Effects of fluorescent dye coating of metal oxide nanoparticles on DNA scission

Cancer cells have in their genomes mutated copies of normal cellular proto-oncogenes that cannot currently be removed or inactivated in the cancers. A major focus of our laboratory is the controlled induction of DNA cleavage through the use of titanium dioxide (TiO₂) nanoparticles. We anticipate that if this DNA cleavage by TiO₂ can be controlled, then it can be used to remove mutant DNA from cancer cells that have activated oncogenes. Titanium dioxide is a semiconductor material that can be formed in to nanoparticles by various methods (for review see, Cozzoi et al, 2003; Kim et al, 2003; Ramakishna et al, 2003; Wu et al, 2002; Zhang et al, 2003; Zhao et al, 2007). Due to its semiconductor properties, illumination of TiO₂ leads to a charge separation and the promotion of electrons from the valence band in to the conduction band of TiO₂. In aqueous solution, this charge separation can lead to the production of both free electrons and free electropositive holes on the surface of TiO₂. The production of free electrons and electropositive holes, can result in the production of reactive oxygen species in the buffer in which nanoparticles have been dispersed at the time of illumination (Fujishima et al., 1972; Blake et al, 1999). In some of the publications investigating this event, cleavage of plasmid DNA was used as an indicator of this process. However, in TiO₂ nanoparticles, similar to the effect within quantum dots, electrons are confined to the nanoparticle and are believed to be unable to separate from it. Thus, only electropositive holes become available for charge recombination on the surface of the nanoparticle since they are still capable of physical separation from the TiO₂ (Rajh et al., 2001, 2002). Previous work by others has established that conjugation of electronic leads, such as dopamine, to the nanoparticle surface results in an extended charge separation where the electropositive holes move in to the ligand—dopamine, and away from the nanoparticle itself (Rajh et al., 2001, 2002). In our laboratory we have investigated TiO₂ surface modification by Alizarin Red S(ARS) (Thurn et al., 2009) and found this conjugationto be as stable as the connection between Alizarin and TiO₂ (Rajh et al., 2002). Since the reaction between Alizarin and TiO₂ was described as having the same stability as the conjugation to dopamine, and since dopamine can be used to separate electropositive holes from the nanoparticles, we could infer that, likewise, ARS should aid in separation of electropositive holes from the TiO₂.

We proceeded to test the ability of TiO₂-Alizarin Red S complexes to introduce reactive oxygen species (ROS) in to anaqueous solution. In the work presented here we particularly focused on the TiO₂-Alizarin Red S complex and on core-shell nanoparticles as a source of TiO₂. Core shell nanoparticles used for this purpose were composed of a core made of iron oxide (Fe₃O₄) coated with a TiO₂ shell with the final size of 6 nM. We have shown in previous studies that the Fe₃O₄ core will permit MR imaging of the nanoparticles and might be useful in diagnostic imaging. Similar to tests performed on TiO₂ nanoparticles, Fe₃O₄@TiO₂ nanoparticles were prepared and tested for their capacity to induce DNA cleavage by reactive oxygen species(Wu et al., manuscript in preparation).

Results

Conjugation of Alizarin Red S(ARS)to TiO2-PNA nanoconjugates

To demonstrate that Alizarin Red S binds to TiO₂-peptide nucleic acid (PNA) nanoconjugates we have used UV-visible light spectroscopy (Thurn et al., 2009) as well as gel electrophoresis (Brown et al., 2008). TiO₂ nanoparticles are unable to migrate through a polyacrylamide gel (Paunesku et al., 2003, Brown et al., 2008). Thus, any molecule bound to the surface of a TiO₂ nanoparticle will also not be able to migrate through a polyacrylamide gel. When free ARS or nanoparticles with conjugated ARS were applied onto a 16% polyacrylamide gel and imaged for Alizarin Red Sby eithera digital camera or a fluoroimager at the appropriate setting (excitation laser 543nm, emission filter 560-615nm) ARS could be visualized (Figure 1). With visible light, the ARS-TiO₂ complex appears as a red band in those wells of the gel where it was conjugated to the TiO₂ nanoparticles (well 3, Figure 1). After additional electrophoresis and imaging of ARS, this dye is abundant only inside the gel and only in those lanes where ARS was not conjugated to the nanoparticles(lanes 1 and 2, Figure 1). In the presence of TiO₂ nanoparticles (lane 3) a small quantity of free Alizarin Red Sis inside the gel, with most of the fluorescent signal still present in the well.

Figure 1. Alizarin red s-coated TiO2 (3nm) nanoparticles.

Lane 1 is free Alizarin red s; lane 2 is a mixture of Alizarin red s and a DNA oligonucleotide; lane 3 is Alizarin red s conjugated nanoparticle ; samples were run on a 16% polyacrylamide gel for approximately two hours and imaged for Alizarin red s in two ways: First, using a digital camera while the gel was still running; Second, using a fluoroimager without separation of gel from the glass plates that contained it.

DMSO sequesters reactive oxygen species (ROS) reducing cleavage

Previous work byothers with TiO₂ release of ROS and plasmid DNA cleavage (Tachikawa et al., 2007)has shown that DMSO is an effective scavenger of TiO₂ produced ROS. To determine if the production of reactive oxygen species by ARS coated nanoparticles could still be achieved using the same approach;DMSO was used to sequester reactive oxygen species from the samples (Figure 2). Since DMSO is a ROS scavenger, the addition of DMSO would prevent ROS from cleaving DNA. If the mechanism by which ARS and ARS-TiO₂ are cleaving DNA is by the production of ROS, then the addition of DMSO would decrease cleavage. In our experimental design, samples were as follows: 119ng pkaede plasmid alone, Alizarin Red S (equal to the same concentration at which the nanoparticles were coated) and plasmid, 6 nm TiO₂ nanoparticles and plasmid, or plasmid and 6 nm TiO₂ nanoparticles that were 60% coated with Alizarin Red S. These samples were then exposed to white light (7 minutes at an intensity of 75 watts by a Fiber-Lite^© High Intensity Illuminator Series 180)or left untreated (control). Both of these treatments were with or without 0.1% DMSO. Samples were then run on a 1.2% agarose gel and stained with Gelstar. Samples containing Alizarin Red S and plasmid showed a decrease in DNA cleavage upon addition of DMSO. Samples that contained uncoated TiO₂ nanoparticles, however, showed no change in DNA cleavage upon the addition of DMSO. The addition of DMSO to samples containing Alizarin Red S coated TiO₂ nanoparticles showed a decrease in DNA cleavage as compared to the same sample without DMSO.

Figure 2. Plasmid Cleavage By Excited Alizarin Red S Coated Nanoparticles is Due to Release of Reactive Oxygen Species.

Samples exposed to either no light or light in the presence or absence of DMSO (0.1%). 119ng of pkaede plasmid was added to 11.76 pico moles TiO2 nanoparticles. TiO2 Nanoparticles were 60% coated with Alizarin Red S. Plasmid cleavage resulting from excitation of alizarin red s-coated TiO2 nanoparticles is due to release of reactive oxygen species. Samples were either illuminated for 5 minutes at 75 watts (L) or not illuminated (θ). (D) Represents the addition of DMSO (0.1% final concentration). (−) represents that no DMSO added to samples.

Cleavage of plasmid DNA with core-shell nanoparticles and ARS and an excessof ARS

In order to pursue new possible applications of TiO₂ nanoparticles, we have decided to prepare core-shell nanoparticlesthat could be used for magnetic resonance imaging (MRI)as well as for DNA cleavage. To investigate the ability of Fe₃O₂@TiO₂ nanoparticles coated with excess Alizarin Red S to result incleavage of plasmid DNA in vitro we preformed an agarose gelelectrophoresis assay(Figure 3). In these studies 6 nm Fe₃O₄@TiO₂ nanoparticles (various concentrations, as indicated) were completely coated with Alizarin Red S (with free Alizarin Red S in excess in the solution, to a final total concentration of ARS at 1.5 mM). As an indicator of DNA cleavage induced by ROS production 14.65nM pKaede plasmid DNA was added to the Alizarin Red S coated nanoparticles and the samples were illuminated and then separated on a 1.2% agarose gel and stained with Gel Star. When the plasmid migrates through the gel its migration is dependent upon its conformation and the concentration of agarose in the gel. In a 1.2% agarose gel, the plasmid that is in a super-coiled conformation will migrate faster than a linearized or nicked (relaxed circular) conformation. When illuminated, and in the presence of excess Alizarin Red S and Fe₃O₄@TiO₂, the plasmid is cleaved into nicked and linear conformations. A concentration of 56.4 nM Fe₃O₄@TiO₂ with 1.5 mM ARS is the minimal concentration of nanoparticles needed under these circumstances to produce detectable plasmid cleavage.

Figure 3. Cleavage of plasmid DNA with excess Alizarin Red S.

All samples contain Alizarin Red S coated Fe₃O₄@TiO₂ nanoparticles (complete coverage by Alizarin Red S with excess Alizarin Red S in solution, 1.5mM final concentration). Samples were either illuminated for 5 minutes at 75 watts (L) or not illuminated (θ). Nicked, linear and super-coiled designates the confirmation of the plasmid.

Cleavage of plasmid DNA with varying concentrations of Alizarin Red S

To determine the extent to which coating of Fe₃O₄@TiO₂ nanoparticles with Alizarin Red S, and having free ARS present in solution, enhance cleavage, Fe₃O₄@TiO₂ nanoparticles were either coated with excess Alizarin Red S (final concentration of 1.5mM) or left uncoated (Figure 3). The same procedure as in Figure 3 was preformed. Samples containing Alizarin Red S coated Fe₃O₄@TiO₂ nanoparticles showed plasmid DNA to be in both a nicked and a linear conformation but it lacked a super-coiled conformation. Samples containing the same molarity of uncoated Fe₃O₄@TiO₂ nanoparticles, on the other hand, showed the presence of only super-coiled and nicked conformations but no linearized DNA. As can be seen from the lack of super-coiled plasmid DNA, and an increase in both nicked and linearized DNA, samples that contained Alizarin Red S showed more cleavage of plasmid DNA than samples that lacked Alizarin Red S.

Discussion

Recent advancesin nanotechnology, have lead to the creation of nanomaterials that acquire novel properties at the nanoscale level. These novel properties may lead to the development of new nanoparticle formulations with applications in medical therapy and diagnostics. In our laboratory, we are working on the development of multi-purpose nanoparticles that can cause illumination dependent DNA cleavage and be used for magnetic resonance imaging as well. The ultimate goal of this work would be to develop a theranostic agent that would cleave DNA in an inducible and sequence specific manner that could be used for imaging and treating cancer cells simultaneously in the patient.

Currently our lab is evaluating the use of TiO₂ shell nanoparticles as a possible theranostic agent. Nano-scaleTiO₂ exhibits unique properties:TiO₂ nanoparticles that are less than 20 nM in size can covalently bind bidentate enediol ligands such as dopamine and Alizarin Red S(Rajh et al., 2001, 2002). TiO₂ is also a wide gap semiconductor that exhibits photo-reactivity. When TiO₂ is conjugated to a biological molecule such as DNA or PNA, via a dopamine linker, it still retains its photo-catalytic activity. This property can be exploited for use in sequence specific DNA damage. When a TiO₂-PNA nanoconjugate is hybridized to a target sequence in a cell, the nanoconjugate can be induced with light energies to cause sequence specific cleavage of cellular DNA (Paunesku et al., 2003). Therefore TiO₂ nanoconjugates can act as inducible endonucleases to target, in a sequence specific manner, genes of interest in a cell. This ability will allow for TiO₂-nanoconjugates to be used as a possible gene therapy to remove unwanted foreign DNA from cells without altering host DNA, such as one finds in cancer cells with mutated oncogenes.

To further extend the DNA cleavage capabilities of TiO₂ we designed experiments to determine if binding Alizarin Red S to TiO₂ nanoparticles increases ROS production in aqueous solution, thus increasing DNA damage. Alizarin Red S is a common textile dye that has been previously found to produce reactive oxygen species (ROS) when illuminated with white light, when in a solution of bulk TiO₂ (Guangming et al., 2000). The reactive oxygen species produced when Alizarin Red S undergoes photooxidation with TiO₂ (bulk) are active oxygen radicals of super oxide (O₂.⁻ ) as well as hydroxyl radicals (.OH). Both of these reactive oxygen species can elicit DNA damage. Since Alizarin Red S is a bidentate endiol ligand, it can be covalently bound to the TiO₂ nanoparticle surface. In this paper we have discussed the potential use of coating TiO₂ and Fe₃O₄@TiO₂ nanoparticles and with Alizarin Red S to enhance DNA damage.

In a series of in vitro experiments we have shown that Alizarin Red S has the ability to bind to TiO₂ nanoparticles in solution and within cells (Thurn et al., 2009, Brown et al., 2008, Figures 1–4). In vitro cleavage assays done using Fe₃O₄@TiO₂ nanoparticles coated with excess Alizarin Red S show that not only does DNA cleavage occur upon illumination, but cleavage of plasmid DNA is enhanced by coating nanoparticles with Alizarin Red S. The proposed mechanism by which Alizarin Red S cleaves DNA is based on the production of reactive oxygen species. To re-confirm the production of ROS as the main cause of plasmid DNA cleavage, we added DMSO to samples containing Alizarin Red S coated TiO₂ nanoparticles and saw a decrease in DNA cleavage. In summary, we have shown through a series of in vitro experiments that the addition Alizarin Red S to TiO₂ or Fe₃O₄@TiO₂ nanoparticles enhances the efficacy of TiO₂ and Fe₃O₄@TiO₂ nanoparticles to cause DNA damage in vitro.

Figure 4. Cleavage of plasmid DNA with varying concentrations of Alizarin Red S.

Samples were either illuminated (L) (Light exposure for 5 minutes at an intensity of 75 watts) or received no light treatment (θ) in either the presence of Alizarin Red S coated Fe3O4@TiO2 nanoparticles ( Alizarin Red S was in excess, 1.5mM) or in the presence of uncoated Fe3O4@TiO2 nanoparticles. Nicked, linear and super-coiled designates the confirmation of the plasmid.