Gene delivery with low molecular weight linear polyethylenimines.
BACKGROUND
Linear polyethylenimine (LPEI) with a molecular weight (MW) of 22 kDa has been described as having a superior ability to induce gene transfer compared to its branched form. However, the transfection efficiency of the polymer cannot be enhanced beyond a certain limit due to cytotoxicity. We explored the potential of utilizing LPEIs with MWs ranging from 1.0 to 9.5 kDa to overcome this limitation.


METHODS
Polyplexes of plasmid DNA encoding for the enhanced green fluorescent protein (EGFP) and various LPEIs were compared concerning their transfection efficiency and cytotoxicity in CHO-K1 and HeLa cells by flow cytometry. The involvement of endolysosomes in LPEI-mediated gene transfer was investigated by applying the proton pump inhibitor bafilomycin A1 and the lysosomotropic agent sucrose. Confocal laser scanning microscopy was applied to assess the size and shape of polyplexes under cell culture conditions, to detect their endolysosomal localization and to observe their translocation to the nucleus.


RESULTS
The transfection efficiency could be altered by varying the MW and the amount of the polymer available for polyplex formation. The highest transfection efficiency (about 44%), i.e. the fraction of EGFP-positive cells, was obtained with LPEI 5.6 kDa, while the cytotoxicity remained low. The colocalization of polyplexes and endolysosomes was observed, and it appeared that the larger polyplexes escaped from the acidic organelles particularly quickly. For LPEI 5.0 and 9.0 kDa, the number of cells and nuclei that had taken up DNA after 6 hours was similar, as determined by flow cytometry.


CONCLUSIONS
Our study suggests that LPEIs with low MWs are promising candidates for non-viral gene delivery, because they are more efficient and substantially less toxic than their higher MW counterparts.
Breunig
Miriam
M
Department of Pharmaceutical Technology, University of Regensburg, Universitaetsstrasse 31, 93040 Regensburg, Germany.
Lungwitz
Uta
U
Liebl
Renate
R
Fontanari
Claudia
C
Klar
Juergen
J
Kurtz
Armin
A
Blunk
Torsten
T
Goepferich
Achim
A
D000818:Animals; D001693:Biological Transport, Active; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000008:administration & dosage*; D004337:Drug Carriers; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D018942:Macrolides / Q000494:pharmacology; D018613:Microscopy, Confocal; D010957:Plasmids; D011094:Polyethyleneimine / Q000009:adverse effects / Q000737:chemistry*; D013395:Sucrose / Q000494:pharmacology; D014162:Transfection
D004337:Drug Carriers; D004791:Enzyme Inhibitors; D018942:Macrolides; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D013395:Sucrose; C040929:bafilomycin A1; D011094:Polyethyleneimine; D004247:DNA