A comparison of the effectiveness of cationic polymers poly-L-lysine (PLL) and polyethylenimine (PEI) for non-viral delivery of plasmid DNA to bone marrow stromal cells (BMSC).
Bone marrow stromal cells (BMSC) represent an important cell phenotype for pursuit of successful gene therapy. Non-viral methods to enable expression of exogenous genes in BMSC will accelerate clinical application of gene therapy, without the concerns associated with the viral means of gene transfer. Towards this end, this study investigated the potential of cationic polymers poly-L-lysine (PLL) and branched polyethylenimine (PEI) as gene carriers for modification of BMSC. Both polymers rapidly (approximately 30 min) condensed a 4.2 kb Enhanced Green Fluorescent Protein (pEGFP-N2) plasmid into 100-200 nm particles. PLL and PEI were both readily internalized with BMSC with >80% of BMSC exhibiting polymer uptake by flow cytometric analysis. The relative uptake of PEI, however, was significantly higher as compared to the PLL. The majority of the BMSC (>60%) exhibited nuclear presence of the polymers as analyzed by fluorescent microscopy. Although both polymers were able to deliver the pEGFP-N2 into the cells under microscopic evaluation, only a small fraction of the cells (<10%) displayed nuclear localization of the plasmid. Consistent with better uptake, PEI gave a higher delivery of pEGFP-N2 into the BMSC, which resulted in a more sustained expression of the model gene EGFP in short-term (7-day) culture. We conclude that both PLL and PEI readily displayed cellular uptake, but PEI was more effective in delivering plasmid DNA intracellularly, which was likely the underlying basis for a more sustained gene expression.
Farrell
Laura-Lee
LL
Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alta., Canada.
Pepin
Joel
J
Kucharski
Cezary
C
Lin
Xiaoyue
X
Xu
Zhenghe
Z
Uludag
Hasan
H
D021581:Active Transport, Cell Nucleus; D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism*; D002412:Cations; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008856:Microscopy, Fluorescence; D010316:Particle Size; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011094:Polyethyleneimine / Q000737:chemistry* / Q000378:metabolism; D011107:Polylysine / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017154:Stromal Cells / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D002412:Cations; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D011107:Polylysine; D011094:Polyethyleneimine