NONENZYMATIC GLYCOSYLATION OF POLY-L-LYSINE - A NEW TOOL FOR TARGETED GENE DELIVERY
NONENZYMATIC GLYCOSYLATION OF POLY-L-LYSINE - A NEW TOOL FOR TARGETED GENE DELIVERY
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DOI：
10.1002/hep.1840200633
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发表时间：
1994-12-01
期刊：
影响因子：
13.5
通讯作者：
MARTINEZHERNANDEZ, A
中科院分区：
文献类型：
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作者：
MARTINEZFONG, D;MULLERSMAN, JE;MARTINEZHERNANDEZ, A
靶向基因递送的基本方法依赖于载体和分子之间的复合物的形成，该复合物将被靶细胞选择性地内化。在肝细胞的情况下，由于肝细胞半乳糖受体的高亲和力和亲合力，脱唾液酸糖蛋白是方便的靶向分子。在该系统中，多聚-L-赖氨酸与脱唾液酸糖蛋白交联，所得缀合物与表达载体（DNA）复合。DNA和聚-L-赖氨酸-去唾液酸糖蛋白之间的静电结合确保了静脉注射的复合物递送到肝脏，在那里它被肝细胞内吞。然而，聚-L-赖氨酸-脱唾液酸糖蛋白复合物往往不稳定，溶解度有限，且碳水化合物含量有限。出于这些原因，我们寻找了一个更简单的替代方案。我们利用已知的还原糖与蛋白质中的ε-氨基还原偶联的能力，并使用乳糖获得具有“暴露”半乳糖的聚-L-赖氨酸。在高pH硼酸盐缓冲液中用氰基硼氢化钠进行糖基化是一种简单、可重复的方法。“乳糖基化"聚-L-赖氨酸已被证明非常稳定，高度可溶，易于与质粒结合。在一组实验中，我们通过在培养物中分离肝癌细胞（HepG 2）来比较去唾液酸胎球蛋白-聚-L-赖氨酸载体复合物与乳糖基化聚-L-赖氨酸载体复合物。对于这些实验，我们使用了含有突变TGF-β 1互补DNA的pRc/巨细胞病毒真核表达载体。在Northern印迹分析中，用乳糖基化聚-L-赖氨酸转染的细胞表达的TGF-β 1信使RNA比用与去唾液酸胎球蛋白-聚-L-赖氨酸偶联的相同质粒转染的细胞多10至20倍。因此，糖基化聚-L-赖氨酸是聚-L-赖氨酸-无唾液酸蛋白复合物的简单、高效的替代物。此外，使用不同的二糖可以允许复合物靶向不同的细胞类型。
The basic approach in targeted gene delivery relies on the formation of a complex between a vector and a molecule that will be selectively internalized by the target cells. In the case of hepatocytes, asialoglycoproteins are convenient targeting molecules because of the high affinity and avidity of the hepatocyte galactose receptor. In this system, poly-L-lysine is cross-linked to an asialoglycoprotein, and the resulting conjugate is complexed with the expression vector (DNA). The electrostatic binding between DNA and poly-L-lysine-asialoglycoprotein ensures delivery of the intravenously injected complex to the liver, where it is subjected to endocytosis by hepatocytes. However, the poly-L-lysine-asialoglycoprotein complexes tend to be unstable, of limited solubility and of searched carbohydrate content. For these reasons we searched for a simpler alternative. We exploited the known capacity of reducing sugars to be reductively coupled to the epsilon-amino groups in proteins and used lactose to obtain poly-L-lysine with ''exposed'' galactose. Glycosylation with sodium cyanoborohydride at high pH in borate buffer is a simple, reproducible procedure. The ''lactosylated'' poly-L-lysine has proved very stable, highly soluble and easily bound to plasmids. In a set of experiments we compared the asialofetuin-poly-L-lysine vector complexes with lactosylated poly-L-lysine vector complexes by transfecting hepatoma cells (HepG2) in culture. For these experiments we used a pRc/cytomegalovirus eukaryotic expression vector containing a mutant TGF-beta 1 complementary DNA. On Northern-blot analysis, cells transfected with lactosylated poly-L-lysine expressed 10 to 20 times more TGF-beta 1 messenger RNA than did cells transfected with the same plasmid coupled to asialofetuin-poly-L-lysine. Therefore glycosylated poly-L-lysine is a simple, highly effective alternative to poly-L-lysine-asialoprotein complexes. Furthermore the use of different disaccharides may permit targeting of the complexes to different cell types.