Polyethylenimine-grafted multiwalled carbon nanotubes for secure noncovalent immobilization and efficient delivery of DNA
Polyethylenimine-grafted multiwalled carbon nanotubes for secure noncovalent immobilization and efficient delivery of DNA
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DOI：
10.1002/anie.200500042
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发表时间：
2005-01-01
影响因子：
16.6
通讯作者：
Leong, KW
中科院分区：
文献类型：
--
作者：
Liu, Y;Wu, DC;Leong, KW
碳纳米管（CNT）的功能化已经以各种方式在生物技术中的许多应用中进行，[1-15]包括用于制备传感器，[2，3]作为细胞生长的支架，[4]成像试剂，[5]和用于药物递送的转运蛋白。[6-8一种方法是通过非共价相互作用[8-13]或共价键将DNA固定到CNT表面上。[3，12-15]由于化学反应和释放DNA的困难，共价键方法可能会损害甚至破坏DNA的功能。[13然而，迄今为止开发的非共价方法可能仅提供DNA在CNT表面上的亚稳态固定。据报道，在凝胶电泳中，共价连接到CNT的DNA的迁移被延迟，但DNA和CNT之间的非共价相互作用并不能完全阻止迁移。[14b]聚乙烯亚胺（PEI）是一种具有高密度胺的聚合物，因此DNA可以安全地固定在多壁碳纳米管（MWNT）的表面上，所述多壁碳纳米管已经通过由这些胺产生的强静电相互作用与PEI官能化。因此，我们采用了一种接枝自的方法来制备聚乙烯亚胺接枝多壁碳纳米管（PEI-g-MWNTs）。DNA已被安全地固定在PEI-g-MWNTs的表面上，如通过在凝胶电泳中DNA的迁移的总抑制所证明的，并且PEI-g-MWNTs显示出用于递送DNA的转染效率，其与PEI（25 K）的转染效率相似或甚至数倍于PEI（25 K），并且比裸DNA的转染效率高几个数量级。在胺官能化的多壁碳纳米管（NH 2-MWNTs）的存在下，通过氮丙啶的阳离子聚合将PEI接枝到多壁碳纳米管表面。NH 2-MWNTs通过在3.0 M硝酸中加热回流将羧酸基团引入到MWNTs的表面上而获得。通过用亚硫酰氯处理将羧酸基团转化为酰氯基团[16]，然后用乙二胺处理。[14]PEI的接枝通过两种机制实现，活化单体机制（AMM）或活化链机制（ACM），通过这两种机制，质子化的氮丙啶单体或增长链的末端亚胺离子基团分别转移到MWNTs表面上的胺。[17]（参见支持信息。）通过在氮气下进行的热重分析（TGA）研究了接枝到MWNTs表面上的PEI的相对量。MWNT在高达600 ° C下是热稳定的（图1A，曲线a），而纯PEI在约500 ° C下完全降解（图1A，曲线d）。在500 ℃下，原始MWNT、NH 2-MWNT和PEI-g-MWNT分别显示出可忽略的约2.3%和10.5%的重量损失，因此PEI-g-MWNT含有约8.2%PEI。接枝PEI后的PEI-g-MWNTs在水中分散性好，6个月后仍保持稳定。然而，NH 2-MWNTs在水中分散性差，并且在几小时内发生沉淀（参见支持信息）。透射电子显微镜（TEM）提供了PEI接枝到MWNTs表面上的直接证据。图1 B显示了多孔碳膜上PEI-g-MWNT的TEM图像：单独分散的MWNT与其它MWNT分离。高分辨率TEM（图1B，插图）表明PEI作为具有不同尺寸的团块而不是作为均匀涂层接枝到MWNT表面上。这种结块是由于羧酸基团、乙二胺和PEI优选地粘附在…
The functionalization of carbon nanotubes (CNTs) has been carried out in various ways for numerous applications in biotechnology,[1–15] including for the preparation of sensors,[2, 3] as scaffolds for cell growth,[4] imaging reagents,[5] and transporters for drug delivery.[6–8, 13, 15] One way is to immobilize DNA onto the surface of CNTs through noncovalent interactions [8–13] or covalent bonds.[3, 12–15] Covalent-bond approaches might compromise and even spoil the functions of DNA owing to chemical reactions and the difficulty in releasing DNA.[13, 14b, 15] Nevertheless, noncovalent approaches developed to date may only provide metastable immobilization of DNA onto the surface of CNTs. It was reported that the migration of DNA linked covalently to CNTs was retarded in gel electrophoresis but noncovalent interactions between DNA and CNTs did not completely prevent migration.[14b] Polyethylenimine (PEI) is a type of polymer with a high density of amines, thus DNA may be immobilized securely onto the surface of multiwalled carbon nanotubes (MWNTs) that have been functionalized with PEI through strong electrostatic interactions arising from these amines. Hence, we have adopted a grafting-from approach to prepare polyethylenimine-graft multiwalled carbon nanotubes (PEI-g-MWNTs). DNA has been immobilized securely onto the surface of PEI-g-MWNTs as demonstrated by the total inhibition of the migration of DNA in gel electrophoresis, and PEI-g-MWNTs showed transfection efficiency for delivery of DNA that was similar to or even several times higher than that of PEI (25 K) and several orders of magnitude higher than that of naked DNA. PEI was grafted onto the surface of MWNTs by performing a cationic polymerization of aziridine in the presence of amine-functionalized MWNTs (NH2–MWNTs). NH2–MWNTs were obtained by introducing carboxylic acid groups onto the surface of MWNTs by heating at reflux in 3.0 m nitric acid. The carboxylic acid groups were transformed into acyl chloride groups by treatment with thionyl chloride [16] followed by treatment with ethylenediamine.[14] The grafting of PEI was realized through two mechanisms, the activated monomer mechanism (AMM) or the activated chain mechanism (ACM), by which protonated aziridine monomers or the terminal iminium ion groups of propagation chains, respectively, are transferred to amines on the surface of MWNTs.[17](see Supporting Information.) The relative amount of PEI grafted onto the surface of MWNTs was investigated by thermogravimetric analysis (TGA) performed under nitrogen. MWNTs were thermally stable up to 6008C (Figure 1A, curve a) whereas pure PEI degraded completely at about 5008C (Figure 1 A, curve d). At 5008C, pristine MWNTs, NH2–MWNTs, and PEI-g-MWNTs showed negligible, about 2.3%, and 10.5% weight losses, respectively, thus PEI-g-MWNTs contained about 8.2% PEI. Grafting with PEI made PEI-g-MWNTs easy to disperse in water, and the resulting suspension was still stable after six months. However, NH2–MWNTs dispersed poorly in water and precipitation occurred within several hours (see Supporting Information). Transmission electron microscopy (TEM) provides direct evidence of grafting of PEI onto the surface of MWNTs. Figure 1 B shows TEM images of PEI-g-MWNTs on a holey carbon film: individually dispersed MWNTs are separated from others. High-resolution TEM (Figure1B, inset) indicates that PEI was grafted onto the surface of MWNTs as lumps with different sizes instead of as a uniform coating. This clumping results from the carboxylic acid groups, the ethylenediamine, and the PEI adhering preferably to …