins; D011094:Polyethyleneimine