Carbon Nanotube Delivery of the GFP Gene into Mammalian Cells

Abstract

Exogenous-gene expression and manipulation in mammalian cells has become a mainstay of biomedical research. Consequently, improving methods for efficient gene transfer to a broad range of cell types is of great interest and remains a high priority. Several classes of transfection methods have been developed, which include traditional cationic moleculemediated agents, such as Lipofectamine 20000 and FuGENE 6, viral-vector systems, and the “gene gun” approach. With the rapid development of nanobiotechnology, a variety of new materials, such as gold nanoparticles, silica nanoparticles, polymers, nanogels, and dendrimers have been investigated as biocompatible transporters. Recently, carbon nanotube—a well-studied nanomaterial— have been investigated for their ability to interact with and affect living systems. For instance, carbon nanotubes have been found to enhance DNA amplification in PCR and affect the growth pattern of neurons. Pantarotto et al. have reported the internalization of fluorescein isothiocynate (FITC) labeled nanotubes and nanotube delivery of the gene that encodes b-galactosidase into cells, with no apparent toxic effects. Kam et al. have studied the mechanism of protein-conjugated carbon nanotube uptake into cells via the endocytic pathway. Here we present our finding that amino-functionalized multiwalled carbon nanotubes (NH2-MWCNTs) are able to interact with plasmid DNA and deliver the green fluorescence protein (GFP) gene into cultured human cells. Our data strongly suggest that carbon nanotubes can be considered as a new carrier for the delivery of biomolecules, such as DNA, proteins, and peptides into mammalian cells. Therefore, this novel system might have potential applications in biology and therapy, including vaccine and gene delivery. In order to increase their biocompatibility, we introduced amino-, carboxyl-, hydroxyl-, and alkyl groups onto the surface of MWCNTs. COOH-MWCNTs were first prepared by nitric / sulfuric acid oxidation, and then NH2and CH3CH2CH2-groups were added. Finally, we obtained four types of MWCNTs with different chemical groups on their surface. Functionalized MWCNTs were observed under an electron microscope and were found to be 60–70 nm in diameter and 1–2 mm in length. Although we did not find a significant difference in size between the NH2-MWCNTs and NH2-MWCNT–DNAs, the latter appeared to have the tendency to aggregate (Figure 1B). In order to test the DNA-binding ability of amino-, carboxyl-, hydroxyl-, and alkyl-group-modified MWCNTs, we incubated them with pEGFPN1-plasmid DNA, and MWCNT–DNA mixtures were analyzed by agarose-gel electrophoresis. The results show that only NH2-MWCNT bound to DNA (Figure 2); since the NH2-MWCNT–DNA complex was too big to run into the