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.

RGD conjugation to polyethyleneimine does not improve DNA delivery to bone marrow stromal cells.

Bone marrow stromal cells (BMSC) modified with therapeutic genes are being actively pursued for gene therapy protocols. To develop safe and effective nonviral methods for BMSC modification, the cationic polymer polyethyleneimine (PEI) has been utilized to condense plasmid DNA for intracellular delivery. This study was conducted to explore the feasibility of increasing the PEI's effectiveness by coupling integrin-binding arginine-glycine-aspartic acid (RGD) peptides to the polymer. BMSC from rats were isolated and expanded in culture for gene transfer studies. In contrast to our expectations, RGD-conjugated PEI did not exhibit an enhanced binding to BMSC. This was the case where the peptides were conjugated to PEI by short, disulfide linkages or long poly(ethylene glycol) linkages. Using a reporter gene for the enhanced green fluorescent protein, the transfection efficiency of RGD-conjugated PEI was also lower than the delivery by the native PEI, which exhibited equivalent transfection efficiency to that of an adenovirus. We conclude that native PEI was sufficient for the transformation of BMSC and that coupling of the integrin-binding RGD-peptides did not improve the effectiveness of this polymer for BMSC transfection.