Abstract
Carbon nanotubes are capable of penetrating the cell membrane and are widely considered as potential carriers for gene or drug delivery. Because the C-C and C=C bonds in carbon nanotubes are nonpolar, functionalization is required for carbon nanotubes to interact with genes or drugs as well as to improve their biocompatibility. In this study, polyethylenimine (PEI)-functionalized single-wall (PEI-NH-SWNTs) and multiwall carbon nanotubes (PEI-NH-MWNTs) were produced by direct amination method. PEI functionalization increased the positive charge on the surface of SWNTs and MWNTs, allowing carbon nanotubes to interact electrostatically with the negatively charged small interfering RNAs (siRNAs) and to serve as nonviral gene delivery reagents. PEI-NH-MWNTs and PEI-NH-SWNTs had a better solubility in water than pristine carbon nanotubes, and further removal of large aggregates by centrifugation produced a stable suspension of reduced particle size and improved homogeneity and dispersity. The amount of grafted PEI estimated by thermogravimetric analysis was 5.08% (w/w) and 5.28% (w/w) for PEI-NH-SWNTs and PEI-NH-MWNTs, respectively. For the assessment of cytotoxicity, various concentrations of PEI-NH-SWNTs and PEI-NH-MWNTs were incubated with human cervical cancer cells, HeLa-S3, for 48 h. PEI-NH-SWNTs and PEI-NH-MWNTs induced cell deaths in a dose-dependent manner but were less cytotoxic compared to pure PEI. As determined by electrophoretic mobility shift assay, siRNAs directed against glyceraldehyde-3-phosphate dehydrogenase (siGAPDH) were completely associated with PEI-NH-SWNTs or PEI-NH-MWNTs at a PEI-NH-SWNT/siGAPDH or PEI-NH-MWNT/siGAPDH mass ratio of 80:1 or 160:1, respectively. Furthermore, PEI-NH-SWNTs and PEI-NH-MWNTs successfully delivered siGAPDH into HeLa-S3 cells at PEI-NH-SWNT/siGAPDH and PEI-NH-MWNT/siGAPDH mass ratios of 1:1 to 20:1, resulting in suppression of the mRNA level of GAPDH to an extent similar to that of DharmaFECT, a common transfection reagent for siRNAs. Our results indicate that the PEI-NH-SWNTs and PEI-NH-MWNTs produced in this study are capable of delivering siRNAs into HeLa-S3 cells to suppress gene expression and may therefore be considered as novel nonviral gene delivery reagents.
Highlights
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 [1]
PEI-NH-CNT suspensions PEI functionalization remarkably increased the degree of dispersibility of single-wall carbon nanotubes (SWNTs) and multiwall carbon nanotubes (MWNTs)
Because agglomeration of carbon nanotubes as a result of van der Waals' interaction tends to increase cytotoxicity [32,33], PEI-NH-CNTs were subjected to centrifugation to remove large aggregates, and the PEI content of PEI-NH-CNTs The amount of PEI introduced to PEI-NH-CNTs during the functionalization procedure was quantified by thermogravimetric analysis (TGA)
Summary
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 [1]. 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. 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 [7,9,16,17,18,19]. Intratumoral administration of cytotoxic siRNAs delivered by aminofunctionalized MWNTs successfully suppressed tumor volume in animal models of human lung cancer [20]
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