Gene expression and internalization following vector adsorption to immobilized proteins: dependence on protein identity and density
Gene expression and internalization following vector adsorption to immobilized proteins: dependence on protein identity and density
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DOI：
10.1002/jgm.1058
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发表时间：
2007-08-01
影响因子：
3.5
通讯作者：
Shea, Lonnie D.
中科院分区：
文献类型：
--
作者：
Bengali, Zain;Rea, Jennifer C.;Shea, Lonnie D.
通过非病毒载体非特异性吸附到蛋白质包被的表面的基因递送可以减少所需的DNA量，并且还增加转基因表达和表达转基因的细胞的数量。表面上的蛋白质介导细胞粘附和载体固定，并起到使两者共定位以增强基因递送的作用。这份报告调查的机制和特异性，其中蛋白质涂层增强基因transfer，并确定如果蛋白质涂层靶向的载体内化通过一个特定的pathway.Methods蛋白质（FBS，BSA，纤连蛋白，胶原蛋白I，和层粘连蛋白）干燥到培养皿上，然后由PEI/DNA复合物吸附表面delivery。采用报告基因来表征转染作为蛋白质身份和密度的函数。载体固定使用放射性标记的质粒进行测量，并在存在和不存在的内吞抑制剂氯丙嗪和genistein.Results纤连蛋白涂层的情况下进行量化的内化产生的PEI/DNA复合物的最大表达，在中间蛋白质密度的最大表达。在使用推注递送的对照研究中的表达与蛋白质身份无关。底物结合是独立的蛋白质的身份，然而，内化是最大的纤连蛋白和胶原蛋白I包被的表面上。抑制小窝介导的内吞作用比网格蛋白介导的内吞作用更能降低基因表达。同样，抑制小窝介导的内吞作用显着降低了细胞内水平的DNA.Conclusions纤连蛋白在中间密度介导的最高水平的转基因表达，可能通过靶向通过小窝介导的内吞内化。底物修饰，如蛋白质的同一性和密度，为修饰生物材料以增强基因表达提供了机会。版权所有（c）2007约翰威利父子有限公司。
Background Gene delivery by non-specific adsorption of non-viral vectors to protein-coated surfaces can reduce the amount of DNA required, and also increase transgene expression and the number of cells expressing the transgene. The protein on the surface mediates cell adhesion and vector immobilization, and functions to colocalize the two to enhance gene delivery. This report investigates the mechanism and specificity by which the protein coating enhances gene transfer, and determines if the protein coating targets the vector for internalization by a specific pathway.Methods Proteins (FBS, BSA, fibronectin, collagen I, and laminin) were dried onto culture dishes, followed by PEI/DNA complex adsorption for surface delivery. Reporter genes were employed to characterize transfection as a function of the protein identity and density. Vector immobilization was measured using radiolabeled plasmid, and internalization was quantified in the presence and absence of the endocytosis inhibitors chlorpromazine and genistein.Results Fibronectin coating yielded the greatest expression for PEI/DNA polyplexes, with maximal expression at intermediate protein densities. Expression in control studies with bolus delivery was independent of the protein identity. Substrate binding was independent of the protein identity; however, internalization was greatest on surfaces coated with fibronectin and collagen I. Inhibition of caveolae-mediated endocytosis reduced gene expression more than clathrin-mediated endocytosis. Similarly, inhibition of caveolae-mediated endocytosis significantly reduced the intracellular levels of DNA.Conclusions Fibronectin at intermediate densities mediated the highest levels of transgene expression, potentially by targeting internalization through caveolae-mediated endocytosis. Substrate modifications, such as the identity and density of proteins, provide an opportunity for modification of biomaterials for enhancing gene expression. Copyright (c) 2007 John Wiley & Sons, Ltd.