Early entry and deformation of macropinosomes correlates with high efficiency of decaarginine-PEG-lipid-mediated gene delivery
Early entry and deformation of macropinosomes correlates with high efficiency of decaarginine-PEG-lipid-mediated gene delivery
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大胞质体的早期进入和变形与十精氨酸-PEG-脂质介导的基因传递的高效率相关
DOI：
10.1002/jgm.2615
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发表时间：
2012
期刊：
影响因子：
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通讯作者：
S.
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文献类型：
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作者：
Kobayashi;S.
研究背景十精氨酸-聚乙二醇-偶联的3，5-双（十二烷氧基）苯甲酰胺/质粒DNA [Arg 10-聚乙二醇（PEG）-脂质/质粒DNA（pDNA）]复合物（R10 B/DNA复合物）是一种有效的非病毒载体，可将pDNA导入人宫颈癌HeLa细胞。以前的报道表明，这些复合物在相对低的R10 B/DNA比例下形成，并显示出高的转基因表达效率。然而，两种不同纳米结构的细胞内行为，导致基因递送的差异，仍有待阐明。方法将以8.5/1或42.5/1的N/P比制备的R10 B/DNA复合物，分别对应于5 μ莫尔25 μ mR 10 B，加入HeLa细胞，并使用电子显微镜（EM）和相关光电子显微镜（CLEM）通过细胞成像检查它们的摄入和随后的细胞内命运。结果EM和CLEM分析显示R10 B/在较低的N/P比下形成的DNA复合物主要通过巨胞饮作用被细胞吸收，而在较高的N/P比下形成的R10 B/DNA复合物结合到突出的膜结构上或通过不同的途径渗透到细胞中。在表达转基因的细胞中，在大胞饮体和细胞质中均观察到R10 B/DNA复合物。此外，这些细胞具有膜破裂的大胞饮体。这些结果表明，通过巨胞饮和随后的破坏macropinosome膜的细胞摄取可能是R10 B介导的基因delivery.ConclusionsWe的关键步骤已经表明，存在的R10 B/DNA复合物在macropinosomes在基因传递的早期阶段与高效率R10 B介导的基因传递。这一发现将为基于寡聚精氨酸介导的载体的更有效的基因递送系统的工程设计提供有价值的见解。版权所有© 2012约翰威利父子有限公司.
BackgroundDecaarginine‐polyethylene glycol‐conjugated 3,5‐bis(dodecyloxy)benzamide/plasmid DNA [Arg10‐polyethylene glycol (PEG)‐lipid/plasmid DNA (pDNA)] complexes (designated R10B/DNA complexes) are efficient nonviral carriers for pDNA delivery into human cervical carcinoma HeLa cells. Previous reports indicated that these complexes formed at a relatively low R10B/DNA ratio and showed high transgene expression efficiency. However, the intracellular behaviour of the two different nanostructures, which leads to differences in gene delivery, remains to be elucidated.MethodsR10B/DNA complexes prepared at a N/P ratio of 8.5/1 or 42.5/1, corresponding to 5 µmor 25 µmR10B, respectively, were added to HeLa cells, and their uptake and subsequent intracellular fate were examined by cell imaging using electron microscopy (EM) and correlative light‐electron microscopy (CLEM).ResultsEM and CLEM analyses revealed that R10B/DNA complexes formed at the lower N/P ratio were mainly taken up by the cells through macropinocytosis, whereas R10B/DNA complexes formed at the higher N/P ratio bound to protruding membrane structures or permeated into the cells by a different pathway. In cells expressing the transgene, R10B/DNA complexes were observed both in macropinosomes and in the cytoplasm. In addition, these cells had macropinosomes with disrupted membranes. These results suggest that cellular uptake through macropinocytosis and subsequent disruption of the macropinosome membrane may be a critical step for R10B‐mediated gene delivery.ConclusionsWe have shown that the existence of R10B/DNA complexes in macropinosomes at the early stages of gene delivery correlates with high efficiency R10B‐mediated gene delivery. This finding will provide valuable insights for the engineering of more efficient gene delivery systems based on oligoarginine‐mediated carriers. Copyright © 2012 John Wiley & Sons, Ltd.