ABSTRACT
Palmitic acid conjugates of poly-L-lysine (PLL-PA) were prepared, and their ability to deliver plasmid DNA into human skin fibroblasts was evaluated in vitro. The conjugates were capable of condensing a 4.7 kb plasmid DNA into 50-200 nm particles (mean +/- SD = 112 +/- 34 nm), which were slightly smaller than the particles formed by PLL (mean +/- SD = 126 +/- 51 nm). Both PLL and PLL-PA were readily taken up by the cells, but PLL-PA delivered the plasmid DNA into a higher proportion of cells. DNA delivery was found to be reduced by endocytosis inhibitor Brefeldin A, suggesting an active mechanism of particle uptake. Using enhanced green fluorescent protein (EGFP) as a reporter gene, PLL-PA was found to give the highest number of EGFP-positive cells among several carriers tested, including polyethyleneimine, Lipofectamine-2000, and an adenovirus. Although some carriers gave a higher percentage of EGFP-positive cells than PLL-PA, they were also associated with higher toxicities. We conclude that PLL-PA is a promising gene carrier for non-viral modification of human fibroblasts.
Subject(s)
DNA/pharmacokinetics , Drug Carriers/chemistry , Fibroblasts/metabolism , Gene Transfer Techniques , Palmitic Acid/chemistry , Polylysine/chemistry , Cells, Cultured , DNA/chemistry , DNA/metabolism , Drug Carriers/chemical synthesis , Drug Carriers/pharmacokinetics , Fibroblasts/chemistry , Humans , Molecular Structure , Palmitic Acid/metabolism , Particle Size , Plasmids/genetics , Polylysine/metabolism , Skin/metabolism , Time FactorsABSTRACT
Nonviral gene carriers are actively explored in gene therapy due to safety concerns of the viral carriers. To design effective gene carriers for modification of bone marrow stromal cells (BMSC), an important cell phenotype for clinical application of gene therapy, cationic polymers polyethyleneimine (PEI), and poly-L-Lysine (PLL) were substituted with palmitic acid (PA) via amide linkages. Depending on the reaction conditions, PEI and PLL was substituted with 2.2-5.2 and 13.4-16.2 PA per polymer chain. The PA substituted polymers displayed slightly lower binding efficiency towards a plasmid containing Enhanced Green Fluorescent Protein (pEGFP) in an agarose gel binding assay. The cell binding of PLL-PA, but not PEI-PA, was particularly enhanced, resulting in higher percentage of the cells displaying a significant polymer uptake. pEGFP delivery into the BMSC was also significantly increased with the PLL-PA (vs. PLL), but not PEI-PA (vs. PEI). The transfection efficiency of PLL-PA was significantly higher ( approximately fivefold) than the unmodified polymer. We conclude that PA substitution on PLL provides an effective carrier for transfection of primary cells derived from the bone marrow.
