Intracellular Trafficking Pathways for Nuclear Delivery of Plasmid DNA Complexed with Highly Efficient Endosome Escape Polymers
Intracellular Trafficking Pathways for Nuclear Delivery of Plasmid DNA Complexed with Highly Efficient Endosome Escape Polymers
复制标题
DOI：
10.1021/bm5008376
复制
发表时间：
2014-10-01
影响因子：
6.2
通讯作者：
Monteiro, Michael J.
中科院分区：
文献类型：
--
作者：
Gillard, Marianne;Jia, Zhongfan;Monteiro, Michael J.
了解核进入的途径可以看到将遗传物质输送到细胞的聚合物设计的巨大改进。在这里，我们使用一种新型的与质粒DNA(PDNA)复合的两嵌段共聚物来确定其细胞进入和核途径。二嵌段共聚物(A-C3)是专门设计用来结合和保护PDNA，在特定的时间释放它，但更重要的是，快速逃离内体。共聚物被HEK293细胞优先通过笼蛋白介导的内吞作用(CME)途径摄取，48h后PDNA进入细胞核，在所有细胞中产生高水平的基因表达，这与商业上可用的聚合物转染剂PEI Max相似。这表明，聚合物必须首先逃离内小体，然后介导pDNA到细胞核的运输，才能发生基因表达。我们发现我们的A-C3聚合物在细胞核内的PDNA含量比PEI Max高，24小时后我们的聚合物提供的PDNA是PEI Max的7倍。我们进一步发现，进入细胞核的主要是通过小的核孔，当核膜受损时，在有丝分裂期间不会发生。核中也发现了聚合物，这一观察结果支持这样的假设，即大的PDNA/聚合物复合体(尺寸类似200 nm)必须在进入核之前解离，聚合物上的阳离子和疏水单体单元可能有助于PDNA通过核孔的主动运输。
Understanding the pathways for nuclear entry could see vast improvements in polymer design for the delivery of genetic materials to cells. Here, we use a novel diblock copolymer complexed with plasmid DNA (pDNA) to determine both its cellular entry and nuclear pathways. The diblock copolymer (A-C3) is specifically designed to bind and protect pDNA, release it at a specific time, but more importantly, rapidly escape the endosome. The copolymer was taken up by HEK293 cells preferentially via the clathrin-mediated endocytosis (CME) pathway, and the pDNA entered the nucleus to produce high gene expression levels in all cells after 48 h, a similar observation to the commercially available polymer transfection agent, PEI Max. This demonstrates that the polymers must first escape the endosome and then mediate transport of pDNA to the nucleus for occurrence of gene expression. The amount of pDNA within the nucleus was found to be higher for our A-C3 polymer than PEI Max, with our polymer delivering 7 times more pDNA than PEI Max after 24 h. We further found that entry into the nucleus was primarily through the small nuclear pores and did not occur during mitosis when the nuclear envelope becomes compromised. The observation that the polymers are also found in the nucleus supports the hypothesis that the large pDNA/polymer complex (size similar to 200 nm) must dissociate prior to nucleus entry and that cationic and hydrophobic monomer units on the polymer may facilitate active transport of the pDNA through the nuclear pore.