Genetic vaccination and gene therapy research could benefit from the application of carbon nanotubes. Functionalized, positively charged, water‐soluble carbon nanotubes are able to penetrate into cells (see figure) and can transport plasmid DNA by formation of noncovalent DNA–nanotube complexes. Such nanotubes can be used as novel nonviral delivery systems for gene transfer.

Carbon nanotubes (CNTs) constitute a class of nanomaterials that possess characteristics suitable for a variety of possible applications. Their compatibility with aqueous environments has been made possible by the chemical functionalization of their surface, allowing for exploration of their interactions with biological components including mammalian cells. Functionalized CNTs (f-CNTs) are being intensively explored in advanced biotechnological applications ranging from molecular biosensors to cellular growth substrates. We have been exploring the potential of f-CNTs as delivery vehicles of biologically active molecules in view of possible biomedical applications, including vaccination and gene delivery. Recently we reported the capability of ammonium-functionalized single-walled CNTs to penetrate human and murine cells and facilitate the delivery of plasmid DNA leading to expression of marker genes. To optimize f-CNTs as gene delivery vehicles, it is essential to characterize their interactions with DNA. In the present report, we study the interactions of three types of f-CNTs, ammonium-functionalized single-walled and multiwalled carbon nanotubes (SWNT-NH3+; MWNT-NH3+), and lysine-functionalized single-walled carbon nanotubes (SWNT-Lys-NH3+), with plasmid DNA. Nanotube-DNA complexes were analyzed by scanning electron microscopy, surface plasmon resonance, PicoGreen dye exclusion, and agarose gel shift assay. The results indicate that all three types of cationic carbon nanotubes are able to condense DNA to varying degrees, indicating that both nanotube surface area and charge density are critical parameters that determine the interaction and electrostatic complex formation between f-CNTs with DNA. All three different f-CNT types in this study exhibited upregulation of marker gene expression over naked DNA using a mammalian (human) cell line. Differences in the levels of gene expression were correlated with the structural and biophysical data obtained for the f-CNT:DNA complexes to suggest that large surface area leading to very efficient DNA condensation is not necessary for effective gene transfer. However, it will require further investigation to determine whether the degree of binding and tight association between DNA and nanotubes is a desirable trait to increase gene expression efficiency in vitro or in vivo. This study constitutes the first thorough investigation into the physicochemical interactions between cationic functionalized carbon nanotubes and DNA toward construction of carbon nanotube-based gene transfer vector systems.

Multiwalled carbon nanotube aqueous dispersions using block copolymers are able to form supramolecular complexes with the aromatic chromophore and anticancer agent doxorubicin via pi-pi stacking and enhance its cytotoxic activity.

Genetische Impfung und Gentherapie könnten von den Eigenschaften von Kohlenstoffnanoröhren profitieren. Funktionalisierte, positiv geladene Kohlenstoffnanoröhren sind wasserlöslich und können in Zellen eindringen (siehe Bild). Sie transportieren Plasmid‐DNA in Form von nichtkovalenten DNA‐Nanoröhren‐Komplexen, was sie zu neuartigen nichtviralen Systemen für den Gentransfer macht.

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Plasmid DNA pRc/CMV HBS encoding the S (small) region of hepatitis B surface antigen (HBsAg) was incorporated by the dehydration-rehydration method into Lipodine liposomes composed of 16 micro moles phosphatidylcholine (PC) or distearoyl phosphatidylcholine (DSPC), 8 micro moles of (dioleoyl phosphatidylethanolamine (DOPE) or cholesterol and 4 micro moles of the cationic lipid 1,2-dioleoyl-3-(trimethylammonium propane (DOTAP) (molar ratios 1 : 0.5 : 0.25). Incorporation efficiency was high (89-93% of the amount of DNA used) in all four formulations tested and incorporated DNA was shown to be resistant to displacement in the presence of the competing anionic sodium dodecyl sulphate molecules. This is consistent with the notion that most of the DNA is incorporated within the multilamellar vesicles structure rather than being vesicle surface-complexed. Stability studies performed in simulated intestinal media also demonstrated that dehydration-rehydration vesicles (DRV) incorporating DNA (DRV(DNA)) were able to retain significantly more of their DNA content compared to DNA complexed with preformed small unilamellar vesicles (SUV-DNA) of the same composition. Moreover, after 4h incubation in the media, DNA loss for DSPC DRV(DNA) was only minimal, suggesting this to be the most stable formulation. Oral (intragastric) liposome-mediated DNA immunisation studies employing a variety of DRV(DNA) formulations as well as naked DNA revealed that secreted IgA responses against the encoded HBsAg were (as early as three weeks after the first dose) substantially higher after dosing with 100 micro g liposome-entrapped DNA compared to naked DNA. Throughout the fourteen week investigation, IgA responses in mice were consistently higher with the DSPC DRV(DNA) liposomes compared to naked DNA and correlated well with their improved DNA retention when exposed to model intestinal fluids. To investigate gene expression after oral (intragastric) administration, mice were given 100 micro g of naked or DSPC DRV liposome-entrapped plasmid DNA expressing the enhanced green fluorescent protein (pCMV.EGFP). Expression of the gene, in terms of fluorescence intensity in the draining mesenteric lymph nodes, was much greater in mice dosed with liposomal DNA than in animals dosed with the naked DNA. These results suggest that DSPC DRV liposomes containing DNA (Lipodine) may be a useful system for the oral delivery of DNA vaccines.

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The intrinsic nonlinear photoluminescence (PL) property of chemically functionalized multi-walled nanotubes MWNTs (f-MWNTs) is reported in this study. f-MWNTs are imaged in fixed lung epithelial cancer cells (A549) and Kupffer cells in vitro, and in subcutaneously implanted solid tumors in vivo, for the first time, using multiphoton PL and fluorescence lifetime imaging (FLIM). Multiphoton imaging in the near-infrared excitation region (∼750-950 nm), employed in this study in a label-free manner, provides sensitivity and resolution optimal to track f-MWNTs within intra-cellular compartments and facilitates tumour imaging and sentinel lymph node tracking in vivo. Wider applications include employing this technique in live imaging of f-MWNTs in biological milieu to facilitate image-guided drug delivery.