Intracellular Localization of Titanium Dioxide-DNA Nanocomposites
G. Woloschak1, T. Paunesku1, K. Thurn1, S. Vogt2, J. Maser2, B. Lai2
1Northwestern
University, Chicago
2Argonne National Laboratory
We are developing TiO2-DNA nanocomposites into a new type of treatment against cancer - a therapy that would be done by inducible gene removal. These nanocomposites are made of 45 angstrom TiO2 nanoparticles, coated with glycidyl isopropyl ether and conjugated to oligonucleotide DNA(s). Within the nanocomposites DNA oligonucleotides retain base-pairing specificity, while the TiO2 nanoparticles exhibit photoreactivity. The assembled TiO2 nanocomposites incorporate both components into a charge separation scheme - excitation of TiO2 (exposure to electromagnetic radiation of energies above 3.2 eV, 390 nm) results in charge separation concluded by irreversible electropositive hole trapping in the sugar molecules of the DNA phosphodiester backbone leading to the cleavage of the DNA (Paunesku et al., 2003). This endonuclease-like activity is: i) excitable by a factor not naturally encountered by the cells in vivo (electromagnetic radiation of energy higher than 3.2 eV); and ii) highly sequence specific - it can be directed toward a single target in a whole genome (due to the high specificity of long oligonucleotide base-pairing).
TiO2-DNA nanocomposites were prepared as described (Paunesku et al., 2003) so that on the surface of each nanoparticle, otherwise covered with glycidyl isopropyl ether, one to three dopamine-modified DNA molecules were attached. In vitro DNA cleavage reactions included mixtures of nanocomposites and radiolabeled complementary oligonucleotides, annealed and incubated for varying ammounts of time. Oligonucleotides used for intracellular experiments were specific either to ribosomal RNA coding sequences in the nucleus (rDNA), for 18SrRNA in particular, or for the DNA of the mitochondrial genome. Rat pheochromocytoma PC12 Tet-ON (Invitrogen/Gibco) and human breast cancer MCF-7/WS8 (American Type Culture Collection) cell lines were grown in 5% CO2 in serum suplemented F12K or RPMI1640 media, respectively. The cells were grown to 80% confluence, serum starved for 16 hours and electroporated with nanocomposites using the Mammozapper™ apparatus (Tritech) following the manufacturer’s instructions. Aliquots of 10 E6 cells were mixed with 5 μl of 10 μM nanocomposites.
In vitro, TiO2 nanocomposites excited by exposure to white light or ionization radiation cause the scission of DNA. Some of the factors influencing reaction of cleavage of DNA are length of time of illumination; temperature of incubation post-illumination; presence of radiolabel on oligonucleotides, concentration of nanocomposites, presence of a mismatch/deletion in DNA attached to TiO2.
For the intracellular treatment we transfected cultured cells by TiO2-oligonucleotide nanocomposites with oligonucleotides specific for ribosomal or mitochondrial DNA, and showed the sequence specific intracellular targeting by nanocomposites. The location of titanium in the cells was mapped by detecting titanium specific K alpha X-ray fluorescence induced at the 2ID-E beamline at the Advanced Photon Source of Argonne National Laboratory, USA.
In vitro data show that the TiO2-DNA nanocomposites can cause scission of DNA in a sequence specific manner. Results of the intracellular experiments showed that the nanocomposites containing nucleus or mitochondria specific oligonucleotides were retained and accumulated specifically primarily in the matching subcellular locations. At the same time, TiO2 nanoparticles without attached DNA were gradually removed from all cellular compartments. This study suggests that it is possible to use the TiO2-DNA nanocomposites for local “treatment” of DNA targets.
