Further investigation of lipid-substituted poly(L-Lysine) polymers for transfection of human skin fibroblasts
Further investigation of lipid-substituted poly(L-Lysine) polymers for transfection of human skin fibroblasts
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DOI：
10.1021/bm800132n
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发表时间：
2008-06-01
影响因子：
6.2
通讯作者：
Jeffery, Andrea
中科院分区：
文献类型：
--
作者：
Abbasi, Meysam;Uludag, Hasan;Jeffery, Andrea
在皮肤成纤维细胞中使用安全的、非病毒的基因传递使基因表达具有刺激伤口愈合和帮助皮肤组织工程努力的潜力。在这项研究中，研究了几种脂质取代的聚l -赖氨酸（PLL）向人皮肤成纤维细胞传递质粒DNA （pEGFP）的能力。虽然天然pll和脂质取代的pll与pEGFP完全配合，但脂质取代较高的聚合物在肝素处理后更容易解离。所有聚合物对DNase 1和DNase 11的体外酶切均有良好的保护作用。使用荧光标记pEGFP的DNA传递研究表明，天然PLL缺乏将pEGFP传递到细胞中的能力，而大多数脂质取代的PLL能够有效地将pEGFP传递到细胞中。脂质取代程度是影响DNA传递效率的重要因素。细胞内的pEGFP在脂质取代聚合物递送后的7天内是完整的。RT-PCR方法表明报告基因GFP转录成功，而用不含功能GFP基因的空白质粒转染细胞时则不是这样。进一步的流式细胞术研究表明，用肉豆蔻酸和硬脂酸代替pll可以成功表达蛋白，硬脂酸的毒性相对较低。我们得出结论，在PLL上取代脂质会产生有效的基因载体，而取代的程度，而不是单个脂质，似乎是有效质粒递送的关键。
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 11 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.