Further investigation of lipid-substituted poly(L-Lysine) polymers for transfection of human skin fibroblasts.
Enabling gene expression in skin fibroblasts using safe, nonviral gene delivery has the potential to stimulate wound healing and aid in skin tissue engineering efforts. In this study, several lipid-substituted poly(L-Lysines) (PLL) were investigated for their ability to deliver a plasmid DNA (pEGFP) to human skin fibroblasts. While native and lipid-substituted PLLs showed complete complexation with pEGFP, polymers with higher lipid substitution were more resilient to dissociation after heparin treatment. All polymers showed good protection of pEGFP against DNase I and DNase II digestion in vitro. DNA delivery studies using fluorescently labeled pEGFP showed that native PLL lacked the ability to deliver pEGFP into cells, whereas most of the lipid-substituted PLLs gave efficient pEGFP delivery into the cells. Extent of lipid substitution was an important factor in DNA delivery efficiency. The intracellular pEGFP was intact after delivery with lipid-substituted polymers up to 7 days. An RT-PCR methodology indicated successful transcription of the reporter GFP gene, which was not the case when the cells were transfected with a blank plasmid containing no functional GFP gene. Further studies with flow cytometry showed that successful protein expression was obtained with PLLs substituted with myristic and stearic acid, the latter displaying a relatively lower toxicity. We conclude that substituting lipids on PLL results in effective gene carriers and the extent of substitution, rather than the individual lipid, appeared to be critical for effective plasmid delivery.
Abbasi
Meysam
M
Department of Biomedical Engineering, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
Uludag
Hasan
H
Incani
Vanessa
V
Hsu
Charlie Yu Ming
CY
Jeffery
Andrea
A
D053938:DNA Fragmentation; D003851:Deoxyribonucleases / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D005347:Fibroblasts / Q000378:metabolism*; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D019814:Myristic Acid / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011107:Polylysine / Q000138:chemical synthesis / Q000737:chemistry*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012867:Skin / Q000378:metabolism*; D013229:Stearic Acids / Q000737:chemistry; D014162:Transfection
D008055:Lipids; D013229:Stearic Acids; C485184:enhanced green fluorescent protein; D019814:Myristic Acid; D049452:Green Fluorescent Proteins; D011107:Polylysine; C031183:stearic acid; D003851:Deoxyribonucleases