Rao Gururaj A, Tsai Roger, Roura Daniel, Hughes Jeffrey A
Department of Pharmaceutics, University of Florida, Gainesville, U.S.A.
J Drug Target. 2008 Jan;16(1):79-89. doi: 10.1080/10611860701733328.
Experiments were conducted to evaluate the utility of a peptide receptor ligand to improve transfection efficiency as part of a polyethylenimine-polyethylene glycol (PEI-PEG) polyplex. The 7-mer peptide (MQLPLAT), targeted toward the fibroblast growth factor 2 (FGF2) receptor, was recently identified using a phage-display library method as possessing a high degree of specificity for the FGF2 receptor without the mutagenicity associated with FGF itself. Two approaches (pre-modification or post-modification) to incorporate the peptide into the PEGylated polyplex were compared in terms of their effect on particle size, surface charge, DNA condensation ability, toxicity, cellular uptake and transfection efficiency.
The peptide was conjugated to branched PEI (25 kDa) via a PEG spacer either before (pre-modified) or after (post-modified) complexation of PEI with DNA. Polyethyleneimine was conjugated to the PEG spacer (N-hydroxy succinimide (NHS) -PEG-maleimide (Mal)) through the NHS group. The FGF2 peptide was synthesized to contain a cysteine at the carboxyl end (MQLPLATC) and conjugated to the PEG spacer via the Maleimide group. Conjugates were evaluated using (1)H NMR, amino acid analysis, and picrylsulfonic acid assay. DNA condensation was evaluated using agarose gel electrophoresis and cellular toxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cellular uptake was measured using flow cytometry and transfection efficiency was determined using a luciferase reporter gene assay.
Both pre- and post-modification approaches led to a decrease in the zeta potential of the resulting polyplexes but did not alter their size. The pre-modification of PEI did not affect its ability to condense DNA. However, polyplexes formed with the pre-conjugated PEI did not improve cell uptake or transfection efficiency. In contrast, polyplexes that were post-modified with the FGF2 peptide resulted in a 3-fold increase in cell uptake and a 6-fold increase in transfection efficiency. Both pre- and post-modified polyplexes resulted in lower toxicity compared with unmodified PEI.
The results indicate that the FGF2 peptide improves transfection efficiency when used as part of post-modified PEI/PEG polyplex. When used with pre-modified PEI/PEG, the beneficial effect of the peptide on transfection is not evident, probably because, in this case, the peptide ligand is not readily accessible to the FGF receptor.
进行实验以评估一种肽受体配体作为聚乙烯亚胺 - 聚乙二醇（PEI - PEG）多聚体的一部分来提高转染效率的效用。最近使用噬菌体展示文库方法鉴定出一种靶向成纤维细胞生长因子2（FGF2）受体的7聚体肽（MQLPLAT），它对FGF2受体具有高度特异性，且没有与FGF本身相关的致突变性。比较了将该肽掺入聚乙二醇化多聚体的两种方法（预修饰或后修饰）对粒径、表面电荷、DNA凝聚能力、毒性、细胞摄取和转染效率的影响。
在PEI与DNA络合之前（预修饰）或之后（后修饰），通过聚乙二醇间隔物将该肽与支链PEI（25 kDa）偶联。聚乙烯亚胺通过N - 羟基琥珀酰亚胺（NHS）基团与聚乙二醇间隔物（N - 羟基琥珀酰亚胺（NHS） - 聚乙二醇 - 马来酰亚胺（Mal））偶联。合成的FGF2肽在羧基末端含有一个半胱氨酸（MQLPLATC）并通过马来酰亚胺基团与聚乙二醇间隔物偶联。使用核磁共振氢谱（¹H NMR）、氨基酸分析和苦味磺酸测定法对偶联物进行评估。使用琼脂糖凝胶电泳评估DNA凝聚，使用3 -（4,5 - 二甲基噻唑 - 2 - 基） - 2,5 - 二苯基四氮唑溴盐（MTT）测定法测定细胞毒性。使用流式细胞术测量细胞摄取，并使用荧光素酶报告基因测定法测定转染效率。
预修饰和后修饰方法均导致所得多聚体的zeta电位降低，但未改变其大小。PEI的预修饰不影响其凝聚DNA的能力。然而，用预偶联的PEI形成的多聚体并未提高细胞摄取或转染效率。相比之下，用FGF2肽进行后修饰的多聚体导致细胞摄取增加3倍，转染效率增加6倍。与未修饰的PEI相比，预修饰和后修饰的多聚体毒性均较低。
结果表明，当作为后修饰的PEI / PEG多聚体的一部分使用时，FGF2肽可提高转染效率。当与预修饰的PEI / PEG一起使用时，该肽对转染的有益作用不明显，可能是因为在这种情况下，肽配体不易被FGF受体识别。
