Increased receptor-mediated gene delivery to the liver by protamine-enhanced-asialofetuin-lipoplexes
Increased receptor-mediated gene delivery to the liver by protamine-enhanced-asialofetuin-lipoplexes
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DOI：
10.1038/sj.gt.3301840
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发表时间：
2003-01-01
期刊：
影响因子：
5.1
通讯作者：
de Ilarduya, CT
中科院分区：
文献类型：
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作者：
Arangoa, MA;Düzgünes, N;de Ilarduya, CT
开发了一种由 DOTAP/Chol 脂质体、脱唾液酸胎球蛋白 (AF)、硫酸鱼精蛋白和 DNA 组成的新型脂质载体。由此产生的鱼精蛋白-AF-脂质复合物显着提高了培养细胞和静脉注射后肝脏中的基因表达水平。行政。含有最佳量 AF（1 微克/微克 DNA）的脂质复合物在 HepG2 细胞中的转染活性比非靶向（纯）复合物高 16 倍。添加游离AF后，其质膜上具有脱唾液酸糖蛋白受体（ASGPr）的细胞的摄取量减少，表明AF-脂质复合物通过ASGPr介导的内吞作用被细胞特异性摄取。在 ASGPr 有缺陷的细胞（即 HeLa 细胞）中进行的转染结果证实了这种机制。通过添加缩合肽硫酸鱼精蛋白，获得了更小的复合物，这进一步增强了 HepG2 细胞和肝脏中 AF 复合物的摄取。鱼精蛋白的最佳量为 0.4 微克/微克 DNA，基因表达量比不存在肽时使用 AF-lipoplex 获得的基因表达量高出约 5 倍，比使用普通传统 lipoplex 获得的基因表达量高出 75 倍。与普通复合物相比，通过尾静脉全身给药的小鼠肝脏中的鱼精蛋白-AF-脂质复合物使荧光素酶基因表达增加了 12 倍。总之，我们的研究结果扩展了之前使用 AF-lipoplex 将 DNA 引入肝细胞的研究范围。靶向和鱼精蛋白浓缩的结合消除了部分肝切除术的需要，而部分肝切除术通常需要在该器官中获得有效的基因递送。由于硫酸谷醇溶蛋白已被证明对人体无毒，因此本文描述的新型肝脏特异性载体可能有助于将临床重要基因递送至该器官。
A novel lipidic vector composed of DOTAP/Chol liposomes, asialofetuin (AF), protamine sulfate and DNA has been developed. The resulting protamine-AF-lipoplexes improved significantly the levels of gene expression in cultured cells and in the liver upon i.v. administration. Lipoplexes containing the optimal amount of AF (1 mug/mug DNA) showed a 16-fold higher transfection activity in HepG2 cells than non-targeted (plain) complexes. The uptake by cells having asialoglycoprotein receptors (ASGPr) on their plasma membrane was decreased by the addition of free AF, indicating that AF-lipoplexes were taken up specifically by cells via ASGPr-mediated endocytosis. Results from transfections performed in cells defective in ASGPr, ie HeLa cells, confirmed this mechanism. By addition of the condensing peptide, protamine sulfate, smaller complexes were obtained, which enhanced even more the uptake of AF-complexes in HepG2 cells and in the liver. The optimal amount of protamine was 0.4 mug/mug DNA, and gene expression was about 5-fold over that obtained with AF-lipoplexes in the absence of the peptide, and 75-fold higher than that with plain conventional lipoplexes. Protamine-AF-lipoplexes increased by a factor of 12 luciferase gene expression in the liver of mice administered systemically via the tail vein, compared to plain complexes. In summary, our findings extend the scope of previous studies where AF-lipoplexes were used to introduce DNA into hepatocytes. The combination of targeting and protamine condensation obviated the need for partial hepatectomy, commonly required to obtain efficient gene delivery in this organ. Since prolamine sulfate has been proven to be non-toxic in humans, the novel liver-specific vector described here may be useful for the delivery of clinically important genes to this organ.