Carbon nanotubes (CNTs) are cylindrical structures formed by graphite sheets with a diameter in the nanometer range and tens to hundreds of micrometers in length . They can be categorized into single-wall carbon nanotubes (SWNTs) and multiwall carbon nanotubes (MWNTs), according to the number of concentric layers of graphite sheets.
Carbon nanotubes are being extensively studied as carriers for gene or drug delivery [2–5]. In order to provide functional groups for the binding of plasmid DNAs, small interfering RNAs (siRNAs), or chemical compounds and to reduce the potential toxicity of pristine carbon nanotubes, functionalization of carbon nanotubes is necessary for their biomedical applications [6–10]. After complexed with nucleotides or chemicals through either covalent or noncovalent binding, functionalized carbon nanotubes may then enter cells by endocytosis [3, 11, 12] or by penetrating directly through the cell membrane [13–15].
To serve as carriers for nonviral gene delivery, as opposed to viral transfection which applies viral vectors to achieve high transfection efficiency, carbon nanotubes are often functionalized with cationic molecules or polymers in order to interact electrostatically with negatively charged siRNAs or plasmid DNAs [7, 9, 16–19]. SWNTs and MWNTs chemically modified with amino groups were capable of delivering plasmid DNAs into A549, HeLa, and CHO cell lines [18, 19]. MWNTs functionalized with polycationic dendron may enhance siRNA delivery and gene silencing in vitro. Furthermore, positively charged SWNTs in complex with telomerase reverse transcriptase siRNAs were shown to suppress tumor growth in animal studies . Intratumoral administration of cytotoxic siRNAs delivered by amino-functionalized MWNTs successfully suppressed tumor volume in animal models of human lung cancer .
Polyethylenimine (PEI), a cationic polymer synthesized in linear or branched form with various molecular weights, is used in several studies to provide a high density of cations on the surface of carbon nanotubes [21, 22]. It was shown that PEI-grafted MWNTs improve the expression of plasmid DNA in human embryonic kidney (HEK 293) and human lung epithelial (A549) cells [22, 23]. Shortened MWNTs of 200 nm in length covalently modified with branched PEI of low molecular weight (600 Da) deliver siRNAs with higher efficacy than a lipid vehicle . Successful delivery of siRNA to human prostate cancer PC-3 cells by PEI-functionalized SWNTs was also reported . Moreover, PEI-modified SWNTs were shown to provide the substrate for neurite outgrowth and branching .
Despite extensive applications, PEI, itself a reagent for nonviral transfection, is cytotoxic, and chemical modification of PEI is required to improve its application as a transfection reagent [23, 26, 27]. It is therefore expected that functionalization of carbon nanotubes with PEI would not only increase their biocompatibility but also reduce the toxicity of PEI. Nevertheless, contradictory conclusions on the toxicity and transfection efficiency of PEI-functionalized carbon nanotubes compared to pure PEI were presented in the literature [21, 23, 24, 28]. In this study, SWNTs and MWNTs were functionalized with PEI for the delivery of siRNAs. The properties and efficiencies of PEI-functionalized SWNTs and MWNTs as nonviral transfection reagents were compared, and whether the functionalization procedure reduces the cytotoxicity of PEI was discussed.