Singlet oxygen mediated DNA degradation by copper nanoparticles: potential towards cytotoxic effect on cancer cells
© Jose et al; licensee BioMed Central Ltd. 2011
Received: 19 December 2010
Accepted: 25 March 2011
Published: 25 March 2011
The DNA degradation potential and anti-cancer activities of copper nanoparticles of 4-5 nm size are reported. A dose dependent degradation of isolated DNA molecules by copper nanoparticles through generation of singlet oxygen was observed. Singlet oxygen scavengers such as sodium azide and Tris [hydroxyl methyl] amino methane were able to prevent the DNA degradation action of copper nanoparticles confirming the involvement of activated oxygen species in the degradation process. Additionally, it was observed that the copper nanoparticles are able to exert cytotoxic effect towards U937 and Hela cells of human histiocytic lymphoma and human cervical cancer origins, respectively by inducing apoptosis. The growth characteristics of U937 and Hela cells were studied applying various concentrations of the copper nanoparticles.
Nanotechnology is one of the most rapidly growing disciplines with a wide range of applications, especially in electronics, information technology, sensor development, catalysis, and biomedical sciences [1–5]. Nanoparticles have a specific capacity for drug loading, efficient photoluminescence ability and are therefore important materials in the targeted delivery of imaging agents and anti-cancer drugs [6–9]. The extremely small size of the nanoparticles makes them to be utilized for potential target oriented delivery of nanomedicines in organs such as the brain, which are normally protected by specialized barriers (such as the blood-brain barrier). If these trends continue with nanomedicines, humans will be continuously benefited using exceedingly improved nanomaterials with diverse properties to act at the interface between nanotechnology and biology .
Continuous demand for new anti-cancer drugs has stimulated chemotherapeutic research based on the use of metals since potential drugs developed in this way may be less toxic and more prone to exhibit anti-proliferative activity against tumors [11, 12]. Transition metal complexes have been extensively studied for their nuclease-like activity using the redox properties of the metal and dioxygen to produce reactive oxygen species to promote DNA cleavage by direct strand scission or base modification . More recent trend in this area has been testing of metal nanoparticles such as gold and platinum nanoparticles for DNA degradation studies [14, 15]. Use of metal nanoparticles can be in particular advantageous in generating singlet oxygen [16, 17]. A recent report by Geddes and coworkers demonstrated that the presence of metal nanoparticles can enhance singlet oxygen generation . The enhanced electromagnetic fields in proximity to metal nanoparticles are the basis for the increased absorption and various computational methods are available to predict the extent of absorption and the relative increase in singlet oxygen generation from photosensitizers [19, 20]. Although a number of well-defined copper (II) complexes exhibited their DNA degradation capabilities [21, 22], there are no reports on in vitro study of DNA degradation using copper nanoparticles (CuNPs). A very recent study by Midander and coworkers reported the effect of CuNPs inducing single stranded breaks in the cultured human lung cells . Earlier studies showed potent cytotoxic, genotoxic and toxicological activities of CuNPs in vivo [23, 24] and in cultured cancer cell lines . However, a systematic study using CuNPs on DNA degradation and cytotoxicity towards different cancer cells are missing till to date to the best of our knowledge.
In this communication, a dose dependent DNA degradation action of copper nanoparticles (CuNPs) on isolated DNA molecules at 37°C is reported. Singlet oxygen scavengers such as sodium azide and Tris [hydroxyl methyl] amino methane were found to prevent the DNA degradation action of CuNPs and this observation confirms the involvement of activated oxygen species in the degradation process. Fluorescence quenching studies and densitometry analysis revealed the affinity of the interaction of DNA with CuNPs and the kinetics of DNA degradation by CuNPs, respectively. This study demonstrates that CuNPs can induce singlet oxygen mediated DNA damage and thus to be considered as potent cytotoxic agent to target cancer cells for the therapeutic applications. In fact, it was observed that the CuNPs could exert cytotoxic effect towards U937 and Hela cell lines of human lymphoma and cervical cancer origins, respectively by inducing apoptosis.
Where KEB = 1.0 × 107 M-1, [EB] = 1.3 μM and [CuNPs]50 is the concentration that cause a 50% quenching of the initial EB fluorescence .
Cytotoxicity of metallic copper nanoparticles, copper oxide nanoparticles and ionic copper on different cells was documented earlier [12, 32, 33]. Studer et al. specifically compared cytotoxic effect of metallic copper nanoparticles, copper oxide nanoparticles and ionic copper on Chinese Hamster Ovary (CHO) cells and Hela cells . It was observed that cytotoxic effect of carbon protected copper nanoparticles (C/Cu) towards CHO cells was less compared to CuO nanoparticles, but was greater than that of CuCl2 . In contrast, Studer et al. found that in case of Hela cells, C/Cu could not exert significant cytotoxicity while both CuO nanoparticles and CuCl2 exerted cytotoxic effect . Interestingly, in our present study, the citrate protected copper nanoparticles were able to show significant cytotoxicity towards both U937 and Hela cells as compared to CuSO4. In addition, U937 and Hela cells, after treatment with CuNPs, exhibited ultra structure and biochemical features that are characteristic of apoptosis, as shown by chromatin condensation and inter nucleosomal DNA fragmentation. The phase-contrast microscopic pictures of altered morphology of U937 and Hela cells which is characteristic of apoptotic cell stage when treated with CuNPs are shown in Figure 4c and 4e. Fluorescent microscopic studies after 4', 6-diamidino-2-phenylindole (DAPI) staining of untreated and CuNPs treated cells clearly exhibited nuclear fragmentation in CuNPs treated U937 and Hela cells which is a hallmark of cellular apoptosis (Figure 4d and 4f). Moreover, CuNPs treated U937 cells displayed a ladder pattern of inter nucleosomal DNA fragmentation on TBE-agarose gel electrophoresis in DNA ladder assay  as shown in Figure 4g (lane 3) which is also another hallmark of apoptosis. All these results demonstrate that treatment with CuNPs induce apoptosis in U937 and Hela cells.
In summary, it was observed for the first time that the copper nanoparticles can initiate the DNA degradation process and also can induce apoptotic cell death in cancer cells. The CuNPs degrade DNA in a singlet oxygen mediated fashion even in the absence of any external agents like hydrogen peroxide or ascorbate. This makes CuNPs as an excellent candidate for targeted therapy. The use of copper nanoparticles as therapeutic agents could be in particular advantageous because human body has an efficient system to deal with metabolism of copper since it is a micronutrient. So the residual copper expected to be produced during the nanoparticle based drug metabolism can be easily managed by the body. Furthermore, this DNA degradation potential and cytotoxic effect of CuNPs can be utilized in designing better and more active cancer drugs by chemically modifying the CuNPs with a number of macromolecules. Current efforts in our laboratory are underway to address these questions and to study the molecular mechanisms of CuNPs mediated cytotoxicity through apoptosis towards cancer cells of different origins.
Authors wish to thank TEM facility of Indian Association for the Cultivation of Science. Authors also want to thank Govuthami Murugesan and Pritha Dasgupta for their assistance. Authors are grateful to the Reviewers for very useful suggestions. Authors also thank Dr. P.S. Ray for valuable suggestions and Mr. Ritabrata Ghosh for technical assistance for confocal microscopy. GPJ and SS thank Council of Scientific and industrial Research, Government of India for research fellowships. SKM thanks Department of Science and Technology, India for financial support.
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