Oxidized ultrashort nanotubes as carbon scaffolds for the construction of cell-penetrating NF-kappaB decoy molecules.
Oligonucleotide (ODN) decoys are synthetic ODNs containing the DNA binding sequence of a transcription factor. When delivered to cells, these molecules can compete with endogenous sequences for binding the transcription factor, thus inhibiting its ability to activate the expression of target genes. Modulation of gene expression by decoy ODNs against nuclear factor-kappaB (NF-kappaB), a transcription factor regulating many genes involved in immunity, has been achieved in a variety of immune/inflammatory disorders. However, the successful use of transcription factor decoys depends on an efficient means to bring the synthetic DNA to target cells. It is known that single-walled carbon nanotubes (SWCNTs), under certain conditions, are able to cross the cell membrane. Thus, we have evaluated the possibility to functionalize SWCNTs with decoy ODNs against NF-kappaB in order to improve their intracellular delivery. To couple ODNs to CNTs, we have exploited the carbodiimide chemistry which allows covalent binding of amino-modified ODNs to carboxyl groups introduced onto SWCNTs through oxidation. The effective binding of ODNs to nanotubes has been demonstrated by a combination of microscopic, spectroscopic, and electrophoretic techniques. The uptake and subcellular distribution of ODN decoys bound to SWCNTs was analyzed by fluorescence microscopy. ODNs were internalized into macrophages and accumulated in the cytosol. Moreover, no cytotoxicity associated with SWCNT administration was observed. Finally, NF-kappaB-dependent gene expression was significantly reduced in cells receiving nanomolar concentrations of SWCNT-NF-kappaB decoys compared to cells receiving SWCNTs or SWCNTs functionalized with a nonspecific ODN sequence, demonstrating both efficacy and specificity of the approach.
Crinelli
Rita
R
Department of Biomolecular Sciences, Biochemistry and Molecular Biology Section, University of Urbino Carlo Bo, 61029 Urbino, PU, Italy. rita.crinelli@uniurb.it
Carloni
Elisa
E
Menotta
Michele
M
Giacomini
Elisa
E
Bianchi
Marzia
M
Ambrosi
Gianluca
G
Giorgi
Luca
L
Magnani
Mauro
M
D001483:Base Sequence; D001667:Binding, Competitive; D001692:Biological Transport; D002234:Carbodiimides / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D005786:Gene Expression Regulation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D037742:Nanotubes, Carbon / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D010084:Oxidation-Reduction
D002234:Carbodiimides; D003432:Cross-Linking Reagents; D008070:Lipopolysaccharides; D016328:NF-kappa B; D037742:Nanotubes, Carbon; D009841:Oligonucleotides