Enhanced delivery of antisense oligonucleotides with fluorophore-conjugated PAMAM dendrimers.
Enhanced delivery of antisense oligonucleotides with fluorophore-conjugated PAMAM dendrimers.
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DOI：
10.1093/nar/28.21.4225
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发表时间：
2000-11
影响因子：
14.9
通讯作者：
Hoon Yoo;Rudy L. Juliano
中科院分区：
文献类型：
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作者：
Hoon Yoo;Rudy L. Juliano
PAMAM树枝状聚合物是已用于将基因和寡核苷酸递送至细胞的阳离子聚合物。然而，很少有人知道树枝状聚合物-核酸复合物的行为，一旦他们到达细胞内部。为了解决这个问题，我们制备了与荧光染料俄勒冈州绿色488缀合的树枝状聚合物。这些与用红色（塔姆拉）荧光团标记的寡核苷酸结合使用，以便通过双色数字荧光显微镜观察树枝状聚合物-寡核苷酸复合物及其组分的亚细胞分布。这些研究中使用的2 '-O：-甲基反义寡核苷酸序列被设计用于纠正插入荧光素酶报告基因的异常内含子处的剪接;因此，反义试剂的有效递送导致报告基因产物的表达。树枝状聚合物-寡核苷酸复合物在摄取到囊泡隔室和最终进入细胞核的过程中保持相关。由于反义化合物的药理学活性在这些条件下表现出来，这表明树枝状聚合物-寡核苷酸复合物具有功能活性。这些研究的令人惊讶的结果是，与未修饰的树枝状聚合物相比，俄勒冈州绿色488缀合的树枝状聚合物是更好的反义化合物递送剂。这表明相对疏水的小分子与PAMAM树枝状聚合物的偶联可以提供增强其作为核酸递送剂的能力的有用手段。
PAMAM dendrimers are cationic polymers that have been used for the delivery of genes and oligonucleotides to cells. However, little is known about the behavior of dendrimer-nucleic acid complexes once they reach the cell interior. To pursue this issue, we prepared dendrimers conjugated with the fluorescent dye Oregon green 488. These were used in conjunction with oligonucleotides labeled with a red (TAMRA) fluorophore in order to visualize the sub-cellular distribution of the dendrimer-oligonucleotide complex and of its components by two-color digital fluorescence microscopy. The 2'-O:-methyl antisense oligonucleotide sequence used in these studies was designed to correct splicing at an aberrant intron inserted into a luciferase reporter gene; thus effective delivery of the antisense agent results in the expression of the reporter gene product. The dendrimer-oligonucleotide complex remained associated during the process of uptake into vesicular compartments and eventual entry into the nucleus. Since the pharmacological activity of the antisense compound was manifest under these conditions, it suggests that the dendrimer-oligonucleotide complex is functionally active. A surprising result of these studies was that the Oregon green 488-conjugated dendrimer was a much better delivery agent for antisense compounds than unmodified dendrimer. This suggests that coupling of relatively hydrophobic small molecules to PAMAM dendrimers may provide a useful means of enhancing their capabilities as delivery agents for nucleic acids.