Nuclear localisation sequence templated nonviral gene delivery vectors: Investigation of intracellular trafficking events, of LMD and LD vector systems
Nuclear localisation sequence templated nonviral gene delivery vectors: Investigation of intracellular trafficking events, of LMD and LD vector systems
复制标题
DOI：
10.1002/cbic.200390049
复制
发表时间：
2003-04-04
期刊：
影响因子：
3.2
通讯作者：
Miller, AD
中科院分区：
文献类型：
--
作者：
Keller, M;Harbottle, RP;Miller, AD
研究了含有核定位序列（NLS）的肽对细胞内DNA运输的影响。在与3 β-[N-（N '，N'-二甲基氨基乙烷）氨基甲酰基]胆固醇/二油酰基-L-α-磷脂酰乙醇胺（DC-Chol/DOPE）脂质体配制之前，我们使用腺病毒护理肽mu和SV 40 NLS肽来缩合质粒DNA（pDNA），以分别得到LMD和LND载体。荧光标记的脂质和肽加上染料标记的pDNA组分被用来研究分裂和S期生长停滞细胞中的基因递送。共聚焦显微镜分析显示细胞内运输事件的差异不大。引人注目的是，在LMD与细胞孵育不到15分钟内，μ肽与细胞的核和核仁缔合，这表明μ肽具有NLS功能。这些NLS特性通过克隆在胞质翻译后定位于细胞核中的μ-β-半乳糖苷酶融合蛋白来证实。在分裂的细胞中，LMD和LND都在与细胞孵育30-45分钟内将pDNA（Cy 3）递送至细胞核。相比之下，在研究的时间段内，仅在生长停滞细胞的细胞质中检测到pDNA，而不在细胞核中。由DC-Chol/DOPE阳离子脂质体和pDNA（Cy 3）制备的LD系统与LMD系统类似，这表明μ肽不能影响该当前LMD制剂中的运输事件，尽管其具有强的NLS能力。我们进一步描述了聚乙二醇（PEG）对细胞摄取的影响。制备了通过将LMD颗粒与荧光标记的PEG分子（0.5、5和10摩尔%荧光素-PEG（5000）-N-羟基琥珀酰亚胺）后冷却获得的“隐形”系统，并显示其被细胞快速内化（分钟），而没有可检测的转基因表达。该结果表明PEG阻断了pDNA的细胞内运输。
The impact of a peptide that contains a nuclear localisation sequence (NLS) on intracellular DNA trafficking was studied. We used the adenoviral care peptide mu and an SV40 NLS peptide to condense plasmid DNA (pDNA) prior to formulation with 3beta-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol/dioleoyl-L-alpha-phosphatidyl ethanolamine (DC-Chol/DOPE) liposomes to give LMD and LND vectors, respectively. Fluorescent-labelled lipid and peptides plus dye-labelled pDNA components were used to investigate gene-delivery in dividing and S-phase growth-arrested cells. Confocal microscopic analyses reveal little difference in intracellular trafficking events. Strikingly, mu peptide associates with nuclei and nucleoli of cells within less than 15 mins incubation of LMD with cells, which suggests that mu peptide has an NLS function. These NLS properties were confirmed by cloning of a mu-beta-galactosidase fusion protein that localises in the nuclei of cells after cytosolic translation. In dividing cells both LMD and LND deliver pDNA(Cy3) to nuclei within 30-45 min incubation with cells. By contrast, pDNA is detected only in the cytoplasm in growth-arrested cells over the period of time investigated, and not in the nuclei. LD systems prepared from DC-Chol/DOPE cationic liposomes and pDNA(Cy3) behove similarly to LMD systems, which suggests that mu peptide is unable to influence trafficking events in this current, LMD formulation, in spite of its strong NLS capacity. We further describe the effect of polyethyleneglycol (PEG) on cellular uptake. "Stealth' systems obtained by post-cooting LMD particles with fluorescent-labelled PEG molecules (0.5, 5 and 10 mol% fluorescein-PEG(5000)-N-hydroxysuccinimide) were prepared and shown to be internalise rapidly (mins) by cells, without detectable transgene expression. This result indicates that PEG blocks, intracellular trafficking of pDNA.